技术领域Technical field
本发明属于抗肿瘤药物技术，特别是涉及稀有糖L-山梨糖在制备治疗肿瘤的药物中的应用。The invention belongs to anti-tumor drug technology, and particularly relates to the application of the rare sugar L-sorbose in the preparation of drugs for treating tumors.
背景技术Background technique
癌症是全球第二大死亡原因，其死亡率仅次于心脑血管疾病，癌症负担在全球范围内逐年继续增长，给个人、家庭、社区和卫生系统造成巨大的身体、情感和经济负担。癌症属于一大类疾病，细胞无法控制地异常生长而超出其正常范围变成恶性肿瘤细胞，侵入身体的邻近部位及扩散到其他器官，肿瘤转移是癌症死亡的主要原因。Cancer is the second leading cause of death in the world, second only to cardiovascular and cerebrovascular diseases. The burden of cancer continues to increase year by year globally, causing huge physical, emotional and economic burdens on individuals, families, communities and health systems. Cancer belongs to a large class of diseases in which cells grow abnormally and uncontrollably beyond their normal range and become malignant tumor cells, invading adjacent parts of the body and spreading to other organs. Tumor metastasis is the main cause of cancer death.
癌症来自正常细胞向肿瘤细胞的多阶段转化过程，该过程通常从癌前病变发展为恶性肿瘤。这些变化是人的遗传因素以及三类外部因素相互作用的结果，包括：物理致癌物，如紫外线和电离辐射等；化学致癌物，如石棉，烟草烟雾的成分，黄曲霉毒素和砷等；生物致癌物，如某些病毒，细菌或寄生虫的感染。目前癌症的主要治疗方式包括手术治疗、放疗、化疗和生物治疗等，但应对各种癌症依旧存在许多治疗方案的局限性。由于多数患者在确诊时已丧失手术最佳时机，因此放、化疗是临床上大部分肿瘤患者接受的主要治疗手段。然而，肿瘤细胞对化疗药物的耐受性常常是化疗失败的重要原因。Cancer results from a multi-stage transformation process of normal cells into tumor cells, which often progresses from precancerous lesions to malignant tumors. These changes are the result of the interaction between human genetic factors and three types of external factors, including: physical carcinogens, such as ultraviolet rays and ionizing radiation; chemical carcinogens, such as asbestos, components of tobacco smoke, aflatoxins and arsenic; biological carcinogens; Carcinogens, such as certain viruses, bacteria, or parasitic infections. Currently, the main treatments for cancer include surgery, radiotherapy, chemotherapy, and biological therapy. However, there are still many limitations in treatment options for various cancers. Since most patients have lost the best opportunity for surgery when they are diagnosed, radiotherapy and chemotherapy are the main treatments for most cancer patients in clinical practice. However, the resistance of tumor cells to chemotherapy drugs is often an important reason for chemotherapy failure.
癌细胞消耗大量葡萄糖作为能量来源，从而允许在细胞过度增殖合成DNA时使用大量氨基酸和核苷酸。利用这一特点，通过提供过量的葡萄糖，肿瘤细胞的有氧糖酵解代谢可以达到非常高的程度，排出大量乳酸，导致肿瘤细胞的酸性微环境。因此，干扰肿瘤细胞的葡萄糖代谢亦可作为一种抗肿瘤的策略。Cancer cells consume large amounts of glucose as a source of energy, allowing large amounts of amino acids and nucleotides to be used as the cells overproliferate to synthesize DNA. Taking advantage of this feature, by providing excess glucose, the aerobic glycolytic metabolism of tumor cells can reach a very high level and excrete a large amount of lactic acid, resulting in an acidic microenvironment of tumor cells. Therefore, interfering with the glucose metabolism of tumor cells can also be used as an anti-tumor strategy.
