小鼠异基因骨髓移植后TLR4/MyD88信号过表达与肠道急性GVHD的相关性

Correlation between overexpression of TLR4/MyD88 signaling and intestinal graft-versus-host disease after allogeneic bone marrow transplantation in mice

目的 探讨异基因骨髓移植后肠道急性移植物抗宿主病（GVHD）小鼠模型中TLR4/MyD88/NF-κB信号通路的表达及其与GVHD的相关性.方法 以雄性C57BL/6小鼠和雌性BALB/c小鼠作为骨髓移植的供、受鼠,受鼠接受致死剂量（8.0 Gy）的全身照射后4～6 h内,输注供鼠来源骨髓细胞（1×10^7个）及脾脏淋巴细胞（1×10^7个,n=12和2×10^7个,n=12）,分别建立轻度和重度小鼠急性GVHD模型,以未接受任何处理的正常BALB/c小鼠作为空白对照（n=6）.以受鼠临床表现、存活时间及小肠病理改变作为肠道GVHD的评价指标;采用实时荧光定量聚合酶链反应、免疫组织化学法和蛋白质印迹法检测各组小鼠小肠组织中TLR4、MyD88和NF-κB p65mRNA及蛋白的表达,并分析其与GVHD进展的相关性.结果 小肠组织中TLR4、MyD88及NF-κB p65的表达均呈上升趋势.重度GVHD小鼠小肠组织中TLR4、NF-κBp65 mRNA水平较正常对照显著升高（P＜0.05）.各组小肠组织中蛋白的表达也有相同的趋势.此外,TLR4、MyD88和NF-κB mRNA间的表达不仅呈正相关,而且其与GVHD的临床评分也呈正相关（R=0.814,P＜0.001;R=0.828,P＜0.001;R=0.568,P=0.034）.结论 肠道GVHD模型小鼠小肠组织中TLR4/MyD88/NF-κB信号通路呈明显的活化状态,基因转录和翻译水平均有增强,且与GVHD病情呈显著的正相关.

Objective To explore the expression of TLR4/MyD88/NF-κB signaling and its correlation with the progression of acute intestinal graft-versus-host disease （iGVHD） after allogeneic bone marrow transplantation in mice.Method Recipient BALB/c female mice were lethally irradiated and were reconstituted within 4-6 h with a transplant of bone marrow cells （1 × 10^7） and different amounts of splenocytes （1 × 1^07,n =12 or 2 × 10^7,n =12） from MHC-mismatched C57BL/6 donors to induce iGVHD,and 6 healthy BALB/c mice served as controls.A globe survey observation of GVHD by survival,clinical manifestation,and histological detection was performed.RT-PCR,immunohistochemistry,and Western blotting technology were used to detect the mRNA and protein levels of TLR4,MyD88 and NF-κB p65 in the small intestine tissue.Result The exacerbation of iGVHD was associated with the increasing dose of allogeneic spleen lymphocytes.The mRNA expression of TLR4,MyD88 and NF-κBp65 was increased as the iGVHD progressed.All of them in severe iGVHD model were significantly increased as compared with the healthy controls （P〈0.05）.The expression of corresponding proteins had the same tendency as mRNA.All of the three genes expression was not only positively correlated with each other,but also with the clinical GVHD score：TLR4 （R =0.814,P〈0.001）,MyD88 （R=0.828,P〈0.001）,and NF-κB p65 （R=0.568,P =0.034）.Conclusion Excessive activation of TLR4/MyD88/NF-κB signaling pathway does exist in iGVHD,and the enhanced levels of gene transcription and translation are positively correlated with the deterioration of iGVHD.