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目的研究白杨素提高肿瘤坏死因子-α(TNF-α)诱导肝癌细胞HepG2凋亡的能力,并对其分子机制进行初步探讨。方法白杨素以不同浓度(10、20、40μmol/L)单独或联合TNF-α(10 ng/mL)处理HepG2细胞后,于普通及荧光倒置显微镜下观察细胞形态变化,获得细胞死亡的定性资料;流式细胞术检测sub-G1峰,分析峰值变化规律,获得细胞死亡的定量资料;并以Western blotting方法检测凋亡标志蛋白caspase-3、caspase-8和PARP原蛋白和相应的裂解产物的变化情况及凋亡抑制蛋白Bcl-xL、cIAPs、xIAP、cFLIP的时间-效应变化规律。结果 形态学观察可发现白杨素联合TNF-α处理HepG2细胞后,与对照组比较细胞出现明显的死亡数量增加,而单独白杨素组、TNF-α组与对照组比较则未观察到明显的细胞减少(P>0.05);流式细胞术分析sub-G1的定量资料也支持这一结果,联合处理组sub-G1值随着白杨素剂量增加而增大,最高达到(27.84±0.54)%,与对照组比较有显著差异(P<0.05),Hochest 33342荧光染色在联合处理组可观察到明显的核固缩细胞增加;Western blotting检测到凋亡标志蛋白caspase-3、caspase-8和PARP原蛋白减少、相应的活化裂解片段出现;全caspase酶抑制剂z-VAD-fmk可有效抑制联合处理组HepG2细胞死亡、sub-G1峰消失比值减少,阻止凋亡标志蛋白caspase-3、caspase-8和PARP的活化降解;TNF-α引起的凋亡抑制蛋白cFLIP-1表达量增加,随联合处理时间延长而明显下调,与对照组比较有明显差异,Bcl-xL、xIAP等其他凋亡抑制蛋白没有明显改变。结论白杨素能够有效提高TNF-α诱导HepG2细胞凋亡的能力,NF-κB调节的凋亡抑制蛋白cFLIP-1表达减少是其重要的分子机制。
Objective To investigate the effect of chrysin on the apoptosis of HepG2 cells induced by tumor necrosis factor-α (TNF-α) and its molecular mechanism. Methods Chlamydia trachomatis HepG2 cells were treated with 10,20 and 40μmol / L of TNF-α alone or in combination with TNF-α (10 ng / mL). The morphological changes of cells were observed under ordinary and fluorescent inverted microscope. The sub-G1 peak was detected by flow cytometry, and the change of peak value was analyzed to obtain the quantitative data of cell death. The protein of apoptosis marker protein caspase-3, caspase-8 and PARP and the corresponding cleavage products were detected by Western blotting And the time-dependent changes of apoptosis-inhibiting protein Bcl-xL, cIAPs, xIAP and cFLIP. Results Morphological observation showed that the number of obvious cell death increased when chrysin was treated with chrysin and TNF-α compared with the control group, while no obvious cell number was observed in chrysin and TNF-α groups (P> 0.05). The quantitative data of sub-G1 by flow cytometry also support this result. The sub-G1 value of the combined treatment group increased with the increase of the dose of chrysin, up to (27.84 ± 0.54)%, Compared with the control group, there was a significant difference (P <0.05). Hochest 33342 staining showed a significant increase of nuclear condensation cells in the combined treatment group. The apoptotic markers caspase-3, caspase-8 and PARP were detected by Western blotting The total caspase inhibitor z-VAD-fmk could effectively inhibit the death of HepG2 cells and decrease the ratio of sub-G1 peak disappearance, which prevented the expression of caspase-3, caspase-8 And PARP. The expression of cFLIP-1, an inhibitor of apoptosis-inducing protein (TNF-α), was increased with the prolongation of treatment time. Compared with the control group, the expression of cFLIP-1 was significantly increased. Other apoptotic inhibitors such as Bcl- No significant changeConclusion Chrysin can effectively improve the ability of TNF-α to induce apoptosis in HepG2 cells, and the decrease of NF-κB-regulated apoptosis protein cFLIP-1 is an important molecular mechanism.