L-山梨糖是稀有糖的一种。国际稀有糖协会(ISRS)定义“稀有糖及其衍生物是自然界中几乎不存在的单糖”，尽管其天然丰度很低，但稀有糖表现出多种生物学功能。在食品工业领域，稀有糖已被公认为是低热量食品的补充剂和非营养性甜味剂。在合成、化妆品和制药等行业也具有巨大的开发潜力。例如，D-阿洛酮糖能够抑制肝脂肪酶活性，从而减少腹部脂肪堆积，且可以抑制血糖的上升，缓解2型糖尿病；D-塔格糖是美国食品和药物管理局(FDA)认证的食品添加剂；D-阿洛糖具有多种生理功能，可作为抗癌药，抗炎药和抗氧化剂；L-果糖是一种非营养性甜味剂、糖苷酶抑制剂，以及家蝇和蚂蚁的杀虫剂。目前研究显示，L-山梨糖可用于合成糖苷酶抑制剂(1-脱氧半乳糖苷霉素)和L-抗坏血酸盐。但是关于其在抗肿瘤制药中的应用未见报道。L-sorbose is a rare sugar. The International Rare Sugar Society (ISRS) defines "rare sugars and their derivatives as monosaccharides that almost do not exist in nature." Despite their low natural abundance, rare sugars exhibit a variety of biological functions. In the food industry, rare sugars have been recognized as supplements and non-nutritive sweeteners for low-calorie foods. It also has huge development potential in industries such as synthesis, cosmetics and pharmaceuticals. For example, D-psicose can inhibit liver lipase activity, thereby reducing abdominal fat accumulation, and can inhibit the rise in blood sugar and alleviate type 2 diabetes; D-tagatose is certified by the U.S. Food and Drug Administration (FDA) Food additive; D-allose has a variety of physiological functions and can be used as an anti-cancer drug, anti-inflammatory drug and antioxidant; L-fructose is a non-nutritive sweetener, glycosidase inhibitor, and houseflies and ants of pesticides. Current research shows that L-sorbose can be used to synthesize glycosidase inhibitors (1-deoxygalactosidase inhibitor) and L-ascorbate. However, there are no reports on its application in anti-tumor pharmaceuticals.
发明内容Contents of the invention
发明目的：针对上述现有技术，本申请提供了L-山梨糖在制备治疗肿瘤的药物中的应用。Purpose of the invention: In view of the above-mentioned prior art, this application provides the application of L-sorbose in the preparation of drugs for treating tumors.
技术方案：本申请公开了L-山梨糖在制备治疗肿瘤的药物中的应用。Technical solution: This application discloses the application of L-sorbose in the preparation of drugs for treating tumors.
本申请采用一定剂量的L-山梨糖作用于人肿瘤细胞，实验结果显示L-山梨糖对肿瘤细胞增殖有抑制作用，通过抑制肿瘤细胞代谢，阻滞细胞周期进展并促进细胞凋亡。This application uses a certain dose of L-sorbose to act on human tumor cells. Experimental results show that L-sorbose has an inhibitory effect on tumor cell proliferation. By inhibiting tumor cell metabolism, it blocks cell cycle progression and promotes cell apoptosis.
本申请还公开了L-山梨糖联合化疗药物在制备抗肿瘤药物中的应用。This application also discloses the use of L-sorbose combined with chemotherapy drugs in the preparation of anti-tumor drugs.
本申请采用L-山梨糖和肿瘤化疗药物联合使用于人肿瘤细胞，结果显示L-山梨糖对化疗药物有协同增效的作用，并经体内实验证实联用增效的同时无毒副作用。This application uses L-sorbose and tumor chemotherapeutic drugs in combination with human tumor cells. The results show that L-sorbose has a synergistic effect on the chemotherapeutic drugs, and in vivo experiments have confirmed that the combination is synergistic and has no toxic side effects.
所述的化疗药物包括但不限于索拉非尼、乐伐替尼、顺铂、多柔比星、紫杉醇等常用临床药物。优选化疗药物是索拉非尼。The chemotherapy drugs include but are not limited to sorafenib, lenvatinib, cisplatin, doxorubicin, paclitaxel and other commonly used clinical drugs. The preferred chemotherapy drug is sorafenib.
本申请所述的肿瘤包括但不限于肝癌、乳腺癌、肺癌、宫颈癌、淋巴瘤、膀胱癌、黑色素瘤等。特别是肝癌。Tumors described in this application include but are not limited to liver cancer, breast cancer, lung cancer, cervical cancer, lymphoma, bladder cancer, melanoma, etc. Especially liver cancer.
