CN103341157A - Application of pingyangmycin combined sodium hyaluronate in medicine for treating venous malformation - Google Patents

Abstract

Translated fromChinese

本发明公开了平阳霉素联合透明质酸钠在治疗静脉畸形药物中的应用，以及一种用于治疗静脉畸形的药物，特别是用于治疗头颈部静脉畸形的药物。其中，透明质酸钠的分子量为600,000～1,500,000道尔顿，平阳霉素和透明质酸钠的浓度分别为10μg/mL和400μg/mL。The invention discloses the application of pingyangmycin combined with sodium hyaluronate in medicine for treating venous malformation, and a medicine for treating venous malformation, especially a medicine for treating head and neck venous malformation. Among them, the molecular weight of sodium hyaluronate is 600,000-1,500,000 Daltons, and the concentrations of pingyangmycin and sodium hyaluronate are 10 μg/mL and 400 μg/mL, respectively.

Description

Translated fromChinese

平阳霉素联合透明质酸钠在治疗静脉畸形药物中的应用Application of pingyangmycin combined with sodium hyaluronate in the treatment of venous malformations

技术领域technical field

本发明涉及平阳霉素联合透明质酸钠在治疗静脉畸形药物中的应用。The invention relates to the application of pingyangmycin combined with sodium hyaluronate in the treatment of venous malformation.

背景技术Background technique

静脉畸形(venous malformation，VM)，过去称为海绵状血管瘤，属于一种低流速脉管畸形，由局部扩张的静脉构成，病变与身体呈比例生长，终身渐近发展，不会自行消退。约40%发生于头颈部，不仅影响面容，若压迫、侵及邻近组织结构，会影响语言、吞咽和呼吸等功能，严重者有窒息死亡的危险。其治疗方法众多，现代医学主张根据患者的具体病情，选择合适的治疗手段。Venous malformation (VM), formerly known as cavernous hemangioma, is a low-velocity vascular malformation consisting of locally dilated veins that grow in proportion to the body, develop gradually throughout life, and will not regress on their own. About 40% of them occur in the head and neck, which not only affects the face, but if it oppresses and invades adjacent tissue structures, it will affect the functions of language, swallowing and breathing. In severe cases, there is a risk of suffocation and death. Its treatment method is numerous, and modern medicine advocates to select the appropriate treatment method according to the patient's specific state of an illness.

静脉畸形的发病机制尚不明确，TIE2受体突变、常染色体9P位点突变、激素水平改变等可能是其发病原因。根据回流静脉的影像学特点，静脉畸形分为4型：I型无明显回流静脉，II型回流静脉正常，III型回流静脉增粗，IV型回流静脉扩张。常见的治疗方法包括手术、硬化、激光、冷冻、电凝和铜针治疗等，这些方法各有优缺点。原则上，应该根据病变的部位、大小、范围、回流速度和技术条件，为患者制订个体化治疗方案。对静脉畸形首先考虑非手术治疗，即保守治疗，最常用的方式为硬化剂局部瘤体内注射。硬化治疗可单独应用，也可与激光、手术等治疗方式联用。常用的硬化剂有平阳霉素、5%鱼肝油酸钠、聚桂醇、无水乙醇等。The pathogenesis of venous malformation is still unclear, and TIE2 receptor mutations, autosomal 9P site mutations, and changes in hormone levels may be the cause. According to the imaging characteristics of the returning veins, venous malformations were divided into 4 types: type I no obvious returning veins, type II returning veins were normal, type III returning veins were thickened, and type IV returning veins were dilated. Common treatment methods include surgery, sclerotherapy, laser, freezing, electrocoagulation, and copper needle therapy, each of which has advantages and disadvantages. In principle, an individualized treatment plan should be formulated for the patient based on the location, size, scope, reflux rate, and technical conditions of the lesion. For venous malformation, non-surgical treatment is considered first, that is, conservative treatment. The most common way is local intratumoral injection of sclerosing agent. Sclerotherapy can be used alone or in combination with laser, surgery and other treatment methods. Commonly used sclerosing agents include pingyangmycin, 5% sodium morrhuate, lauromacrogol, and absolute ethanol.

平阳霉素（pingyangmycin，PYM）硬化治疗是目前最常用的方法之一，平阳霉素是从平阳链球菌中分离出来的一种抗生素类抗肿瘤药物，是博莱霉素A5的一种，也是一种成熟的抗肿瘤药物。由于其安全、简单和有效的特点，瘤体内平阳霉素注射已被广泛用于治疗面颈部静脉畸形。目前普遍认为，PYM治疗静脉畸形的主要机制是：与血管内皮细胞DNA结合，引起DNA链的断裂，从而抑制血管内皮细胞的代谢，致内皮细胞萎缩、变性；损伤血管内皮细胞及管壁，诱导血管平滑肌细胞和内皮细胞增生，使管壁变厚、管腔变狭窄最终闭锁。Pingyangmycin (PYM) sclerotherapy is one of the most commonly used methods at present. Pingyangmycin is an antibiotic antineoplastic drug isolated from Streptococcus pingyang. It is a kind of bleomycin A5 and also A mature antitumor drug. Due to its safety, simplicity and effectiveness, intratumoral pingyangmycin injection has been widely used in the treatment of facial and neck venous malformations. At present, it is generally believed that the main mechanism of PYM in the treatment of venous malformations is: combining with the DNA of vascular endothelial cells, causing DNA chain breaks, thereby inhibiting the metabolism of vascular endothelial cells, causing endothelial cells to atrophy and degeneration; Vascular smooth muscle cells and endothelial cells proliferate, resulting in thickening of the vessel wall, narrowing of the lumen and eventually atresia.

平阳霉素对静脉畸形血管内皮细胞的破坏作用是我们希望其发生的“有利”作用，当局部应用该药后，它不可避免地对血管内皮细胞以外的所有接触到的多种细胞产生同样的作用，即“不利”作用，或称副作用。另外，平阳霉素偶可产生严重的过敏反应，甚至造成患者死亡。大剂量应用平阳霉素可以引起肺纤维化。在头颈部等血流丰富的部位，单纯应用平阳霉素由于其在病灶部位停留时间短，造成使用剂量大，而产生副作用的风险也增大。The destructive effect of Pingyangmycin on the vascular endothelial cells of venous malformation is the "favorable" effect we hope it will have. Effect, that is, "adverse" effect, or side effect. In addition, Pingyangmycin can occasionally cause severe allergic reactions, and even cause death of patients. Large doses of Pingyangmycin can cause pulmonary fibrosis. In areas with abundant blood flow, such as the head and neck, the simple application of Pingyangmycin results in a large dosage due to its short residence time in the lesion, and the risk of side effects also increases.

发明内容Contents of the invention

本发明的目的是为克服上述现有技术的不足，提供平阳霉素联合透明质酸钠在治疗静脉畸形药物中的应用，以及一种用于治疗静脉畸形的药物，特别是用于治疗头颈部静脉畸形的药物。The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, to provide the application of Pingyangmycin combined with sodium hyaluronate in the treatment of venous malformation medicine, and a kind of medicine for the treatment of venous malformation, especially for the treatment of head and neck Drugs for venous malformations.

为实现上述目的，本发明采用下述技术方案：To achieve the above object, the present invention adopts the following technical solutions:

平阳霉素（PYM）联合透明质酸钠（HA）在治疗静脉畸形药物中的应用，其中，所述透明质酸钠的分子量为600,000～1,500,000道尔顿。Application of pingyangmycin (PYM) combined with sodium hyaluronate (HA) in the treatment of venous malformations, wherein the molecular weight of the sodium hyaluronate is 600,000-1,500,000 Daltons.

所述的平阳霉素和透明质酸钠的浓度分别为10μg/mL和400μg/mL。The concentrations of the pingyangmycin and sodium hyaluronate are 10 μg/mL and 400 μg/mL, respectively.

一种用于治疗静脉畸形的药物，它含有作为活性成分的平阳霉素和透明质酸钠，以及药物可接受的载体；其中，所述透明质酸钠的分子量为600,000～1,500,000道尔顿，平阳霉素和透明质酸钠的浓度分别为10μg/mL和400μg/mL。A medicine for treating venous malformation, which contains pingyangmycin and sodium hyaluronate as active ingredients, and a pharmaceutically acceptable carrier; wherein, the molecular weight of the sodium hyaluronate is 600,000 to 1,500,000 Daltons, The concentrations of pingyangmycin and sodium hyaluronate were 10 μg/mL and 400 μg/mL, respectively.

