| Objective:The rat uterine ligament injury model was established by observing the histological changes of mechanical damage of rat uterine ligament at a different time and determining the time of scar formation.Human endometrial mesenchymal stem cells(hEMSCs)were transplanted into damaged uterine ligaments.By detecting the expression of LOX and Fibulin-5 protein,the repair effect of hEMSCs on damaged ligaments was analyzed.Methods:1.Thirty-six SD female non-pregnant rats were randomly divided into six groups,6 in each group.One group was the control group without any treatment.The other five groups of rats underwent mechanical injury of a unilateral uterine ligament,and the injured ligament tissues were taken at 3 days,6 days,9 days,12 days and 15 days after the operation,respectively.By observing the histological changes to understand the repair process,the formation time of the damaged ligament scar was determined.2.HEMSCs suspension was locally injected into the damaged uterine ligaments of rats,and the presence of EMSCs was observed by immunofluorescence technology after the sampling of the determined time point of scar formation.3.Thirty-six SD female rats were randomly divided into 3 groups,12 in each group.In the experimental group,a suspension of hEMSCs with a concentration of 10^6/L was injected 50 ul after mechanical injury of the uterine ligament.In the scar group,partial injection of PBS solution after mechanical injury of the uterine ligament.The control group,they didn’t do anything.Histological changes of specimens in different groups were observed by HE staining at the determined time points of scar formation,and the expression differences of LOX and Fibulin-5 proteins between different groups were detected by immunohistochemistry and Western Blot.Results:1.The histological changes of the damaged uterine ligaments at different time were observed by HE staining.The results showed that the scars were formed in the 12-day group.In the 12-day group,compared with the 3-day,6-day,and 9-day groups,inflammatory cells and neovascularization were significantly reduced,with a large amount of fiber hyperplasia and dense fibrous connective tissue.Compared with the control group,the 12-day group had irregular structure and disordered fiber arrangement.2.Immunofluorescence results showed that human EMSCs could be observed in 12 days of injured ligament tissue.3.Histological changes of the specimens in each group 12 days after ligament injury in rats have observed: the ligament tissue structure of the control group was regular,and the fibers were closely arranged.In the scar group,the ligament tissue was loose and irregular,with the disorderly arrangement of fibers and tissue fibrosis.There were a large number of blood vessels in the ligament tissue of the experimental group,and the arrangement of fibers was more regular than that of the scar group,but the connective tissue was still loose.4.The expression levels of LOX and Fibulin-5 protein in ligament tissues of the control group,scar group,and experimental group were detected by immuneohistochemistry: LOX test results(0.041±0.012),(0.0072±0.0008),(0.058±0.014),Fibulin-5 test results(0.059±0.038),(0.0013±0.0021),(0.057±0.011),the two proteins were compared between the groups: the control group and the scar group,the scar group,and the experimental group were statistically different(P<0.05).5.Western Blot was used to detect LOX and Fibulin-5 protein expression in ligament tissues of control group,scar group and experimental group: LOX test results(1.436±0.051),(1.134±0.071),(1.862±0.127),Fibulin-5 test results(0.814±0.033),(0.412±0.096),(1.146±0.137),the two proteins were compared between the groups: the control group,and the scar group,the scar group and the experimental group were statistically different(P<0.05).Conclusion:1.Scar formation occurred 12 days after ligament injury in rats.2.Stem cells were observed 12 days after transplantation of human EMSCs into damaged ligament tissue.3.Human EMSCs may differentiate into fibroblasts under the induction of damaged ligaments,promote the repair of damaged ligaments in rats,reduce scar formation,secrete LOX and Fibulin-5,and the damaged ligaments may be improved in maintaining elasticity. |
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