本申请还公开了一种抗肿瘤药物组合物，其包含L-山梨糖，以及药学上可接受的载体。This application also discloses an anti-tumor pharmaceutical composition, which contains L-sorbose and a pharmaceutically acceptable carrier.
所述抗肿瘤药物组合物在制备抗肿瘤药物中的应用也在本申请保护范围内。The application of the anti-tumor pharmaceutical composition in preparing anti-tumor drugs is also within the protection scope of this application.
此外，L-山梨糖可以直链形式或环状形式施用而不影响治疗效果。治疗中可使用多种给药方法，包括口服给药、静脉注射、肌肉内给药注射和瘤内注射等。In addition, L-sorbose can be administered in linear or cyclic form without affecting the therapeutic effect. A variety of administration methods can be used in treatment, including oral administration, intravenous injection, intramuscular injection, and intratumoral injection.
有益效果：本申请首次公开了L-山梨糖在制备治疗抗肿瘤药物中的应用，其通过抑制肿瘤细胞的代谢，抑制了肿瘤细胞的增殖，阻滞其细胞周期的进展并促进细胞的凋亡。并且，L-山梨糖与化疗药物联用能明显增强化疗药物的治疗作用，减少化疗药物用药剂量，降低毒副作用。Beneficial effects: This application discloses for the first time the application of L-sorbose in the preparation of anti-tumor drugs. By inhibiting the metabolism of tumor cells, it inhibits the proliferation of tumor cells, blocks the progression of their cell cycle and promotes cell apoptosis. . Moreover, the combination of L-sorbose and chemotherapy drugs can significantly enhance the therapeutic effect of chemotherapy drugs, reduce the dosage of chemotherapy drugs, and reduce the toxic and side effects.
附图说明Description of drawings
图1是不同稀有糖对癌细胞生长的影响结果图；Figure 1 is a graph showing the effects of different rare sugars on cancer cell growth;
图2是L-山梨糖对肝癌细胞株生长抑制曲线图；Figure 2 is a graph showing the inhibitory effect of L-sorbose on the growth of liver cancer cell lines;
图3是L-山梨糖对肝癌细胞细胞周期的影响；Figure 3 shows the effect of L-sorbose on the cell cycle of liver cancer cells;
图4是L-山梨糖对肝癌细胞细胞凋亡的影响；Figure 4 shows the effect of L-sorbose on apoptosis of liver cancer cells;
图5是L-山梨糖对癌细胞HIF-1α及其能量代谢相关靶基因表达的影响；Figure 5 shows the effect of L-sorbose on the expression of HIF-1α and its energy metabolism-related target genes in cancer cells;
图6是不同稀有糖与索拉非尼联用对癌细胞生长影响；Figure 6 shows the effects of combining different rare sugars with sorafenib on the growth of cancer cells;
图7是L-山梨糖与索拉非尼联用增强索拉非尼体外抑制癌细胞生长的作用；Figure 7 shows that the combination of L-sorbose and sorafenib enhances the effect of sorafenib on inhibiting the growth of cancer cells in vitro;
图8是L-山梨糖与索拉非尼联用增强体内肿瘤治疗作用。Figure 8 shows that the combination of L-sorbose and sorafenib enhances the tumor treatment effect in vivo.
具体实施方式Detailed ways
下面结合实施例对本申请技术方案作详细说明。The technical solution of the present application will be described in detail below with reference to examples.
实施例所用所有稀有糖均购于梯希爱(上海)化成工业发展有限公司，细胞系来源于中国医学科学院基础医学研究所细胞资源中心。All rare sugars used in the examples were purchased from TiCIA (Shanghai) Chemical Industry Development Co., Ltd., and the cell lines were obtained from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.