本发明的有益效果：Beneficial effects of the present invention:

本发明为了减缓平阳霉素的快速吸收，加入透明质酸钠，两者的联合使得平阳霉素在注射病灶较长时间内维持有效浓度，减少平阳霉素的总量，降低其副作用。另外，两者的联合能够增强平阳霉素对静脉畸形关键成分——血管内皮细胞的选择性治疗作用，增强治疗效果。In order to slow down the rapid absorption of pingyangmycin, the present invention adds sodium hyaluronate, and the combination of the two can maintain the effective concentration of pingyangmycin in the injection lesion for a long time, reduce the total amount of pingyangmycin, and reduce its side effects. In addition, the combination of the two can enhance the selective therapeutic effect of Pingyangmycin on vascular endothelial cells, the key component of venous malformation, and enhance the therapeutic effect.

透明质酸是一种广泛存在于动物和人体的生理活性物质，在人皮肤、关节滑膜液、脐带、房水及眼玻璃体中均有分布，是一种可天然降解、可吸收的生物医学材料，它具有高粘弹性、可塑性、渗透性、独特的流变学特性以及良好的生物相容性。同时，其在细胞增殖、迁移、血管生成、肿瘤发生等过程中都起着重要作用。透明质酸及其钠盐可以促进药物吸收，提高药物的生物利用度。根据透明质酸的分子量大小，可以将其分为天然大分子透明质酸和透明质酸寡糖片段，正常组织中存在的多为天然大分子量透明质酸，透明质酸寡糖片段为病理条件下天然大分子透明质酸分解而来。高分子量透明质酸发挥结构支撑与抗炎、抑制免疫反应和抗新生血管形成的作用，低分子量透明质酸具有促进炎症发生、刺激机体免疫和促进血管新生。因此，本发明选用大分子量透明质酸钠与平阳霉素联合用药。Hyaluronic acid is a physiologically active substance that widely exists in animals and humans. It is distributed in human skin, joint synovial fluid, umbilical cord, aqueous humor and eye vitreous. It is a biomedical product that can be naturally degraded and absorbed. material, it has high viscoelasticity, plasticity, permeability, unique rheological properties and good biocompatibility. At the same time, it plays an important role in cell proliferation, migration, angiogenesis, tumorigenesis and other processes. Hyaluronic acid and its sodium salt can promote drug absorption and improve drug bioavailability. According to the molecular weight of hyaluronic acid, it can be divided into natural macromolecular hyaluronic acid and hyaluronic acid oligosaccharide fragments. Most of the hyaluronic acid in normal tissues is natural large molecular weight hyaluronic acid, and hyaluronic acid oligosaccharide fragments are pathological conditions. It comes from the decomposition of natural macromolecule hyaluronic acid. High-molecular-weight hyaluronic acid plays the role of structural support and anti-inflammation, inhibiting immune response and anti-angiogenesis, while low-molecular-weight hyaluronic acid can promote inflammation, stimulate the body's immunity and promote angiogenesis. Therefore, the present invention selects high molecular weight sodium hyaluronate and pingyangmycin for use in combination.

不同组织来源的内皮细胞在形态、功能、抗原表达和代谢特征等方面存在差异，本发明一方面通过组织块培养原代细胞的方法，从病理诊断为静脉畸形组织中培养静脉畸形内皮细胞，从而反映该类疾病内皮细胞的生理生化特性，进而准确客观的反映药物的药理作用，另一方面通过平阳霉素与透明质酸钠以合适配比进行临床治疗，初步观察其治疗头颈部静脉畸形的效果。Endothelial cells from different tissue sources have differences in morphology, function, antigen expression, and metabolic characteristics. On the one hand, the present invention cultures venous malformation endothelial cells from tissues diagnosed as venous malformation through the method of culturing primary cells from tissue blocks, thereby Reflect the physiological and biochemical characteristics of the endothelial cells of this type of disease, and then accurately and objectively reflect the pharmacological effects of the drug. On the other hand, the clinical treatment of pingyangmycin and sodium hyaluronate in an appropriate ratio is initially observed in the treatment of head and neck venous malformations. Effect.

将所培养的静脉畸形内皮细胞接种在Matrigel基质胶上体外模拟血管形成实验，模拟体内相似的基质环境，由内皮细胞在Matrigel形成血管样网络结构多少来判断待测物促进还是抑制血管生成。培养12h（小时）后便观测到静脉畸形内皮细胞即在Matrigel基质上形成了血管样网状结构，24h时形成了稳定的网络结构，48h时只有空白对照组依然成管，而透明质酸钠处理组、平阳霉素处理组、平阳霉素联合透明质酸钠处理组所形成的管腔已经消退。对24h时成血管的情况进行统计，结果显示透明质酸可以抑制静脉畸形内皮细胞的血管生成作用，而当其联合平阳霉素应用时，抑制静脉畸形内皮细胞成管的作用比单独应用平阳霉素效果更佳。这证明了大分子透明质酸钠的抑制血管生成作用，而其联合平阳霉素时，大分子透明质酸钠包被平阳霉素分子，与静脉畸形内皮细胞上的受体相结合，从而携带平阳霉素穿透细胞膜，让更多的平阳霉素能够进入胞质，进而两者可以联合干扰其中的信号传导通路，而平阳霉素也可以更快捷地作用于核内的DNA，凋亡结果证明了这一点。The cultured venous malformation endothelial cells were inoculated on Matrigel to simulate the angiogenesis experiment in vitro, simulating a similar matrix environment in vivo, and the degree of vascular-like network structure formed by the endothelial cells on Matrigel was used to determine whether the analyte promoted or inhibited angiogenesis. After culturing for 12 hours (hours), it was observed that the venous malformation endothelial cells formed a blood vessel-like network structure on the Matrigel matrix, and a stable network structure was formed at 24 hours, and only the blank control group still formed tubes at 48 hours, while sodium hyaluronate The lumen formed in the treatment group, pingyangmycin treatment group, and pingyangmycin combined with sodium hyaluronate treatment group has subsided. The statistics of angiogenesis at 24 hours showed that hyaluronic acid can inhibit the angiogenesis of venous malformation endothelial cells, and when it is combined with Pingyangmycin, the effect of inhibiting venous malformation endothelial cell tube formation is better than that of Pingyangmycin alone. Vegetarian effect is better. This proves the angiogenesis-inhibiting effect of macromolecular sodium hyaluronate, and when it is combined with pingyangmycin, macromolecular sodium hyaluronate coats pingyangmycin molecules, which bind to receptors on venous malformation endothelial cells, thereby carrying Pingyangmycin penetrates the cell membrane, allowing more Pingyangmycin to enter the cytoplasm, and then the two can jointly interfere with the signal transduction pathway, and Pingyangmycin can also act on DNA in the nucleus more quickly, resulting in apoptosis It proves it.

本发明的平阳霉素和透明质酸钠以合适的浓度（透明质酸钠400μg/mL，平阳霉素10μg/mL）作用于静脉畸形内皮细胞后，与单独应用平阳霉素相比，静脉畸形内皮细胞的增殖及成血管能力降低，凋亡增强。联合应用平阳霉素和透明质酸钠能显著地抑制静脉畸形内皮细胞的增殖、成血管能力，促进其凋亡，诱发静脉畸形的消退。联合两者用药，必将对临床上静脉畸形的治疗带来新的契机。After the pingyangmycin and sodium hyaluronate of the present invention act on the endothelial cells of venous malformation at appropriate concentrations (sodium hyaluronate 400 μg/mL,pingyangmycin 10 μg/mL), compared with the application of pingyangmycin alone, the venous malformation The proliferation and angiogenesis ability of endothelial cells were reduced, and the apoptosis was enhanced. Combined application of pingyangmycin and sodium hyaluronate can significantly inhibit the proliferation and angiogenesis of venous malformation endothelial cells, promote their apoptosis, and induce the regression of venous malformation. Combining the two drugs will surely bring new opportunities for the clinical treatment of venous malformations.

