| ObjectiveBone marrow was obtained from the rabbits rabbit femoral with puncture,adherent method was used to isolate and culture the rabbit bone marrow mesenchymal stem cells(BMSCs)and flow cytometry was performed to detect the positive and negative surface markers of rabbit BMSCs to ensure target seed cells;capacity of differentiation of the target cells were also detected by osteogenic,adipogenic,cartilage to determine the target seed.To construct and verify the TGF-?1,Sox-9 gene sequence,pLVX-IRES-Puro shuttle vector was used to construct TGF-?1(pLVX-TGF-?1),and Sox-9(p LVX-Sox-9)vector.The feasibility of transfection was determined.Combined with TGF-?1,Sox-9 to co-transfect rabbit BMSCs to detect the target gene and downstream genes,analysising the feasibility and advantages of seed cells after transfection.With Courtman acellular methods to handling porcine annulus to get acellular annulus scaffolds,the effect of decellularising methods.Co-culture the co-transfected stem cells and acellular scaffolds to perform experimental study of tissue engineering annulus fibrosus.MethodsObtain the rabbit BMSCs with the method of direct way.Be sure of the BMSCs through the surface mark and induced differentiation into three different mesoderm cells.Compose the TGF-?1 and Sox-9 gene fragment and make sure of their gene order.Establish the TGF-?1 Lentivirus vector(pLVX-TGF-?1)and Sox-9 lentivirus vector(p LVX-Sox-9).Determine the TGF-?1 and Sox-9 expression in mRNA level through Real-time PCR.Divide BMSCs into four groups:A.blank vector group;B.singly transfected by TGF-?1 lentivirus;C.singly transfected by Sox-9 lentivirus;D.Co-transfected by both TGF-?1 and Sox-9 lentivirus.After 2 weeks,measure the TGF-?1 and Sox-9 protein expression of group A?B?C?D and the expression of ACAN?Collagen I?Collagen II?Collagen X and Sox-9 in mRNA level through Western blot and Realtime PCR respcetively.With improvement of Courtman protocol of decellularising cells to prepare the scaffolds,GAG,DNA and Hyp content were detected to analyze the effect the decellularised annulus fibrosus scaffolds.Co-culture the co-transfected stem cells and acellular scaffolds to perform experimental study of tissue engineering annulus fibrosus.Phalloidin,DAPI and confocal microscopy were used to detect growth status of the co-transfected cells in the acellular scaffold annulus.ResultThe cells got through the all bone marrow adherence method are able to differentiate into at least three kinds of mesoderm cells:osteoblast.chondroblast and adipocyte.The cells also express cell surface marker of CD29,CD44,CD90 according to flow cytometry detection.Gene order sequencing demonstrate that the gene fragments are correct.The amplified DNA by PCR contain TGF-?1 and Sox-9 gene fragments tested by electrophoresis.Two weeks after culture of Co-transfected BMSCs,the shape of most cells changed obviously.Western blot showed ACAN and Sox-9 proteins at a higher level than BMSCs.The expression level of ACAN,Collagen I,Collagen II and Sox-9 gene were much higher than control group,and the expression level of Collagen II in Co-transfected group was higher compared to solo-transfected groups.The expression of Collagen X in Sox-9 transfected group and Co-transfected group were obviously lower than TGF-?1 transfected group and control group.With improvement of Courtman protocol of decellularising cells to prepare the scaffolds,The level of cell proliferation and collagen type ? expression was significantly higher than normal BMSCs.Transgenic rabbit BMSCs can be uniformly distributed in the surface of the scaffolds and grow well.ConclusionIt is pertinent to prepare the BMSCs by direct way.Lentivirus vector of TGF-?1 and Sox-9 could transfect the BMSCs and express the TGF-?1 and Sox-9.BMSCs transfected by TGF-?1 and Sox-9 get higher expression of ACAN,Collagen I,Sox-9 and lower expression of Collagen X,what demonstrated that both TGF-?1 and Sox-9 are able to induce the BMSCs into annulus fibrosus-like cells(AFCs). |
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