间充质干细胞条件培养基对HPV18型阳性人子宫颈癌HeLa细胞凋亡的影响

Effect of umbilical cord-derived mesenchymal stem cell-conditioned medium on apoptosis of HPV18-infected HeLa cell lines

目的观察人脐带间充质干细胞条件培养基(MSC-CM)对HPV18型阳性人子宫颈癌HeLa细胞(HPV18 HeLa细胞)凋亡相关基因的调控作用,以及对HPV18 HeLa细胞凋亡的影响。方法制备MSC-CM,设置空白组、低剂量组和高剂量组,配制相应浓度分别为0%、20%和60%的MSC-CM处理HPV18 HeLa细胞,使用CCK-8法、克隆形成实验检测细胞活力和克隆形成能力;磷脂结合蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染法凋亡染色和流式细胞术检测细胞凋亡的变化;实时荧光定量PCR检测凋亡相关基因mRNA表达量。结果与空白组相比,在低剂量组或高剂量组的MSC-CM作用下,HPV18 HeLa细胞的生长活性和克隆形成能力均降低,细胞凋亡率升高(P均<0.05),且高剂量组作用效果均比低剂量组明显(P均<0.05)。与空白组相比,HPV18 HeLa细胞在低剂量或高剂量组MSC-CM的作用下,HPV18 HeLa细胞中p53、胱天蛋白酶-3(Caspase-3)、Caspase-9、Bax的mRNA表达量升高,而B淋巴细胞瘤-2(Bcl-2)的mRNA表达量降低(P均<0.05),高剂量组比低剂量组的作用效果更明显(P<0.05)。结论MSC-CM能诱发HPV18 HeLa细胞凋亡,可能与p53、Caspase-3、Caspase-9、Bax以及Bcl-2等基因表达差异相关。

Objective To evaluate the effect of umbilical cord-derived mesenchymal stem cellconditioned medium(MSC-CM)on regulating the apoptosis-related genes and determine the impact upon the apoptosis of human papillomavirus type18-infected HeLa cell lines.Methods Primary cervical cancer HeLa cells were divided into the blank,low-dose and high-dose groups.HeLa cells were cultured and treated with 0%,20%and 60%of MSC-CM in three groups,respectively.The cell viability and clone formation ability of HeLa cells were detected by CCK-8 assay and clone formation assay.The apoptosis of HeLa cells was detected by Annexin V-Fluorescein isothiocyanate/Propidium iodide(V-FITC/PI)staining and flow cytometry.The expression levels of p53,Bax,Caspase-3,Caspase-9 and Bcl-2 mRNA in HeLa cells were quantitatively measured by RT-PCR.Results In the low-and high-dose groups,the cell viability and clone formation ability were significantly decreased,whereas the apoptosis rate was significantly elevated compared with those in the blank group(all P<0.05).The effects in the high-dose group were more evident than those in the lowdose group(all P<0.05).The expression levels of p53,Bax,Caspase-3 and Caspase-9 mRNA in the lowand high-dose groups were significantly up-regulated,whereas that of Bcl-2 mRNA was significantly downregulated compared with those in the blank group(all P<0.05).The effects in the high-dose group were more pronounced than those in the low-dose group(all P<0.05).Conclusions Under the effect of MSC-CM,the apoptosis of HPV18 cervical HeLa cells can be induced,probably associated with the differential expression patterns of p53,Bax,Bcl-2,Caspase-3 and Caspase-9 mRNA.