JS-K安全性的初步评价及对HBV阳性肝癌HepG2.2.15细胞的抗肿瘤研究

发布时间：2018-05-13 20:25

本文选题：JS-K + 一氧化氮　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:初步探讨JS-K抗HBV阳性肝癌的效应及相关机制。方法:MTT实验和克隆形成实验将用于检测JS-K是否抑制细胞增殖,划痕实验和Transwell实验将用于细胞迁移能力的检测。流式细胞术将用于细胞周期和细胞凋亡的检测,免疫荧光和Western blot将用于研究JS-K是否引起细胞凋亡和DNA损伤。结果:JS-K抑制Hep G2.2.15细胞的增殖并具有浓度依赖性,抑制细胞克隆形成和迁移,阻滞细胞周期在G2期,诱导细胞凋亡,增加γH2AX绿色荧光蛋白的表达。诱导DNA损伤相关蛋白p-H2AX,p-CHK1,p-CHK2,ATM,p-ATR和凋亡相关蛋白Cleaved Caspase-3,Cleaved Caspase-7,Cleaved PARP的表达增高。结论:JS-K对HBV阳性肝癌细胞的增殖有抑制作用,能诱导细胞发生DNA损伤和细胞凋亡。所以,JS-K是一个潜在的抗HBV阳性肝癌的药物。目的:对具有抗肿瘤效应的一氧化氮前体药JS-K进行初步的安全性评价。方法:雄性昆明小鼠40只,随机分为溶媒(等容量羧甲基纤维素钠,CMC)对照组、JS-K低(2.5 mg/kg)、中(5 mg/kg)和高剂量(10 mg/kg)组。连续口服给药3个月,观察小鼠的生长状况、体重,计算肝肾脏器指数,检测肝肾功能及肝肾组织病理学。另取10只小鼠随机分为生理盐水对照组和JS-K 5 mg/kg组,每天皮下注射给药,连续5 d,检测小鼠骨髓和外周血造血干细胞的比率。结果:JS-K口服或皮下注射给药与对照组小鼠比较,给药和对照组动物在试验期间均生长状态良好、体重增加;肝和肾脏指数及肝肾功能与对照组之间无明显差异(P0.05),均在正常范围;HE染色未发现JS-K产生明显肝肾毒性;小鼠骨髓造血干细胞中阳性细胞的比率,对照组2.19%,JS-K组1.98%;外周血造血干细胞中阳性细胞比率,对照组为0.11%,JS-K组0.13%,两组间无明显差异(P0.05)。结论:小鼠连续口服JS-K 3个月(最高剂量10 mg/kg)对肝肾无明显毒性,对小鼠的生长未见明显影响;小鼠连续皮下注射JS-K(5 mg/kg)5 d,对骨髓造血功能没有明显的影响。
[Abstract]:Objective: to investigate the effect of JS-K on HBV positive liver cancer and its related mechanism. Methods JS-K assay and clone formation assay were used to detect whether JS-K inhibited cell proliferation, while scratch test and Transwell assay were used to detect cell migration ability. Flow cytometry will be used to detect cell cycle and apoptosis, and immunofluorescence and Western blot will be used to study whether JS-K induces apoptosis and DNA damage. Results the cell proliferation of Hep G2.2.15 cells was inhibited in a concentration-dependent manner, cell clone formation and migration were inhibited, cell cycle was blocked in G2 phase, apoptosis was induced and the expression of 纬 H2AX green fluorescent protein was increased. The expression of p-H _ 2AXT _ (p-CHK _ (1) -CHK _ (2) -ATR and the apoptosis-related protein (Cleaved) Caspase-3 (Caspase-3) and Caspase-7 (Caspase-7) were induced to increase the expression of PARP. ConclusionJS-K can inhibit the proliferation of HBV positive hepatoma cells and induce DNA damage and apoptosis. So JS-K is a potential anti-HBV-positive drug for liver cancer. Objective: to evaluate the safety of nitric oxide precursor JS-K with anti-tumor effect. Methods: forty male Kunming mice were randomly divided into 2. 5 mg / kg of JS-K group and 10 mg / kg of high dose group. After oral administration for 3 months, the growth status, body weight, liver and kidney organ index, liver and kidney function and liver and kidney histopathology of mice were observed. Another 10 mice were randomly divided into normal saline control group and JS-K 5 mg/kg group. The ratio of hematopoietic stem cells in bone marrow and peripheral blood was measured by subcutaneous injection daily for 5 days. Results compared with the control mice, the mice in the control group and the control group grew well and gained weight during the trial period. There was no significant difference in liver and kidney index and liver and kidney function between the control group and the control group (P 0.05). No obvious hepatorenal toxicity was found by HE staining in normal range, and the ratio of positive cells in bone marrow hematopoietic stem cells of mice was not found by HE staining. The ratio of positive cells in peripheral blood stem cells in the control group was 0.11 and 0.13 in JS-K group, and there was no significant difference between the two groups (P 0.05). Conclusion: oral administration of JS-K for 3 months (maximum dose 10 mg / kg) has no obvious toxicity on liver and kidney, but has no obvious effect on the growth of mice, while continuous subcutaneous injection of JS-K(5 mg/kg)5 d has no obvious effect on hematopoietic function of bone marrow in mice.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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