The Experimental Research Of Effect Of Hypoxia On Nucleus Pulposus-like Cells Differention Of Human Nucleus Pulposus Derived Mesenchymal Stem Cells

Chapter I In vitro Isolation,culture and identification ofhuman nucleus pulposus derived mesenchymal stem cellsObjective : To isolate, culture and identifyhuman nucleus pulposus derived mesenchymal stem cells in vitro and study their biologicalcharacteristics.Methods:Nucleus pulposus samples from 10 patients with lumbar degenerative disease were collected to extract NP-MSCs by digestion of 0.05 % Type II collagenase for 4 hours.During primary culturing and passaging,the morphological changes of NP-MSCs were observed with inverted microscope.Flow cytometry assaying were used to detect the expression of cell surface markers such as CD29, CD105, CD34, CD45 and HLA-DR.Afterwards,human NP-MSCs were induced by specific induding medium under monolayer culture conditions to detect its differentiating potentials towards adipose,osteocyte and chondrocyte.Result: MSCs from human degenerated nucleus pulposus tissue can be extracted by digestion of 0.05% Type II collagenase.It took an average of 3 days for primary NP-MSCs to achieve adherence and about 30 days to reach a confluence of 80%.After 3 passagings,the polygonal cells gradually changed into spindle-like cells,which demonstrated a swirling clustering capability.They had positive expression of CD29,CD105 and negative or low expression of CD34,CD45,HLA-DR.The cells had positive results of Alizarin red S,Safranin’O staining and negative results of Oil red staining after an induction period of 21 days.Conclusion:NP-MSCs can be isolated from human degenerated disc tissue by use of 0.05% Type II collagenase.They had the multilineage differentiation poetentials such as osteocyte and chondrocyte,but they had a defective adipogenic differentiational capability.Chapter II Biological effects of hypoxia on differention fromhuman nucleus pulposus derived mesenchymal stem cells tonucleus pulposus-like cellsObjective:To explore biological effects of cell differentiation from human nucleus pulposus derived mesenchymal stem cells(NP-MSCs) to nucleus pulposus-like cells under conditions of low oxygen concentration. Methods:NP-MSCs were cultured under medium containing transforming growth factor-β3(TGF-β3) in different oxygen concentrations(5%,21%).The m RNA expression of HIF-lα,SOX-9,Aggrecan and Type II collagen were detected by method of real-time fluorescence PCR at time point of 7 and 14 days.The expression of Type II collagen in differentiated cells was detected by method of immunofluorescence after 14 days.The results between groups were compared by independent-samples t test.Results:At 7 days,there was statistically significant difference on m RNA expression of HIF-lα and SOX-9 between hypoxic group and normoxic group(P<0.05).After 14 days,there was statistically significant difference on m RNA expression of HIF-lα,SOX-9,Aggrecan and Type II collagen between hypoxic group and normoxic group(P<0.05). And the expression of Type II collagen was higher in normoxic group.Conclusion: human nucleus pulposus derived mesenchymal stem cells can be differentiated towards nucleus pulposus-like,which could be promoted by hypoxia.