技术领域Technical Field
本发明涉及生物医药领域，特别涉及一种用于骨修复的药物组合物。The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition for bone repair.
背景技术Background technique
创伤和疾病等原因导致的骨组织缺损是临床常见且较难处理的问题，如何促进骨组织缺损的修复一直是相关领域学者的研究重点。临床治疗骨-软骨全层缺损的方法有：微裂纹、软骨刮削、关节置换、马赛克成形术、软骨下骨钻孔和假关节置换等。然而，这些治疗方式具有供体来源有限、治疗部位的病变、移植体的松脱和假体有限以及耐久性差等局限性。Bone tissue defects caused by trauma and disease are common and difficult to treat clinical problems. How to promote the repair of bone tissue defects has always been the research focus of scholars in related fields. Clinical methods for treating full-thickness bone-cartilage defects include microcracks, cartilage scraping, joint replacement, mosaicplasty, subchondral bone drilling, and pseudoarthrosis replacement. However, these treatment methods have limitations such as limited donor sources, lesions at the treatment site, loosening of the graft, limited prosthesis, and poor durability.
随着生物技术的发展，新得的生物技术不断应用于骨修复，例如有研究通过经皮注射骨生长因子，直接将高效的骨诱导剂输送至骨折、骨缺损及骨不连接部位，具有创伤小，适应症广等优点，针对临床中大段粉碎性骨折患者，在闭合复位后向骨折部位注入骨生长因子能够大大提高骨折愈合率；而对于骨不连患者，在内固定或外固定完善的情况下，采取局部注射骨生长因子，可进一步扩大了非手术治疗的适应范围，但是该方法需要对骨生长因子进行提取纯化，纯化工艺复杂，同时，在纯化保存以及使用过程中，骨生长因子容易失效，并且使用时，有效时间短，需要持续给予注射才能起效。还有研究利用组织工程技术为骨修复，通过间充质干细胞与支架材料复合后进行成骨诱导分化，但利用间充质干细胞进行修复，常常受到局部微环境的调节，炎性细胞因子如肿瘤坏死因子和白细胞介素1等常常抑制间充质干细胞向成骨细胞分化，影响骨再生修复，同时干细胞增生容易引发肿瘤。With the development of biotechnology, new biotechnology is constantly being applied to bone repair. For example, some studies have used percutaneous injection of bone growth factor to directly deliver highly effective bone inductors to fractures, bone defects and bone nonunion sites. This has the advantages of less trauma and a wide range of indications. For patients with large and medium-sized comminuted fractures in clinical practice, injecting bone growth factor into the fracture site after closed reduction can greatly improve the fracture healing rate; and for patients with bone nonunion, local injection of bone growth factor can further expand the scope of non-surgical treatment when internal fixation or external fixation is complete. However, this method requires extraction and purification of bone growth factor, and the purification process is complicated. At the same time, during the purification, storage and use process, bone growth factor is prone to failure, and when used, the effective time is short and requires continuous injection to be effective. There are also studies using tissue engineering technology for bone repair, in which mesenchymal stem cells are combined with scaffold materials and then induced to differentiate into osteoblasts. However, the use of mesenchymal stem cells for repair is often regulated by the local microenvironment. Inflammatory cytokines such as tumor necrosis factor and interleukin 1 often inhibit the differentiation of mesenchymal stem cells into osteoblasts, affecting bone regeneration and repair. At the same time, stem cell proliferation can easily induce tumors.
发明内容Summary of the invention
本发明的具体实施方式提供一种克服以上所述问题的用于骨修复的药物组合物，具体方案如下：The specific embodiment of the present invention provides a pharmaceutical composition for bone repair that overcomes the above-mentioned problems. The specific scheme is as follows:
一种用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含骨修复基因。A pharmaceutical composition for bone repair, comprising self-replicating mRNA, wherein the self-replicating mRNA contains a bone repair gene.
可选的，所述骨修复基因为BMP基因或VEGFA基因。Optionally, the bone repair gene is a BMP gene or a VEGFA gene.
可选的，所述自复制mRNA为BMP2基因自复制mRNA、VEGFA基因自复制mRNA或BMP2与VEGFA双基因自复制mRNA。Optionally, the self-replicating mRNA is BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, or BMP2 and VEGFA dual gene self-replicating mRNA.
可选的，所述组合物还包括脂质体，所述自复制mRNA分散于脂质体中。Optionally, the composition further comprises liposomes, and the self-replicating mRNA is dispersed in the liposomes.
可选的，所述脂质体由DOTAP、HSPC、Chol和PEG-DSPE组成。Optionally, the liposome consists of DOTAP, HSPC, Chol and PEG-DSPE.
可选的，所述脂质体包含阳离子穿膜肽。Optionally, the liposomes contain a cationic membrane-penetrating peptide.
可选的，所述组合物还包括水凝胶，所述自复制mRNA与脂质体负载于所述水凝胶上。Optionally, the composition further comprises a hydrogel, and the self-replicating mRNA and liposomes are loaded on the hydrogel.
可选的，所述水凝胶为甲基丙烯酸酐化明胶水凝胶。Optionally, the hydrogel is methacrylic anhydride-treated gelatin hydrogel.
可选的，所述水凝胶中，甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％。Optionally, in the hydrogel, the weight percentage of methacrylic anhydride gelatin is 3wt% to 9wt%.
可选的，所述BMP2基因自复制mRNA的序列如SEQ ID NO.1所示，所述VEGFA基因自复制mRNA的序列如SEQ ID NO.2所示，所述BMP2与VEGFA双基因自复制mRNA的序列如SEQ IDNO.3所示。Optionally, the sequence of the BMP2 gene self-replicating mRNA is shown as SEQ ID NO.1, the sequence of the VEGFA gene self-replicating mRNA is shown as SEQ ID NO.2, and the sequence of the BMP2 and VEGFA dual gene self-replicating mRNA is shown as SEQ ID NO.3.
可选的，所述药物组合物为骨损伤部原位注射制剂。Optionally, the pharmaceutical composition is an in situ injection preparation for the bone injury site.
