| Part I Effect of TIPE2 modification on proliferation,migration,and secretion ability of immunosuppression-related factors in h AMSCsAim:To investigate the effects of TIPE2 overexpression gene modification on the proliferation,migration,and immunosuppression-related factor secretion ability of h AMSCs.Laying the foundation for in vivo experiments on TIPE2 overexpression of h AMSCs.Method:(1)Identification of h AMSCs by microscopic observation,flow cytometry and immunofluorescence;(2)Selection of the optimal multiplicity of infection by flow cytometry,then observation of viral transfection by inverted fluorescence microscopy,and verification of TIPE2 overexpression by Western Blot;(3)Plotting of untransfected h AMSCs(h AMSCs),null-virus-transfected h AMSCs(GFP-h AMSCs),and TIPE2overexpressed h AMSCs(TIPE2-h AMSCs)by CCK-8 assay to plot the proliferation viability curves.The migratory ability of the three cells was detected by Transwell migration assay;(4)After the three cells were cultured in normal medium or inflammation-inducing medium which supplemented with interferon-γ(10 ng/ml)and tumor necrosis factor-α(10 ng/ml),the indoleamine 2,3 dioxygenase(IDO),transforming growth factor-β(TGF-β)and interleukin 10(IL-10)gene expression levels and secretion levels were measured by q RT-PCR and ELISA.Result:(1)h AMSCs were shuttle-shaped,grew in a swirling pattern and adherence to plastic surfaces,with high expression of CD73 and CD90,and almost no expression of CD11b,CD34 and CD45.Immunofluorescence showed high expression of vimentin,which was consistent with the characteristics of mesenchymal stem cells.(2)The appropriate MOI value was 180,and stronger green fluorescence was seen in GFP-h AMSCs and TIPE2-h AMSCs after infection,and no green fluorescence expression was seen in h AMSCs;WB results showed that compared with h AMSCs and GFP-h AMSCs,the TIPE2 protein level in TIPE2-h AMSCs was significantly higher in TIPE2-h AMSCs.(3)The proliferation viability curves showed that the proliferation ability of GFP-h AMSCs and TIPE2-h AMSCs was decreased compared with h AMSCs,while there was no significant difference between TIPE2-h AMSCs and GFP-h AMSCs;Transwell migration assay showed that the migration ability of TIPE2-h AMSCs was significantly higher compared with h AMSCs and GFP-h AMSCs(p<0.05).q RT-PCR results showed that the gene expression levels of IDO and IL-10 were increased in TIPE2-h AMSCs compared with GFP-h AMSCs in both normal culture and inflammatory factor-induced states(p<0.05);ELISA results showed that the IDO levels in the medium of TIPE2-h AMSCs in normal culture were significantly higher compared with GFP-h AMSCs compared to GFP-h AMSCs(p<0.05),and the levels of IDO and IL-10 in the medium of TIPE2-h AMSCs compared to GFP-h AMSCs were significantly increased after incubation in inflammation-inducing medium(p<0.05).Conclusion:(1)Adenoviral transfection successfully constructed GFP-h AMSCs and TIPE2-h AMSCs.(2)TIPE2 overexpression did not significantly inhibit the proliferative activity of h AMSCs and improved the cell migration ability of h AMSCs and the secretion ability of immunosuppression-related factors.Part II Effect of TIPE2 overexpression on the induction effect of immune tolerance in h AMSCs in allogeneic heart transplanted model and a preliminary investigation of related mechanisms:Aim:To investigate the effect of TIPE2 overexpression on the induction of immune tolerance in h AMSCs in a mice allogeneic heart transplantation model and to conduct a preliminary exploration of the mechanism of its effect.Method:(1)A mice heterotopic cervical heart transplantation model was established with C57BL/6 as the donor and Balb/c as the recipient.12 model mice in each group were divided into control group,GFP-h AMSCs group,and TIPE2-h AMSCs group according to the random number table method,,and received PBS(0.3 ml),GFP-h AMSCs suspension(5×10~5/0.3 ml),and TIPE2-h AMSCs suspension(5×10~5/0.3 ml)on days 1 and 4 after model establishment,respectively.