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目的研究核因子-κB(NF-κB)抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)联合TNF-α对宫颈癌HeLa细胞生长及凋亡的影响,并初步探讨其作用机制。方法应用噻唑蓝(MTT)法检测不同浓度PDTC、TNF-α及两者联合作用对HeLa细胞生长抑制率的影响;采用光镜及DAPI法观察细胞形态学变化及凋亡情况;Westernblot法检测HeLa细胞A20和caspase-3蛋白的表达。结果 PDTC(50,100μmol/L)或TNF-α(30,60,120mg/L)可明显抑制细胞的增殖;低浓度PDTC(25μmol/L)不影响细胞增殖,但与TNF-α联合应用与单用TNF-α比较,细胞增殖抑制率明显增加(P<0.01);光镜与DAPI染色结果显示,不同浓度药物组的细胞均有明显的细胞凋亡特征,且联合用药组细胞凋亡率增高(P<0.01);PDTC(25μmol/L)与TNF-α(60mg/L)联合用药与单用TNF-α(60mg/L)或PDTC(50μmol/L)比较,胞质A20蛋白表达无明显变化,但caspase-3蛋白的表达明显增加(P<0.05)。结论 PDTC可增强TNF-α对宫颈癌HeLa细胞的毒性作用,其机制可能与PDTC抑制TNF-α诱导的核转录因子NF-κB的活性,最终上调凋亡蛋白caspase-3表达有关。
Objective To investigate the effects of pyrrolidine dithiocarbamate (PDTC) combined with TNF-α on the growth and apoptosis of cervical cancer HeLa cells and to explore its possible mechanism. Methods MTT method was used to detect the effect of different concentrations of PDTC, TNF-αand their combination on the growth inhibition rate of HeLa cells. Morphological changes and apoptosis were observed by light microscopy and DAPI method. The expression of HeLa Cell A20 and caspase-3 protein expression. Results PDTC (50,100μmol / L) or TNF-α (30,60,120mg / L) significantly inhibited cell proliferation. Low concentrations of PDTC (25μmol / L) did not affect cell proliferation. (P <0.01). The results of light microscopy and DAPI staining showed that the cells in different concentration groups had obvious apoptosis characteristics, and the apoptosis rate of the combination group was increased (P <0.01). Compared with TNF-α alone (60mg / L) or PDTC (50μmol / L), the combination of PDTC (25μmol / L) , But the expression of caspase-3 protein was significantly increased (P <0.05). Conclusion PDTC can enhance the cytotoxicity of TNF-α on cervical cancer HeLa cells. The mechanism may be related to PDTC inhibiting TNF-α-induced NF-κB activity and eventually up-regulating the expression of caspase-3.