Differential Differentiation Of HAMSCs Transfected With Scleraxis Gene Into Ligament Cells By Different Induction Methods

Objective: To investigate the effect of CTGF combined with hACLFs Transwell coculture system on the differentiation of Scx-hAMSCs towards ligament cells in vitro.Methods: 1)The hAMSCs were isolated from placental amniotic membrane by the method of enzymatic digestion and conducted heirloom culture,the hAMSCs passed to the 3rd generation were subjected to cell phenotype identification and induced differentiation of three lineages,and the effect of directed differentiation of hAMSCs to bone,cartilage and lipid cells was examined.The hACLFs were isolated from the stump of human anterior cruciate ligament by enzymatic digestion,and the expression of type I and type III collagen was detected by immunofluorescence staining;to detect the proliferation ability of hAMSCs and hACLF by using the method of CCK-8 and compare them with each other.2)The target plasmid and lentivirus packaging plasmid were co-transfected into HEK293 T cells to package the lentivirus,and concentrated to the finished lentivirus,and transfected with 3rd generation hAMSCs after titer determination.q PCR and Westen Blot were performed to verify the Scx overexpression of the transfected cells.3)Combined CTGF and Transwell co-culture system was used for induction of differentiation,and the cells were divided into 6 groups according to different culture protocols:(A)hAMSCs induced by CTGF group,(B)hAMSCs+hACLFs Transwell co-culture group,(C)Scx-hAMSCs group,(D)Scx-hAMSCs induced by CTGF group(E)Scx-hAMSCs+ hACLFs Transwell co-culture group(F)Scx-hAMSCs induced by CTGF+ hACLFs Transwell co-culture group.The cell density ratio of the two groups in the co-culture group was 1:1.The proliferation ability of each group was determined by CCK-8 method,and the ligament-related genes and protein expression of each group were examined at 7,10 and 14 days,respectively.Results: 1)The number of hAMSCs obtained by enzymatic digestion had a high digital,grew in a shuttle shape against the wall,and were rotated and arranged;The cell phenotype is meets the identification criteria of MSCs,which can differentiate into osteoblastic,chondrocytic and lipid cells after targeted induction,and has the ability of tri-lineage differentiation,which is line with MSCs characteristics.The number of primary hACLFs extracted by enzymatic digestion method was small,but increased after expansion,with a long shuttle-shaped regular arrangement;the result of immunofluorescence staining showed the expression of type I and III collagen;CCK-8 method showed that the proliferation ability of hAMSCs in vitro was significantly stronger than that of hACLFs,the difference was statistically significant(P < 0.05).2)The constructed plasmids were transformed and expanded by de-endotoxinization extraction,mixed with Lenti-Mix respectively and formulated into Lenti-Mix-DNA transfection system,which transfected HEK293 T cells and packaged with lentivirus,and infected with hAMSCs after lentivirus concentration.q PCR and Westen Blot results both showed that the Scx overexpression group cells in SCX expression was significantly higher than that of the null group(P<0.05).3)The result of CCK-8 showed that the cell proliferation level of the experimental group CTGF induced was significantly higher than that of the other groups(P<0.05).Immunofluorescence showed that the expression levels of collagen type III and fibronectin in each group increased positively with time,The expression levels of typeⅲ collagen and fibrin were higher in CTGF co-culture group at the 14 d.The q PCR results showed that the m RNA expression of type III collagen and fibronectin in each group was significantly up-regulated at the 14 d(P<0.05),and the m RNA expression in the CTGF and co-culture groups was higher than the Scx-hAMSCs group.Western blot results showed that the expression of type I and type III collagen in the co-culture group was higher than the hAMSCs were induced by CTGF group and the ScxhAMSCs group at the same time point.Picric acid Sirius scarlet staining showed that collagen deposition increased significantly in each group at 14 d,more collagen was secreted in the Scx-hAMSCs induced by CTGF+ hACLFs Transwell co-culture group.Conclusion: 1)The hAMSCs have the phenotypic characteristics of MSCs,with the ability of trilineage differentiation and stronger proliferation ability than hACLFs in vitro.2)Lentiviral vectors can transfect Scx gene into hAMSCs and overexpress it in host cells.3)CTGF and Transwell co-culture system have synergistic effects in promoting the differentiation of hAMSCs to ligament cells,and the combination of the two has excellent effects;among them,CTGF is more effective in maintaining cell activity and promoting cell proliferation,while Transwell co-culture is more effective in inducing cell differentiation.