论文部分内容阅读
目的:研究夫拉平度(FP)增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导非小细胞肺癌细胞SPC-A1凋亡的作用及其分子机制。方法:采用TRAIL、FP单独及联合作用的方法诱导细胞凋亡,MTT法检测各处理组细胞的增殖及活性;蛋白质印迹法检测细胞凋亡相关基因蛋白水平的表达;定量PCR检测TRAIL受体及髓系细胞白血病-1(Mcl-1)、Fas相关死亡结构域样IL-1β转化酶样抑制蛋白(FLIP)、X染色体连锁的细胞凋亡抑制因子(XIAP)和Livin等凋亡基因的转录水平表达变化。结果:100nmol/L FP处理组(F)细胞存活率为(80.26±2.43)%,100ng/mL TRAIL处理组(T)为(90.49±1.60)%,FP和TRAIL联合组(F+T)为(47.48±1.41)%,联合组的细胞存活率低于单一处理组,F=406.08,P=0.000。FP和TRAIL合用时,FLIP和XIAP分别降为对照组的4%和15%,Mcl-1与Livin的表达量甚至低于可检测水平。Caspase-3和Caspase-8的活性片段约为对照组的4倍。细胞色素c由线粒体向细胞质的释放增加约7倍,而Bcl-2家族蛋白Bcl-2、Bax、Bak和Bcl-xl的表达量基本不变。FP作用后引起DR4的mRNA表达水平(2.51±0.14)上升,P<0.05。但FP和TRAIL单独作用并未引起Mcl-1、FLIP、XIAP和Livin在转录水平的表达变化。联合组加入广谱Caspase抑制剂Z-VAD-FMK后,细胞存活率由(58.26±1.75)%上升至(88.17±2.65)%,P=0.003。说明Z-VAD-FMK可明显抑制联合处理组的凋亡效应。结论:FP能显著增强TRAIL诱导SPC-A1细胞凋亡的作用,此协同促凋亡效应与抗凋亡基因Mcl-1、FLIP、XIAP和Livin的蛋白水平表达降低以及Caspase活性增强和线粒体损伤有关,同时涉及内源性(线粒体)及外源性(非线粒体)凋亡信号通路的共同作用。
AIM: To investigate the effect of bupivacaine (FP) on the apoptosis of SPC-A1 cells induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its molecular mechanism. Methods: The apoptosis of cells was induced by TRAIL and FP alone or in combination. The proliferation and activity of cells were detected by MTT assay. The protein expression of apoptosis related genes was detected by Western blotting. The expressions of TRAIL receptor, Mcl-1, FLIP, XIAP, and Livin in Fas-related death domain The level of expression changes. Results: The survival rate of the cells treated with 100nmol / L FP was (80.26 ± 2.43)%, while the cells treated with 100ng / mL TRAIL were (90.49 ± 1.60)%. The FP + TRAIL combined group (47.48 ± 1.41)%. The cell survival rate of the combined group was lower than that of the single treatment group (F = 406.08, P = 0.000). When FP and TRAIL were combined, FLIP and XIAP were reduced to 4% and 15% of the control group respectively, and the expression levels of Mcl-1 and Livin were even lower than the detectable level. The active fragments of Caspase-3 and Caspase-8 were about 4 times of the control group. The release of cytochrome c from mitochondria to the cytoplasm increased about 7-fold, while the expression of Bcl-2 family proteins Bcl-2, Bax, Bak and Bcl-xl remained essentially unchanged. The level of mRNA expression of DR4 (2.51 ± 0.14) increased after FP treatment, P <0.05. However, FP and TRAIL alone did not cause changes in the expression of Mcl-1, FLIP, XIAP and Livin at the transcriptional level. The cell viability increased from (58.26 ± 1.75)% to (88.17 ± 2.65)%, P = 0.003 after adding the broad spectrum Caspase inhibitor Z-VAD-FMK in combination group. The results showed that Z-VAD-FMK can significantly inhibit the apoptosis effect of the combined treatment group. CONCLUSION: FP can significantly enhance the apoptosis of SPC-A1 cells induced by TRAIL. The synergistic pro-apoptotic effect is related to the decreased expression of anti-apoptotic genes Mcl-1, FLIP, XIAP and Livin as well as enhanced activity of Caspase and mitochondrial damage , While involving both endogenous (mitochondrial) and extrinsic (non-mitochondrial) apoptotic signaling pathways.