论文部分内容阅读
目的探讨焦脱镁叶绿酸甲酯(methyl pyropheophorbide-a,Mppa)介导的光动力疗法(Mppa-PDT)抑制人卵巢癌SKOV3细胞活性、触发其凋亡的机制。方法将处于对数生长期的人卵巢癌SKOV3细胞随机分为Mppa-PDT处理组(加光敏剂Mppa和LED光照处理)和对照组(空白对照、仅加光敏剂Mppa单药对照、仅接受LED单光对照)。Mppa-PDT作用于人卵巢癌SKOV3细胞后,CCK-8法检测细胞活性;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;DAPI染色观察细胞凋亡的核的形态学改变;DCFH-DA染色观察细胞内活性氧(ROS)的产生;单细胞凝胶电泳观察DNA损伤情况;蛋白质印迹法检测p53、Caspase-3、Bax、Bcl-2蛋白的表达变化。结果 (1)Mppa-PDT能显著抑制人卵巢癌SKOV3细胞的活性,其抑制作用具有一定的剂量依赖性;(2)Mppa-PDT诱导的人卵巢癌SKOV3细胞凋亡率高于空白对照、单药对照和单光对照组(P<0.05),且空白、单药、单光3组对照组间凋亡率差异无统计学意义(P>0.05);(3)Mppa-PDT作用于人卵巢癌SKOV3细胞后,DAPI细胞核荧光染色可见细胞核深染的凋亡细胞;DCFH-DA荧光染色发现Mppa-PDT组细胞内ROS水平高于3个对照组;单细胞凝胶电泳显示Mppa-PDT组的DNA损伤情况高于3个对照组;蛋白质印迹法检测发现p53、Caspase-3、Bax蛋白表达升高,Bcl-2蛋白表达降低(P<0.05)。结论 Mppa介导的光动力疗法能够抑制人卵巢癌SKOV3细胞活性并诱发其凋亡,且伴随有DNA损伤及线粒体凋亡途径的激活。
Objective To investigate the mechanism by which Mppa-PDT inhibits the activity of human ovarian cancer cell line SKOV3 and triggers its apoptosis. Methods Human ovarian cancer SKOV3 cells in logarithmic growth phase were randomly divided into Mppa-PDT treatment group (Mppa and LED light-sensitive agent) and control group (blank control, photosensitizer Mppa alone control only, only LED Single light control). Cell viability was determined by CCK-8 assay after Mppa-PDT treatment on human ovarian cancer cell line SKOV3; apoptosis rate was detected by flow cytometry with AnnexinⅤ-FITC / PI double staining; morphological changes of apoptotic nuclei were observed by DAPI staining; The generation of reactive oxygen species (ROS) was observed by DCFH-DA staining. The DNA damage was observed by single cell gel electrophoresis. The protein expression of p53, Caspase-3, Bax and Bcl-2 was detected by Western blotting. Results (1) Mppa-PDT could significantly inhibit the activity of human ovarian cancer SKOV3 cells in a dose-dependent manner. (2) The apoptosis rate of MOVa-PDT induced SKOV3 cells was higher than that of blank control (P <0.05). There was no significant difference in the apoptotic rate between the control group and blank control group (P> 0.05). (3) The effect of Mppa-PDT on human ovary After the cells were stained with DAPI, the apoptotic cells in the nucleus were observed by fluorescence staining. The level of ROS in Mppa-PDT group was higher than that in the control group by DCFH-DA staining. The single cell gel electrophoresis showed that Mppa-PDT group The DNA damage was higher than that of 3 control groups. The protein expression of p53, Caspase-3 and Bax was increased and the expression of Bcl-2 protein was decreased by Western blot (P <0.05). Conclusion Mppa-mediated photodynamic therapy can inhibit the activity of SKOV3 cells and induce apoptosis in human ovarian cancer, accompanied by DNA damage and the activation of mitochondrial apoptotic pathway.