慢病毒介导的hTERT-shRNA联合光动力治疗对宫颈癌细胞SiHa的影响

Inhibitive effects of lenti virus-mediated hTERT RNA interference and TMPyP4-PDT on cervix cancer SiHa cells

目的探讨慢病毒介导的人端粒酶逆转录酶(hTERT)联合四甲基吡啶卟啉(TMPy P4)及光动力疗法对宫颈癌细胞Si Ha的影响。方法实验分4组:空白对照组、单纯基因治疗组、单纯光动力治疗组、联合治疗组。体外化学合成Lenti-hTERT-shRNA并转染Si Ha细胞,确定最适感染复数(MOI)值,成功稳定转染后给予浓度为7.5μmol/L的TM Py P4行光动力治疗。RT-PCR法检测hTERT、p53、HPV-16 E6、Caspase-8 mRNA的表达;免疫荧光检测hTERT蛋白的表达;细胞增殖及细胞毒性(CCK-8)检测各组中人宫颈癌细胞Si Ha的抑制作用;Annexin V-PE/7-AAD双染试剂盒检测各组细胞的凋亡率;Transwell实验检测各组细胞侵袭能力的改变。结果与空白对照组比较,其他3组hTERT、HPV-16 E6、Caspase-8 mRNA的表达水平均下降,p53mRNA的表达水平升高,hTERT蛋白表达水平相应下降,其中联合治疗组最明显(P<0.05);联合治疗组较其他组明显抑制Si Ha细胞的增殖(P<0.05);流式细胞学Annexin V-PE/7-AAD和Transwell实验检测结果显示,联合治疗组较单纯基因治疗组和单纯光动力治疗组的凋亡率明显升高,细胞侵袭能力明显减弱(P<0.05)。结论慢病毒介导的hTERT-shRNA联合光动力治疗明显抑制宫颈癌Si Ha细胞的增殖和侵袭能力,并促进细胞凋亡。

Objective To investigate the effects of lenti-hTERT-shRNA combined with photodynamic therapy mediated by meso-5,10,15,20-Tetrakis-（Nmethyl-4-pyridyl）（TMPyP4）on cervical cancer SiHa cells in vitro.Methods Four groups were enrolled in the experiment,including the blank control group,gene therapy group,PDT group and com-bined treatment group.Lenti-hTERT-shRNA was synthesized and SiHa cells were transfected,then the optimum multi-plicity of infection （MOI）was confirmed.After that,the cells were treated with 7 .5μmol/L TMPyP4 and irradiation （blue laser,4 J/cm2 ）.The proliferative activity of SiHa cells was assessed by Cell Counting Kit （CCK-8）assay;the expressions of hTERT,p53,HPV-16 E6,and Caspase-8 mRNA were detected by real-time PCR,and the hTERT pro-tein was tested by immunofluorescence （IF）;the apoptosis was examined by AnnexinV-PE/7-AAD double dye kit;the ability of cellular invasion was assessed by Transwell experiment.Results Compared with the blank control group,the other three groups had decreased the expressions of hTERT,HPV-16 E6,and Caspase-8 mRNA,but increased the ex-pression of p53 mRNA.The down-regulation of hTERT protein was the most significant in the combined treatment group （P〈0.05）.In the combined treatment group,the cellular survivability and invasion ability of SiHa cells were in-hibited more markedly than in the other groups （P〈0.05 ）,while the apoptosis rate increased more significantly （P〈0.05）.Conclusion Lenti virus-mediated hTERT RNA interference combined with photodynamic therapy of TMPyP4＆nbsp;can suppress the abilities of proliferation and invasion,and enhance the apoptosis rate of cervix cancer SiHa cells.