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
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摘要:
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100 μL含50 μg 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 mRNA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。
Abstract: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 50μg 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 serund 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.
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