The Study Of The Cartilage Of The Rat Bone Marrow Mesenchymal Stem Cells Transfected With The Gene Sox9

Objective:Degenerative disc disease is a serious threat to human health and quality of life, The normal intervertebral disc`s phenotype is quite similar to chondrocyte`s. In this research, the lentivirus-mediated gene transfection is the gene transfer technology which we used to import the Sox9 gene into the SD rat bone marrow mesenchymal stem cells(BMSCs),After incubation,we studied the effect of cartilage by the related indicators such as detecting the gene and protein expression. In this study we want to provide the seed cells for the subsequent artificial biological disc reconstruction research.Methods:BMSCs was extracted from SD rat bone marrow cavity. After then we subcultured them.We observed the morphology of the bone marrow mesenchymal stem cell we subcultured,and measured the adherence rate of them. The lentiviral which contains the Sox9-eGFP plasmid is packaged by the means of using the transfection technology of 293 T cells,in this step,we will test the best viral multiplicity of infection(MOI) value. Then we got the lentiviral gene transfected BMSCs. We then cultured the viral transfection BMSCs in a complete medium viral.After that we paid close attention to the growth state of the cells every day under a microscope.After a period of 14-day incubation,the qRT-PCR and Westem Blot technologies were used to detect the expression of the target gene,such as the Sox9 protein and collagenⅡ.Results: The adherence rate of the BMSCs extracted from SD rat bone marrow is high, the cells are long fusiform in shape, are polar ordered.After subculture the viability of the cells is good,the volume of cells is normal,the cell nucleus is clear.There is no large number of particles produced in the cytoplasm of the cells.The cells have strong growth capacity. When the 9th generation spread,the aging of the cells occurs.When the cells spread to 12 generations,the signs of aging are very obvious.The qRT-PCR results shows: In the control group, no-load group and the experimental group,the expression of sox9 gene is showed up.The relative expression of Sox9 mRNA of the samples of the three groups:1.064 ug / ul, 1.434 ug / ul, 1.456 ug / ul.The result of the two of the two comparisons of the three groups is statistically significant(P <0.01).The Westem Blot results shows : The result of the relative expression of the Sox9 protein in three groups is: control group 5351.69,no-load group 5536.347, experimental group 11103.782.The statistical method used. The results are statistically significant(P <0.05);In the experimental group,the expression of collagenII shows up. There are no expressions of this protein in the other groups.Conclusion: BMSCs can be used as an ideal seed cells in cartilage tissue engineering application.The extraction of this kind of cells is easy. The BMSCs is easy to culture and to perform the amplification in vitro; The method which used lentivirus as the carrier to transfect the sox9 gene to the rat BMSCs is good. The transfected cells have efficient and stable target gene expressions.We can prove the BMSCs begin to change into chondrocytes in our experiment. The bone marrow mesenchymal stem cells transfected with the Sox9 gene can be used as seed cells for the study of bio- artificial disc preparation.