本发明具体实施方式的用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含BMP基因或VEGFA基因的骨修复基因，所述药物组合物有利于维持促骨修复基因长时间内高表达，对骨创伤位置少量少批次注射便可进行有效修复。The pharmaceutical composition for bone repair according to a specific embodiment of the present invention comprises self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene such as a BMP gene or a VEGFA gene. The pharmaceutical composition is beneficial for maintaining high expression of the bone repair promoting gene for a long time, and can effectively repair bone trauma sites by injecting a small amount in small batches.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
图1为实施例中3wt％,5wt％,7wt％,和9wt％浓度的GelMA制备的水凝胶球的显微镜观察及组织切片内部表征结果；FIG1 is a microscopic observation and internal characterization result of tissue sections of hydrogel spheres prepared with 3wt%, 5wt%, 7wt%, and 9wt% concentrations of GelMA in the embodiment;
图2为实施例中脂质体、短链BMP2基因mRNA与脂质体复合,自复制BMP2基因mRNA与脂质体复合的混合物的SEM观察形态图；2 is a SEM morphological diagram of a mixture of liposomes, short-chain BMP2 gene mRNA complexed with liposomes, and self-replicating BMP2 gene mRNA complexed with liposomes in the embodiment;
图3为实施例中BMP4基因自复制mRNA、TGFB基因自复制mRNA、BMP2基因自复制mRNA、VEGFA基因自复制mRNA、以及BMP2与VEGFA双基因自复制mRNA体外细胞实验结果；3 is the results of in vitro cell experiments on BMP4 gene self-replicating mRNA, TGFB gene self-replicating mRNA, BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, and BMP2 and VEGFA dual gene self-replicating mRNA in the embodiments;
图4为实施中骨修复的实验效果图。FIG. 4 is a diagram showing the experimental effect of bone repair during implementation.
具体实施方式Detailed ways
本发明的具体实施方式提供一种用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含骨修复基因。本发明的发明人研究发现，当骨创伤位置原位施用所述药物组合物后，促骨修复基因会在长的时间内得到持续显著表达，少量少批次注射便可对骨损伤进行有效修复。A specific embodiment of the present invention provides a pharmaceutical composition for bone repair, the composition comprising self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene. The inventors of the present invention have found that when the pharmaceutical composition is administered in situ at the site of bone injury, the bone repair promoting gene will be continuously and significantly expressed for a long time, and a small amount of injection in a small batch can effectively repair the bone injury.
本发明具体实施方式的药物组合物，所述自复制mRNA包括自复制序列以及骨修复基因序列，在一些具体实施方式种，所述自复制序列来源于正链ssRNA病毒，具体例如委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan Equine Encephalitis Virus，VEEV)，辛德毕斯病毒(Sin-dbis virus)、屈曲病毒(Chikungunya virus)、东方马脑脊髓炎病毒(Easternequine encephali-tis virus)、西方马脑脊髓炎病毒(Western equineencephalitisvirus)、马雅鲁病毒(Mayarovirus)、生里基森林病毒(Semliki forest virus)、委内瑞拉马脑脊髓炎病毒(Venezuelan equine encephalitisvirus)等，特别的为用委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan Equine Encephalitis Virus，VEEV)。In a pharmaceutical composition according to a specific embodiment of the present invention, the self-replicating mRNA includes a self-replicating sequence and a bone repair gene sequence. In some specific embodiments, the self-replicating sequence is derived from a positive-strand ssRNA virus, such as TC83 Venezuelan Equine Encephalitis Virus (VEEV), Sin-dbis virus, Chikungunya virus, Eastern equine encephali-tis virus, Western equine encephalitis virus, Mayarovirus, Semliki forest virus, Venezuelan equine encephalitis virus, etc., especially TC83 Venezuelan Equine Encephalitis Virus (VEEV).
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述自复制mRNA包含BMP基因或VEGFA基因，在一些具体实施方式中，所述自复制mRNA为BMP2基因自复制mRNA，在一些具体实施方式中，所述自复制mRNA为VEGFA基因自复制mRNA，在一些具体实施方式中所述自复制mRNA为BMP2与VEGFA双基因自复制mRNA，所述实施方式的自复制mRNA，特别是当自复制mRNA为BMP2与VEGFA双基因自复制mRNA时，具有更为显著的促骨修复基因表达。The pharmaceutical composition of the specific embodiments of the present invention, in some specific embodiments, the self-replicating mRNA contains BMP gene or VEGFA gene, in some specific embodiments, the self-replicating mRNA is BMP2 gene self-replicating mRNA, in some specific embodiments, the self-replicating mRNA is VEGFA gene self-replicating mRNA, in some specific embodiments, the self-replicating mRNA is BMP2 and VEGFA dual gene self-replicating mRNA, the self-replicating mRNA of the embodiments, especially when the self-replicating mRNA is BMP2 and VEGFA dual gene self-replicating mRNA, has more significant gene expression that promotes bone repair.
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述BMP2基因自复制mRNA的序列如SEQ ID NO.1所示，所述VEGFA基因自复制mRNA的序列如SEQ ID NO.2所示，所述BMP2与VEGFA双基因自复制mRNA的序列如SEQ ID NO.3所示。In some specific embodiments of the pharmaceutical composition of the present invention, the sequence of the BMP2 gene self-replicating mRNA is shown as SEQ ID NO.1, the sequence of the VEGFA gene self-replicating mRNA is shown as SEQ ID NO.2, and the sequence of the BMP2 and VEGFA dual gene self-replicating mRNA is shown as SEQ ID NO.3.
本发明具体实施方式的药物组合物，为提高所述自复制mRNA的生物活性，在一些具体实施方式中，所述组合物还包括脂质体，所述自复制mRNA分散于脂质体中，通过所述脂质体作为所述药物组合物的递送载体。在一些具体实施方式中，所述脂质体的脂质为磷脂，例如氢化大豆卵磷脂(HSPC)，卵磷脂、磷脂酰乙醇胺、鞘磷脂、脑磷脂、心磷脂，在一些具体实施方式中所述脂质还包括阳离子脂质，例如1，2-二肉豆蔻酰-3-三甲基铵丙烷(DMTAP)、1，2-二油烯氧基-3-(三甲基氨基)丙烷(DOTAP)、N-[1-(2，3-二(十四烷氧基))丙基]-N，N-二甲基-N-羟乙基溴化铵(DMRIE)、N-[1-(2，3-二油烯氧基)丙基]-N，N-二甲基-N-羟乙基-溴化铵(DORIE)、N-[1-(2，3-二油烯氧基)丙基]-N，N，N-三甲基氯化铵(DOTMA)、3β[N-(N′，N′-二甲基氨基乙烷)氨基甲酰基]胆固醇(DC-Chol)和二甲基-二(十八烷基)溴化铵(DDAB)，在一些具体实施方式中，所述脂质还包括固醇，例如胆固醇(Chol)、胆固醇半琥珀酸酯、胆固醇硫酸酯或胆固醇的任何其他的衍生物，在一些具体实施方式中，所述脂质体还包括脂质聚合物，例如，PEG-DSPE(二硬脂酰磷脂酰乙醇胺)。作为更为有效递送的脂质体，在一具体实施方式中，所述脂质体由DOTAP、HSPC、Chol和PEG-DSPE组成，为进一步提高递送效率，在一些具体实施方式中，所述脂质体中还包含阳离子穿膜肽(CPPs)。The pharmaceutical composition of the specific embodiment of the present invention, in order to improve the biological activity of the self-replicating mRNA, in some specific embodiments, the composition also includes liposomes, the self-replicating mRNA is dispersed in the liposomes, and the liposomes are used as the delivery carrier of the pharmaceutical composition. In some specific embodiments, the lipid of the liposome is a phospholipid, such as hydrogenated soybean lecithin (HSPC), lecithin, phosphatidylethanolamine, sphingomyelin, cephalin, cardiolipin, and in some specific embodiments, the lipid also includes a cationic lipid, such as 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP), N-[1-(2,3-di(tetradecyloxy))propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl -ammonium bromide (DORIE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 3β[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol) and dimethyl-di(octadecyl)ammonium bromide (DDAB). In some embodiments, the lipid also includes a sterol, such as cholesterol (Chol), cholesterol hemisuccinate, cholesterol sulfate or any other derivative of cholesterol. In some embodiments, the liposome also includes a lipid polymer, such as PEG-DSPE (distearoylphosphatidylethanolamine). As a more effective delivery liposome, in one embodiment, the liposome is composed of DOTAP, HSPC, Chol and PEG-DSPE. To further improve the delivery efficiency, in some embodiments, the liposome also includes a cationic transmembrane peptide (CPPs).
