Experimental Study On Chondrocyte Differentiation Induced By Psoralen Combined With TGF-?1

Objective: To establish an effective bone marrow stem cell culture and identification system;to explore the optimal growth concentration of psoralen for bone marrow mesenchymal stem cells;To observe the effect of psoralen combined with transforming growth ?1(TGF-?1)in inducing rat bone marrow mesenchymal stem cells(BMSCs)to differentiate into chondrocytes.Provide experimental basis for the animal experiment of cell tissue engineering technology to treat knee joint cartilage injury in rats,and then explore the potential and practical value of Chinese medicine in tissue engineering repair technology;At the same time,it provides a reliable basic experimental basis for the TCM efficacy of Psoralen "Invigorate kidney qi,strengthen bones and muscles".Method: Experiment 1:Extraction,purification and identification of SD rat bone marrow mesenchymal stem cells:using rat femur whole bone marrow extraction + cell adhesion method to separate and extract rat bone marrow mesenchymal stem cells,and culture and expand rat BMCS in vitro;It was identified by the study of its morphological structure and flow cytometry.Experiment 2:Different concentration gradients of psoralen culture medium were configured to cultivate bone marrow mesenchymal stem cells,and the effect of culture medium containing different concentrations of psoralen on cell growth was detected by CCK-8 method.Experiment 3: Psoralen combined with transfer growth factor ?1 in vitro induces bone marrow mesenchymal stem cells to differentiate into chondrocytes.In vitro induction and grouping,set up normal control group(LG-DEME,10% FBS,double antibodies),psoralen group(10umol/L psoralen,LG-DEME,10% FBS,double antibodies),psoralen Adipin combined with TGF-?1 group(10umol/L psoralen,10ng/m LTGF-?1,LG-DEME,10% FBS,double antibodies),positive induction group(10ng/m LTGF-?1,LG-DEME,10% FBS,double antibody).After 14 days,the induced cell morphology was observed with an optical microscope,and type II collagen immunocytochemical staining was performed;RT-PCR was used to detect the m RNA expression of cartilage differentiation marker genes type II collagen and Sox9;Western-blot was used to detect type II collagen And Sox9 expression level.Result: 1.After the primary culture,the fluid was changed for the first time to remove the non adherent cells.Most of the cells were spindle-shaped and triangular,and a small part was circular.After generation,the cells are purified and have uniform morphology,and impurity cells are rare.2.Flow cytometry identification of BMSCs showed that CD90 and CD44 were highly expressed in the cultured cells,but CD43 and CD45 were rarely expressed.3.CCK-8 method can promote the growth and proliferation of BMSCs,especially the concentration of 10 umol / L is the best.4.Identification of BMSCs after directional induction to chondrocytes: 4.1 Observed by inverted microscope: Bone marrow mesenchymal stem cells increase in volume after two weeks of induction,showing polygons,triangles,and stars with multiple protrusions.4.2 Type II collagen immunocytochemical staining: No positive cells were seen in the normal control group,positive cells were seen in the other three groups,and the cytoplasm was brown-red.4.3 Western-blot results: Compared with the normal control group,the expression of type ? collagen and Sox9 increased in the other three groups(P<0.05),and the combination group had the best effect(P<0.01);Compared with the psoralen group and positive Compared with the induction group,the expression of type ? collagen in the combination group was increased(P<0.05).4.4 RT-PCR results: Compared with the normal control group,the expression of type II collagen gene and Sox9 gene in the other three groups increased(P < 0.05),among which the combined group had the best effect(P < 0.01);compared with the psoralen group and the positive induction group,the expression of type II collagen gene in the combined group increased(P < 0.05).Conclusion 1.The whole bone marrow adherence method has high adherence efficiency,short first passage time,continuous proliferation ability and strong cell viability.2.CD44 and CD90 are two important surface markers of MSCs.CD44 and CD90 of BMSCs are positive.Therefore,it can be used as a practical method to identify BMSCs.3.The proliferation of BMSCs was enhanced by the stimulation of psoralen at different concentrations and time periods,and the best effect was 10?mol/L.4.Psoralen combined with TGF-?1 can promote the expression of type II collagen of rat bone marrow mesenchymal stem cells and induce them to differentiate into chondrocytes.And the effect is better than the effect of using psoralen alone.It provides an experimental basis for further in vivo experiments using traditional Chinese medicine in tissue engineering technology to treat osteoarthritis in rats.