组织学上，静脉畸形由衬有内皮细胞的无数血窦组成，由此以培养自人静脉畸形组织的静脉畸形内皮细胞（human vascular malformation endothelial cells,HVMECs）作为研究模型，通过联合应用平阳霉素和透明质酸的钠盐（透明质酸钠）对其进行处理，观察其在细胞增殖、血管生成、凋亡方面的作用，由此直观地观察到平阳霉素和透明质酸钠对静脉畸形的治疗效果。Histologically, venous malformation is composed of numerous sinusoids lined with endothelial cells. Therefore, human vascular malformation endothelial cells (HVMECs) cultured from human venous malformation tissue were used as a research model. Treat it with the sodium salt of hyaluronic acid (sodium hyaluronate), observe its effect on cell proliferation, angiogenesis, and apoptosis, and thus intuitively observe that pingyangmycin and sodium hyaluronate have a positive effect on venous malformation. the therapeutic effect.

用组织块培养法培养静脉畸形内皮细胞，倒置相差显微镜下观察所培养细胞的生长情况和形态特征，用内皮细胞特异性抗体兔抗人vWF（1:150）和兔抗人CD34（1:150）进行鉴定。用MTT比色法描记细胞生长曲线的变化，筛选平阳霉素和透明质酸钠分别作用于静脉畸形内皮细胞的最佳浓度。按照所确定的最佳药浓度，将所培养的细胞分别用透明质酸钠、平阳霉素、平阳霉素联合透明质酸钠进行处理，用MTT比色法检测药物对静脉畸形内皮细胞增殖的影响，用Tube formation法观测药物对静脉畸形内皮细胞成血管能力的影响，用TUNEL试剂盒，检测药物对静脉畸形内皮细胞凋亡的影响。The venous malformation endothelial cells were cultured by the tissue block culture method, and the growth and morphological characteristics of the cultured cells were observed under an inverted phase-contrast microscope. The endothelial cell-specific antibodies rabbit anti-human vWF (1:150) and rabbit anti-human CD34 (1:150) ) for identification. The changes of the cell growth curve were traced by MTT colorimetry, and the optimal concentrations of pingyangmycin and sodium hyaluronate acting on the venous malformation endothelial cells were screened. According to the determined optimal drug concentration, the cultured cells were treated with sodium hyaluronate, pingyangmycin, and pingyangmycin combined with sodium hyaluronate, and the effects of the drugs on the proliferation of venous malformation endothelial cells were detected by MTT colorimetry. Influence, the effect of drugs on the angiogenesis ability of venous malformation endothelial cells was observed by Tube formation method, and the effect of drugs on the apoptosis of venous malformation endothelial cells was detected by TUNEL kit.

组织块法培养的细胞vWF、CD34染色呈阳性表达，证实所培养细胞为静脉畸形内皮细胞。用透明质酸钠、平阳霉素单独作用于静脉畸形内皮细胞时，最佳有效浓度分别为400μg/mL、10μg/mL。MTT比色法显示透明质酸钠联合平阳霉素能更加有效抑制静脉畸形内皮细胞的增殖。Tube formation观测药物作用后静脉畸形内皮细胞成血管能力改变，结果显示平阳霉素联合透明质酸钠作用较单独应用平阳霉素细胞成血管能力下降，且成管持续时间减短，管壁消退快，与空白对照组相比有明显差异。用TUNEL试剂盒观测药物作用后静脉畸形内皮细胞凋亡能力改变，结果显示平阳霉素联合透明质酸钠作用较单独应用平阳霉素凋亡加剧，与空白对照组相比有显著差异。The staining of vWF and CD34 in cells cultured by tissue block method was positive, which confirmed that the cultured cells were endothelial cells of venous malformation. When sodium hyaluronate and pingyangmycin act on endothelial cells of venous malformation alone, the optimal effective concentrations are 400 μg/mL and 10 μg/mL, respectively. MTT colorimetry showed that sodium hyaluronate combined with pingyangmycin could more effectively inhibit the proliferation of venous malformation endothelial cells. Tube formation observed the changes in the angiogenesis ability of venous malformation endothelial cells after drug action, and the results showed that the angiogenesis ability of Pingyangmycin combined with sodium hyaluronate was lower than that of Pingyangmycin alone, and the duration of tube formation was shortened, and the wall of the tube disappeared faster , which were significantly different from the blank control group. TUNEL kit was used to observe the changes in the apoptosis ability of venous malformation endothelial cells after drug action. The results showed that the effect of pingyangmycin combined with sodium hyaluronate was more severe than that of pingyangmycin alone, and there was a significant difference compared with the blank control group.

平阳霉素联合透明质酸钠对50余例头颈部静脉畸形进行治疗，发现临床效果极其显著。Pingyangmycin combined with sodium hyaluronate treated more than 50 cases of head and neck venous malformations, and the clinical effect was found to be extremely significant.

附图说明Description of drawings

图1a是不同质量浓度平阳霉素作用不同时间对HVMECs的增殖作用效果，图1b是不同质量浓度透明质酸钠作用不同时间对HVMECs的增殖抑制效果；Figure 1a is the effect of different mass concentrations of pingyangmycin on the proliferation of HVMECs at different times, and Figure 1b is the effect of different mass concentrations of sodium hyaluronate on the proliferation of HVMECs at different times;

图2是加入药物后的OD值曲线；Fig. 2 is the OD value curve after adding medicine;

图3是细胞干扰前后的生长曲线；Fig. 3 is the growth curve before and after cell interference;

图4是药物干扰前后细胞成血管能力的差异；Figure 4 is the difference in the ability of cells to form angiogenesis before and after drug interference;

图5是药物干扰前后细胞凋亡能力的差异；Figure 5 is the difference in apoptosis ability before and after drug interference;

图6是组织块法培养静脉畸形内皮细胞，呈铺路石样贴壁；Figure 6 shows the culture of venous malformation endothelial cells by the tissue block method, showing paving stone-like adherence;

图7是第一代静脉畸形内皮细胞，成纤维样外观，胞浆均匀清晰，核圆形或卵圆形，并有不同程度的增大；Figure 7 is the first generation of venous malformation endothelial cells, with a fibroblast-like appearance, uniform and clear cytoplasm, round or oval nuclei, and different degrees of enlargement;

图8是静脉畸形内皮细胞，内皮细胞标志物vwf染色呈阳性表达，主要表达于胞质，细胞核表面未见明显表达；Figure 8 is a venous malformation endothelial cell, the endothelial cell marker vwf staining is positive, mainly expressed in the cytoplasm, and there is no obvious expression on the surface of the nucleus;

图9是静脉畸形内皮细胞，内皮细胞标志物CD34染色呈阳性表达，主要表达于胞质，细胞核表面未见明显表达；Figure 9 shows the endothelial cells of venous malformation. The staining of endothelial cell marker CD34 is positive, mainly expressed in the cytoplasm, and no obvious expression is seen on the surface of the nucleus;

图10是经不同药物处理48h后静脉畸形内皮细胞生长情况，（a）control组，细胞呈指数增长，大量增殖，细胞间接触超过80%，达传代的密度；（b）HA处理组，细胞生长缓慢，增殖收到一定影响；（c）PYM处理组，细胞生长停滞，大量死亡，可见碎裂的细胞残片；（d）PYM联合HA处理组，尚存少量存活的细胞；Figure 10 shows the growth of venous malformation endothelial cells after 48 hours of treatment with different drugs. (a) In the control group, the cells showed exponential growth and proliferated in large quantities, and the contact between cells exceeded 80%, reaching the density of passage; Growth was slow, and proliferation was affected to a certain extent; (c) PYM treatment group, cell growth stagnation, a large number of death, and fragmented cell fragments were seen; (d) PYM combined with HA treatment group, there were still a small number of surviving cells;