本发明具体实施方式的药物组合物，为实现药物的负载和缓慢释放，在一些具体实施方式中，所述组合物还包括水凝胶，所述自复制mRNA与脂质体负载于所述水凝胶上，所述水凝胶具有亲水性聚合物链，在一些具体实施方式中，所述水凝胶与细胞具有相容性，例如可以是明胶、甲基丙烯酸酐化明胶、海藻酸钠、丝素、壳聚糖和胶原蛋白等等，在一些具体实施方式中，所述水凝胶为甲基丙烯酸酐化明胶水凝胶，为兼顾既水凝胶球小的塌陷率以及大的脂质体吸附比例，在一些具体实施方式中，所述水凝胶中，甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％。The pharmaceutical composition of the specific embodiment of the present invention, in order to achieve drug loading and slow release, in some specific embodiments, the composition also includes a hydrogel, the self-replicating mRNA and liposomes are loaded on the hydrogel, the hydrogel has a hydrophilic polymer chain, in some specific embodiments, the hydrogel is compatible with cells, for example, it can be gelatin, methacrylic anhydride gelatin, sodium alginate, silk, chitosan and collagen, etc. In some specific embodiments, the hydrogel is a methacrylic anhydride gelatin hydrogel, in order to take into account both the small collapse rate of the hydrogel balls and the large liposome adsorption ratio, in some specific embodiments, the weight percentage of methacrylic anhydride gelatin in the hydrogel is 3wt% to 9wt%.
本发明具体实施方式的药物组合物，所述组合物适用于各种创伤或疾病等原因导致的骨组织缺损，例如骨折、骨缺损或骨不连接部位等等。The pharmaceutical composition according to a specific embodiment of the present invention is suitable for treating bone tissue defects caused by various traumas or diseases, such as fractures, bone defects or bone non-union sites.
本发明具体实施方式的药物组合物，所述骨修复包括骨的再生或者形成。In the pharmaceutical composition according to a specific embodiment of the present invention, the bone repair includes bone regeneration or formation.
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述药物组合物为骨损伤部原位注射制剂，使用时，将所述药物组合物原位注射至所述骨损伤部位。In some specific embodiments, the pharmaceutical composition of the specific embodiment of the present invention is an in situ injection preparation for the bone injury site. When used, the pharmaceutical composition is injected in situ into the bone injury site.
以下通过具体实施例对本发明做进一步说明。The present invention is further described below by means of specific examples.
实施例Example
水凝胶制备Hydrogel preparation
称取20g明胶分散于200mlPBS(磷酸缓冲盐溶液)(0.01M)中，500mL烧瓶或烧杯中，置于水浴锅中加热搅拌，使明胶温度达到60℃，溶解呈澄清状态(约30min)。用注射器抽取16mlMA(甲基丙烯酸酐)，去除气泡，用微量注射泵以0.25ml/min的速度将MA缓慢加入明胶中，注意避光。加完MA后，水浴锅中继续反应2小时，后加200mlPBS终止反应。反应终止15min后，将GelMA(甲基丙烯酸酐化明胶)分装入透析袋中(MWCO 3500)，38℃透析过夜，7000rpm15min离心除去不溶物，取上清继续透析2～3天。用真空泵抽滤，滤掉超过滤膜(0.22微米)孔径的大分子。将抽滤好的GelMA分装到多个10cm的平皿中，并置于负80冰箱中冷冻保存。低温冻干得到产物GelMA。Weigh 20g gelatin and disperse it in 200ml PBS (phosphate buffered saline) (0.01M), place it in a 500mL flask or beaker, and heat and stir it in a water bath until the gelatin temperature reaches 60°C and dissolves in a clear state (about 30min). Use a syringe to extract 16ml MA (methacrylic anhydride), remove bubbles, and slowly add MA to the gelatin at a rate of 0.25ml/min using a microinjection pump, paying attention to avoid light. After adding MA, continue to react in a water bath for 2 hours, and then add 200ml PBS to terminate the reaction. 15min after the reaction is terminated, divide GelMA (methacrylic anhydride gelatin) into dialysis bags (MWCO 3500), dialyze overnight at 38°C, centrifuge at 7000rpm for 15min to remove insoluble matter, and take the supernatant and continue dialysis for 2 to 3 days. Use a vacuum pump to filter out large molecules with an ultrafiltration membrane (0.22 micron) pore size. The filtered GelMA was divided into several 10 cm dishes and stored in a -80°C refrigerator. The product GelMA was obtained by freeze drying.
将3wt％,5wt％,7wt％,和9wt％浓度的GelMA溶于双蒸水后37℃加热，使用微流控装置制备微球，-30℃冰冻后蓝光照射5min。，如图1显微镜观察可以看出甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％时，可以保证既水凝胶球小的塌陷率以及大的脂质体吸附比例，特别时为5wt％时可以保证水凝胶球在最小的塌陷率的情况下实现最大的脂质体吸附比例。GelMA with concentrations of 3wt%, 5wt%, 7wt%, and 9wt% was dissolved in double distilled water and heated at 37℃, and microspheres were prepared using a microfluidic device, and then frozen at -30℃ and irradiated with blue light for 5min. As shown in Figure 1, when the weight percentage of methacrylic anhydride gelatin is 3wt% to 9wt%, it can ensure a small collapse rate of the hydrogel sphere and a large liposome adsorption ratio, especially when it is 5wt%, it can ensure that the hydrogel sphere achieves the maximum liposome adsorption ratio under the condition of the minimum collapse rate.
