邱杰洪?张昌林?李田

【摘要】目的 觀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。

【关键词】人乳头瘤病毒18型;宫颈癌;HeLa细胞;间充质干细胞;细胞凋亡

Effect of umbilical cord-derived mesenchymal stem cell-conditioned medium on apoptosis of HPV18-infected HeLa cell lines Qiu Jiehong， Zhang Changlin， Li Tian. Department of Gynecology and Pelvic Floor Disorders Center， the Seven Affiliated Hospital of Sun Yat-sen University， Shenzhen 518107， China

Corresponding author， Li Tian， E-mail： sandylitian@ 126. com

【Abstract】Objective To evaluate the effect of umbilical cord-derived mesenchymal stem cell-conditioned 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 low-dose group （all P < 0.05）. The expression levels of p53， Bax， Caspase-3 and Caspase-9 mRNA in the low- and high-dose groups were significantly up-regulated， whereas that of Bcl-2 mRNA was significantly down-regulated 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.

【Key words】Human papillomavirus type18;Cervical cancer;HeLa cell;Mesenchymal stem cells;

Apoptosis

人乳头瘤病毒（HPV）是一种闭合双链DNA病毒[1]。在HPV感染导致的众多上皮病变中，子宫颈癌與HPV的持续感染高度相关[2]。目前约90%的子宫颈癌由高危型HPV感染引起，其中69.1%的子宫颈癌是由HPV16和HPV18引起的，这对全球女性的健康造成了严重的不良影响[3]。目前大部分HPV疫苗以预防HPV感染为主，主要保护HPV阴性人群，且不能终身预防[4]。因此，探索一种靶向清除HPV阳性子宫颈癌细胞的治疗方案，成为了目前子宫颈癌防治的迫切需求。

人脐带间充质干细胞（MSC）是一种多能干细胞，因取材广泛、增殖迅速，在细胞治疗领域备受关注[5]。研究显示，MSC能增加血循环中未成熟CD4+ T淋巴细胞数目，逆转HIV-1持续感染的结局[6]。乙型肝炎终末期肝病患者输注MSC，能降低HBV的病毒复制水平[7]。同时，MSC的抗癌特性引起了研究人员的关注[8]。……