半边旗二萜类化合物5F诱导人翼状胬肉成纤维细胞凋亡及对caspase-3表达的研究

Experimental Research of Apoptosis in Pterygium Fibroblasts Induced by 5F and Effect on the Expression of Caspase-3

目的观察半边旗二萜类化合物5F(5F)对体外培养的人翼状胬肉成纤维细胞(HPF)的致凋亡作用及其对凋亡相关的半胱氨酸蛋白酶3(caspase-3)表达的影响。方法采用5F不同药物浓度组(0,8,32,128 mg.L-1)作用不同时间处理细胞。噻唑蓝比色法检测5F对细胞增殖的影响;Hoechst33258荧光染色法观察细胞凋亡;流式细胞术检测细胞周期及凋亡;RT-PCR检测细胞内凋亡相关基因caspase-3的转录水平;Western blot检测细胞caspase-3蛋白表达;化学比色法检测细胞凋亡过程中caspase-3活性的变化。结果给药组细胞的生长与对照组相比均受到显著的抑制(P<0.01),细胞增殖抑制率呈剂量和时间的依赖关系;Hoechst33258荧光染色显示,给药组部分细胞呈典型的凋亡表现;流式细胞术检测在32 mg.L-1浓度作用下,12 h即出现“凋亡峰”,其峰值达(12.48±1.29)%;RT-PCR检测发现细胞内凋亡相关基因caspase-3 mRNA表达上调;Western blot检测细胞caspase-3蛋白表达水平升高;caspase-3活性检测显示,其活性呈剂量和时间依赖性增高,5F浓度达到128 mg.L-1时,其活性为对照组的3.52倍。结论5F可显著地抑制HPF增殖、诱导细胞凋亡、升高caspase-3活性,并呈明显的量效与时效关系;其诱导细胞凋亡的作用机制可能与升高caspase-3活性,促进caspase-3基因转录和蛋白翻译,使细胞内caspase-3增加从而促进细胞凋亡有关。

OBJECTIVE To observe the induced apoptosis effect of 5F on HPF cultured in vitro and the expression of caspase-3. METHODS Cell were treated with different concentration of 5F（0,8,32 and 128 mg·L^-1）with different time. The effect of 5F on cell proliferation was detected by thiazole blue chromatomentry. Cell apoptosis was observed by Hoechst33258 fluorescent staining;flow cell cycle and apoptosis were investigated by flow cytometry.The transcription level of caspase-3 were determined by RT-PCR;The expression of caspase-3 protein was detected by Western blot;The change of caspase-3′s activity during the process of cell apoptosis was valued by chemistry chromatometry. RESULTS Compared with the control group, the cell growth in experimental group was inhibited significantly （ P 〈 0.01）, and the cell proliferation inhibition rate showed a dose- and time- dependent manner. Hoechst33258 fluorescent staining displayed that cells in experimental group partly showed typical apoptosis. Under the effect of 5F with the concentration of 32.18 mg·L^-1, the peak of apoptosis was reached at 12 h and was （ 12.48±1.29） % ; RT-PCR showed that the expression of caspase-3 mRNA in cells was increased. Western blot showed that the expression of caspase-3 protein was increased. The caspase-3 activity was increased dose- and time- dependerthy. The activity was 3.52 times as control group when the concentration of 5F was 128 mg·L^-1. CONCLUSION 5F can inhibit the proliferation of PHF, induce cell apoptosis, increases the activity of caspase-3, and show a dose- and time- dependent manner. The mechanism of 5F inducing cell apoptosis may relate with the increasing of caspase-3 activity, facilitating of transcription of caspase-3 and protein translation, increasing of casepase-3 in order to accelerate the cell apoptosis.