脂质体制备Liposome preparation
用电子分析天平精密量取DOTAP(SigmaAldrich)、HSPC(SigmaAldrich)、Cholesterol(SigmaAldrich)和mPEG2000-DSPE(上海子起生物科技有限公司)，289w(阳离子穿膜肽)(参考文献Acta BIomaterialia 63(2017)123-134Discerning thecomposition of penetratin for safe penetration from cornea to retina合成)分别使用三氯甲烷溶解定容，各配制10mg/mL的溶液1.5mL，按以下量：DOTAP(MW＝698.5):0.706mg/70.6uL，HSPC(MW＝78.3):10mg/mL,7.92mg/792uL，Chol(MW＝386):3.52mg/352uL，DSPE.PEG2000(MW＝2750):1.667mg/166.7uL，289w 2.701mg/270.1uL分别加入上述溶液进入25mL烧瓶中，接旋转蒸发仪，使用标口夹夹紧，水浴38℃，打开旋转开关，打开真空泵，调整真空阀，缓慢抽真空。待烧瓶底部形成脂质膜后计20min，关闭旋转开关，调整烧瓶位置，擦干瓶底外部水，打开真空阀，关闭真空泵，移液器于瓶底缓慢滴入1.5mL纯水，打开超声细胞破碎仪，调整参数功率20-40％，3min工作2s停1s，洗净超声头，将超声头没过烧瓶底部液面，进行水化，注意观察底部脂质膜水化情况，使用1mL针管抽取溶液分别经过0.45μm和0.22μm滤器滤入2mL EP管中，封口膜封闭，4℃保存。DOTAP (Sigma Aldrich), HSPC (Sigma Aldrich), Cholesterol (Sigma Aldrich), mPEG2000-DSPE (Shanghai Ziqi Biotechnology Co., Ltd.), 289w (cationic penetratin) (reference Acta Biomaterialia 63 (2017) 123-134) were accurately measured using an electronic analytical balance. retina synthesis) were dissolved in chloroform to make up to volume, and 1.5 mL of 10 mg/mL solution was prepared respectively, according to the following amounts: DOTAP (MW = 698.5): 0.706 mg/70.6 uL, HSPC (MW = 78.3): 10 mg/mL, 7.92 mg/792 uL, Chol (MW = 386): 3.52 mg/352 uL, DSPE.PEG2000 (MW = 2750): 1.667 mg/166.7 uL, 289w 2.701 mg/270.1 uL were added into a 25 mL flask, connected to a rotary evaporator, clamped with a standard clamp, placed in a water bath at 38 ° C, turned on the rotary switch, turned on the vacuum pump, adjusted the vacuum valve, and slowly evacuated. After a lipid film is formed on the bottom of the flask for 20 minutes, turn off the rotary switch, adjust the position of the flask, wipe off the external water on the bottom of the flask, open the vacuum valve, turn off the vacuum pump, slowly drip 1.5 mL of pure water into the bottom of the flask with a pipette, turn on the ultrasonic cell disruptor, adjust the parameter power to 20-40%, work for 3 minutes and stop for 1 second, wash the ultrasonic head, submerge the ultrasonic head above the liquid surface at the bottom of the flask for hydration, pay attention to the hydration of the lipid film at the bottom, use a 1 mL syringe to extract the solution, filter it through 0.45 μm and 0.22 μm filters respectively into a 2 mL EP tube, seal it with a sealing film, and store it at 4°C.
自复制mRNA制备Self-replicating mRNA production
利用SimpliconTM RNA质粒，分别构建了表达BMP4基因、TGFB基因、BMP2基因、VEGFA基因以及BMP2与VEGFA双基因的质粒模板。对构建好的质粒模板进行测序验证(利用二代测序仪进行基因序列测序)，并对验证好的质粒模板利用QRT-PCR方法进行扩增,利用限制性核酸内切酶分别对60ug BMP4、TGFB、BMP2、VEGFA、BMP2与VEGFA基因质粒线性化处理，并沉淀纯化得到的线性化BMP4、TGFB、BMP2、VEGFA、BMP2与VEGFA基因模板为30-50ug。利用线性化模板，进行T7启动子体外转录试剂盒体外转录，在37℃恒温条件下转录1-2小时，然后在37℃恒温条件下进行5’端加帽和3’端加尾修饰，修饰后的mRNA用转录纯化试剂盒进行纯化，最后用去离子水进行洗脱，得到BMP4、TGFB、BMP2、VEGFA、BMP2及VEGFA双基因自复制mRNA(Self-amplifying mRNA)溶液。Using SimpliconTM RNA plasmid, plasmid templates expressing BMP4 gene, TGFB gene, BMP2 gene, VEGFA gene and BMP2 and VEGFA double gene were constructed. The constructed plasmid templates were sequenced and verified (using second-generation sequencer for gene sequence sequencing), and the verified plasmid templates were amplified using QRT-PCR method. 60ug BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA gene plasmids were linearized using restriction endonucleases, and the linearized BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA gene templates obtained by precipitation and purification were 30-50ug. The linearized template was used to perform in vitro transcription using a T7 promoter in vitro transcription kit, and the transcription was performed at a constant temperature of 37°C for 1-2 hours. Then, the 5' end capping and 3' end tailing were performed at a constant temperature of 37°C. The modified mRNA was purified using a transcription purification kit and finally eluted with deionized water to obtain BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA dual gene self-replicating mRNA (self-amplifying mRNA) solutions.
自复制mRNA和脂质体复合制备Preparation of self-replicating mRNA and liposome complex
将以上制备获得的自复制mRNA溶液500ul(浓度1ug/ul)继续溶解在500ul无血清培养基，将500ul脂质体溶液(浓度10mg/ml)溶解在500ul无血清培养液中，自复制mRNA与脂质体混合液以1：1比例共孵育10min得到自复制mRNA/脂质体混合物。500ul of the self-replicating mRNA solution (concentration 1ug/ul) prepared above was further dissolved in 500ul of serum-free culture medium, 500ul of liposome solution (concentration 10mg/ml) was dissolved in 500ul of serum-free culture medium, and the self-replicating mRNA and liposome mixture were incubated at a ratio of 1:1 for 10min to obtain a self-replicating mRNA/liposome mixture.
作为对比，按以上方式，制备短链BMP2基因mRNA与脂质体复合后的混合物。As a comparison, a mixture of short-chain BMP2 gene mRNA and liposomes was prepared in the above manner.
将脂质体、短链BMP2基因mRNA与脂质体复合后的混合物、自复制BMP2基因mRNA与脂质体复合的混合物进行SEM观察，其形体如图2所示，如图2，采用所述脂质体与自复制BMP2基因mRNA复合后的混合物大小与混合前的脂质体基本一致的形貌，表明所述脂质体对自复制BMP2基因mRNA具有优异的负载作用。Liposomes, a mixture of short-chain BMP2 gene mRNA and liposomes, and a mixture of self-replicating BMP2 gene mRNA and liposomes were observed by SEM, and their shapes are shown in Figure 2. As shown in Figure 2, the size of the mixture after the liposomes and self-replicating BMP2 gene mRNA are complexed is basically the same as the morphology of the liposomes before mixing, indicating that the liposomes have an excellent loading effect on the self-replicating BMP2 gene mRNA.
