【摘要】 目的：评价光动力疗法(PDT)对Ki-67不同表达水平宫颈癌细胞株HeLa的体外抑制作用。方法：通过RNA干扰技术构建Ki-67不同表达水平的HeLa细胞株，分别为空白对照组、阴性对照组(转染阴性对照siRNA)和Ki-67敲低组(转染Ki-67 siRNA)。使用Western blot法检测Ki-67蛋白表达，验证敲低效果。利用CCK-8试剂盒和划痕试验评估各组细胞增殖活性和迁移率。分别对各组细胞进行PDT处理(使用浓度为3 mmol/L的光敏剂5-ALA，在激光波长630 nm、能量15 J/cm~2、最高输出功率2 W条件下，对各组细胞分别孵育6 h)，观察PDT处理后细胞形态变化，并再次检测细胞增殖活性和迁移率。结果：Western blot检测结果显示Ki-67敲低组细胞的Ki-67蛋白表达水平较两个对照组减弱(P<0.05)。CCK-8试剂盒检测结果显示，Ki-67敲低组细胞在第1～5天各时间点的D(450)均低于空白对照组(P<0.05)。划痕试验显示，Ki-67敲低组细胞迁移率低于空白对照组(P<0.05)。PDT处理后，空白对照组和Ki-67敲低组细胞增殖活性均明显下降，在第4和第5天，Ki-67敲低组细胞的增殖抑制率高于空白对照组(P<0.05)。划痕试验结果显示，PDT处理24 h后，空白对照组和Ki-67敲低组细胞的迁移率均降低，且空白对照组下降更为明显(P<0.05)。显微镜下观察，PDT处理后细胞形态发生类似细胞焦亡的变化，包括细胞体积减小、细胞核移至边缘、细胞另一侧凸出一大囊泡。结论：PDT对宫颈癌HeLa细胞的增殖迁移产生抑制作用，且对Ki-67高表达的HeLa细胞的抑制作用更强，PDT处理后细胞形态发生类似细胞焦亡的变化。更多还原

【Abstract】 OBJECTIVE: To evaluate inhibitory effects of photodynamic therapy(PDT) on the cervical cancer cell lines,HeLa,with different Ki-67 expression levels. METHODS:HeLa cells with different Ki-67 expression levels were constructed by RNA interference technology: Blank control group, Negative control group(treated with negative control siRNA) and Ki-67 knockdown group(treated with Ki-67 siRNA). Western blot was used to detect Ki-67 protein expression to verify the knockdown effect. CCK8 assay and wound healing assay were used to assess cell proliferation rate and wound healing rate in each group. PDT treatment was performed on each group of cells(the cells in each group were incubated with 5-ALA photosensitizer at a concentration of 3 mmol/L for 6 hours under the conditions of laser wavelength 630 nm,energy 15 J/cm~2,and maximum output power 2 W). Morphological changes were observed post-PDT,and cell proliferation rate and wound healing rate were re-evaluated. RESULTS:Western blot analysis showed that the grayscale of the Ki-67 protein expression band in the Ki-67 knockdown group was weaker than that in the two control groups(P<0.05). CCK-8 assay results showed that the D(450) of the Ki-67 knockdown group was lower than that of the blank control group at each time point from day 1 to day 5(P<0.05). The scratch test showed that the migration rate of the Ki-67 knockdown group was lower than that of the blank control group(P<0.05). After PDT treatment, the proliferation activity of the cells in the blank control group and the Ki-67 knockdown group was significantly decreased. On days 4 and 5,the proliferation inhibition rate of the cells in the Ki-67 knockdown group was higher than that in the blank control group(P<0.05). At 24 h after PDT treatment,the scratch test results showed that the migration rate of the cells in the blank control group and the Ki-67 knockdown group was decreased,and the decrease in the blank control group was more significant(P<0.05).After PDT treatment,morphological observations under a microscope showed that the cell morphology underwent changes similar to cell pyroptosis,including a decrease in cell volume,movement of the cell nucleus to the edge,and a large vesicle bulging out from the other side of the cell. CONCLUSION:PDT had an inhibitory effect on the proliferation and migration of cervical cancer HeLa cells,and its inhibitory effect on cells with high expression of Ki-67 was stronger. After PDT,cell morphology showed changes like those of pyroptosis.更多还原