Effects Of PD-L1 Gene Silencing In Adipose Tissue-derived Mesenchymal Stem Cells On Acute Rejection In Rat Liver Transplantation

Objective: To construct a lentiviral vector for RNA interference (RNAi) of the PD-L1 gene, and to silence PD-L1 gene of adipose tissue-derived mesenchymal stem cells (ADSCs); To study the effect and mechanism of preoperative infusion of adipose tissue-derived mesenchymal stem cells of PD-L1 gene silencing on acute rejection in rat liver transplantation.Methods: A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting the PD-L1 gene were designed and inserted into the linearized pFU-GW-iRNA vector using gene recombination technology. The recombinant plasmid and a lentivirus packaging mix were co-transfected into 293T cells to obtain packaged lentivirus particles. ADSCs were transfected by the produced virus. Model of SD rat to Wister rat liver transplantation was set up. Recipient Wistar rats were randomly divided into four groups, 10 in each group: control group (A group), the donor group (B group), third-party group (C group), third-party transfected group (D group). Seven days before liver transplantation, in the control group, recipient Wistar rats received 1ml PBS; in the experimental group, recipient Wistar rats received 1ml PBS of 1×107 ADSCs from donor SD rats, third-party SD rats and third-party SD rats with PD-L1 gene silencing. Rats were killed seven days after liver transplantation. The liver function, IL-2 and IL-10 levels and liver pathological changes were tested in each group. The apoptosis of liver was detected by TUNEL. The expression levels of Bcl-2 and Bax proteins were detected by Western blot.Results: The recombinant plasmid was identified by DNA sequencing. The sequence of PD-L1 interference was inserted into pFU-GW-iRNA carrier correctly. A lentiviral vector for RNA interference (RNAi) of the PD-L1 gene was constructed successfully, and the titer of virus was 5×108TU/ml. Bright green fluorescence in the ADSCs was observed under the fluorescence microscope after 48h transfected with the virus. The best multiplicity of infection (MOI) was 20. The levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin and IL-2 of other groups were significantly reduced compared with A group (P<0.05), they were higher in D group than in C group. The level of interleukin-10 was significantly increased(P<0.05), it was lower in D group than in C group. Liver pathology revealed a severe acute rejection in liver graft of A group, a mild acute rejection in B and C groups, moderate acute rejection in D group. TUNEL staining showed that a lot of apoptotic cells were observed on tissue sections of liver graft, while there were some apoptotic cells in B and C groups, many apoptotic cells were observed in D group. Western blot results showed that: the expression of Bcl-2 protein was low in A group and was high in B and C groups; it was lower in D group than in C group. The expression of Bax protein was high in A group and was low in B and C groups; it was higher in D group than in C group.Conclusion: PD-L1 gene RNAi lentiviral vector plasmid was constructed successfully. PD-L1 gene of ADSCs was successfully knocked down. Acute rejection in rat liver transplantation was less inhibited by preoperative infusion of ADSCs with PD-L1 gene silencing than infusion of ADSCs. PD-1/PD-L1 was an important pathway which ADSCs down-regulate the immune response.