图11是药物干扰前后细胞成血管图；（a）12h时，Control组已成血管规则，管腔完整；HA组已成血管规则，管腔完整，PYM组和HA联合PYM组细胞依然杂乱无规则分布；（b）24h时，四组均已成管，但Control组和HA组成血管规则且管腔比较完整，Control组所形成的结点数更多和管壁更厚，而PYM组和PYM+HA组细胞大部分聚集成堆，仅有微弱的成血管能力；（c）48h时，观察已有大量细胞死亡，已成血管的管壁开始萎缩，NC组、HA组依然保持着一定的血管形状，而PYM组、PYM+HA组细胞聚堆萎缩显著，已形成的管腔亦消退明显；Figure 11 is the diagram of cell angiogenesis before and after drug interference; (a) At 12 hours, the control group had regular blood vessels with a complete lumen; the HA group had regular blood vessels and a complete lumen, while the cells in the PYM group and the HA combined with PYM group were still messy Regular distribution; (b) At 24 hours, all four groups had formed tubes, but the control group and HA formed regular vessels with relatively complete lumens, and the Control group formed more nodes and thicker tube walls, while the PYM group and PYM Most of the cells in the +HA group aggregated into piles, with only weak angiogenesis ability; (c) At 48 hours, a large number of cells were observed to die, and the wall of the blood vessel began to shrink, while the NC group and HA group still maintained a certain degree The shape of blood vessels, while the cells in PYM group and PYM+HA group shrank significantly, and the formed lumen also disappeared significantly;

图12是TUNEL法检测细胞凋亡，正常细胞核被染成蓝色，凋亡细胞核呈黄色至棕色，（a）control组，少量细胞出现凋亡状态；（b）HA处理组，约半数细胞出现凋亡状态；（c）PYM处理组，仅少量细胞染色核正常；（d）PYM联合HA处理组，无正常细胞存在。Figure 12 is the detection of cell apoptosis by TUNEL method. Normal cell nuclei are stained blue, and apoptotic cell nuclei are yellow to brown. (a) In the control group, a small number of cells appear in apoptotic state; (b) in the HA treatment group, about half of the cells appear Apoptotic state; (c) PYM treatment group, only a small number of cells with normal staining nuclei; (d) PYM combined with HA treatment group, no normal cells existed.

具体实施方式Detailed ways

下面结合附图和实施例对本发明进行进一步的阐述，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and not limiting its content.

实施例1：Example 1:

本发明所涉及的一种用于治疗静脉畸形的药物的制备：The preparation of a kind of medicine that is used for the treatment of venous malformation involved in the present invention:

取平阳霉素0.1mg，透明质酸钠4mg，加入制备注射剂的药物可接受的载体，配制得到10mL注射剂。由于瘤体中的液体不可能完全抽出来而被置换成该浓度的药物，可以适当调整平阳霉素和透明质酸钠的用量，使进入到瘤体内的平阳霉素和透明质酸钠的实际浓度约为10μg/mL和400μg/mL。Take pingyangmycin 0.1 mg, sodium hyaluronate 4 mg, add pharmaceutically acceptable carrier for preparation of injection, and prepare 10 mL injection. Because the liquid in the tumor cannot be completely pumped out and replaced with drugs of this concentration, the dosage of Pingyangmycin and sodium hyaluronate can be adjusted appropriately so that the actual amount of Pingyangmycin and sodium hyaluronate entering the tumor The concentration is about 10μg/mL and 400μg/mL.

透明质酸钠为市购的玻璃酸钠注射液，山东博士伦福瑞达制药有限公司，批准文号：国药准字H10960136。Sodium hyaluronate is commercially available sodium hyaluronate injection, Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd., approval number: National Drug Accreditation H10960136.

下面通过药效学实验来进一步说明其药物活性。The following pharmacodynamic experiments will further illustrate its drug activity.

试验例Test case

1.主要仪器设备1. Main equipment

超净工作台（哈尔滨东联电子技术开发有限公司）；Ultra-clean workbench (Harbin Donglian Electronic Technology Development Co., Ltd.);

超纯水及除菌系统（MILLI-Q，法国）；Ultrapure water and sterilization system (MILLI-Q, France);

高压蒸汽灭菌器（HIRAYAMA，日本）；Autoclave (HIRAYAMA, Japan);

高速低温离心机（THERMO，德国）；High-speed low-temperature centrifuge (THERMO, Germany);

电热恒温水槽（上海精宏实验设备有限公司）；Electric constant temperature water tank (Shanghai Jinghong Experimental Equipment Co., Ltd.);

恒温CO₂细胞培养孵箱（德国Heraeus公司）；Constant temperature_CO2 cell culture incubator (Germany Heraeus);

制冰机，低温冰箱（SANYO，日本）；Ice machine, low temperature refrigerator (SANYO, Japan);

4℃，-20℃冰箱（合肥美菱股份有限公司）；4℃, -20℃ refrigerator (Hefei Meiling Co., Ltd.);

倒置相差显微镜及照相系统（LEICA，德国）；Inverted phase contrast microscope and camera system (LEICA, Germany);

荧光显微镜及照相系统（LEICA，德国）；Fluorescence microscope and camera system (LEICA, Germany);

电子分析天平（METTLER TOLEDO，瑞士）；Electronic analytical balance (METTLER TOLEDO, Switzerland);

电热恒温干燥箱（上海精宏实验设备有限公司）；Electric constant temperature drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.);

低氧恒温细胞培养箱（THERMO，美国）；Hypoxic constant temperature cell incubator (THERMO, USA);

微量移液器（GIBCO）；Micropipette (GIBCO);

测重器、精密天平、PH计（METTLER TOLEDO）；Weighing device, precision balance, PH meter (METTLER TOLEDO);

紫外/可见光分光光度仪（美国Biochrom公司）；UV/visible spectrophotometer (Biochrom, USA);

STS-8A脱色摇床（QILINBEIER）。STS-8A decolorization shaker (QILINBEIER).

常规外科器械：眼科剪，眼科镊，刀片，持针器，止血钳，探针，刀片等；Conventional surgical instruments: ophthalmic scissors, ophthalmic forceps, blades, needle holders, hemostats, probes, blades, etc.;

一次性培养皿，一次性培养瓶、培养板、15mL离心管、50mL离心管（NUNC）；Disposable culture dish, disposable culture bottle, culture plate, 15mL centrifuge tube, 50mL centrifuge tube (NUNC);

2.主要试剂及配制2. Main reagents and preparation

（1）ECM培养基，购自ScienCell公司。(1) ECM medium, purchased from ScienCell Company.

（2）胎牛血清FBS，购自ScienCell公司。(2) Fetal bovine serum FBS was purchased from ScienCell.

（3）质量浓度0.25%胰蛋白酶，购自GIBCO公司。(3) Trypsin with a mass concentration of 0.25% was purchased from GIBCO.

（4）PBS缓冲液，购自Sergene公司。(4) PBS buffer, purchased from Sergene Company.

（5）DMSO，购自SIGMA公司。(5) DMSO, purchased from SIGMA Company.

（6）DAB显色液，购自gene tech。(6) DAB chromogenic solution, purchased from Gene Tech.

（7）HRP标记山羊抗兔IgG，工作浓度为1:3000，购自gene tech。(7) HRP-labeled goat anti-rabbit IgG, the working concentration is 1:3000, purchased from Gene Tech.

（8）vWF兔抗人多克隆抗体，ready to use，购自gene tech。(8) vWF rabbit anti-human polyclonal antibody, ready to use, purchased from Gene Tech.

（9）CD34兔抗人多克隆抗体，ready to use，购自gene tech。(9) CD34 rabbit anti-human polyclonal antibody, ready to use, purchased from Gene Tech.

（10）TUNEL凋亡检测试剂盒，购自Roche公司。(10) TUNEL Apoptosis Detection Kit, purchased from Roche Company.

（11）MTT溶液，用前配成5mg/mL的溶液，购自SIGMA公司。(11) MTT solution, made into a 5 mg/mL solution before use, purchased from SIGMA Company.

Matrigel基质胶，购自美国碧迪公司。Matrigel Matrigel was purchased from American Bidi Company.