自复制mRNA和脂质体复合物的水凝胶负载制备Preparation of hydrogel loading of self-replicating mRNA and liposome complexes
将2000ul以上自复制mRNA与脂质体复合后的混合物放在15ml离心管A中，将以上制备的5wt％的GelMA水凝胶产物2000ul置于15ml离心管B中，将离心管A中的自复制mRNA与脂质体复合后的混合物加入离心管B中，冰上孵育30min。Place 2000ul of the above self-replicating mRNA and liposome complex mixture in 15ml centrifuge tube A, place 2000ul of the above prepared 5wt% GelMA hydrogel product in 15ml centrifuge tube B, add the self-replicating mRNA and liposome complex mixture in centrifuge tube A to centrifuge tube B, and incubate on ice for 30min.
细胞实验Cell experiments
在六孔培养皿中体外培养骨髓间充质干细胞(BMSC)，设置GFP mRNA处理为对照组，将上述获得的水凝胶负载的BMP4基因自复制mRNA(Sa-BMP4)、TGFB基因自复制mRNA(Sa-TGFB)、BMP2基因自复制mRNA(Sa-BMP2)、VEGFA基因自复制mRNA(Sa-VEGF)、BMP2与VEGFA双基因自复制mRNA(Sa-BMP2+VEGF)作为实验组，每组设置三个重复实验孔，每孔转入1ug对应mRNA，通过qRT-PCR观察骨修复主要指标Runx2、OPN和OCN的表达情况，选取第五天的表达情况结果如图3所示，根据图3的结果显示，相对于空白样，BMP4以及TGFB基因自复制mRNA在第五天的Runx2、OPN和OCN表达略有提升，而BMP2基因自复制mRNA、VEGFA基因自复制mRNA、BMP2与VEGFA双基因自复制mRNA在第五天均具有显著的Runx2、OPN和OCN表达，特别是BMP2与VEGFA双基因自复制mRNA具有最为显著的表达。Bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro in six-well culture dishes. GFP mRNA treatment was set as the control group, and the hydrogel-loaded BMP4 gene self-replicating mRNA (Sa-BMP4), TGFB gene self-replicating mRNA (Sa-TGFB), BMP2 gene self-replicating mRNA (Sa-BMP2), VEGFA gene self-replicating mRNA (Sa-VEGF), and BMP2 and VEGFA dual gene self-replicating mRNA (Sa-BMP2+VEGF) obtained above were used as experimental groups. Three replicate experimental wells were set up in each group, and 1ug of corresponding mRNA was transferred into each well. The main indexes of bone repair, Run, were observed by qRT-PCR. The expression of Runx2, OPN and OCN on the fifth day were shown in Figure 3. According to the results in Figure 3, compared with the blank sample, the expression of BMP4 and TGFB gene self-replicating mRNA on the fifth day was slightly increased, while BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, BMP2 and VEGFA double gene self-replicating mRNA all had significant Runx2, OPN and OCN expression on the fifth day, especially BMP2 and VEGFA double gene self-replicating mRNA had the most significant expression.
骨修复实验Bone repair experiment
1)建立大鼠股骨远端骨缺损模型1) Establishment of distal femoral bone defect model in rats
根据每只实验动物体重，使用戊巴比妥钠+吸入异氟烷复合麻醉。大鼠仰卧固定于手术操作台，右侧后肢膝关节内侧周围备皮，常规消毒，铺巾。取膝关节内侧纵行切口，逐层进入，暴露股骨远端，使用直径3mm克氏针由股骨远端内侧面水平进针，构建直径3mm的骨隧道，随后根据分组注入生理盐水或不同材料，使用骨蜡封闭隧道口。反复冲洗后逐层关闭切口。术后放回单笼饲养。According to the weight of each experimental animal, sodium pentobarbital + inhaled isoflurane combined anesthesia was used. The rat was fixed on the operating table in supine position, and the skin around the medial knee joint of the right hind limb was prepared, routinely disinfected, and draped. A longitudinal incision was made on the medial side of the knee joint, and the distal femur was exposed layer by layer. A 3mm diameter Kirschner wire was used to insert the needle horizontally from the medial side of the distal femur to construct a 3mm diameter bone tunnel. Then, physiological saline or different materials were injected according to the grouping, and the tunnel opening was sealed with bone wax. After repeated flushing, the incision was closed layer by layer. After surgery, it was returned to a single cage for breeding.
2)注射样准备2) Preparation of injection samples
sham：生理盐水sham: saline solution
Sa-mRNA/Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载BMP2与VEGFA双基因自复制mRNA和脂质体复合物；Sa-mRNA/Lip@GelMA: hydrogel loaded with BMP2 and VEGFA dual gene self-replicating mRNA and liposome complex prepared by the above method of preparing hydrogel loading of self-replicating mRNA and liposome complex;
Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式,其中脂质体中不混合有自复制mRNA，制备的水凝胶负载脂质体；Lip@GelMA: hydrogel-loaded liposomes prepared by the above self-replicating mRNA and liposome complex hydrogel loading method, wherein the liposomes are not mixed with self-replicating mRNA;
mRNA/Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载短链mRNA和脂质体复合物,其中的自复制mRNA替换为短链BMP2基因mRNA与短链VEGFA基因mRNA(1mol:1mol)混合。mRNA/Lip@GelMA: A hydrogel-loaded short-chain mRNA and liposome complex prepared according to the above hydrogel-loaded preparation method of self-replicating mRNA and liposome complex, in which the self-replicating mRNA is replaced by a mixture of short-chain BMP2 gene mRNA and short-chain VEGFA gene mRNA (1 mol:1 mol).
mRNA/Lip(-CPPs)@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载BMP2与VEGFA双基因自复制mRNA和脂质体复合物,但其中脂质体中不含阳离子穿膜肽(CPPs)。mRNA/Lip(-CPPs)@GelMA: The hydrogel loaded with BMP2 and VEGFA dual gene self-replicating mRNA and liposome complex prepared by the above method of preparing hydrogel loading of self-replicating mRNA and liposome complex, but the liposome does not contain cationic transmembrane peptides (CPPs).
3)骨修复分析3) Bone repair analysis
抽取150ul以上步骤2)中的注射样，对步骤1)中的大鼠股骨远端骨缺损模型进行关节损伤部位原位注射，间隔2周后进行第二次注射，2周和4周后时间点均获取小鼠关节进行组织固定，取样microCT分析，结果如图4所示。根据图4的结果可知，sham样与Lip@GelMA样无骨修复作用，mRNA/Lip(-CPPs)@GelMA样与mRNA/Lip@GelMA样仍存在有空洞，而Sa-mRNA/Lip@GelMA样的骨骺处骨质愈合最好，骨质增生紧致，表明BMP2与VEGFA双基因自复制mRNA由包含有阳离子穿膜肽的脂质体负载具有十分优异的骨修复作用。150ul of the injection sample in step 2) was extracted and injected into the joint injury site of the rat distal femoral bone defect model in step 1) in situ, and the second injection was performed after an interval of 2 weeks. The mouse joints were obtained at 2 weeks and 4 weeks later for tissue fixation, and the samples were analyzed by microCT. The results are shown in Figure 4. According to the results in Figure 4, the sham sample and the Lip@GelMA sample had no bone repair effect, the mRNA/Lip(-CPPs)@GelMA sample and the mRNA/Lip@GelMA sample still had cavities, and the epiphyseal bone healing of the Sa-mRNA/Lip@GelMA sample was the best, and the bone hyperplasia was compact, indicating that the BMP2 and VEGFA dual gene self-replicating mRNA loaded by liposomes containing cationic transmembrane peptides has a very excellent bone repair effect.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
序列表Sequence Listing
<110> 上海唯可生物科技有限公司<110> Shanghai Weike Biotechnology Co., Ltd.
