Study On The Biological Characteristics Of Human Umbilical Cord Derived Mesenchymal Stem Cells And It Transplant To Promote Healing Of Defective Epidermis

Objective:To establish the methods of isolation , purification and expansion of human umbilical cord derived mesenchymal stem cells, (hUC-MSCs). By studying the basic biological characteristics of hUC-MSCs and its capacity of inducation and sign by green fluorescent protein (GFP)in vitro and effect on promote coalescent for skin in vivo to provide another cells source for tissue engineering and to provide a new way for cutaneous deficiency result by fire burn and tumor etc.Methods:The study was divided into three parts:1. hUC-MSCs were isolated, purification and expanded from human umbilical cord after enzyme digested. The basic biological characteristics of hUC-MSCs were investigated by the morphology investigation,growth curve drawing of proliferation and passage cultures, unit-fibroblast like colony-forming (CFU-F) assay. Immunophenotype were also detected by flow cytometre (FCM) and to compare with hUC-MSCs.2.To extract plasmid from Bacillus coil 109 which was transfected by GFP-N2, lipofecamine 2000 act as mediation to transfect hUC-MSCs,and calculate the efficiency of transfectiong after 24h and 96h.set up negative control.3.Transplant the hUC-MSCs which was signed by GFP-N2 to athymic mouse who coloboma some skin.and to detect the henosis of skin and the distribution of hUC-MSCs through histology , immunofluorescence.Results:The primary hUC-MSCs adhered to culture flask within 24-48h, formed unit-fibroblast like colonies in culture by 5-7d days.By 12-16 days primary expansion, hUC-MSCs can be passaged. The passaged hUC-MSCs proliferated quite rapidly and the number of hUC-MSCs in logarithemic growth period can be doubled within 29h. hUC-MSCs has more stronger reproductive activity than hUCB-MSCs. The immunophenotypic analysis showed that hUC-MSCs and hUCB-MSCs are all expressed CD13, CD29,CD105 and CD166, but negative expression of CD14, CD34, CD45 and CD31.2.24h later,the rate of green fluorescence of hUC-MSCs transfected by GFP-N2 is thirty-seven percent by 24h,and with the time going,the rate of green fluorescence is more higher.3. The skin henosis of experimental group athymic mouse are better than control group,the texture and layer of experimental group are more clear than control group through histology , and hUC-MSCs are distributed in skin through immunofluorescence.Conclusion:1. A simple method to isolate and amply culture-expand hUC-MSCs from human umbilical cord was established successfully. The number of harvested hUC-MSCs was appropriate for therapeutic purpose.hUC-MSCs had the basic biological and phenotypic characteristics of MSCs, and its capacity of high proliferation was quite outstanding, more than hUCB-MSCs.2.The lipofecamine 2000 as mediation to transfect GFP-N2 to hUC-MSCs is a good tracer technique,and it can be used to experiment of cells labeling.3. hUC-MSCs transplanted in vivo can promote defective skin to healing, and this result provide theory for clinical treat.