3.实验方法：3. Experimental method:

3.1细胞培养3.1 Cell culture

A：组织块法培养细胞A: Tissue block method to culture cells

1）用无菌PBS缓冲液冲洗手术切除静脉畸形标本（来自头颈部静脉畸形手术切除的新鲜组织）至清亮、无混浊及脱落血性组织，用眼科剪及镊子去除多余皮肤及脂肪组织；1) Rinse surgically resected venous malformation specimens (fresh tissue from head and neck venous malformation surgery) with sterile PBS buffer until clear, free of turbidity and exfoliated bloody tissue, and remove excess skin and fat tissue with ophthalmic scissors and tweezers;

2）修剪成1～2cm³组织块，放入37℃的质量浓度0.25%胰蛋白酶溶液中，水浴振荡消化10～20分钟；2) Trim it into^1-2cm3 tissue pieces, put them in 0.25% trypsin solution at 37°C, shake and digest in a water bath for 10-20 minutes;

3）加入含胎牛血清的培养液终止消化，并将消化后的组织块修剪成1mm³；3) Add culture medium containing fetal bovine serum to stop digestion, and trim the digested tissue pieces to 1mm³ ;

4）将修剪好的组织块接种于培养瓶中，将培养瓶放于37℃孵箱中倒置培养10分钟；4) Inoculate the trimmed tissue pieces into a culture bottle, and place the culture bottle upside down in a 37°C incubator for 10 minutes;

5）小心向培养瓶中加入培养基，轻摇培养瓶，不要使组织块漂浮，液面正好盖过组织块；5) Carefully add the culture medium to the culture bottle, shake the culture bottle lightly, do not make the tissue pieces float, and the liquid level just covers the tissue pieces;

6）放入孵箱（37℃，体积分数5%CO₂）中培养，3～4天换液，5～6天去除组织块，7～9天传代。6) Culture in an incubator (37°C, 5% CO₂ ), replace the medium in 3 to 4 days, remove tissue pieces in 5 to 6 days, and pass passage in 7 to 9 days.

B：细胞传代B: Cell passage

1）弃掉培养瓶中的培养液，用无菌PBS冲洗2～3遍，加入1mL0.25%胰蛋白酶溶液培养，置于37℃约1分钟，于倒置显微镜下观察细胞的形态变化。当细胞突起回缩，形态变圆，细胞间隙增大，同时有少量脱壁细胞时，立即终止消化，吸出消化液并加入少许ECM完全培养基洗净消化液，吸除液体，加入ECM培养基；1) Discard the culture medium in the culture bottle, wash it with sterile PBS 2 to 3 times, add 1 mL of 0.25% trypsin solution to incubate, place at 37°C for about 1 minute, and observe the morphological changes of the cells under an inverted microscope. When the cell protrusions retract, the shape becomes round, the intercellular space increases, and there are a small amount of detachment cells, stop the digestion immediately, suck out the digestion solution and add a little ECM complete medium to wash the digestion solution, absorb the liquid, and add ECM medium ;

2）从培养瓶底部的一边至另一边依次反复吹打瓶壁上的细胞，使细胞脱离瓶壁后制成单细胞悬液，吹打时动作轻柔，尽可能不产生气泡；2) Blow and beat the cells on the bottle wall repeatedly from one side of the bottom of the culture bottle to the other side, so that the cells are separated from the bottle wall and made into a single-cell suspension. When blowing and blowing, the action is gentle, and no air bubbles are generated as much as possible;

3）将获得的单细胞悬液转移至离心管中，进行离心分离（800转/分转速离心7分钟），弃上清液，加入完全ECM培养基，将细胞团片轻轻吹打为单细胞悬液，尽可能不产生气泡；以5×10⁵/mL接种于培养瓶和培养板中，进行传代并实验。3) Transfer the obtained single-cell suspension to a centrifuge tube, perform centrifugation (800 rpm for 7 minutes), discard the supernatant, add complete ECM medium, and gently blow the cell pellets into single cells Suspension, without generating air bubbles as much as possible; inoculate culture flasks and culture plates at 5×10⁵ /mL for passage and experiment.

C：细胞冻存与复苏：C: Cell cryopreservation and recovery:

（一）细胞冻存(1) Cell cryopreservation

1）配制胎牛血清与DMSO体积比为9:1的冻存培养液；1) Prepare a frozen culture medium with a volume ratio of fetal bovine serum and DMSO of 9:1;

2）取对数生长期的细胞，用胰蛋白酶把单层生长的细胞消化下来，将细胞移至15mL离心管中，离心1000rpm，5min；2) Take the cells in the logarithmic growth phase, digest the monolayer-growing cells with trypsin, transfer the cells to a 15mL centrifuge tube, and centrifuge at 1000rpm for 5min;

3）去除胰蛋白酶及旧培养液，加入适量配制好的冻存培养液，用吸管轻轻吹打使细胞均匀并计数，调节冻存液中细胞的最终密度为5×10⁶/mL～1×10⁷/mL，将细胞分装入冻存管中，每管1～1.5mL；3) Remove trypsin and old culture medium, add an appropriate amount of prepared frozen culture medium, blow gently with a pipette to make the cells uniform and count, and adjust the final density of cells in the frozen medium to 5×10⁶ /mL～1× 10⁷ /mL, divide the cells into cryopreservation tubes, 1-1.5 mL per tube;

4）在冻存管上标明细胞的名称，冻存时间及操作者；4) Mark the name of the cells, the time of freezing and the operator on the cryopreservation tube;

5）将冻存管放入程序降温盒中，然后放入-80℃冰箱中过夜，取出冻存管，移入液氮容器内。5) Put the cryopreservation tube into the programmed cooling box, then put it in a -80°C refrigerator overnight, take out the cryopreservation tube, and move it into a liquid nitrogen container.

（二）细胞复苏(2) Cell recovery

1）从液氮容器中取出冻存管，直接浸入37℃温水中，并不时摇动令其尽快融化；1) Take out the cryotube from the liquid nitrogen container, immerse it directly in warm water at 37°C, and shake it from time to time to melt it as soon as possible;

2）从37℃水浴中取出冻存管，打开盖子，用吸管吸出细胞悬液，加到离心管并滴加10倍以上培养液，混匀后离心，1000rpm，5min；2) Take out the cryopreservation tube from the 37°C water bath, open the lid, suck out the cell suspension with a straw, add it to the centrifuge tube and drop more than 10 times of the culture medium, mix well and centrifuge at 1000rpm for 5min;

3）弃去上清液，加入完全ECM培养基重悬细胞，计数，调整细胞密度，接种培养瓶，37℃培养箱静置培养；3) Discard the supernatant, add complete ECM medium to resuspend the cells, count, adjust the cell density, inoculate the culture flask, and culture in a 37°C incubator;

4）第二天更换一次培养液，继续培养，细胞传代（2～3天换液、5～6天传代），随时观察与拍照。4) Replace the culture medium on the second day, continue to culture, and subculture the cells (change the medium on 2-3 days, passage on 5-6 days), observe and take pictures at any time.

D.HVMECs的形态学观察及鉴定D. Morphological observation and identification of HVMECs

（一）形态学观察：倒置相差显微镜下观察HVMECs的生长情况和形态特征。(1) Morphological observation: The growth and morphological characteristics of HVMECs were observed under an inverted phase-contrast microscope.

（二）免疫组化EnVision法鉴定(2) Identification by immunohistochemical EnVision method

1）将原代HVMECs接种至24孔板中制作细胞爬片，待细胞密度约70%时，取出爬片，PBS冲洗3次；1) Inoculate the primary HVMECs into a 24-well plate to make cell slides. When the cell density is about 70%, take out the slides and wash them with PBS for 3 times;

2）质量浓度4%多聚甲醛室温下固定10min，PBS冲洗3次；2) Fix with 4% paraformaldehyde at room temperature for 10 minutes, wash with PBS for 3 times;

3）质量浓度0.3%H₂O₂与甲醇等体积混合液室温下孵育10min，去除内源性过氧化物酶，PBS冲洗3次；3) Incubate at room temperature for 10 minutes with a mixture of 0.3% H₂ O₂ and methanol at room temperature to remove endogenous peroxidase, and wash with PBS for 3 times;

4）质量浓度10%羊血清封闭10min，分别加入兔抗人vWF（1:150）和鼠抗人CD34（1:150），37℃下孵育2h，PBS冲洗3次；4) Block with 10% goat serum for 10 minutes, add rabbit anti-human vWF (1:150) and mouse anti-human CD34 (1:150), incubate at 37°C for 2 hours, wash with PBS three times;

5）加入HRP标记的山羊抗兔IgG，37℃下孵育1h，PBS冲洗后，DAB显色，苏木素复染，体积浓度为0.5%的盐酸酒精（浓盐酸配比体积浓度比为75%的酒精）分化，体积浓度为5%氨水返蓝，封片，拍照。5) Add HRP-labeled goat anti-rabbit IgG, incubate at 37°C for 1 hour, wash with PBS, develop color with DAB, counterstain with hematoxylin, and use 0.5% hydrochloric acid alcohol (concentrated hydrochloric acid with a volume concentration ratio of 75% alcohol) ) differentiation, the volume concentration was 5% ammonia water and turned blue, mounted and photographed.