<120> 一种用于骨修复的药物组合物<120> A pharmaceutical composition for bone repair
<130> MP21018097<130> MP21018097
<141> 2021-11-11<141> 2021-11-11
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<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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cggctgggcc caccccaaga cacggttccc ttcggggaga acacccggag aaggaggagg 120cggctgggcc caccccaaga cacggttccc ttcggggaga acacccggag aaggaggagg 120
tgaagaaagg caacagaagc ccagtcgctg ctccaggtcc ctcggacaga gctttttcca 180tgaagaaagg caacagaagc ccagtcgctg ctccaggtcc ctcggacaga gctttttcca 180
tgtggagact ctctcaatgg acgtgccccc tagtgcttct tagacggact gcggtctcct 240tgtggagact ctctcaatgg acgtgccccc tagtgcttct tagacggact gcggtctcct 240
aaaggtcgac catggtggcc gggacccgct gtcttctagt gttgctgctt ccccaggtcc 300aaaggtcgac catggtggcc gggacccgct gtcttctagt gttgctgctt ccccaggtcc 300
tcctgggcgg cgcggccggc ctcattccgg agctgggccg caagaagttc gccggggcat 360tcctgggcgg cgcggccggc ctcattccgg agctgggccg caagaagttc gccggggcat 360
ccggccgccc cttgtcccgg ccttcggacg acgtcctcag cgagtttgag ttgaggctgc 420ccggccgccc cttgtcccgg ccttcggacg acgtcctcag cgagtttgag ttgaggctgc 420
tcagcatgtt tggcctgaag cagagaccca cccccagcaa ggacgtcgtg gtgcccccct 480tcagcatgtt tggcctgaag cagagaccca cccccagcaa ggacgtcgtg gtgcccccct 480
atatgctcga cctgtaccgc cggcactcgg gccagccagg agcgcccgcc ccagaccacc 540atatgctcga cctgtaccgc cggcactcgg gccagccagg agcgcccgcc ccagaccacc 540
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agggacacac caaccatggg tttgtggtgg aagtggccca cttagaggag aagccaggtg 960agggacacac caaccatggg tttgtggtgg aagtggccca cttagaggag aagccaggtg 960
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aggtaagacc actgctagtg acttttggcc acgacggaaa aggacatcca ctccacaaac 1080aggtaagacc actgctagtg acttttggcc acgacggaaa aggacatcca ctccacaaac 1080
gagaaaagcg tcaagccaaa cacaaacagc ggaagcgtct taagtccagc tgcaaaaggc 1140gagaaaagcg tcaagccaaa cacaaacagc ggaagcgtct taagtccagc tgcaaaaggc 1140
accctttgta tgtggacttc agtgatgtgg ggtggaatga ctggatcgtg gcccctccag 1200accctttgta tgtggacttc agtgatgtgg ggtggaatga ctggatcgtg gcccctccag 1200
gctatcatgc cttttactgc catggggaat gtccttttcc cctggctgat cacctgaact 1260gctatcatgc cttttactgc catggggaat gtccttttcc cctggctgat cacctgaact 1260
ccaccaacca tgccatagtg cagactctgg taaactctgt gaattccaaa atccctaagg 1320ccaccaacca tgccatagtg cagactctgg taaactctgt gaattccaaa atccctaagg 1320
catgctgtgt ccccactgag cttagcgcaa tctccatgtt gtacctagat gaaaacgaaa 1380catgctgtgt ccccactgag cttagcgcaa tctccatgtt gtacctagat gaaaacgaaa 1380
aggttgtgct aaaaaactat caggacatgg ttgtggaggg ttgcgggtgt cgctagcaca 1440aggttgtgct aaaaaactat caggacatgg ttgtggaggg ttgcgggtgt cgctagcaca 1440
gcaagaacaa a 1451gcaagaacaa a 1451
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ctgacggaca gacagacaga caccgccccc agccccagcg cccacctcct cgccggcggg 60ctgacggaca gacagacaga caccgccccc agccccagcg cccacctcct cgccggcggg 60
cagccgacgg tggacgcggc ggcgagccgc gagcaggagc cgaagcccgc gcccggaggc 120cagccgacgg tggacgcggc ggcgagccgc gagcaggagc cgaagcccgc gcccggaggc 120
ggggtggagg gggtcggggc tcgcgggatt gcacggaaac ttttcgtcca acttctgggc 180ggggtggagg gggtcggggc tcgcgggatt gcacggaaac ttttcgtcca acttctgggc 180
tcttctctct ccggagtagc cgtggtctgc gccgcaggag gcaaaccgat cggagctggg 240tcttctctct ccggagtagc cgtggtctgc gccgcaggag gcaaaccgat cggagctggg 240
agaagtgcta gctcgggcct ggagaagccg gggcccgaga agagagggga gaaagagaag 300agaagtgcta gctcgggcct ggagaagccg gggcccgaga agagagggga gaaagagaag 300
gaagaggaga gggggccgca gtgggcgctc ggctctcggg agccgggctc atggacgggt 360gaagaggaga gggggccgca gtgggcgctc ggctctcggg agccgggctc atggacgggt 360
gaggcggcgg tgtgcgcaga cagtgctcca gccgcgcgcg cgccccaggc cccggcccgg 420gaggcggcgg tgtgcgcaga cagtgctcca gccgcgcgcg cgccccaggc cccggcccgg 420
gcctcggttc cagaagggag aggagcccgc caaggcgcgc aagagagcgg gctgcctcgc 480gcctcggttc cagaagggag aggagcccgc caaggcgcgc aagagagcgg gctgcctcgc 480
agccgagccg gagagggagc gcgagccgcg ccggccccgg acgggcctct gaaaccatga 540agccgagccg gagagggagc gcgagccgcg ccggccccgg acgggcctct gaaaccatga 540
actttctgct ctcttgggtg cactggaccc tggctttact gctgtacctc caccatgcca 600actttctgct ctcttgggtg cactggaccc tggctttact gctgtacctc caccatgcca 600
agtggtccca ggctgcaccc acgacagaag gggagcagaa agcccatgaa gtggtgaagt 660agtggtccca ggctgcaccc acgacagaag gggagcagaa agcccatgaa gtggtgaagt 660
tcatggacgt ctaccagcgc agctattgcc gtccaattga gaccctggtg gacatcttcc 720tcatggacgt ctaccagcgc agctattgcc gtccaattga gaccctggtg gacatcttcc 720
aggagtaccc cgatgagata gagtatatct tcaagccgtc ctgtgtgccc ctaatgcggt 780aggagtaccc cgatgagata gagtatatct tcaagccgtc ctgtgtgccc ctaatgcggt 780
gtgcgggctg ctgcaatgat gaagccctgg agtgcgtgcc cacgtcggag agcaacgtca 840gtgcgggctg ctgcaatgat gaagccctgg agtgcgtgcc cacgtcggag agcaacgtca 840
