脉冲电场联合野生型p53基因诱导人宫颈癌Hela细胞凋亡

背景:在前期研究基础上,设想将基因电转染作为脉冲电场治疗恶性肿瘤的辅助方法。目的:探讨脉冲电场联合抑癌基因野生型p53基因诱导人宫颈癌hela细胞发生凋亡及相互作用。方法:采用空质粒及含有野生型p53的质粒转染Hela细胞得到Hela-vector、Hela-p53细胞,将Hela细胞、Hela-vector和Hela-p53细胞分别施加固定脉宽100μs、频率1Hz、脉冲数8个、电场强度为1500V/cm的脉冲电场处理。结果与结论:相同参数脉冲电场作用于各组细胞12h后,MTT结果显示Hela-p53组细胞吸光度明显低于Hela组(P<0.05);流式细胞仪及RT-PCR检测结果显示Hela-p53组的早期凋亡率和p53mRNA表达较Hela组明显增加;Westernbolt及激光共聚焦显微镜结果显示与Hela组比较,Hela-p53组细胞Bax蛋白表达明显上升,而Bcl-2蛋白则明显下降,Casepase-3相对荧光强度明显增强。表明野生型p53基因对宫颈癌Hela细胞生长有明显的抑制作用,野生型p53基因可以提高脉冲电场诱导宫颈癌Hela细胞凋亡的作用。

Combined effects of pulse electric fields and wild-type p53 gene on apoptosis of Hela cells

BACKGROUND:There have been lots of experimental works of animals and cells on pulses electric fields treating tumors.But electric fields combined with gene transfer had been seldom studied.Wild-type p53 gene combined with pulses electric fields(PEFs) can induce apoptosis of carcinoma cells.OBJECTIVE:To investigate apoptosis of Hela cells induced by pulse electric fields combined with wild-type p53 gene and the mutual effect between them.METHODS:PEFs with 100 μs duration,1 Hz frequency,8 pieces pulses and 1 500 V/cm electric intensity were performed on Hela cells,Hela-vector and Hela-p53 cells.p53 mRNA expression was detected by RT-PCR.The inhibitory effect on Hela cells growth was detected by MTT assay.Hela cells apoptosis was detected by flow cytometry at 12 hours after PEFs were performed.The expression levels of Bax and Bcl-2 were detected by western blot analysis.Expression of casepase-3 in cells was stained with immunofluorescence and detected by laser scanning confocal microscopy.RESULTS AND CONCLUSION:Apoptotic rate of Hela,Hela-vector and Hela-p53 were(6.48±2.06)%,(7.22±2.25)% and(17.41±2.62)%,respectively.The apoptotic rate was significantly higher in the wild-type p53 gene combined with PEFs group than in the other two groups.At the same time,p53 and caspase-3 mRNA expression was significantly higher in the wild-type p53 gene combined with PEFs group than in the other groups.Bax/Bcl-2 expression ratio was significantly higher in the wild-type p53 gene combined with PEFs group than in the Hela cell group and Hela-vector group as detected by western blot analysis.Hela cells were inhibited by wild-type p53 gene transfer and wild-type p53 gene could increase Hela cells apoptosis induced by PEFs.