An Experimental Study On Differential Expression Of ChM-I And CTGF Protein In MSCs And Chondrocyte

Background and Objective: Cartilage is one of the most important biomaterials in otorhinolaryngology. Because it has a limited capacity for self-repair when the defects are caused. Many kinds of biomaterials mean no effective now. Therefore the study on cartilage tissue engineering is becoming hot spot in otorhinolaryngology. And the biological factors become more and more hot because of its important effect in inducing MSCs into chondrocyte.The previous studies of our group screened differentially expressed genes between SD rat bone mesenchymal stem cells(MSCs) and hyaline cartilage cells /elastic cartilage cells and explore the gene expression pattern and molecular mechanism of them. It suggested that the expression of chondromodulin-I(ChM-I) and connective tissue growth factor(CTGF) genes in hyaline cartilage cells/elastic cartilage cells was obviously higher than that in MSCs. It indicate that the two genes may play an important role in the forming of cartilage , and maybe a key gene of inducing MSCs into cartilage. In the present study, we validate the differential expression of ChM-I and CTGF protein in MSCs, hyaline cartilage cells and elastic cartilage cells by Western blot assay. It will lay a solid foundation in accurately choosing biological factors that to induce MSCs into hyaline cartilage cells /elastic cartilage cells.Methods:MSCs were obtained from infancy SD rat, separated by gradient centrifugation method, and purified by monolayer culture. The hyaline cartilage cells and elastic cartilage cells were isolated from SD rat auricular cartilage and costal cartilage by enzyme digestion. Total protein was extracted from the cells of 2nd- 3rd passage, and then apply Western blot assay to observe the expression of ChM-I and CTGF in protein level. Quantified analysis of western blot was performed later.Results:1.We can isolate MSCs by density gradient centrifugation combined with attachment method. MSCs showed spindle cells in shape while cultured in vitro.We find the phenotype of cells was similar to the characteristics of MSCs.2. Western blot analysis showed : (1) the average expression of ChM-I protein level in hyaline cartilage cells was obviously higher than that in MSC(sP<0.05); The average expression of ChM-I protein level in elastic cartilage cells was obviously higher than that in MSCs(P<0.05); But the average expression of ChM- I protein level in hyaline cartilage cells compared with that in elastic cartilage cells, there was has no significant deviation(P>0.05). (2) the average expression of CTGF protein level in hyaline cartilage cells was obviously lower than that in MSCs(P<0.05); The average expression of CTGF protein level in elastic cartilage cells was obviously lower than that in MSCs(P<0.05); But the average expression of CTGF protein level in hyaline cartilage cells compared with that in elastic cartilage cells, there was has no significant deviation(P>0.05).Conclusion:1.Density gradient centrifugation combined with attachment method can be used to isolate and differentiate MSCs of rat.2. The expression of ChM-I is unanimous at both protein level and gene level. The high expression of ChM-I protein in chondrocyte indicates that it will be one of the important biological factors which is able to induce the differentiation of MSCs into chondrocyte.3. The expression of CTGF is not unanimous at either protein level or gene level, the functional mechanism in the forming of cartilage require further experiments . At present , CTGF is not recommended as a biological factor to induce the differentiation of MSCs into chondrocyte.