Therapeutic Effect And Mechanisms Of Human Umbilical Cord Mesenchymal Stem Cells Knocking Down EPCR On Pulmonary Fibrosis Induced By Bleomycin In Mice

Objective:To observe the therapeutic effect of human umbilical cord mesenchymal stem cells knocking down the EPCR gene on mice with pulmonary fibrosis and to explore their possible mechanisms.Methods:Three siRNAs were synthesized according to the mRNA sequence of human EPCR and were transferred into hUC-MSCs.Western blot was conducted to analyze the suppression efficiency of three siRNAs to screen for an optimum siRNA.Then the shRNA targeting the optimum siRNA sequence was synthesized,cloned into lentiviral vector and packaged by lentivirus.HUC-MSCs were infected with the recombinant lentivirus.The efficiency of knocking down was detected both at mRNA and protein levels.The C57BL/6 mice were divided in five groups randomly:blank control group(Ctrl),model control group(B),HUC-MSCs group,shRNA-EPCR-MSCs group and sh RNA-NC-MSCs group.Mice were intraperitoneally anesthetized with 10%chloral hydrate,and non-invasive tracheal intubation was given to 50?L saline in the control group,and the remaining group was equal volume of bleomycin 3mg/kg.For the hUC-MSCs group,shRNA-EPCR-MSCs group and shRNA-NC-MSCs group,5*10~5/50?L cell suspension were given by tracheal intubation and the same amount of sterile saline were given by tracheal intubation for other groups at the seventh day after bleomycin administration.The mice were sacrificed and the lung tissues were collected at twenty-first day after modeling.The pulmonary pathological tissues were used for hematoxylin-eosin and Masson trichrome staining.Ashcroft score was used to calculate the severity of pulmonary structure destroy and the degree of fibrosis.The collagen concentration in the lung tissues was measured by Sircol soluble collagen assay to evaluate the degree of lung fibrosis in different groups of mice.Construction of TGF-?1induced stem cells to fibroblast differentiation model in vitro,inducing shRNA-EPCR-MSCs and shRNA-NC-MSCs,respectively.The expression levels of?-SMA and Col?were detected by Real-time PCR and Western blot for evaluation of differentiative capacity of hUC-MSCs into fibroblasts.Results:Lentiviral vector that targeting to the optimal siRNA sequence of EPCR and can down-regulate the mRNA(P<0.001)and protein(P<0.001)expression of EPCR was constructed successfully.Compared to model control group,the middle survival time of mice in B+shRNA-EPCR-MSCs group was prolonged(P<0.05).Pulmonary pathological improvement in the B+sh RNA-NC-MSCs group mice.Ashcroft score also decreased(P<0.05),and collagen deposition in the lung was significantly reduced(P<0.05).And knocking down of EPCR in hUC-MSCs decreased the mRNA and protein expression(P<0.05)of Col?.TGF-?1 could induce up-expression of?-SMA and Col?in mRNA(P<0.05)and protein level(P<0.05)and transform hUC-MSCs into fibroblasts.However,knockdown of EPCR partially inhibits the expression of?-SMA and Col I induced by TGF-?1 in mRNA(P<0.05)and protein level(P<0.05).Conclusion:shRNA-NC-MSCs could ameliorate bleomycin induced mice pulmonary fibrosis and may be associated with knockdown of EPCR to reduce the expression of Col I in hUC-MSCs and suppress its differentiation capacity into fibroblasts.