| Objective:To establish methods of culture and amplification and identidy of bone marrow mesenchymal stem cell in vitro; and to study on the effect of cell cycle and apoptosis and survivin expression on human mesenchymal stem cells from bone marrow (BM-MSC) pretreated with varying doses of X-rays.Methods:The cells of bone marrow were harvested by bone marrow aspiration on the posterior superior iliac spine of health adult donors. The mononuclear cells were separated by percol separating medium(r=1.073g/ml)and then adherent cultured in DMEM-LG containing 15%FBS.The traits of shape were observed by inverted microscope,the surface markers of human mesenchymal stem cells from bone marrow were determined by FACS. The experiments were assigned to group A (control group),group B (the dose of X-irradiation was 5cGy),group C (the dose of X-irradiation was 10cGy) and group D (the dose of X-irradiation was 20cGy). The P4 BM-MSC were exposed to X rays at the dose of 5cGy, 10cGy and 20cGy(dose rate 0.1 Gy/min,radiation distan was 100cm).The proliferation of cells was tested with MTT assay,and then the cell growth curve was drawn according to OD from one to seven days.The cells were harvested at 24h,48h and 72h after treating by low of X-rays.The cell cycle and apoptosis(marker with AnnexinV and PI)were determined by FACS;The expression of survivin mRNA was detected by RT-PCR assays;The damage to DNAof BM-MSC induced by X-irradiation were detected with mononuclearcell gel electrophoresis assays.Results:The cells shape was observed by inverted microscope, we found that the BM-MSC were homogeneous,spindle-shaped and fibroblast-like cells.The cells can be propagated and extensively amplified in vitro. The FACS results show that the BM-MSC express CD44 and CD13 (93.37%),without expressing marker of hemopoietic stem cells(CD117,CD45,CD38,CD3).we found from MTT that the proliferation of BM-MSC was not suppressed in groups B,C and D, and the cell grow cability and growth times of BM-MSC treated with varying doses of X-rays were obviously increased.The results of cell cycle show that the G0/G1 stage cells gradually decrease after radiating with dose X-rays of 5cGy,10cGy,20cGy,compared with the same time control group, The percent of G0/G1 had obviously decreased at 48h,72h, (P<0.05),the percent of G0/G1 stage cells of lOcGy at 72h was the lowest.Compared with control group, the percent of S stage cells were gradually increased at 24h,48h,72h in C group and D group,which had obviously increased at 48h and 72h in C group and D group, (P<0.05),but it was not obviously change in B group.Compared with same time control group,the percent of G2/M was not change between A group and B group, the percent of G2/M were obviously increased at 24h and 48h in C group and D group,(P<0.05),however,it was decreased at 72h,and the percent Of G2/M stage cells of 20cGy at 72h was the lowest, (P<0.05);The result of apoptosis indicated that the percent of apoptosis cells had increased tendency at 24h in B group and C group and D group, (P<0.05), while had decreased tendency at 48h,72h, The apoptosis percentages was the lowest at 72h,especially in 10cGy and 20cGy groups, (P<0.05). The expression level of survivin mRNA in BM-MSC was gradually increased after exposure to X-irradiation for 24h and 48h in groups B,C and D,while decreased tendency at 72h,but still higher than that in A group(p< 0.005). The level expression of survivin of BM-MSC was came to peak at 48h after exposure to X-irradiation.The comet experiment indicated the degree of damage to DNA of BM-MSC induced X-irradiation was depending on the dose of X-rays(A group,comet rate 2%,B group comet rate 14%,C group comet rate 33%,D group comet rate 39%).Conclusion:We found from MTT that the proliferation of BM-MSC was not suppressed while the cell growth rates and growth times of BM-MSC treated with varying doses of X-rays were obviously increased than control group. The results of cell cycle and apoptosis show BM-MSC were promoted transformed from G0/G1 stage to S stage,and the percent of S stage cells was increased than control, the percentages of apoptosis was transient enhance in the early 24h (P<0.05), and soon here decreased,these results indicated that the low dose of X-irradiation has hormesis effect on BM-MSC. The low dose of X-irradiation had promoted expression of survivin and the express of survivin was came to peak at 48h after pretreated with X-rays,it indicated the surviving had a important of role to resist apopotosis induced with low dose of X-irradiation. The comet experiment indicated the degree of damage to DNA of BM-MSC induced X-irradiation was depending on the dose of X-rays. |
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