| Objectives⑴To construct 4-1BB specific small hairpin RNA(shRNA) expression plasmids as well as lentiviral vector,and observe the expression of 4-1BB costimulatory molecule on rat lymphocytes inhibited by small interfering RNA(siRNA).⑵To investigate the role of blockade of 4-1BB/4-1BBL costimulatory pathway with RNA interference(RNAi) in acute rejection of rat orthotopic liver transplantation.Methods⑴According to the mechanism of RNAi, 4 expression sequences of shRNA target to rat lymphocyte 4-1BB were designed(shRNA441,shRNA540,shRNA621,shRNA758), and inserted into expression vector pGPU6. The recombined plasmid vectors(p441,p540,p621,p758) were identified by restriction enzyme analysis and sequencing,then, transfected into rat lymphocytes with electroporation. The changes of 4-1BB mRNA level and gene expression were determined by semi-quantitative Reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry(FCM)at 48 h post transfection, meanwhile, one of the recombined plasmids was judged optimization for following experiments.⑵The sequence of U6 + shRNA was obtained from the optimization recombined plasmid by PCR amplification, and cloned to lentivirus tansfer vector pcDNA-CMV- Lentivector. identified with restriction endonuclease digestion and sequencing, recombined tansfer vector pLVs/shRNA and other package plasmids were cotransfected to 293T cells with the help of lipofectamine 2000 to produce Lentivirus(LVs). Lymphocytes from BN rats as well as Lewis rats were respectively infected by LVs and pretreated by mitomycin C(MMC),then,the mixed lymphocyte reaction(MLR) were performed. RT-PCR, FCM and Western blot assay were employed to assess the expression of 4-1BB. Apoptosis was monitored by FCM also.The cell reproductive activity were detected by MTT and 3H-TdR.cytokine such as IL-2,IL-10 and IFN-γlevels were assayed by commercial ELISA kits.⑶24 cases of orthotopic liver transplantation were performed in Lewis to BN rats by using"the two-cuff technique",and all rats were divided into experimental contrast group, negative control group and interference group randomly. The BN rats of 3 groups were injected saline, empty LVs and recombinant LVs via the dorsal penis vein respectively before operation. On the 5th day after transplantation, 3 rats in each group were killed. The lymphocytes were obtained from spleen to detect the 4-1BB expression by RT-PCR and FCM. blood samples were drawn from inferior vena cava for biochemistry tests and cytokine assay. liver lobes were excised to study the pathological changes. Postoperative survival time of the rats in each group was recorded also.Results⑴The interferential plasmid p441,p540,p621 and p758 were successfully constructed identified with restriction endonuclease digestion and sequencing. Relative expression of 4-1BB on activated T lymphocytes subjected to those plasmid were (33.4±2.3)%,(53.0±2.3)%,(62.1±3.1)% and (40.9±1.5)% respectively,while (67.2±1.5)% in control group. Silencing effect of p441 was the best.⑵Recombined lentivirus tansfervector LVs441 was also identified by restriction enzyme analysis and sequencing. The lentivirus was produced successfully by 293T cells with titer of 5×106 TU/ml,even 3×108 TU/ml if concentrating was carried out.90% T cells could be infected by LVs441, expression of 4-1BB was decreased(p<0.01), and the apoptosis T cell numbers increased in interference group, T cells reproductive activity and cytokine levels in interference group were lower than those in control group.⑶In vivo,the expression of 4-1BB in interference group was lower than those in control group,and levels of IL-2,IFN-γ,ALT,AST,LDH were decreased too.Banff scores calculated by the degree of acute cellular rejection in two groups were 7.11±0.78 and 5.89±1.05(p=0.0384),while the mean survival time were (11.00±4.28) and (12.75±5.57) days respectively(p<0.05).conclusions⑴siRNA could decrease the expression of 4-1BB mRNA and protein, induce T cells to apoptosis. furthermore, the proliferation of rat T cells and the levels of cytokine production were inhibited.⑵RNAi could inhibit T cell costimulatory pathway, prevent acute rejection,and prolong survival.⑶Electroporation technique was an effective method for transfecting plasmid to some difficult-transfection cells,such as T lymphocytes.⑷Lentivirus was an appropriate and efficient vector to deliver siRNA into T lymphocytes.
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