ctatgcagat catgcggatc aaacctcacc aaagccagca cataggagag atgagcttcc 900ctatgcagat catgcggatc aaacctcacc aaagccagca cataggagag atgagcttcc 900
tgcagcatag cagatgtgaa tgcagaccaa agaaagatag aacaaagcca gaaaatcact 960tgcagcatag cagatgtgaa tgcagaccaa agaaagatag aacaaagcca gaaaatcact 960
gtgagccttg ttcagagcgg agaaagcatt tgtttgtcca agatccgcag acgtgtaaat 1020gtgagccttg ttcagagcgg agaaagcatt tgtttgtcca agatccgcag acgtgtaaat 1020
gttcctgcaa aaacacagac tcgcgttgca aggcgaggca gcttgagtta aacgaacgta 1080gttcctgcaa aaacacagac tcgcgttgca aggcgaggca gcttgagtta aacgaacgta 1080
cttgcagatg tgacaagcca aggcggtga 1109cttgcagatg tgacaagcca aggcggtga 1109
<210> 3<210> 3
<211> 2626<211> 2626
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 3<400> 3
tcgcccggcg gatccagtct tgccgccgcc tccagcccgg tcacctctct gttccttggc 60tcgcccggcg gatccagtct tgccgccgcc tccagcccgg tcacctctct gttccttggc 60
cggctgggcc caccccaaga cacggttccc ttcggggaga acacccggag aaggaggagg 120cggctgggcc caccccaaga cacggttccc ttcggggaga acacccggag aaggaggagg 120
tgaagaaagg caacagaagc ccagtcgctg ctccaggtcc ctcggacaga gctttttcca 180tgaagaaagg caacagaagc ccagtcgctg ctccaggtcc ctcggacaga gctttttcca 180
tgtggagact ctctcaatgg acgtgccccc tagtgcttct tagacggact gcggtctcct 240tgtggagact ctctcaatgg acgtgccccc tagtgcttct tagacggact gcggtctcct 240
aaaggtcgac catggtggcc gggacccgct gtcttctagt gttgctgctt ccccaggtcc 300aaaggtcgac catggtggcc gggacccgct gtcttctagt gttgctgctt ccccaggtcc 300
tcctgggcgg cgcggccggc ctcattccgg agctgggccg caagaagttc gccggggcat 360tcctgggcgg cgcggccggc ctcattccgg agctgggccg caagaagttc gccggggcat 360
ccggccgccc cttgtcccgg ccttcggacg acgtcctcag cgagtttgag ttgaggctgc 420ccggccgccc cttgtcccgg ccttcggacg acgtcctcag cgagtttgag ttgaggctgc 420
tcagcatgtt tggcctgaag cagagaccca cccccagcaa ggacgtcgtg gtgcccccct 480tcagcatgtt tggcctgaag cagagaccca cccccagcaa ggacgtcgtg gtgcccccct 480
atatgctcga cctgtaccgc cggcactcgg gccagccagg agcgcccgcc ccagaccacc 540atatgctcga cctgtaccgc cggcactcgg gccagccagg agcgcccgcc ccagaccacc 540
ggctggagag ggcagccagc cgcgccaaca ccgtgcgcag cttccatcac gaagaagcca 600ggctggagag ggcagccagc cgcgccaaca ccgtgcgcag cttccatcac gaagaagcca 600
tcgaggaact tccagaaatg agtgggaaaa cgtcccgacg cttcttcttc aatttaagtt 660tcgaggaact tccagaaatg agtgggaaaa cgtcccgacg cttcttcttc aatttaagtt 660
ctgtccctac tgatgagttt ctcacatctg cggagctcca gatttttcgg gaacaaatgc 720ctgtccctac tgatgagttt ctcacatctg cggagctcca gatttttcgg gaacaaatgc 720
aggaagcttt gggaaatagt agtttccagc accgaattaa tatttatgaa attataaagc 780aggaagcttt gggaaatagt agtttccagc accgaattaa tatttatgaa attataaagc 780
ctgccacagc cagctcaaaa tttcctgtga ccagactatt ggacaccagg ttagtgactc 840ctgccacagc cagctcaaaa tttcctgtga ccagactatt ggacaccagg ttagtgactc 840
agaacacaag tcagtgggag agctttgatg tcaccccggc tgtgatgcga tggacagcac 900agaacacaag tcagtgggag agctttgatg tcaccccggc tgtgatgcga tggacagcac 900
agggacacac caaccatggg tttgtggtgg aagtggccca cttagaggag aagccaggtg 960agggacacac caaccatggg tttgtggtgg aagtggccca cttagaggag aagccaggtg 960
tctccaagag acatgtgagg attagcaggt ctttgcacca agatgaacac agctggtctc 1020tctccaagag acatgtgagg attagcaggt ctttgcacca agatgaacac agctggtctc 1020
aggtaagacc actgctagtg acttttggcc acgacggaaa aggacatcca ctccacaaac 1080aggtaagacc actgctagtg acttttggcc acgacggaaa aggacatcca ctccacaaac 1080
gagaaaagcg tcaagccaaa cacaaacagc ggaagcgtct taagtccagc tgcaaaaggc 1140gagaaaagcg tcaagccaaa cacaaacagc ggaagcgtct taagtccagc tgcaaaaggc 1140
accctttgta tgtggacttc agtgatgtgg ggtggaatga ctggatcgtg gcccctccag 1200accctttgta tgtggacttc agtgatgtgg ggtggaatga ctggatcgtg gcccctccag 1200
gctatcatgc cttttactgc catggggaat gtccttttcc cctggctgat cacctgaact 1260gctatcatgc cttttactgc catggggaat gtccttttcc cctggctgat cacctgaact 1260
ccaccaacca tgccatagtg cagactctgg taaactctgt gaattccaaa atccctaagg 1320ccaccaacca tgccatagtg cagactctgg taaactctgt gaattccaaa atccctaagg 1320
catgctgtgt ccccactgag cttagcgcaa tctccatgtt gtacctagat gaaaacgaaa 1380catgctgtgt ccccactgag cttagcgcaa tctccatgtt gtacctagat gaaaacgaaa 1380
aggttgtgct aaaaaactat caggacatgg ttgtggaggg ttgcgggtgt cgctagcaca 1440aggttgtgct aaaaaactat caggacatgg ttgtggaggg ttgcgggtgt cgctagcaca 1440
gcaagaacaa aggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg 1500gcaagaacaa aggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg 1500
aggagaaccc tggacctctg acggacagac agacagacac cgcccccagc cccagcgccc 1560aggagaaccc tggacctctg acggacagac agacagacac cgcccccagc cccagcgccc 1560
acctcctcgc cggcgggcag ccgacggtgg acgcggcggc gagccgcgag caggagccga 1620acctcctcgc cggcgggcag ccgacggtgg acgcggcggc gagccgcgag caggagccga 1620
agcccgcgcc cggaggcggg gtggaggggg tcggggctcg cgggattgca cggaaacttt 1680agcccgcgcc cggaggcggg gtggaggggg tcggggctcg cgggattgca cggaaacttt 1680
tcgtccaact tctgggctct tctctctccg gagtagccgt ggtctgcgcc gcaggaggca 1740tcgtccaact tctgggctct tctctctccg gagtagccgt ggtctgcgcc gcaggaggca 1740
