人脐带源间充质干细胞条件培养液对人肝癌HepG2细胞生长的影响

Effects of human umbilical cord-derived mesenchymal stem cells on cell growth of human hepatocellular carcinoma HepG2 cell

目的 探讨人脐带源间充质干细胞（HUC-MSC）条件培养液（CM）对人肝癌HepG2细胞生长的影响.方法 选择2011年2月至2014年1月于中国人民解放军第105医院妇产科分娩的35例足月妊娠剖宫产健康胎儿脐带为研究对象,取其用于分离、培养并扩增HUC-MSC.传代扩增至第3代HUC-MSC融合达80％时,更换培养液,继续培养24 h后收集上清液,即为HUC-MSC-CM备用.将对数生长期的人肝癌HepG2细胞按照随机数字表法随机分成相等的3份,分别于含50％,20％及不含HUC-MSC-CM的培养中培养,并分别纳入高含量组、低含量组和空白对照组.采用倒置显微镜观察人肝癌HepG2细胞生长状态,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）法和细胞划痕愈合实验分别检测3组人肝癌HepG2细胞增殖和迁移情况,并采用流式细胞术（FCM）法检测各组细胞的细胞周期变化情况.本研究遵循的程序符合中国人民解放军第105医院人体试验委员会所制定的伦理学标准,得到该委员会批准,脐带获取前,均征得孕妇知情同意,并与之签署临床研究知情同意书.结果 ①所分离的原代HUC-MSC培养至第3代后,细胞形态开始比较均一.②MTT法检测结果显示,人肝癌HepG2细胞培养24,48和72 h时,3组相对吸光度（A）值比较,差异均有统计学意义（F=21.330,6.223,9.345;P=0.004,0.032,0.027）,且在这3个时间点,高含量组和低含量组相对A值均显著高于空白对照组,差异均有统计学意义（P＜0.05）,高含量组相对A值亦显著高于低含量组,差异亦均有统计学意义（P＜0.05）.③划痕愈合实验结果显示,3组人肝癌HepG2细胞过河时间比较,差异有统计学意义（F=12.860,P=0.025）,且高含量组人肝癌HepG2细胞过河时间短于低含量组和空白对照组,差异均有统计学意义（P＜0.05）,低浓度组人肝癌HePG2细胞过河时间亦短于对照组,差异亦有统计学意义（P＜0.05）.④FCM法检测人肝癌HepG2细胞周期结果显示,3组G0/G1期和S期人肝癌HepG2细胞比例比较,差异均有统计学意义（F=30.600,30.590;P＜0.05）.且与空白对照组相比,高含量组和低含量组人肝癌HepG2细胞G0/G1期细胞比例显著降低,S期细胞比例显著增加,差异均有统计学意义（P＜0.01）;且高含量组G0/G1期细胞比例比低含量组下降更显著,而S期细胞比例显著增加,差异均有统计学意义（P＜0.05）.结论 HUC-MSC-CM可促进入肝癌HepG2细胞增殖和迁移,并可促进人肝癌HepG2细胞周期G1/S转换.

Objective To investigate the effect of human umbilical cord-derived mesenchymal stem cells （HUC-MSC） condition medium （CM） on cell growth of hepatocellular carcinoma HepG2 cell.Methods From February 2011 to January 2014,a total of 35 cases of full-term pregnancy healthy fetuses＇ umbilical cords were chose into this study.All the fetuses were delivered through uterine-incision in the 105th Hospital of People＇s Liberation Army.Mesenchymal stem cells were derived from human umbilical cord and expanded in vitro.When the confluence of the third generation reached 80％,the medium was changed to fresh medium.After 24-hour incubation,the supernatant HUC-MSC-CM was collected.Then the HepG2 cells in logarithmic growth phase were divided into 3 equal groups randomly by random number table method.They were cultured with 5 ％ fetal calf serum high glucose （HG）-DMEM fresh medium alone as the control group,and the experimental group were cultured with 25％ HUC-MSC-CM together with 5％ fetal calf serum HG-DMEM （low concentration group） and 50％ MSC-CM together with 5％ fetal calf serum HG-DMEM （high concentration group）,respectively.HepG2 morphology was observed by inverted microscope.The abilities of proliferation were detected by 3-（4,5-dimethyl-thiazol-2）-2,5-diphenyl tetrazolium bromide （MTT） method,and the migration of HepG2 cells in each group were detected by wound healing assay.The cell cycle distribution was analyzed by flow cytometry （FCM）.The study protocol was approved by the Ethical Reviews Board of Investigation in Human Being of the 105th Hospital of People＇s Liberation Army.Informed consent was obtained from all participants.Results ① The morphology of isolated primary HUC-MSC was uniform when cultured into the third generation.②MTT assay showed that when the HepG2 cells were cultured 24,48 and 72 h,the differences of relative absorbance （A） values in the three groups were statistically significant （F=21.330,6.223,9.345;P=0.004,0.032,0.027）.And the relative A values of HepG2 cells in two experimental groups significantly increased than that in control group at these three time points,and the differences were statistically significant （P ＜ 0.05）;the relative A values of HepG2 cells in high concentrations group was also significantly higher than in low concentration group,and the difference was also statistically significant （P＜ 0.05）.③）Wound healing assay showed that the difference of healing time of HepG2 cell in three groups was statistically significant （F=12.860,P=0.025）,and the healing time in high concentration group was shorter than that in the low concentration group and control group,the differences were statistically significant （P＜0.05）;the healing time in the low concentration group was shorter than that in the control group,the difference was statistically significant （P ＜ 0.05）.④ FCM assay showed that there were significant differences among the percentage of cells in G0/G1 phase and S phase in three groups （F =30.600,30.590;P＜0.05）.And compared with control group,the percentage of HepG2 cells in G0/G1 phase of high concentration group and low concentration group were significantly decrease,and the percentage of cells in S phase were higher,all the differences were statistically significant （P＜0.01）.The percentage of cells in G0/G1 phase of the high concentration group decreased more significantly than that in low concentration group,while the percentage of cells in S phase increased more significantly,the differences were statistically significant （P ＜ 0.05）.Conclusions HUC-MSC-CM could promote the proliferation and migration of HepG2 cells,and it can accelerate cell cycle G1/S phase transition.