| Objective: To research the separation, in vitro culture and expanding method of mesenchymal stem cells (MSCs). Study the biological characteristics of MSCs and result of DAPI labeling MSCs. This study was designed to investigate the effect of traditional Chinese medicine on the therapeutic potency of MSCs transplantation in a rat model of limb ischemia.Methods: MSCs were isolated and purified from rats using density centrifugation and anchoring culture , then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for expanding. Observed the morphous and growth velocity of MSCs, identify the phenotype of MSCs, draw the growth curve of MSCs and determine the mitotic index and cloning efficiency of MSCs. Observed the survival rate and growth state of MSCs underwent freeze thawing. Also observe the efficiency of DAPI labeling MSCs and impact of DAPI on MSCs. Rats (n=24) were ligatured the right femoral artery and its branches and divided randomly into three groups (n=8, each). 24 h after the operation, rats in the MSCs+traditional Chinese medicine group and in the MSCs group were injected with 0.4 ml of DMEM loaded with MSCs (1×107 cells) intramuscularly into eight points of an ischemic area (0.05 ml each). Rats in the control group were injected with only the same dose DMEM. Traditional Chinese medicine was given twice a day for 3 weeks in the MSCs+traditional Chinese medicine group. Laser Doppler Perfusion Imaging (LDPI) and immunohistochemistry were performed to examine the effects of the treatments at 22 days after the ligation.Results: MSCs got together at 9th～12th day .The passage MSCs proliferated fast. MSCs were purified at 3rd generation. MSCs grew in whirlpool. Through detection by flow cytometry, the expressions of CD44 and CD29 were positive, the exprssions of CD11b and CD45 were negative. As the passage increases, the cloning efficiency and proliferative ability of MSCs decrease. The survival rates of MSCs thawed after frozen 1 month and 6 months were 90% and 85% respectively. The efficiency of DAPI labeling MSCs is 100%. The fluor colouration didn't weaken. DAPI had no influence on the morphous and growth of MSCs. The blood perfusion was more sufficient was higher in the MSCs+traditional Chinese medicine group and the MSCs group than that in the control group. The MSCs+ traditional Chinese medicine group demonstrated a further increase compared with the MSCs group. Immunohistochemistry and computer imaging analysis found that VEGF was upregulated in the MSCs+traditional Chinese medicine group compared to the other two groups.Conclusions: MSCs can be harvested using density centrifugation and anchoring culture. In vitro expanding of MSCs is feasible. Freeze thawing has no obvious influence on MSCs. DAPI can label the nucleus of MSCs effectively and has no obvious influence on the morphous and growth of MSCs.Combined treatment with MSCs transplantation and traditional Chinese medicine is superior to MSCs transplantation alone in the treatment of limb ischemia. |
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