实施例1：L-山梨糖对体外癌细胞生长的抑制Example 1: Inhibition of cancer cell growth in vitro by L-sorbose
选用不同的稀有糖分别刺激人肝癌细胞Huh7和HepG2、乳腺癌细胞MCF7和肺癌细胞A549后考察细胞活力变化。选用不同浓度的L-山梨糖分别刺激人肝癌细胞Huh7和HepG2，并计算IC50。Different rare sugars were used to stimulate human liver cancer cells Huh7 and HepG2, breast cancer cells MCF7 and lung cancer cells A549 respectively to examine changes in cell viability. Different concentrations of L-sorbose were used to stimulate human liver cancer cells Huh7 and HepG2 respectively, and the IC50 was calculated.
按5×104个细胞/mL的密度将细胞按每孔100μL体积接种于96孔板内，将96孔板放置于细胞培养箱内培养过夜。细胞贴壁24h后给予50mM的稀有糖标准品D-塔格糖(D-Tagatose)、L-塔格糖(L-Tagatose)、D-山梨糖(D-Sorbose)、L-山梨糖(L-Sorbose)、D-阿洛糖(D-Allose)和L-果糖(L-Fructose)刺激细胞24h或不同剂量(0、5、10、25、50和100mM)的L-山梨糖刺激细胞24h、48h和72h。给药孵育结束后，进行CCK-8实验，先用完全培养基按1:10的比例稀释一定体积的CCK-8试剂，之后吸去孔内的含药旧培养基，每孔加入100μL稀释后的CCK-8工作液，选择3个空白孔，弃去含有旧D-PBS，加入配好的CCK-8工作液，作为Blank(用于扣除孔背景值)，之后将96孔板放回培养箱孵育45min-1h。孵育结束后，在450nm波长下检测OD450nm值，按照公式[(加药组-Blank)/(对照组-Blank)]×100％计算细胞存活率。The cells were seeded into a 96-well plate at a density of 5×104 cells/mL in a volume of 100 μL per well, and the 96-well plate was placed in a cell culture incubator and cultured overnight. After 24 hours of cell attachment, 50mM rare sugar standards D-Tagatose (D-Tagatose), L-Tagatose (L-Tagatose), D-Sorbose (D-Sorbose), and L-sorbose (L -Sorbose), D-Allose (D-Allose) and L-Fructose (L-Fructose) stimulated cells for 24h or different doses (0, 5, 10, 25, 50 and 100mM) of L-sorbose stimulated cells for 24h , 48h and 72h. After the administration and incubation, perform the CCK-8 experiment. First dilute a certain volume of CCK-8 reagent with complete culture medium at a ratio of 1:10, then absorb the old drug-containing culture medium in the wells, and add 100 μL of diluted culture medium to each well. CCK-8 working solution, select 3 blank wells, discard the old D-PBS, add the prepared CCK-8 working solution as Blank (used to subtract the well background value), and then put the 96-well plate back into culture Incubate for 45min-1h. After the incubation, the OD450nm value was detected at a wavelength of 450nm, and the cell survival rate was calculated according to the formula [(drug added group-Blank)/(control group-Blank)] × 100%.
实验结果如图1所示，根据图示可见，50mM的L-山梨糖对人肝癌细胞Huh7、HepG2、乳腺癌细胞MCF-7和肺癌细胞A549均有一定程度的生长抑制作用，且除MCF7细胞外，L-山梨糖在其余细胞上的抑制作用均强于D-Allose。如图2所示，L-山梨糖作用于Huh7细胞IC50为33.82mM(24h)、27.32mM(48h)和30.88mM(72h)；L-山梨糖作用于HepG2细胞IC50为27.68mM(24h)、34.89mM(48h)和22.60mM(72h)。The experimental results are shown in Figure 1. According to the figure, 50mM L-sorbose has a certain degree of growth inhibitory effect on human liver cancer cells Huh7, HepG2, breast cancer cells MCF-7 and lung cancer cells A549, except for MCF7 cells. In addition, the inhibitory effect of L-sorbose on other cells was stronger than that of D-Allose. As shown in Figure 2, the IC50 of L-sorbose on Huh7 cells is 33.82mM (24h), 27.32mM (48h) and 30.88mM (72h); the IC50 of L-sorbose on HepG2 cells is 27.68mM (24h). ), 34.89mM (48h) and 22.60mM (72h).