3.2选定HA、PYM抑制HVMECs的最佳浓度3.2 Select the optimal concentration of HA and PYM to inhibit HVMECs

将HVMECs以2×10⁴个/mL浓度接种于96孔板，每孔200μL，培养48h后更换培养液。处理组分为PYM处理组和HA处理组，每个处理组加入5个不同浓度梯度（PYM：1、5、10、20、50μg/mL；HA:10、100、200、500、1000μg/mL）的药物，并设空白对照组（未加药物），培养不同时间（0、12、24、48、72h）后，每孔加入MTT溶液（5mg/mL）20μL，继续孵育4h，吸弃培养液，加入150μL二甲基亚砜（DMSO），振荡10min，于酶联免疫检测仪上测波长为490nm的吸光度（OD）值，实验重复3次，取3次数据的平均值，选取合适的PYM和HA作用与HVMECs的浓度。HVMECs were inoculated in 96-well plates at a concentration of 2×10⁴ cells/mL, 200 μL per well, and the culture medium was replaced after 48 h of culture. The treatment groups were divided into PYM treatment group and HA treatment group, and five different concentration gradients were added to each treatment group (PYM: 1, 5, 10, 20, 50 μg/mL; HA: 10, 100, 200, 500, 1000 μg/mL ), and set up a blank control group (no drug added), after culturing for different time (0, 12, 24, 48, 72h), add 20μL of MTT solution (5mg/mL) to each well, continue to incubate for 4h, and discard the culture solution, add 150 μL dimethyl sulfoxide (DMSO), shake for 10 minutes, measure the absorbance (OD) value at a wavelength of 490 nm on an enzyme-linked immunosorbent assay instrument, repeat the experiment 3 times, take the average value of the 3 times of data, and select a suitable The effect of PYM and HA on the concentration of HVMECs.

3.3MTT法检测药物处理后各组的增殖能力及活力3.3MTT method to detect the proliferation ability and activity of each group after drug treatment

A.实验分组A. Experimental grouping

1）取生长稳定的HVMECs进行实验，共分为4组：1) HVMECs with stable growth were used for experiments, and they were divided into 4 groups:

第1组：HVMECs培养组；Group 1: HVMECs culture group;

第2组：HA干扰HVMECs培养组；Group 2: HA interference HVMECs culture group;

第3组：PYM干扰HVMECs培养组；Group 3: PYM interference HVMECs culture group;

第4组：HA联合PYM干扰HVMECs培养组；Group 4: HA combined with PYM interfered with HVMECs culture group;

2）各组细胞均以1×10³/孔接种于96孔培养板内，每组5孔；培养基为完全ECM培养基；每2～3天更换一次培养基，培养3天后，自第四天开始分别取12h，24h，48h，72h的时间段对三组细胞进行MTT检测，共4次。2) Cells in each group were inoculated in 96-well culture plates at 1×10³ /well, 5 wells in each group; the medium was complete ECM medium; the medium was replaced every 2 to 3 days, and after 3 days of culture, the From the beginning of four days, the three groups of cells were tested for MTT at time periods of 12h, 24h, 48h, and 72h, a total of 4 times.

B.MTT法检测各组细胞的增殖能力及其活力B. MTT method to detect the proliferation ability and viability of cells in each group

吸取孔内旧培养基，用不含FBS的ECM培养基冲洗后，更换培养基为含5%FBS的ECM培养基，同时每孔加入质量浓度0.5%的MTT20μL，继续在37℃细胞培养箱培养4h后，吸去孔内培养液，于每孔中加入150μL的DMSO，震荡10min，在分光光度仪490nm波长下测定吸光值（OD值）。所测OD值用均数±标准差表示，绘制各组细胞生长曲线，采用SPSS统计软件处理，用SPSS软件进行方差分析和q检验，P<0.05为差异有统计学意义。Aspirate the old medium in the well, wash it with ECM medium without FBS, replace the medium with ECM medium containing 5% FBS, and add 20 μL of MTT with a mass concentration of 0.5% to each well, and continue to culture in a 37°C cell culture incubator After 4 hours, the culture medium in the wells was sucked off, 150 μL of DMSO was added to each well, shaken for 10 minutes, and the absorbance value (OD value) was measured at a wavelength of 490 nm by a spectrophotometer. The measured OD values were expressed as mean ± standard deviation, and the cell growth curves of each group were drawn, processed with SPSS statistical software, and SPSS software was used for analysis of variance and q test. P<0.05 was considered statistically significant.

3.4Tube Formation方法：3.4Tube Formation method:

1）将Matrigel基质胶4℃过夜；1) Put Matrigel overnight at 4°C;

低温下将200μL/孔Matrigel基质胶平铺孔底，共6孔，分4组，每组3个复孔。At low temperature, 200 μL/well of Matrigel was spread on the bottom of the wells, a total of 6 wells were divided into 4 groups, and each group had 3 replicate wells.

第1组：HVMECs培养组；Group 1: HVMECs culture group;

第2组：HA干扰HVMECs培养组；Group 2: HA interference HVMECs culture group;

第3组：PYM干扰HVMECs培养组；Group 3: PYM interference HVMECs culture group;

第4组：HA联合PYM干扰HVMECs培养组；Group 4: HA combined with PYM interfered with HVMECs culture group;

2）放入37℃恒温孵箱中30～60分钟；2) Put it in a constant temperature incubator at 37°C for 30-60 minutes;

3）每孔接种10万个细胞，加培养基1mL；3) Inoculate 100,000 cells per well and add 1 mL of culture medium;

4）37℃培养，第1组用于对比观察，第2组与第3组在随时镜下观察照片，并对24h成管量化后进行统计学分析。4) Cultivate at 37°C. Group 1 is used for comparative observation. Group 2 and Group 3 observe the photos under the microscope at any time, and perform statistical analysis after quantifying the 24-hour tube formation.

3.5TUNEL凋亡检测方法：3.5 TUNEL apoptosis detection method:

实验分组：Experimental group:

第1组：HVMECs培养组；Group 1: HVMECs culture group;

第2组：HA干扰HVMECs培养组；Group 2: HA interference HVMECs culture group;

第3组：PYM干扰HVMECs培养组；Group 3: PYM interference HVMECs culture group;

第4组：HA联合PYM干扰HVMECs培养组；Group 4: HA combined with PYM interfered with HVMECs culture group;

1）提前一天种板，待细胞融合率达50%时即可进行凋亡检测；1) Plant the plate one day in advance, and perform apoptosis detection when the cell fusion rate reaches 50%;

2）质量浓度4%的多聚甲醛固定1h，PBS冲洗5min×3次；2) Fix with 4% paraformaldehyde for 1 hour, rinse with PBS for 5 minutes x 3 times;

3）质量浓度3%H₂O₂阻断液室温作用10min，PBS冲洗5min×3次；3) Blocking solution with a mass concentration of 3% H₂ O₂ acts at room temperature for 10 minutes, rinses with PBS for 5 minutes x 3 times;

4）加渗透液（质量浓度0.1%Triton-X100柠檬酸钠缓冲液）4℃20min，PBS冲洗5min×3次；4) Add permeate solution (mass concentration 0.1% Triton-X100 sodium citrate buffer solution) at 4°C for 20 min, wash with PBS for 5 min x 3 times;

5）加50μL TUNEL反应混合液，加上覆盖膜，湿盒中37℃作用1h，PBS冲洗5min×3次；5) Add 50 μL of TUNEL reaction mixture, add cover film, act in a wet box at 37°C for 1 hour, wash with PBS for 5 minutes×3 times;

6）质量浓度3%的小牛血清蛋白37℃封闭20min，PBS冲洗5min×3次；6) Block with 3% bovine serum albumin at 37°C for 20 minutes, wash with PBS for 5 minutes x 3 times;

7）加50μL转换POD溶液，加上覆盖膜，湿盒中37℃作用30min，PBS冲洗5min×3次；7) Add 50 μL of converted POD solution, add a cover film, act in a wet box at 37°C for 30 minutes, rinse with PBS for 5 minutes×3 times;

8）加50μL DAB底物（Roche公司），15℃-25℃湿盒中避光显色10min，PBS冲洗5min×3次；8) Add 50 μL of DAB substrate (Roche Company), develop color in a humid chamber at 15°C-25°C for 10 minutes in the dark, wash with PBS for 5 minutes×3 times;

9）常规进行苏木素复染，盐酸酒精分化，氨水返蓝。封片，拍照。9) Routinely carry out hematoxylin counterstaining, hydrochloric acid alcohol differentiation, and ammonia water to turn blue. Seal the film and take a picture.

