Experimental Research Of Differentiation Of Human Amniotic Mesenchymal Stem Cells Into Ligament Cells In Vitro

Objective Discuss whether h AMSCs possesses the characteristic of mesenchymal stem cells, and could be differentiated by ligament cells in vitro after induction.Methods The h AMSCs were separated through enzyme digestion, the cells at passage 3 were divided into 4 groups:A, B, C And D, and phenotypic characteristics of h AMSCs was tested through flow cytometry; the cellular activities and proliferation were examined by an inverted contrast microscope and SRB; the capacity of cells in each group to secrete ligament specific proteins was detected through IHC; and m RNA expressions of ligament specific proteins were measured by real-time fluorescence quantification PCR.Results The flow cytometry result indicates that h AMSCs expresses mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B and C grow like spindle-shaped fibroblasts when observed under an inverted contrast microscope,The SRB result indicates that the cells in each group reach the peak of growth curve on about the sixth day, Also, the IHC results indicates: after induction training for 7,14 d and 21 d, respectively, expressions of type I collagen in group A, B, and C were significantly higher than that of group D; and the expression of type I collagen in group B was gradually increased over time, with statistically significant differences(P<0.001). After induction training for 14 d and 21 d, respectively, expressions of type III collagen in group A, B, and C were significantly higher than that of group D; and expressions of type III collagen in group A and C were gradually increased over time,with statistically significant differences(P<0.001). After induction training for 7 d,expressions of Tenascin-C were not detected in any of the groups. But after induction training for 14 d and 21 d, expression of Tenascin-C was not detected in group D,while expressions of Tenascin-C in group A and C were gradually increased over time,with statistically significant differences(P<0.05). The real-time fluorescence?quantification PCR results revealed: on 21 d, m RNA expressions of type I and type III collagen were significantly higher in group C than that of group A, m RNA expressions of type I and type III collagen were also significantly higher in group C than that of group B; m RNA expression of Tenascin-C was significantly higher in group B than that of group A, while m RNA expression of Tenascin-C was significantly higher in group C than that of group B, with statistically significant differences(P<0.05). m RNA expressions of type I collagen and Tenascin-C in group A and C were gradually increased over time, while m RNA expressions of type III collagen in group C showed a trend of increase, with statistically significant differences(P<0.001).Conclusion The h AMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. Through in vitro induction, the expression of ligament peculiar genes is improved and the synthesis of ligament peculiar proteins also strengthened. As a result, it can be regarded as one of ligament tissue engineering seed cell sources.