实施例2：L-山梨糖对癌细胞的细胞周期阻滞Example 2: Cell cycle arrest of cancer cells by L-sorbose
给予不同浓度的L-Sorbose刺激肝癌细胞Huh7和HepG2，利用流式细胞术检测L-Sorbose对细胞周期的影响。Different concentrations of L-Sorbose were given to stimulate liver cancer cells Huh7 and HepG2, and flow cytometry was used to detect the effect of L-Sorbose on the cell cycle.
按2×105个细胞/mL的密度分别将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱内培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用胰酶消化，1400rpm，离心3min，加入1mL D-PBS轻柔清洗细胞，1400rpm，离心3min，重复清洗两次。Working Solution的配制：1sample：500μL Assay Buffer+25μL PI Solution+2.5μL RNase Solution。在细胞团块中加入0.5mLWorking Solution重悬，4℃避光孵育30min。赋予结束后斡旋振荡使细胞分散，37℃避光孵育30min。然后斡旋振荡，流式细胞仪检测细胞周期。The cells were seeded into a 6-well plate with a volume of 2 mL per well at a density of 2 × 105 cells/mL, and placed in a cell culture incubator for overnight culture. After the cells adhered for 24 hours, different doses (0, 12.5, 25 and 50mM) of L-Sorbose were given to stimulate the cells for 24 hours. After administration and incubation, cells in each group were digested with trypsin, centrifuged at 1400 rpm for 3 min, 1 mL of D-PBS was added to gently wash the cells, centrifuged at 1400 rpm for 3 min, and washed twice. Preparation of Working Solution: 1sample: 500μL Assay Buffer+25μL PI Solution+2.5μL RNase Solution. Add 0.5mL Working Solution to the cell pellet to resuspend, and incubate at 4°C in the dark for 30 minutes. After the infusion, the cells were dispersed by vortexing and incubated at 37°C in the dark for 30 min. Then the cells were oscillated and the cell cycle was detected by flow cytometry.
实验结果如图3所示，根据图示可见，L-Sorbose对Huh7和HepG2有细胞周期阻滞作用，将细胞周期阻滞于S期，且呈现剂量依赖性。The experimental results are shown in Figure 3. According to the diagram, L-Sorbose has a cell cycle arresting effect on Huh7 and HepG2, arresting the cell cycle in the S phase in a dose-dependent manner.
实施例3：L-山梨糖促进癌细胞凋亡Example 3: L-sorbose promotes apoptosis of cancer cells
给予不同浓度的L-Sorbose刺激肝癌细胞Huh7和HepG2，利用流式细胞术检测L-Sorbose对细胞凋亡的影响。Different concentrations of L-Sorbose were given to stimulate liver cancer cells Huh7 and HepG2, and flow cytometry was used to detect the effect of L-Sorbose on cell apoptosis.
按2×105个细胞/mL的密度将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用不含EDTA的胰酶消化后，1400rpm离心3min，加入1mLD-PBS轻柔地清洗细胞，1400rpm，离心3min，重复清洗两次。1×Annexin V BindingSolution的配制：将10×Annexin V Binding Solution用超纯水稀释为1×Annexin VBinding Solution。在细胞团块中加入500μL Annexin V Binding Solution。取100μL悬液于新的EP管中，加入5μL Annexin V，FITC结合物，再加入5μL PI Solution。室温避光培养15min。各EP管中分别加入400μL Annexin V Binding Solution，1h内流式细胞仪检测。设置对照组：①未染色细胞；②Annexin V，FITC染色细胞(无PI)；③PI染色细胞(无AnnexinV，FITC)。The cells were seeded into a 6-well plate with a volume of 2 mL per well at a density of 2×105 cells/mL, and placed in a cell culture incubator for overnight culture. After the cells adhered for 24 hours, different doses (0, 12.5, 25 and 50mM) of L-Sorbose were given to stimulate the cells for 24 hours. After administration and incubation, cells in each group were digested with trypsin without EDTA, centrifuged at 1400 rpm for 3 min, 1 mL of D-PBS was added to gently wash the cells, centrifuged at 1400 rpm for 3 min, and washed twice. Preparation of 1×Annexin V BindingSolution: Dilute 10×Annexin V Binding Solution with ultrapure water to 1×Annexin VBinding Solution. Add 500 μL Annexin V Binding Solution to the cell pellet. Take 100 μL of the suspension into a new EP tube, add 5 μL of Annexin V, FITC conjugate, and then add 5 μL of PI Solution. Incubate at room temperature in the dark for 15 minutes. Add 400 μL of Annexin V Binding Solution to each EP tube, and perform flow cytometry detection within 1 hour. Set the control group: ① unstained cells; ② Annexin V, FITC-stained cells (without PI); ③ PI-stained cells (without AnnexinV, FITC).
