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目的利用转基因技术构建人类白细胞抗原(HLA)-B*1301转基因小鼠,探讨三氯乙烯诱导迟发型超敏反应效应。方法以无特定病原体级C57BL/6雌性小鼠为实验动物。采用显微注射法构建HLA-B*1301转基因小鼠,流式细胞仪检测其脾淋巴细胞HLA-B*1301蛋白表达。野生型和转基因小鼠各16只,均设1周、2周TCE组和1周、2周对照组,每组4只,TCE组以腹腔注射联合皮肤涂抹的方式进行TCE染毒,对照组予橄榄油处理。染毒结束后,采用流式微球分析法检测各组小鼠周围血血清细胞因子表达水平,观察小鼠皮肤和肝脏病理变化,并分离小鼠脾淋巴细胞,分别予终浓度为13.14、29.88和5.00 mg/L的TCE、三氯乙醇(TCOH)和刀豆蛋白A(Con A)处理后采用5-乙炔基-2’脱氧尿嘧啶核苷掺入法测定脾淋巴细胞增殖水平。结果构建的转基因小鼠可正常表达HLA-B*1301蛋白。白细胞介素(IL)-6表达水平在小鼠类型、染毒处理和染毒时间之间存在交互效应(P<0.01)。IL-4表达水平在小鼠类型和染毒处理之间存在交互效应(P<0.05)。野生型小鼠和转基因小鼠的1周、2周TCE组IL-6表达水平分别高于同类型小鼠的1周、2周对照组[(9.78±3.18)vs(3.40±0.26)ng/L,(12.59±4.79)vs(3.56±1.68)ng/L;(10.41±3.08)vs(2.70±0.49)ng/L,(4.04±0.82)vs(2.96±1.12)ng/L;P<0.01];转基因小鼠的1周和2周TCE组IL-4表达水平分别低于同类型小鼠的1周和2周对照组[(3.87±0.42)vs(5.33±1.69)ng/L,(3.45±0.35)vs(4.44±0.33)ng/L,P<0.05,P<0.01]。转基因小鼠2周TCE组IL-6和IL-4表达水平均低于野生型小鼠的2周TCE组[(4.04±0.82)vs(10.41±3.08)ng/L,(3.45±0.35)vs(4.90±0.71)ng/L,P<0.01],并低于转基因小鼠的1周TCE组[(4.04±0.82)vs(12.59±4.79)ng/L,(3.45±0.35)vs(3.87±0.42)ng/L,P<0.01]。Con A处理后,脾淋巴细胞增殖水平在小鼠类型、染毒处理和染毒时间之间存在交互效应(P<0.05);分别经TCE和TCOH处理后,脾淋巴细胞增殖水平在小鼠类型、染毒处理和染毒时间的主效应和交互效应上均无统计学意义(P>0.05)。连续在体TCE染毒2周后,4只野生型小鼠中有1只皮肤出现轻微病理学改变;4只转基因小鼠中有1只出现背部皮肤组织和肝脏组织病理改变,均较野生型小鼠明显。结论首次构建了HLA-B*1301转基因小鼠,并且能正常表达HLA-B*1301蛋白。转基因小鼠对TCE的免疫毒性作用较野生型小鼠更敏感,经在体连续TCE染毒后,其机体免疫功能呈现先增强后抑制的现象。
OBJECTIVE: To construct human leukocyte antigen (HLA) -B * 1301 transgenic mice by using transgenic technology and investigate the effect of trichlorethylene on delayed type hypersensitivity. Methods Female C57BL / 6 mice without specific pathogen were used as experimental animals. HLA-B * 1301 transgenic mice were constructed by microinjection. The expression of HLA-B * 1301 protein in splenic lymphocytes was detected by flow cytometry. TCE group was given 1 week, 2 weeks TCE group and 1 week, 2 weeks control group, with 4 mice in each group. The TCE group was treated with TCE by intraperitoneal injection combined with dermal smear, while the control group To olive oil treatment. After the exposure, the expression of cytokines in peripheral blood serum of mice in each group were detected by flow cytometry, the pathological changes of skin and liver in mice were observed, and the spleen lymphocytes of mice were isolated and the final concentrations were 13.14, 29.88 and The proliferation of splenic lymphocytes was measured by 5-ethynyl-2’-deoxyuridine incorporation after 5.00 mg / L TCE, TCOH and Con A treatments. Results The constructed transgenic mice can express HLA-B * 1301 protein normally. The interleukin (IL) -6 expression level had an interaction effect (P <0.01) between mouse type, exposure time and exposure time. IL-4 expression level had interaction effect between mouse type and exposure (P <0.05). The expression of IL-6 in TCE group was higher than that in the same type of mice for 1 week and 2 weeks respectively ([9.78 ± 3.18 vs 3.40 ± 0.26] ng / L, (12.59 ± 4.79) vs (3.56 ± 1.68) ng / L, (10.41 ± 3.08) vs (2.70 ± 0.49) ng / L, (4.04 ± 0.82) vs (2.96 ± 1.12) ng / ]. The levels of IL-4 in the TCE group were lower than those in the control group [(3.87 ± 0.42) vs (5.33 ± 1.69) ng / L, 3.45 ± 0.35) vs (4.44 ± 0.33) ng / L, P <0.05, P <0.01]. The expression of IL-6 and IL-4 in TCE group was lower than that in wild-type mice [(4.04 ± 0.82) vs (10.41 ± 3.08) ng / L, (3.45 ± 0.35) vs (4.90 ± 0.71) ng / L, P <0.01] and lower than those in the 1 week TCE group [(4.04 ± 0.82) vs (12.59 ± 4.79) ng / L, 0.42) ng / L, P <0.01]. Con A treatment, spleen lymphocyte proliferation level in mouse type, exposure and exposure time between interaction effect (P <0.05); respectively, after TCE and TCOH treatment, the level of splenic lymphocyte proliferation in the mouse type There were no significant differences in the main effects and interaction effects between exposure and exposure time (P> 0.05). After two consecutive weeks of exposure to TCE in vivo, there was a slight pathological change in one of the four wild-type mice; one of the four transgenic mice showed pathological changes in the skin and liver on the back compared to the wild-type Mice obviously. Conclusion HLA-B * 1301 transgenic mice were constructed for the first time and expressed HLA-B * 1301 protein normally. The immunotoxicity of transgenic mice was more sensitive to TCE than that of wild type mice. After continuous TCE exposure, the immune function of the transgenic mice first increased and then inhibited.