aaccgatcgg agctgggaga agtgctagct cgggcctgga gaagccgggg cccgagaaga 1800aaccgatcgg agctgggaga agtgctagct cgggcctgga gaagccgggg cccgagaaga 1800
gaggggagaa agagaaggaa gaggagaggg ggccgcagtg ggcgctcggc tctcgggagc 1860gaggggagaa agagaaggaa gaggagaggg ggccgcagtg ggcgctcggc tctcgggagc 1860
cgggctcatg gacgggtgag gcggcggtgt gcgcagacag tgctccagcc gcgcgcgcgc 1920cgggctcatg gacgggtgag gcggcggtgt gcgcagacag tgctccagcc gcgcgcgcgc 1920
cccaggcccc ggcccgggcc tcggttccag aagggagagg agcccgccaa ggcgcgcaag 1980cccaggcccc ggcccgggcc tcggttccag aagggagagg agcccgccaa ggcgcgcaag 1980
agagcgggct gcctcgcagc cgagccggag agggagcgcg agccgcgccg gccccggacg 2040agagcgggct gcctcgcagc cgagccggag agggagcgcg agccgcgccg gccccggacg 2040
ggcctctgaa accatgaact ttctgctctc ttgggtgcac tggaccctgg ctttactgct 2100ggcctctgaa accatgaact ttctgctctc ttgggtgcac tggaccctgg ctttactgct 2100
gtacctccac catgccaagt ggtcccaggc tgcacccacg acagaagggg agcagaaagc 2160gtacctccac catgccaagt ggtcccaggc tgcacccacg acagaagggg agcagaaagc 2160
ccatgaagtg gtgaagttca tggacgtcta ccagcgcagc tattgccgtc caattgagac 2220ccatgaagtg gtgaagttca tggacgtcta ccagcgcagc tattgccgtc caattgagac 2220
cctggtggac atcttccagg agtaccccga tgagatagag tatatcttca agccgtcctg 2280cctggtggac atcttccagg agtaccccga tgagatagag tatatcttca agccgtcctg 2280
tgtgccccta atgcggtgtg cgggctgctg caatgatgaa gccctggagt gcgtgcccac 2340tgtgccccta atgcggtgtg cgggctgctg caatgatgaa gccctggagt gcgtgcccac 2340
gtcggagagc aacgtcacta tgcagatcat gcggatcaaa cctcaccaaa gccagcacat 2400gtcggagagc aacgtcacta tgcagatcat gcggatcaaa cctcaccaaa gccagcacat 2400
aggagagatg agcttcctgc agcatagcag atgtgaatgc agaccaaaga aagatagaac 2460aggagagatg agcttcctgc agcatagcag atgtgaatgc agaccaaaga aagatagaac 2460
aaagccagaa aatcactgtg agccttgttc agagcggaga aagcatttgt ttgtccaaga 2520aaagccagaa aatcactgtg agccttgttc agagcggaga aagcatttgt ttgtccaaga 2520
tccgcagacg tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct 2580tccgcagacg tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct 2580
tgagttaaac gaacgtactt gcagatgtga caagccaagg cggtga 2626tgagttaaac gaacgtactt gcagatgtga caagccaagg cggtga 2626
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210012767.0ACN114191445B (en) | 2022-01-06 | 2022-01-06 | Pharmaceutical composition for bone repair |
| PCT/CN2022/140988WO2023130973A1 (en) | 2022-01-06 | 2022-12-30 | Pharmaceutical composition for bone repair |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210012767.0ACN114191445B (en) | 2022-01-06 | 2022-01-06 | Pharmaceutical composition for bone repair |
| Publication Number | Publication Date |
|---|---|
| CN114191445A CN114191445A (en) | 2022-03-18 |
| CN114191445Btrue CN114191445B (en) | 2024-07-02 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210012767.0AActiveCN114191445B (en) | 2022-01-06 | 2022-01-06 | Pharmaceutical composition for bone repair |
| Country | Link |
|---|---|
| CN (1) | CN114191445B (en) |
| WO (1) | WO2023130973A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114191445B (en)* | 2022-01-06 | 2024-07-02 | 上海唯可生物科技有限公司 | Pharmaceutical composition for bone repair |
| CN116098856B (en)* | 2023-01-16 | 2024-06-21 | 四川大学华西医院 | Photocurable hydrogel composite nucleic acid delivery system and medicine for treating spinal cord injury |
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| CN111569148A (en)* | 2020-04-14 | 2020-08-25 | 杭州医学院 | Composite hydrogel for promoting bone repair and preparation method and application thereof |
| CN114191445B (en)* | 2022-01-06 | 2024-07-02 | 上海唯可生物科技有限公司 | Pharmaceutical composition for bone repair |
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| Caroline M et al..Combinatorial Gene Therapy Accelerates Bone Regeneration: Non-Viral Dual Delivery of VEGF and BMP2 in a Collagen-Nanohydroxyapatite Scaffold.《Advanced Healthcare Materials》.2014,第4卷(第2期),223-227.* |
| Combinatorial Gene Therapy Accelerates Bone Regeneration: Non-Viral Dual Delivery of VEGF and BMP2 in a Collagen-Nanohydroxyapatite Scaffold;Caroline M et al.;《Advanced Healthcare Materials》;第4卷(第2期);223-227* |
| 双效骨诱导性的有机-无机水凝胶的构建及其骨再生研究;程若昱 等;上海交通大学学报（医学版）;第38卷(第8期);901-909* |
| Publication number | Publication date |
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| WO2023130973A1 (en) | 2023-07-13 |
| CN114191445A (en) | 2022-03-18 |
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| PE01 | Entry into force of the registration of the contract for pledge of patent right | Denomination of invention:A drug combination for bone repair Granted publication date:20240702 Pledgee:Minhang Branch of Shanghai Rural Commercial Bank Co.,Ltd. Pledgor:Shanghai Weike Biotechnology Co.,Ltd. Registration number:Y2024310001060 | |
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