实验结果如图4所示，根据图示可见，L-Sorbose能促进肝癌细胞Huh7和HepG2的细胞凋亡。The experimental results are shown in Figure 4. According to the figure, L-Sorbose can promote apoptosis of liver cancer cells Huh7 and HepG2.
实施例4：L-山梨糖对癌细胞缺氧诱导因子HIF-1α及其代谢相关靶基因表达的影响Example 4: Effect of L-sorbose on the expression of hypoxia-inducible factor HIF-1α and its metabolism-related target genes in cancer cells
不同剂量(0、12.5、25和50mM)的L-山梨糖分别作用于肝癌细胞24h后，采用Western Blot方法检测肝癌细胞中HIF-1α及其下游代谢相关靶基因(HIF-1α、HK2、PKM2、LDHA)的蛋白表达变化。After different doses (0, 12.5, 25 and 50mM) of L-sorbose were applied to liver cancer cells for 24 hours, the Western Blot method was used to detect HIF-1α and its downstream metabolism-related target genes (HIF-1α, HK2, PKM2) in the liver cancer cells. , LDHA) protein expression changes.
按2×105个细胞/mL的密度将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用不含EDTA的胰酶消化后，1400rpm离心3min，D-PBS清洗细胞3次，在L-山梨糖处理后的人肝癌细胞中加入蛋白裂解液和蛋白酶抑制剂，于冰上低温裂解30min，4℃，15000rpm离心10min，收集上清液，定量后取等量蛋白样品进行SDS-PAGE电泳分离，半干法转膜，5％脱脂奶粉封闭2h，加入抗体，4℃过夜孵育。PBST洗涤后加入HRP标记的二抗，室温孵育1h，洗涤后加入化学发光液显色，成像拍照。The cells were seeded into a 6-well plate with a volume of 2 mL per well at a density of 2×105 cells/mL, and placed in a cell culture incubator for overnight culture. After the cells adhered for 24 hours, different doses (0, 12.5, 25 and 50mM) of L-Sorbose were given to stimulate the cells for 24 hours. After administration and incubation, the cells in each group were digested with EDTA-free trypsin, centrifuged at 1400 rpm for 3 minutes, and washed three times with D-PBS. Protein lysis solution and Protease inhibitors were lysed at low temperature on ice for 30 minutes, centrifuged at 15,000 rpm for 10 minutes at 4°C, and the supernatant was collected. After quantification, an equal amount of protein samples were taken for SDS-PAGE electrophoresis separation, semi-dry transfer to membrane, and blocked with 5% skimmed milk powder for 2 hours. Add antibody and incubate overnight at 4°C. After washing with PBST, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. After washing, add chemiluminescence solution for color development and image and take pictures.
实验结果如图5所示，根据图示可见，不同剂量的L-山梨糖刺激肝癌细胞24h后，能够不同程度地抑制HIF-1α、HK2、PKM2、LDHA的蛋白表达，且呈剂量依赖性。The experimental results are shown in Figure 5. According to the figure, different doses of L-sorbose can inhibit the protein expression of HIF-1α, HK2, PKM2, and LDHA to varying degrees after stimulating liver cancer cells for 24 hours in a dose-dependent manner.