10）选定区域，计算一定面积的区域中凋亡细胞的个数及比率；ANOVA及q检验检测4组细胞凋亡差异。10) Select an area, calculate the number and ratio of apoptotic cells in a certain area; ANOVA and q test detect the difference in apoptosis among the 4 groups.

4.实验结果4. Experimental results

4.1静脉畸形内皮细胞形态及鉴定：4.1 Morphology and identification of venous malformation endothelial cells:

组织块法培养静脉畸形内皮细胞，细胞贴壁后表现为铺路石样（图6），胞浆均匀清晰，核圆形或卵圆形，并有不同程度的增大，集簇生长（图7）；利用免疫细胞化学的方法检测两种内皮细胞标志性抗原vWF和CD34，均呈阳性表达（图8～9），证实所培养的细胞为静脉畸形内皮细胞。The tissue block method was used to culture venous malformation endothelial cells. After the cells adhered to the wall, they appeared as paving stones (Figure 6), with uniform and clear cytoplasm, round or oval nuclei, and different degrees of enlargement, clustered growth (Figure 7 ); two endothelial cell marker antigens vWF and CD34 were detected by immunocytochemistry, both of which were positively expressed (Fig. 8-9), confirming that the cultured cells were venous malformation endothelial cells.

4.2选定HA、PYM抑制HVMECs的最佳浓度4.2 Select the optimal concentration of HA and PYM to inhibit HVMECs

不同质量浓度平阳霉素作用不同时间对HVMECs的增殖作用效果见图1（a）。在相同平阳霉素质量浓度作用下，随着作用时间的延长，细胞的增殖抑制率逐渐增加；而在同一作用时间段，随质量浓度的增大，细胞的增殖抑制率亦逐渐增加，显示出剂量依赖效应和时间依赖效应，本实验的平阳霉素组验证了这一点。在平阳霉素浓度≥10μg/mL时，对HVMECs的增殖有明显的抑制作用，因此本实验联合透明质酸钠作用于HVMECs时所用的平阳霉素浓度为10μg/mL。The effect of different concentrations of pingyangmycin on the proliferation of HVMECs at different times is shown in Figure 1 (a). Under the same mass concentration of pingyangmycin, as the action time prolongs, the cell proliferation inhibition rate gradually increases; while in the same action time period, with the increase of mass concentration, the cell proliferation inhibition rate also gradually increases, showing that The dose-dependent effect and time-dependent effect were verified by the Pingyangmycin group in this experiment. When the concentration of pingyangmycin is ≥10 μg/mL, it has obvious inhibitory effect on the proliferation of HVMECs. Therefore, the concentration of pingyangmycin used in this experiment combined with sodium hyaluronate to act on HVMECs is 10 μg/mL.

不同质量浓度透明质酸钠作用不同时间对HVMECs的增殖抑制效果见图1（b），同样显示出剂量依赖效应和时间依赖效应，在高浓度时能轻微抑制HVMEVCs增殖，从400μg/mL时开始出现抑制效果，因此联合用药时选用的HA的浓度为400μg/mL。The inhibitory effect of different mass concentrations of sodium hyaluronate on the proliferation of HVMECs at different times is shown in Figure 1(b), which also shows dose-dependent and time-dependent effects, and can slightly inhibit the proliferation of HVMECs at high concentrations, starting from 400 μg/mL Inhibitory effect occurs, so the concentration of HA used in combination is 400 μg/mL.

4.3MTT法检测细胞增殖活力及生存能力：4.3MTT method to detect cell proliferation activity and viability:

自加入药物后开始计时，分别检测0h,12h,24h,48h,72h的四组之间的OD值，并描记曲线，如图2。Start timing after adding the drug, respectively detect the OD values among the four groups of 0h, 12h, 24h, 48h, and 72h, and trace the curve, as shown in Figure 2.

自加入药物后开始计时，分别检测0h,12h,24h,48h,72h的四组之间的OD值，并计算其对HVMECs的抑制率，如表1。The time was started after adding the drug, and the OD values among the four groups of 0h, 12h, 24h, 48h, and 72h were detected respectively, and the inhibition rate on HVMECs was calculated, as shown in Table 1.

表1.不同的药物随时间对HVMECs增殖的平均抑制率Table 1. The average inhibition rate of different drugs on the proliferation of HVMECs over time

以上结果显示：The above results show:

0h：各组之间无明显差别。0h: There is no significant difference between the groups.

12h：PYM+HA组、PYM组与Control组、HA组相比差异明显，***P<0.001；PYM+HA组和PYM组之间差异无统计学意义。12h: There were significant differences between the PYM+HA group and the PYM group compared with the Control group and the HA group, ***P<0.001; there was no statistically significant difference between the PYM+HA group and the PYM group.

24h：PYM+HA组、PYM组与Control组、HA组相比差异明显，***P<0.001；PYM+HA组和PYM组之间有统计学差异，*P<0.05。24h: There were significant differences between the PYM+HA group and the PYM group compared with the Control group and the HA group, ***P<0.001; there was a statistical difference between the PYM+HA group and the PYM group, *P<0.05.

48h：PYM+HA组、PYM组与Control组、HA组相比差异明显，***P<0.001；PYM+HA组和PYM组之间有较24h差异更加明显，*P<0.05。48h: The difference between PYM+HA group, PYM group and Control group, HA group was significantly different, ***P<0.001; the difference between PYM+HA group and PYM group was more obvious than that at 24h, *P<0.05.

72h：PYM+HA组、PYM组与Control组、HA组相比差异明显，***P<0.001；PYM+HA组和PYM组之间有较48h差异更加明显，*P<0.05。72h: The difference between PYM+HA group, PYM group and Control group, HA group was significantly different, ***P<0.001; the difference between PYM+HA group and PYM group was more obvious than that at 48h, *P<0.05.

平阳霉素联合透明质酸钠较单独应用平阳霉素对静脉畸形内皮细胞的抑制随时间愈加明显。The inhibition of pingyangmycin combined with sodium hyaluronate on venous malformation endothelial cells is more obvious with time than that of pingyangmycin alone.

描记细胞干扰前后的生长曲线，如图3，细胞生长状况见图10。The growth curves before and after cell interference were traced, as shown in Figure 3, and the growth status of the cells was shown in Figure 10.

综上可知，细胞培养液中添加了PYM后，细胞增殖能力受抑制，细胞活力及生存能力下降，PYM和HA联合应用时效果更明显，随着时间推移，显示出HA的缓释。In summary, after adding PYM to the cell culture medium, the cell proliferation ability is inhibited, and the cell viability and viability are reduced. The effect is more obvious when PYM and HA are combined, and the sustained release of HA is shown over time.

4.4药物干扰前后细胞成血管能力的差异：4.4 Differences in the angiogenesis ability of cells before and after drug interference:

我们将12h、24h、48h两组的成管情况罗列出，如图11，12h时，Control组已成血管规则，管腔完整；HA组已成血管规则，管腔完整，PYM组和HA联合PYM组细胞依然杂乱无规则分布。We listed the tube formation status of the two groups at 12h, 24h, and 48h, as shown in Figure 11. At 12h, the control group had regular blood vessels and a complete lumen; the HA group had regular blood vessels and a complete lumen, and the PYM group and HA The cells in the combined PYM group were still disorderly and irregularly distributed.

24h时，四组组均已成管，但Control组和HA组成血管规则且官腔比较完整，Control组所形成的结点数更多和管壁更厚，而PYM组和PYM+HA组细胞大部分聚集成堆，仅有微弱的成血管能力；At 24 hours, the four groups had formed tubes, but the Control group and HA formed regular blood vessels and relatively complete official lumens. The Control group formed more nodes and thicker tube walls, while the PYM group and the PYM+HA group had most of the cells Gathered in piles with only weak angiogenesis ability;

48h时，观察已有大量细胞死亡，已成血管的管壁开始萎缩，Control组、HA组依然保持着一定的血管形状，而PYM组、PYM+HA组细胞聚堆萎缩更厉害，已形成的管腔亦消退明显。At 48 hours, it was observed that a large number of cells had died, and the walls of the blood vessels began to shrink. The Control group and the HA group still maintained a certain shape of blood vessels, while the PYM group and the PYM+HA group shrank more severely. Lumen also subsided significantly.

