热休克蛋白抑制剂17-DMAG在体外对人肝星状细胞生长增殖及细胞凋亡的作用研究

发布时间：2018-02-12 15:21

  本文关键词： HSP90 17-DMAG 人肝星状细胞LX2 细胞凋亡 α-SMA　出处：《青岛大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的研究HSP90抑制剂17-DMAG在体外对人LX2肝星状细胞的生长增殖及凋亡机制。方法体外培养人LX2肝星状细胞作为受试细胞,实验设计为对照组和17-DMAG处理组,以24h为作用时间点,采用细胞毒试验(MTT法)观察并计算不同浓度(100.250.500.1000nmol/L)和作用时间(24h、48h、72h)的17-DMAG对LX2细胞的生长抑制率并计算IC50值；应用流式细胞仪Annexin V-FITC／PI试剂双染法检测药物作用48h、72h作用后LX2肝星状细胞的凋亡率；Western blot检测药物作用48h、72h作用后α-SMA的表达,分析灰度值并与对照组进行对比。结果LX2肝星状细胞在DMEM培养液中生长良好,并且当细胞浓度5.0×104m1时,培养4-5天后,细胞进入平台期；17-DMAG台抑制人LX2肝星状细胞生长,且在100-1000nmol/L浓度范围内及1-5天的时间范围内呈浓度和时间依赖性(p0.01)并且能够抑制细胞增殖,随药物浓度(100-1000nmol/L)加大和作用时间(24h、48h、72h)延长,存活细胞数量逐渐减少,并计算在24h、48h、72h的IC50值为：757.380.12lnmol/L:流式细胞仪PI双染法证实400nmol/L的17-DMAG可诱导LX2细胞凋亡,与24h、48h对照组(3.07±0.72、7.50±0.95)比较,17-DMAG处理组细胞凋亡率分别为(46.68±2.46、62.84±2.73)明显增加(p0.01); Westernblot研究结果表明经过17-DMAG处理作用48h、72h,可使LX2肝星状细胞α-SMA表达减少,与对照组比较差异有统计学意义(p0.05)。结论17-DMAG在100-1000nmol/L浓度范围内及1-5天的时间范围内具有量效和时效关系；17-DMAG能够抑制LX2细胞增殖,并且流式细胞仪PI双染法证实400nmol/L的17-DMAG可以诱导该细胞凋亡,这与降低α-SMA表达有关。
[Abstract]:Objective to study the growth, proliferation and apoptosis mechanism of HSP90 inhibitor 17-DMAG on human LX2 hepatic stellate cells in vitro. Methods Human LX2 hepatic stellate cells were cultured in vitro. MTT assay was used to observe and calculate the growth inhibition rate of 17-DMAG (100.250.500.1000nmol / L) and the time of treatment (24h ~ 48h ~ 72h) on LX2 cells, and to calculate the IC50 value. The apoptosis rate of hepatic stellate cells of LX2 was detected by flow cytometry with Annexin V-FITC / Pi reagent double staining method. The expression of 伪 -SMA in hepatic stellate cells of LX2 was detected by Western blot after 48 h and 72 h treatment. Results LX2 hepatic stellate cells grew well in DMEM medium, and when the cell concentration was 5.0 脳 10 ~ (4) m ~ (1), after 4-5 days of culture, the growth of human LX2 hepatic stellate cells was inhibited by entering the platform-phase 17-DMAG platform. In the range of 100-1000 nmol / L and 1-5 days, the cell proliferation was inhibited in a concentration and time-dependent manner (p0.01). With the increase of drug concentration of 100-1000 nmol / L and the prolongation of the time of treatment, the number of viable cells gradually decreased. The IC50 value was calculated to be: 1 757.380.12 lnmol / L at 24 h after 48 h. Flow cytometry Pi double staining showed that 400nmol / L 17-DMAG could induce apoptosis of LX2 cells. The apoptotic rate of 17-DMAG group was 46.68 卤2.46 卤62.84 卤2.73, respectively, and the expression of 伪 -SMA in LX2 hepatic stellate cells was decreased after treatment with 17-DMAG for 48 h and 72 h after treatment with 17-DMAG for 48 h and 72 h, respectively, and the apoptotic rate was 46.68 卤2.46 and 62.84 卤2.73, respectively. Conclusion 17-DMAG can inhibit the proliferation of LX2 cells in the range of 100-1000 nmol / L and in the time range of 1-5 days. Flow cytometry Pi double staining showed that 400nmol / L 17-DMAG could induce apoptosis of the cell, which was related to the decrease of 伪 -SMA expression.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【共引文献】

中国博士学位论文全文数据库 前1条

1 徐磊;非酒精性脂肪性肝病与内分泌代谢紊乱的关系的研究[D];浙江大学;2014年

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