实施例5：L-山梨糖与索拉非尼联用体外抑制癌细胞生长Example 5: Combination of L-sorbose and sorafenib inhibits the growth of cancer cells in vitro
选用25mM不同稀有糖与2μM索拉非尼联用刺激人肝癌细胞Huh7和HepG2后检测细胞活力变化。选用12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用刺激人肝癌细胞Huh7和HepG2后检测细胞活力变化。25mM different rare sugars were used in combination with 2μM sorafenib to stimulate human liver cancer cells Huh7 and HepG2 and then detect changes in cell viability. 12.5mM and 25mM L-sorbose were used in combination with 2μM and 4μM sorafenib to stimulate human liver cancer cells Huh7 and HepG2 and then detect changes in cell viability.
按5×104个细胞/mL的密度将细胞按每孔100μL体积接种于96孔板内，置于细胞培养箱培养过夜。细胞贴壁24h后，给于25mM的稀有糖标准品D-Tagatose、L-Tagatose、D-Sorbose、L-Sorbose、D-Allose、L-Fructose和D-Allulose与2μM索拉非尼联用，或选用12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用联用刺激人肝癌细胞Huh7和HepG224h。给药孵育结束后，进行CCK-8实验，先用完全培养基按1:10的比例稀释一定体积的CCK-8试剂，之后去除孔内的含药旧培养基，每孔加入100μL稀释后的CCK-8工作液，选择3个空白孔加入配好的CCK-8工作液，作为Blank(用于扣除孔背景值)，之后将96孔板放回培养箱继续孵育45min-1h。CCK-8工作液孵育结束后，从培养箱中取出待检测96孔板，在450nm波长下检测每个孔的OD450nm值，按照公式[(加药组-Blank)/(对照组-Blank)]×100％计算细胞存活率。The cells were seeded into a 96-well plate with a volume of 100 μL per well at a density of 5×104 cells/mL, and cultured in a cell culture incubator overnight. After the cells adhered for 24 hours, 25mM rare sugar standards D-Tagatose, L-Tagatose, D-Sorbose, L-Sorbose, D-Allose, L-Fructose and D-Allulose were given in combination with 2μM sorafenib. Or use 12.5mM and 25mM L-sorbose in combination with 2μM and 4μM sorafenib to stimulate human liver cancer cells Huh7 and HepG224h. After the administration and incubation, conduct the CCK-8 experiment. First, dilute a certain volume of CCK-8 reagent with complete culture medium at a ratio of 1:10. Then remove the old drug-containing culture medium in the wells and add 100 μL of diluted CCK-8 reagent to each well. CCK-8 working solution, select 3 blank wells and add the prepared CCK-8 working solution as Blank (used to subtract the well background value), then put the 96-well plate back into the incubator and continue incubating for 45min-1h. After the CCK-8 working solution is incubated, take out the 96-well plate to be tested from the incubator, and detect the OD450nm value of each well at a wavelength of 450nm, according to the formula [(medication group-Blank)/(control group-Blank)] ×100% to calculate cell viability.
实验结果如图6所示，根据图示可见，除L-山梨糖外，其他稀有糖在25mM时与2μM索拉非尼联用无增效作用。如图7所示，12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用均能明显增强索拉非尼对癌细胞生长的抑制作用，且在低剂量12.5mM的L-山梨糖与2μM索拉非尼联用已具有明显的抑制癌细胞生长的作用。The experimental results are shown in Figure 6. According to the figure, except for L-sorbose, other rare sugars have no synergistic effect when combined with 2 μM sorafenib at 25mM. As shown in Figure 7, the combination of 12.5mM and 25mM L-sorbose with 2μM and 4μM sorafenib can significantly enhance the inhibitory effect of sorafenib on cancer cell growth, and at the low dose of 12.5mM L-sorbose The combination of sorbose and 2 μM sorafenib has a significant inhibitory effect on the growth of cancer cells.
实施例6：L-山梨糖与索拉非尼联用增强肿瘤治疗作用Example 6: Combination of L-sorbose and sorafenib enhances tumor treatment effect
体内药效学研究通过裸鼠皮下接种Huh7肝癌细胞构建裸鼠肝癌移植瘤小鼠模型考察L-山梨糖体内对肝癌细胞的抑制作用。In vivo pharmacodynamic study: nude mice were subcutaneously inoculated with Huh7 liver cancer cells to construct a nude mouse liver cancer xenograft tumor mouse model to examine the inhibitory effect of L-sorbose on liver cancer cells in vivo.