进行三次独立重复实验，以每张图中成管有效结点（三条管壁相交视为有效结点）为计数单位，相对量化后进行分析，t检验具有统计学意义；*P<0.05，如图4。Three independent repeated experiments were carried out, and the effective nodes of tube formation in each picture (the intersection of three tube walls was regarded as an effective node) was used as the counting unit, and the analysis was performed after relative quantification, and the t test was statistically significant; *P<0.05, as shown in Figure 4 .

综上可知，HA单独作用于HVMECs时能轻度抑制其成血管，当其与PYM联合时比单独应用PYM，能明显抑制HVMECs的成血管能力。In summary, HA alone can slightly inhibit the angiogenesis of HVMECs, and when it is combined with PYM, it can significantly inhibit the angiogenesis ability of HVMECs compared with PYM alone.

4.5药物干扰前后细胞凋亡能力的差异：4.5 Differences in apoptosis ability before and after drug interference:

TUNEL法检测细胞凋亡，正常细胞核被染成蓝色，凋亡细胞核呈黄色至棕色。四组细胞凋亡图片如图12。比较四组细胞同等视野下核黄染细胞的个数/全部细胞个数得出的比率，进行统计学分析。t检验分析结果显示，Control组与HA组相比无明显差异，没有统计学意义P>0.05。PYM组与HA联合PYM组差异明显，***P<0.001，如图5。Cell apoptosis was detected by TUNEL method, normal cell nuclei were stained blue, and apoptotic cell nuclei were stained yellow to brown. Figure 12 shows the pictures of cell apoptosis in the four groups. Statistical analysis was performed by comparing the ratio of the number of nuclear yellow stained cells/the number of all cells in the same field of view of the four groups of cells. The t-test analysis results showed that there was no significant difference between the Control group and the HA group, and there was no statistical significance P>0.05. There was a significant difference between the PYM group and the HA combined with PYM group, ***P<0.001, as shown in Figure 5.

由上述初步证明，PYM联合HA作用于静脉畸形内皮细胞后，静脉畸形内皮细胞凋亡增多，细胞生存能力下降。From the above preliminary evidence, after PYM combined with HA acted on venous malformation endothelial cells, the apoptosis of venous malformation endothelial cells increased and the cell viability decreased.

5.结论5 Conclusion

5.1合适浓度的平阳霉素联合透明质酸比单独的平阳霉素能更加有效地抑制静脉畸形内皮细胞的增殖能力、成血管能力，促进静脉畸形内皮细胞的凋亡。5.1 Appropriate concentrations of pingyangmycin combined with hyaluronic acid can more effectively inhibit the proliferation and angiogenesis of venous malformation endothelial cells than pingyangmycin alone, and promote the apoptosis of venous malformation endothelial cells.

5.2平阳霉素联合透明质酸更加有效地作用于静脉畸形内皮细胞的机制，可能是透明质酸包被平阳霉素作用于静脉畸形内皮细胞表面受体CD44，使平阳霉素更多的进入细胞，从而导致内皮细胞增殖及成血管能力的下降，凋亡能力增强。5.2 The mechanism by which Pingyangmycin combined with hyaluronic acid acts more effectively on venous malformation endothelial cells may be that hyaluronic acid-coated pingyangmycin acts on the surface receptor CD44 of venous malformation endothelial cells, allowing more pingyangmycin to enter the cells , resulting in the decrease of endothelial cell proliferation and angiogenesis ability, and the enhancement of apoptosis ability.

申请人用平阳霉素联合透明质酸钠治疗头颈部血管畸形50余例，取得了极其显著的临床效果。与过去单纯应用平阳霉素相比，治疗次数明显减少，治疗效果显著提高。The applicant used Pingyangmycin combined with sodium hyaluronate to treat more than 50 cases of head and neck vascular malformations, and achieved extremely significant clinical effects. Compared with the simple application of Pingyangmycin in the past, the number of treatments is significantly reduced, and the treatment effect is significantly improved.

以两个病例为例进行说明，是从50余例患者中挑选的位置比较表浅，容易肉眼观察的患者（绝大多数病变位置深在，肉眼难以直接观察）。Taking two cases as examples to illustrate, the patients were selected from more than 50 patients whose location was relatively superficial and easy to observe with the naked eye (most lesions were located deep and difficult to directly observe with the naked eye).

病例一：男，60岁，左面部肿胀约15年，低头及从事体力劳动时肿胀加剧，近年来左侧头痛。肿胀及头痛进行性加剧。CT及MRI检查发现左侧面部血管畸形广泛破坏骨质及软组织，临床诊断为左面部血管畸形。利用本发明配方比的平阳霉素联合透明质酸钠治疗一次，方法如下：先将一支平阳霉素（8mg）用8mL生理盐水溶解，得到1mg/mL的平阳霉素溶液，抽取0.1～0.2mL溶液待用。再从一支（2mL，20mg）透明质酸钠液体中抽取0.4～0.8mL，然后抽取适量注射用生理盐水，使总体积达到10mL，充分混匀。用7号半头皮针经皮刺入瘤腔内，尽量抽尽其内的血液，保持头皮针在瘤腔内的位置不变，根据手术前核磁共振成像或者CT成像检查的结果，初步估计病变总体积，并参考抽出的血液体积，得出进行治疗体位下的病变体积。将上述配置好的硬化剂按照这一体积量注入瘤体内。一个月后，头痛及肿胀完全消失，获得临床痊愈。Case 1: Male, 60 years old, had left facial swelling for about 15 years. The swelling intensified when he lowered his head and engaged in physical labor, and had headaches on the left side in recent years. Swelling and headache progressively intensified. CT and MRI examinations revealed that the left facial vascular malformation extensively destroyed bone and soft tissue, and the clinical diagnosis was left facial vascular malformation. Use Pingyangmycin combined with sodium hyaluronate in the formula ratio of the present invention for one treatment, the method is as follows: first dissolve a piece of Pingyangmycin (8 mg) with 8 mL of physiological saline to obtain a 1 mg/mL Pingyangmycin solution, and extract 0.1 to 0.2 mL solution is ready for use. Then draw 0.4-0.8mL from a tube (2mL, 20mg) of sodium hyaluronate liquid, and then take an appropriate amount of normal saline for injection to make the total volume reach 10mL, and mix well. Use a No. 7 semi-scalp needle to percutaneously penetrate the tumor cavity, drain the blood as much as possible, keep the position of the scalp needle in the tumor cavity unchanged, and preliminarily estimate the lesion according to the results of MRI or CT imaging before surgery. The total volume, and with reference to the volume of blood drawn, yields the volume of the lesion in the treatment position. The sclerosing agent configured above was injected into the tumor according to this volume. One month later, the headache and swelling disappeared completely, and clinical recovery was obtained.

病例二：女，17岁，舌静脉畸形，病变范围占舌体4/5，闭口困难，严重影响吞咽、语言及社会交往。利用本发明配方比的平阳霉素联合透明质酸钠治疗2次，每月1次，每次治疗方法同病例一。2个月后，获得痊愈。Case 2: female, 17 years old, tongue vein malformation, lesions accounted for 4/5 of the tongue, difficulty closing mouth, seriously affecting swallowing, language and social interaction. Utilize the pingyangmycin combined with sodium hyaluronate of the formula ratio of the present invention to treat 2 times, once a month, and each treatment method is the same as case one. After 2 months, he recovered.

上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. On the basis of the technical solution of the present invention, those skilled in the art can make various Modifications or variations are still within the protection scope of the present invention.

Claims (3)

1. the application of Bleomycin A5 associating hyaluronate sodium in treatment venous malformation medicine is characterized in that wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton.

2. application according to claim 1 is characterized in that, the concentration of described Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.

3. a medicine that is used for the treatment of venous malformation is characterized in that, it contains as the Bleomycin A5 of active component and hyaluronate sodium, and the medicine acceptable carrier; Wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton, and the concentration of Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.

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