补体调节蛋白CD46在聚肌胞苷酸加重三氯乙烯致敏小鼠肝损伤中的表达规律

Expression of complement regulatory protein CD46 in poly I:C exacerbated liver immune injury in trichloroethylene sensitized mice

[目的]研究补体调节蛋白CD46在聚肌胞苷酸(poly I∶C)加重三氯乙烯(TCE)致敏小鼠免疫性肝损伤中的表达和转录水平。[方法]6~8周的雌性BALB/c小鼠36只,随机分为空白对照组(4只),溶剂对照组(4只),TCE组(15只),TCE+poly I∶C组(13只),适应性喂养1周,于第1、4、7、10天作致敏处理,第17、19天作激发处理,TCE+poly I∶C组于末次激发前3 h腹腔注射100μL含50μg poly I∶C的液体。末次激发24 h后进行皮肤评分,48 h后取小鼠血清及肝脏,试剂盒检测丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST),HE染色法观察小鼠肝脏病理,免疫组化法检测小鼠肝脏CD46和C3片段的表达,RT-PCR法检测肝脏CD46 m RNA表达水平。[结果]空白对照组与溶剂对照组小鼠背部皮肤无水肿与红斑,TCE处理组致敏率33.3%(5/15),TCE+poly I∶C处理组致敏率38.5%(5/13),差异无统计学意义。与溶剂对照组比较,TCE致敏组和TCE+poly I∶C致敏组肝脏系数升高,差异有统计学意义(P<0.01)。血清ALT和AST值在空白对照组、溶剂对照组以及未致敏组之间的差异均无统计学意义;与溶剂对照组相比,TCE致敏组和TCE+poly I∶C致敏组ALT和AST值升高,TCE+poly I∶C致敏组ALT和AST值相对TCE致敏组升高,差异有统计学意义(P<0.01)。肝脏病理检测发现TCE致敏组小鼠细胞形态异常,部分区域出现细胞水肿,而TCE+poly I∶C致敏小鼠肝脏出现大面积的细胞空泡样变性,肝细胞浆疏松且伊红染色变浅。小鼠肝脏免疫组化显示TCE致敏组C3片段少量沉积,TCE+poly I∶C组C3片段大量沉积,空白对照组、溶剂对照组与未致敏组则未发现。小鼠肝脏免疫组化显示CD46在肝脏细胞膜部位表达,空白对照组、溶剂对照组以及未致敏组高表达,TCE致敏组表达减少,而TCE+poly I∶C致敏组CD46表达极少。肝脏CD46转录水平在空白对照组、溶剂对照组以及未致敏组之间差异均无统计学意义;与溶剂对照组相比,TCE致敏组和TCE+poly I∶C致敏组CD46转录水平下降,TCE+poly I∶C致敏组CD46的转录水平较TCE致敏组下降,差异有统计学意义(P<0.01)。[结论]CD46可能在poly I∶C加重TCE致敏小鼠免疫性肝损伤中发挥重要作用。

[Objective] To study the expression and transcription of complement regulatory protein CD46 in immune liver injury of trichlorethylene （TCE） sensitized mice exacerbated by poly I ： C. [Methods] Thirty-six female BALB/c mice （6-8 weeks old） were randomly divided into blank control group （n=4）, solvent control group （n=4）, TCE treatment group （n=15）, and TCE＋poly I ： C treatment group （n=13）. Sensitization treatment was administered on day 1, 4, 7, and 10 after adaptive feeding for one week, and challenge on day 17 and 19. The mice in the TCE＋poly I：C treatment group were given intraperitoneal injection of 50 μg poly I ： C at 3 h before last challenge treatment. Mice skin reactions were observed and scored at 24 h after the last challenge. ALT and AST were detected by commercial kit from serum and liver samples at 48 h after challenge. Histological damage of liver was observed by HE staining. Expressions of CD46 and C3 fragment were observed by immunohistochemistry, mRNA expressions of CD46 were detected by RT-PCR. [Results] There was no skin erythema or edema in the blank control group and the solvent control group. The sensitization rate was 33.3% （5/15） in the TCE treatment group and 38.5% （5/13） in the TCE＋poly I：C treatment group, with no statistical differences. The liver/body coefficients in the TCE sensitized group and the TCE＋poly I ： C sensitized group were significantly higher than that in the solvent control group （P〈0.01）. No difference in the serum levels of ALT and AST was observed among the blank control group, the solvent control group, and the non-sensitized group. Compared with the solvent control group, the serum levels of ALT and AST in the TCE sensitized group and the TCE＋poly I：C sensitized group were increased （P 〈 0.01）. Compared with the TCE sensitized group, the serum levels of ALT and AST in the TCE＋poly I：C sensitized group were higher （P〈0.01）. Pathology examination of liver showed abnormal cell morphology and cell edema in the TCE sensitized group, and a large area of vacuolar degeneration of cells was observed in the TCE＋poly I ： C sensitized group, leading to liver cell cytoplasm loose and eosin staining lighter. Immunohistochemistry results showed little C3 fragment in TCE sensitized group, but largely deposited in the TCE＋poly I ： C group, and none in the blank control group, the solvent control group, and the non-sensitized group. The liver immunohistochemical assay results showed high expression of CD46 in liver cell surface in the blank control group, the solvent control group, and the non-sensitized group, reduced expression in the TCE sensitized group, and minimal in the TCE＋poly I ： C sensitized group. There was no difference in transcriptions of CD46 among the blank control group, the solvent control group, and the non-sensitized group. However, compared with the solvent control group, the transcription was decreased in the TCE sensitized group and the TCE＋poly I ： C sensitized group. Compared with the TCE sensitized group, the transcriptions of CD46 in the TCE＋poly I ： C sensitized group were decreased （P〈0.01）. [Conclusion] CD46 may play an important role in poly I ： C exacerbated immune liver injury in TCE sensitized mice