四周龄雄性SPF级Balb/c nu小鼠，先适应性饲养一周，保持自由摄水摄食状态。培养人肝癌细胞株Huh7细胞，用D-PBS调整细胞浓度为1.0×107/100μL，接种前将细胞悬液轻轻混匀，每只裸鼠腋下靠近背部一侧皮下接种100μL Huh7细胞悬液。正常饲养并每天密切观察裸鼠皮下接种处肿瘤大小。接种四天左右，肿瘤体积达到约50mm3时，将裸鼠随机分为4组，采用灌胃的方式给予L-山梨糖和索拉非尼。模型组每天灌胃生理盐水，L-山梨糖组每天灌胃20％的L-山梨糖(200μL/20g)，索拉非尼组灌胃5mg/mL索拉非尼(50mg/kg)，联用组灌胃20％的L-山梨糖(200μL/20g)和5mg/mL索拉非尼(50mg/kg)。每天观察裸鼠生长状态，每隔一天记录一次裸鼠体重，并用游标卡尺精密测定肿瘤大小，绘制裸鼠肿瘤变化曲线图和裸鼠体重变化曲线图。给药四周结束后处死动物，小心剥离完整肿瘤并称瘤重，并将肿瘤组织分成四份，一份置于10％福尔马林中固定，三份用液氮速冻后转移至-80℃冰箱保存。Four-week-old male SPF grade Balb/c nu mice were first adaptively raised for one week and maintained with free access to water and food. Culture human liver cancer cell line Huh7 cells, adjust the cell concentration to 1.0×107 /100 μL with D-PBS, mix the cell suspension gently before inoculation, and inoculate 100 μL Huh7 cell suspension subcutaneously under the armpit of each nude mouse near the back. liquid. The nude mice were raised normally and the tumor size at the subcutaneous inoculation site of nude mice was closely observed every day. About four days after inoculation, when the tumor volume reached about50mm3 , the nude mice were randomly divided into 4 groups, and L-sorbose and sorafenib were administered by gavage. The model group was gavaged with normal saline every day, the L-sorbose group was gavaged with 20% L-sorbose (200 μL/20g) every day, and the sorafenib group was gavaged with 5 mg/mL sorafenib (50 mg/kg). The control group was orally administered 20% L-sorbose (200 μL/20g) and 5 mg/mL sorafenib (50 mg/kg). Observe the growth status of nude mice every day, record the body weight of nude mice every other day, accurately measure the tumor size with a vernier caliper, and draw the nude mouse tumor change curve and the nude mouse body weight change curve. After four weeks of administration, the animals were sacrificed, and the intact tumors were carefully peeled off and weighed. The tumor tissue was divided into four parts, one part was fixed in 10% formalin, and the third part was quickly frozen in liquid nitrogen and then transferred to a -80°C refrigerator for storage. .
实验结果如图8所示，根据图示可见，由裸鼠肿瘤体积变化曲线及肿瘤重量变化可知，与模型组相比，L-山梨糖组和索拉非尼组均能不同程度的抑制肿瘤体积的生长，抑制肿瘤重量，且联用组抑制效果最好。由裸鼠体重变化曲线可知，在整个实验过程中小鼠体重变化较为平缓，状态良好。The experimental results are shown in Figure 8. According to the figure, it can be seen from the nude mouse tumor volume change curve and tumor weight change that compared with the model group, both the L-sorbose group and the sorafenib group can inhibit tumors to varying degrees. The growth of volume and tumor weight were inhibited, and the combination group had the best inhibitory effect. It can be seen from the body weight change curve of nude mice that the body weight of the mice changed relatively slowly during the entire experiment and they were in good condition.
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| CN110870869A (en)* | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Pharmaceutical composition comprising carbohydrate nutrients and conventional ineffective compounds and use thereof |
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| （美）阿波斯托利亚-玛蒂亚·钦巴瑞多等.《癌症转化医学研究中的靶向治疗》.上海科学技术出版社,2017,(第1版),第81-82页.* |
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