| Objective: Cord mesenchymal stem cells has the function of self-renewal,multi-directional differentiation and immunosuppression.It has been proved by many clinical trials that it has good therapeutic effect on many diseases.UC-MSCs has the advantages of convenient access,good genetic stability,low immunogenicity and strong tissue repair ability,and its clinical application prospects are very considerable.This study aims to establish a method of human UC-MSCs preparation based on serum-free culture system,identify the biological characteristics and functions of the cells,and evaluate the curative effect of UC-MSCs by using diabetic foot mouse model,and lay a foundation for the research and development of stem cell drugs for diabetic foot diseases.Method: 1.UC-MSCs was prepared by using culture system without serum,animal source and limited chemical composition.The density of inoculation and concentration of wall promoting substances were optimized by CCK-8 method.The cell morphology was observed under microscope,the cell wall and growth were recorded.The proliferation activity of cells was measured by CCK-8 method.The cell surface markers were detected by flow cytometry.The differentiation potential of UC-MSCs was identified by oil red-O staining,alizarin red staining and alxinlan staining respectively.The immunosuppressive function of UC-MSCs and lymphocyte was evaluated by co culture test.2.C57 mice were used to establish diabetic model of foot ischemia foot ulcer.The efficacy test was carried out by local injection around the focus and UC-MSCs injection by tail vein respectively.After injection of stem cells,take photos every three days,record the wound ulceration or healing,and record the duration of 18 days.During the period,the number of independent standing of mice was measured by the small animal autonomous activity instrument every five days.This was the method of measuring the number of independent standing of mice every five days In addition,the blood glucose was monitored for five weeks in the treatment group of UC-MSCs after intravenous injection.The pancreas of mice was taken at the end of the experiment to observe the changes of tissue structure by HE staining.Result: 1.1% of the materials were determined as the best concentration for the application of wall promoting substances,and 12000 / cm2 density was the best density of UC-MSCs.The results showed that the positive expression rates of CD73,CD90 and CD105 on the surface of the cells were 99.9 ± 0.59%,100.0 ± 0.00% and 97.4 ± 2.19%respectively,and the positive expression rates of CD34,CD45 and HLA DR were 0.80 ±0.76%,1.00 ± 0.29% and 0.10 ± 0.26% respectively.Under the specific conditions,it can be successfully differentiated into adipocytes,osteoblasts and chondrocytes.In addition,the cells have obvious inhibitory effect on lymphocyte proliferation,and the inhibition rate can reach more than 75%.2.UC-MSCs can significantly promote wound healing and improve the ability of independent standing activity in diabetic foot model mice.The effect of tail vein injection group is more obvious.In addition,the blood glucose level of model mice in the tail vein injection group is significantly reduced and the pancreatic tissue structure is improved obviously.Conclusion: 1.UC-MSCs prepared in this study conform to the criteria of the identification of mesenchymal stem cells determined by the International Association of cell therapy in terms of morphological characteristics,proliferation ability,surface marker expression and differentiation potential.In addition,it also has a significant inhibitory effect on lymphocyte proliferation.The results show that UC-MSCs with good biological function can be prepared by using the preparation scheme of this study.2.UC-MSCs can promote wound healing and improve diabetic symptoms in diabetic foot mice,and has great potential to improve and treat human related refractory diseases,and has great clinical application value. |
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