摘要
AIM:To investigate the function of PU.1-silenced semimature dendritic cells(DCs)and the possibility of utilizing cell immunity in rat intestinal transplantation.METHODS:DCs were isolated from the bone marrow of F344 rats and cultured using the adherent method.The PU.1 gene was knocked down in DCs using small interfering RNAs(siRNAs)for 24 h,and the cells were then incubated with lipopolysaccharide for 48 h.The PU.1 siRNA that had the highest silencing efficiency was screened using reverse transcription-polymerase chain reaction and Western blot for further study.The tolerance capacity was analyzed and compared between PU.1-silenced DCs(siRNA PU.1 group),negative control-silenced DCs(siRNA NC group)and immature DCs(control group)both in vitro and in vivo.CONCLUSION:Blocking expression of the PU.1 gene in vitro led to a reduction in DC maturation and an increased tolerance capability.PU.1-silenced DCs expressed moderate levels of major histocompatibility complex(MHC)-Ⅱand low levels of co-stimulatory molecules,and produced more interleukin(IL)-10,but less IL-12.Compared with the negative control group,surface molecules cluster of differentiation 80(CD80),CD86 and MHC-Ⅱin the siRNA PU.1 group were 27.0%±5.6%,23.6%±4.8%and 36.8%±6.8%,respectively,and showed a significantly lower trend(P<0.05).In vivo treatment of recipients with PU.1-silenced DCs injected before intestinal transplantation(siRNA PU.1group),significantly prolonged allograft survival and resulted in better tissue histopathology compared with the siRNA NC group and control group.Mean survival time after transplantation was 14.3±3.3 d in the siRNA PU.1 group(P<0.05).CONCLUSION:PU.1-silenced semi-mature DCs induced partial immune tolerance both in vitro and in vivo,which could be used as a new strategy to promote transplantation tolerance.
AIM: To investigate the function of PU.1-silenced semi-mature dendritic cells (DCs) and the possibility of utilizing cell immunity in rat intestinal transplantation.
METHODS: DCs were isolated from the bone marrow of F344 rats and cultured using the adherent method. The PU.1 gene was knocked down in DCs using small interfering RNAs (siRNAs) for 24 h, and the cells were then incubated with lipopolysaccharide for 48 h. The PU.1 siRNA that had the highest silencing efficiency was screened using reverse transcription-polymerase chain reaction and Western blot for further study. The tolerance capacity was analyzed and compared between PU.1-silenced DCs (siRNA PU.1 group), negative control-silenced DCs (siRNA NC group) and immature DCs (control group) both in vitro and in vivo.
RESULTS: Blocking expression of the PU.1 gene in vitro led to a reduction in DC maturation and an increased tolerance capability. PU.1-silenced DCs expressed moderate levels of major histocompatibility complex (MHC)-II and low levels of co-stimulatory molecules, and produced more interleukin (IL)-10, but less IL-12. Compared with the negative control group, surface molecules cluster of differentiation 80 (CD80), CD86 and MHC-II in the siRNA PU.1 group were 27.0% ± 5.6%, 23.6% ± 4.8% and 36.8% ± 6.8%, respectively, and showed a significantly lower trend (P < 0.05). In vivo treatment of recipients with PU.1-silenced DCs injected before intestinal transplantation (siRNA PU.1 group), significantly prolonged allograft survival and resulted in better tissue histopathology compared with the siRNA NC group and control group. Mean survival time after transplantation was 14.3 ± 3.3 d in the siRNA PU.1 group (P < 0.05).
CONCLUSION: PU.1-silenced semi-mature DCs induced partial immune tolerance both in vitro and in vivo, which could be used as a new strategy to promote transplantation tolerance.
基金
Supported by a grant from the Natural Science Foundation of Jiangsu Province,China,No.BK2008237
