Long Time Trypsin Treat Impact On Bovine Fetal Bone Marrow Mesench Ymal Stem Cells

Bone marrow contains a variety of adult pluripotent stem cells.The current isolated methods of bone marrow mesenchymal stem cells,decided that,the isolated mesenchymal stem cells may contain other types of pluripotent stem cells. These pluripotent stem cells, not onlyhave the potency of differentiate, but also repair tissues when the bodyunder stress, such as tissue damage, they will rapid proliferation, differentiation, and ultimately repair tissues.Trypsin, thatused in cell culture frequently,has strong ability of digestion and low price,is one kind of incision enzyme. Long time trypsin treat will excessive damage the membrane protein on the surface of cell membrane,caused cell damage and induced cell AP optosis.Generally,it should not be too long when we digest the adherent cells.But researchershad found and isolated Muse cells from fibroblasts and mesenchymal after long time trypsin digested them.however there is rarely reported about biological features of the adherent cells after long time trypsin treated. Experiment proves that expect enrichs the multipotency stem cells of the somatic cells after long time trypsin treated,it also improves the stress-tolerance ability like Muse cells of the survived cells. Given the current production of transgenic animals, i PS and other donor-induced cell proliferation, pluripotency, pressure receptivity,cell activity and other aspects of the increasinglyhigh demand. Research of biological properties of the cells that survived after long time trypsin treat is very necessary.This experiment, bone marrow mesenchymal stem cellshas been isolated from bovine fetal bone marrow by adherent culture.And they were identified by flow cytometry and their differentiation experiments. Digested the bovine fetal bone marrow mesenchymal stem cells(BMSCs)10 h by 0.25% trypsin solution.And then observed morphology drew cell growth curve, AP staining, quantitative PCR, Immunofluorescence staining to analyz and research biological properties of the cells(LTT-BMSCs)that survived after digestion.Found long trypsin treated would contribute to the enrichment of small volume cells and improving pluripotency gene expression. Finally,cultured LTT-BMSCs by the culture medium that were add 0 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml basic fibroblast growth factor(b FGF) respectively,to explore the impact of b FGF to the pluripotency gene expression and proliferation of LTT-BMSCs. This study laid a foundation for betterunderstanding the impact of LTT method to bone marrow mesenchymal stem cells on biological characteristics.1. Isolation cultivation and identification of bovine fetal bone marrow mesenchymal stem cells( BMSCs)In this study, bone marrow mesenchymal stem cellshad been isolated from bovine fetal bone marrow by adherent culture.And they were identified by flow cytometry and their differentiation experiments. Showed that isolated cells were mesenchymal stem cells. And in bone marrow mesenchymal stem cells CD105, CD73, CD90 expression ashigh as97.4%,84.4% and 91.4%, CD34 expression was 3.5%. The results of immunofluorescence staining and induce differentiation experiments proved the pluripotent of bovine bone marrow mesenchymal stem cells.2. Study on the impact of long time trypsin treatment( LTT) to the biological characteris of bone marrow mesenchymal stem cellsFirst,digested bone marrow mesenchymal stem cells with 0.25% trypsin for for different time period.Selected the processing time that pluripotency gene expression improved favorable by quantitative PCR, and cell counts. Trypsin treated bone marrow mesenchymal stem cells 10 h,then studied the biological characteristics of LTT-BMSCs by observed morphology, growth curve, alkaline phosphatase staining, quantitative PCR, I mmunofluorescence staining, Brdu staining and karyotype analysis.Found the LTT methodhelps to enriched smaller cells in bone marrow mesenchymal stem cells, improved the relative expression of pluripotent genes and promoting cell proliferation.Culturedhigher generation LTT-BMSCs, with culture media contain different concentrations of b FGF. Deter mined that 20 ng/ml b FGF was mosthelpful to maintain the cell proliferation and pluripotency gene expression of LTT-BMSCs.