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

Abstract

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 ①The 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). ③Wound 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). ④FCM 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. Key words: Mesenchymal stem cells; Carcinoma, hepatocellular; Cell proliferation; Cell cycle; Culture media, conditioned