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目的观察移植肝脏中Kupffer细胞(KCs)和T淋巴细胞(T lymphocytes,TLCs)中Fas、FasL基因和蛋白的表达,以及KCs对侵入肝脏内的淋巴细胞的细胞毒性作用。方法肝移植后0、48h分离肝移植组、氯化钆(GdCl3)组动物肝脏的KCs和TLCs,并对TLCs进一步分类;用RT-PCR法测定KCs和TLCs中Fas、FasL基因表达,用免疫组化、激光扫描共聚焦显微镜和Western blot蛋白印迹等方法测定KCs和TLCs中Fas、FasL蛋白质表达;观察不同培养条件下淋巴细胞凋亡与KCs的相互作用。结果GdCl3组分离出的KCs显著少于肝移植组,淋巴细胞数量及CD8+T细胞显著多于肝移植组(P<0.05),免疫组化和激光共聚焦显微镜显示GdCl3组FasL阳性KCs显著少于肝移植组(P<0.05),2组Fas阳性淋巴细胞无显著差异(P>0.05);RT-PCR和Western blot显示KCs中FasL mRNA及蛋白表达GdCl3组较弱(P<0.05),2组淋巴细胞中Fas、FasLmRNA无显著差异(P>0.05)。GdCl3组中分离出的KCs对TLCs杀伤率明显低于肝移植组(P<0.05),抗FasL抗体对KCs杀伤力有抑制作用。结论KCs能表达FasL蛋白,并通过Fas、FasL途径杀伤移植肝脏中的淋巴细胞,诱导移植肝脏产生免疫耐受。GdCl3能阻止KCs中FasL基因和蛋白表达,抑制肝移植中免疫耐受的产生。
Objective To observe the expression of Fas and FasL gene and protein in Kupffer cells (KCs) and T lymphocytes (T lymphocytes) in the grafted liver and the cytotoxicity of KCs on lymphocytes invaded the liver. Methods The hepatic KCs and TLCs in liver transplantation group and GdCl3 group were isolated at 0,48 h after liver transplantation and the TLCs were further classified. The expression of Fas and FasL gene in KCs and TLCs was detected by RT-PCR, The expression of Fas and FasL protein in KCs and TLCs was detected by laser confocal microscopy and Western blotting. The interaction between lymphocyte apoptosis and KCs in different culture conditions was observed. Results The number of KCs isolated from GdCl3 group was significantly less than that from liver transplantation group. The number of lymphocytes and CD8 + T cells in liver transplantation group were significantly more than those in liver transplantation group (P <0.05). Immunohistochemistry and confocal laser scanning microscopy showed that the number of FasL positive KCs in GdCl3 group was significantly less There was no significant difference in Fas positive lymphocytes between the two groups (P> 0.05). The expression of FasL mRNA and protein in KCs was lower than that in GdCl3 group (P <0.05) by RT-PCR and Western blot. There was no significant difference in Fas, FasL mRNA in lymphocytes (P> 0.05). The killing rates of TLCs isolated from GdCl3 group were significantly lower than that of the liver transplantation group (P <0.05). Anti-FasL antibody could inhibit the killing of KCs. Conclusion KCs can express FasL protein and kill lymphocytes in the liver by Fas and FasL pathway to induce immune tolerance in transplanted liver. GdCl3 can prevent FasL gene and protein expression in KCs and inhibit immune tolerance in liver transplantation.