ABSTRACT
To investigate the effects and mechanism of bone marrow mesenchymal stem cell (MSC)modified by cytotoxicity T lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig)gene on the rejection of orthotopic liver transplantation (OLT)in rats. Methods MSC was infected with recombinant adenoviruses (Ad)5 containing CTLA4-Ig gene. After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h,the total proteins were extracted. The protein expression of CTLA4-Ig was assessed by Western-blot.The suppression to lymphocyte proliferation by MSC and transgenic MSC were tested by cell counting kit (CCK)-8 analysis. Forty models of acute rejection after OLT in rats were established by modified Kamada’s two-cuff technique,with male Lewis and BN rats serving as liver donors and recipients respectively. Forty recipient rats were randomly divided into 4 groups with 10 rats in each group including control group (group A, only saline solution was injected into portal venous during transplantation),MSC group (group B,MSC was injected into portal venous during transplantation),transgenic MSC group (group C,transgenic MSC was injected into portal venous during transplantation),immunosuppressant group [group D,saline solution was injected into portal venous during transplantation,and ciclosporin (CsA)was administered intramuscularly at a dose of 1.5 mg /(kg·d) for 8 days]. On the 9 th day after operation,5 rats were killed randomly in every group,then the levels of interleukin (IL)-2,interferon (IFN)-γ,IL-4 in peripheral blood were measured and the pathological changes and rejection expression of liver tissues were observed by light microscope. The survival condition of other 5 rats in 4 groups was observed. Results After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h,the protein expression of CTLA4-Ig gene in MSC infected with Ad5-CTLA4-Ig could be detected by Western-blot.When the ratios of MSC∶peripheral blood monouclear cell (PBMC)were 1∶10 and 1∶20,the rates of suppression to lymphocyte proliferation were 85.60% and 76.69% respectively.When the ratios of transgenic MSC∶PBMC were 1∶10 and 1∶20,the rates of suppression to lymphocyte proliferation were 90.50% and 84.20% respectively. Compared with MSC,MSC infected with Ad5-CTLA4-Ig had stronger effect on suppression to lymphocyte proliferation (P <0.05 ). The survival time after liver transplantation of rats in group A,B,C,D was (13 ±3),(41 ±6),(90 ±15),(102 ±18)d respectively.There were significant differences among group A,B,C (P<0.05 )and there was no significant difference between group C and D (P>0.05 ). Compared with group A,the serum levels of IL-4 in group B and C were significantly higher (P<0.05 ). The serum levels of IL-4 in group C were significantly higher than that in group B (P<0.05 ). There was no significant difference in the serum levels of IL-4 between group C and D (P>0.05 ). Compared with group A,the serum levels of IL-2 and IFN-γin group B and C were lower significantly (P<0.05 ). The serum levels of IL-2 and IFN-γin group C were significantly lower than those in group B (both in P<0.05 ). There were no significant differences in the serum levels of IL-2 and IFN-γbetween group C and D (P>0.05 ). The pathological result of liver tissues of rats showed that the grafts of group A developed severe rejection,and the grafts of group B developed moderate rejection. And the grafts of group C and D developed slight rejection. Conclusions MSC infected with recombinant Ad5-CTLA4-Ig can inhabit the rejection in liver transplantation,and the effect is superior to MSC alone.
ABSTRACT
Objective To study the mechanism of cytotoxic T lympocytes in the acute rejection of xenogenic liver transplantation(XLT). Methods Hamster to rat orthotopic liver transplantation model was performed with three cuff method. In XLT,lymphocytes including CD8 and CD4 subsets were observed by histology and immunohistochemistry; the expression of perforin and Fas- L was observed by immunohistochemistry; and apoptotic cells of XLT were observed in situ end-labelling of fragmented DNA. Results In the XLT, T cell infiltration firstly ocurred on the 2nd day posttansplantation and located at the portal triads; the infiltrating lymphocytes proliferated increased with time and reached the peak on the 5th to 6th days.Perforin and Fas-ligand were expressed on the 4th day after XLT, and peaked on the 5th to 6th day,and the expression of perforing was still higher than the expression of Fas-ligand. Conclusions T lymphocyte participates in the acute rejection of XLT through the expression of perforin and Fas-ligand, which leads to apoptosis or necrosis of hepatocytes.