雷公藤甲素对肝细胞增殖和凋亡的影响

Effects of Triptolide on Proliferation and Apoptosis of Human Hepatocytes

摘要:

目的: 研究雷公藤甲素对人源性肝细胞(L-02细胞)增殖和凋亡的影响,为临床应用雷公藤甲素治疗肝脏疾病,防治雷公藤甲素肝毒性的不良反应提供实验依据。 方法: 体外培养L-02细胞,用10,30,50 nmol·L^-1的雷公藤甲素处理细胞48 h后,采用台盼蓝染色后细胞计数的方法和BrdU掺入后流式细胞仪检测的方法检测细胞的生长和增殖能力;PI单染后流式细胞仪检测细胞周期中细胞分布的百分率;AV/PI双染后流式细胞仪检测细胞的凋亡率;Western blot检测细胞中p21和p53的蛋白表达含量。 结果: 与对照组相比,10,30,50 nmol·L^-1的雷公藤甲素处理组,细胞数目从(2.72±1.14)×10⁵个/mL分别减少到(1.95±0.38)×10⁵,(1.44±0.44)×10⁵,(6.97±0.47)×10⁴个/mL;BrdU阳性细胞数从(7.67±0.51)%分别减少到(5.67±0.29)%,(4.05±0.14)%,(2.51±0.15)%;阻滞在G₁期的细胞百分率从65.39%分别上调至68.47%,71.19%,83.98%;细胞凋亡率从(20.54±1.44)%分别上调至(28.02±0.93)%,(36.79±0.49)%,(43.35±1.12)%;p21和p53的蛋白表达水平上调,呈量效性关系。 结论: 雷公藤甲素可以通过上调p21的表达,将细胞阻滞在G₁期,抑制L-02细胞的生长和增殖;同时通过上调p53的表达诱导L-02细胞的凋亡。

Abstract:

Objective:To study the effects of triptolide on proliferation and apoptosis of human hepatocytes(L-02 cells), providing experimental basis for the clinical application of triptolide in treatment of liver diseases and the prevention of its liver toxicity. Method: Culture L-02 cells in vitro and treat the L-02 cells with 10, 30, 50 nmol·L^-1 triptolide respectively for 48 h. Detect the cellular growth and proliferation using trypan blue staining and Brdu incorporation, detect cell cycle distribution using PI staining,detect the percentage of cellular apoptosis using AV/PI double staining,detect the expression levels of p21 and p53 using western blot analysis. Result: Compared with the control, after treated with 10, 30, 50 nmol·L^-1 triptolide, the cell number was reduced from (2.72±1.14)×10⁵ cells/mL to (1.95±0.38)×10⁵,(1.44±0.44)×10⁵,(6.97±0.47)×10⁴ cells/mL respectively;the percentage of Brdu positive cells was reduced from (7.67±0.51)% to (5.67±0.29)%, (4.05±0.14)%, (2.51±0.15)% respectively;the percentage of cells arrested in G₁ stage was increased from 65.39% to 68.47%, 71.19%,83.98% respectively;the percentage of apoptotic cells was increased from (20.54±1.44)% to (28.02±0.93)%,(36.79±0.49)%,(43.35±1.12)% respectively; the expression level of p21 and p53 protein were both up-regulated, and showed a dose-effect relationship. Conclusion: Triptolide could inhibit L-02 cells proliferation through up-regulation of p21 expression thus arresting cells in G₁ stage, and induce cellular apoptosis through up-regulation of p53 expression in L-02 cells.