(2)intraperitoneal injection of DIL fluorescent probe labeled GFP-h AMSCs and TIPE2-h AMSCs into the heart transplantation model mice,and observation of DIL fluorescence using the in vivo fluorescence imaging system on day 3after injection distribution in model mice.Recording the survival time of each group of transplanted hearts and plotting the survival curve.The transplanted heart,peripheral blood and spleen were recovered on postoperative day 7 for the following tests:a.Hematoxylin and eosin(H&E)stained sections were prepared to observe the cardiac pathological changes in the transplanted hearts;b.ELISA was performed to detect the serum levels of IL-10 and TGF-βin mice;c.Flow cytometry was performed to detect the peripheral blood and spleen levels of helper T cells in mice.and spleen,and the percentage of regulatory T cells(Treg)in CD4+T cells,and calculate the Th1/Th2 and Th17/Treg ratios;d.WB detection of p38 and p-p38 expression in transplanted heart tissues,and preliminary exploration of signaling pathway of TIPE2-h AMSCs to attenuate heart transplant rejection.Result:(1)In vivo fluorescence imaging system showed similar distribution of intraperitoneally injected GFP-h AMSCs and TIPE2-h AMSCs,and observation of each organ after dissection revealed that both cells could be homed to the transplanted heart on day 3 after intraperitoneal injection;(2)Compared with the Control group,the length of survival of the transplanted heart was significantly prolonged in the GFP-h AMSCs group(p<0.05),and further prolonged in the TIPE2-h AMSCs group compared with the GFP-h AMSCs group(p<0.05).(3)H&E stained sections showed a large amount of inflammatory cell infiltration,myocardial cell necrosis,interstitial edema,myocardial structural disorder,and myocardial fiber rupture in the Control group of transplanted hearts.The Inflammatory cell infiltration,myocardial interstitial edema and hemorrhage,and myocardial cell necrosis were alleviated in the GFP-h AMSCs compared with the Control group.A small amount of inflammatory cell infiltration could be seen in the TIPE2-h AMSCs group,and myocardial cell necrosis and interstitial hemorrhage were significantly improved compared with the GFP-h AMSCs group.(4)ELISA results showed that the serum levels of IL-10 as well as TGF-βwere significantly higher in the TIPE2-h AMSCs group compared with the GFP-h AMSCs group(p<0.05).(5)Splenocyte flow cytometric results showed that proportion of Th1 and Th17 cells in splenic lymphocytes was significantly lower(p<0.05,p<0.05)and the proportion of Treg cells was significantly higher(p<0.05),and the Th1/Th2 and Th17/Treg ratio of splenic lymphocytes was significantly lower(p<0.05,p<0.05)in the GFP-h AMSCs group compared with the Control group;compared with GFP-h AMSCs group,TIPE2-h AMSCs significantly upregulated splenic Th2 percentage(p<0.05),downregulated Th1/Th2 and Th17/Treg ratio in spleen(p<0.05,p<0.05),induced shift of splenic lymphocyte Th1/Th2 balance to Th2and Th17/Treg balance to Treg.Peripheral blood cell flow cytometric results showed that the proportion of Th1 cells was significantly lower,the proportion of Th2 and Treg cells was significantly higher(p<0.05,p<0.05),and showed lower Th1/Th2 and Th17/Treg ratio(p<0.05,p<0.05)in the GFP-h AMSCs group compared with the Control group.Compared with the GFP-h AMSCs group,TIPE2-h AMSCs significantly down-regulated the ratio of Th1 and Th17 cells(p<0.05,p<0.05),up-regulated the proportion of Th2 and Treg cells in peripheral blood lymphocytes(p<0.05,p<0.05),down-regulated the Th1/Th2ratio in peripheral blood lymphocytes(p<0.05),induced a shift of peripheral blood lymphocyte Th1/Th2 balance toward Th2.(6)WB results showed that p38 phosphorylation levels were downregulated in the GFP-h AMSCs group compared to the Control group(p<0.05).And the p38 phosphorylation level was further down-regulated in the TIPE2-h AMSCs group compared with the GFP-h AMSCs group(p<0.05).Conclusion:(1)h AMSCs injected intraperitoneally can home to the transplanted heart far from the neck;(2)h AMSCs prolonged the survival of the transplanted heart and reduced the extent of rejection injury,upregulated the levels of immunosuppressive-related factors in serum,and induced the formation of immune tolerance by regulating the ratio of CD4+T cells subsets in peripheral blood and spleen,while TIPE2 overexpression further enhanced the immune tolerance-inducing effect of h AMSCs,and the mechanism may be related to the inhibition of p38 phosphorylation in heart tissue. |
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