| Objective:1.To investigate the expression of ACE2 gene in h AMSCs,changes of cellular function and the paracrine action of stem cells after ACE2 gene transfection into h AMSCs.2.The therapeutic effect of human amniotic mesenchymal stem cells transfected with ACE2 gene on pulmonary arterial hypertension was observed through a rat model of pulmonary arterial hypertension(PAH).Methods:1.ACE2-h AMSCs were prepared,and the expression changes of ACE2 protein in human amniotic mesenchymal stem cells transfected with ACE2 are detected by Western blot(WB).Quantitation real-time polymerase chain reaction is used to detect the m RNA expression changes of VEGF-A,FGF-2,Ang-1,SDF-1,e NOS and TNF-a in human amniotic mesenchymal stem cells transfected with ACE2.Whether the cell function of h AMSCs transfected with ACE2 gene is enhanced was determined by cell migration assay and tubule formation assay.2.Choose healthy,male,6-week-old SD rats,and are randomly divided into normal control group(Control group),MCT-induced pulmonary arterial hypertension group(PAH group),the treatment group of human amniotic mesenchymal stem cells(h AMSCs group)and the treatment of human amniotic mesenchymal stem cells transfected with ACE2(ACE2-h AMSCs group).The preparation of PAH model:one-time intraperitoneal injection of monocrotaline(MCT)50mg/Kg,control group intraperitoneal injection of the same volume of saline solution.Building model three weeks later,detect whether the building successfully.On the 2nd day after the successful modeling.The ACE2 group and the ACE2-h AMSCs group are injected with 1×10~6 cells diluted with 0.5m L saline,in the tail vein of each group at one time,once a week,a total of 2 weeks.Control group and PAH group are injected with the same amount of saline.The m PAP is measured after 2 weeks of intervention,and the right ventricular hypertrophy index is measured,HE staining of lung tissue.Western Blot and QPCR test the proteins and genes expression levels of ACE2,Ang II,Apelin,GCP-2,α-actin in the lung tissue of rats in each group.ELISA detecte the expressions of TNFα,IL-6,IL-10,IL-17 and IL-23 in the serum in each group.Results:1.In vitro cell test results(1)FACS analysis showed that h AMSCs and ACE2-h AMSCs showed MSC-specific characteristics,expressed CD29 surface antigen,and the expression rate was 94.15%;they did not express CD45 surface antigen,and the expression rate was 2.79%.(2)The cell migration and tubule formation experiments showed that:after 24hours of cell culture,the cell migration ability of ACE2-h AMSCs was enhanced by68%compared with pure h AMSCs,and the difference was statistically significant(p<0.05).After 15 hours of cell culture,the tubule formation ability of ACE2-h AMSCs increased by 125%compared with pure h AMSCs,and the difference was statistically significant(p<0.05).(3)The expression of ACE2 protein detected by WB:Compared with pure h AMSCs(0.36±0.02),the level of ACE2 protein detected in ACE2-h AMSCs(0.83±0.10)was significantly increased,and the difference was statistically significant(p<0.05).(4)QPCR detects the m RNA expression of pro-angiogenic factors,inflammatory factors,and vasodilatory substances:Compared with pure h AMSCs,ACE2-h AMSCs up-regulated pro-angiogenic factors and pro-vasodilation factors,including VEGF-A,FGF-2,Ang-1,SDF-1,and e NOS,and the difference was statistically significant(p<0.01).2.Interventional effect of ACE2-h AMSCs on cardiopulmonary tissue in pulmonary hypertension rats(1)Compared with PAH group and h AMSCs group,m PAP and RV/(LV+S)of ACE2-h AMSCs group were significantly decreased,and the difference was statistically significant(p<0.01).(2)HE staining of lung tissue specimens:Under light microscope(×200),compared with PAH group and h AMSCs group,ACE2-h AMSCs group significantly reduced inflammatory cell infiltration,significantly reduced lumen stenosis,and significantly thickened pulmonary arteriole walls improve.(3)The protein expression of ACE2,Ang II and Apelin in lung tissue of each group was detected by Western Blot:compared with the PAH group,the ACE2 and Apelin protein expressions in the ACE2-h AMSCs group were significantly increased,and the difference was statistically significant(p<0.05).(4)QPCR detection of GCP-2,ACE2,α-actin m RNA expression in lung tissue of rats in each group:Compared with the control group,the results showed that the expression levels of ACE2 and GCP-2 m RNA in the PAH group were significantly decreased,and the expression ofα-actin was significantly increased,and the difference was statistically significant(p<0.01);Compared with PAH group,the m RNA expression levels of ACE2 and GCP2 in lung tissue of ACE2-h AMSCs group were significantly increased,whileα-actin was significantly decreased,and the difference was statistically significant(p<0.05).(5)ELISA test results in serum of rats in each group:Compared with PAH group and h AMSCs group,TNFα,IL-6,IL-23 and IL-17 in ACE2-h AMSCs group were significantly decreased,and IL-10 was significantly increased,the difference was statistically significant(p<0.01).Conclusions:1.After transfection with the ACE2 gene,the tubule formation ability and cell migration ability of h AMSCs were improved,and the paracrine effect of h AMSCs was also promoted.2.ACE2-h AMSCs can significantly reduce pulmonary mean arterial pressure and improve pulmonary vascular and right ventricular remodeling in MCT-induced PAH.3.The h AMSCs transfected with ACE2 up-regulated the expression level of Apelin in lung tissue,down-regulated the expression level ofα-actin,and at the same time increased the expression level of anti-inflammatory factors and decreased the expression level of pro-inflammatory factors in serum.These changes were associated with increased expression of ACE2 and decreased expression of Ang II in lung tissue.Stable overexpression of ACE2 promotes the transformation of the ACE-Ang II-AT1R axis into the ACE2-Ang-(1-7)-Mas axis in RAS,which expands blood vessels,improves endothelial cell function,regulates the levels of pro-and anti-inflammatory factors,Anti-fibrotic effect. |
