摘要
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 establish human leukocyte antigen（ HLA）-B＊1301 transgenic mice by transgenic technique and explore the trichloroethylene（ TCE）-induced delayed hypersensitivity effect on the model. Methods Specific pathogen-free C57BL/6 female mice were used as the laboratory animals. HLA-B＊1301 transgenic mice were established by microin-jection. The expression of HLA-B＊1301 protein of spleen lymphocyte was determined by flow cytometry. Wild type and＆amp;nbsp;HLA-B＊1301 transgenic mice were divided into one-week,two-week TCE groups and one-week,two-week control groups respectively. Each group included 4 mice. TCE groups were exposed to TCE by intraperitoneal injection and back skin expo-sure while control groups were treated with olive oil. After the treatment,cytokine expression in serum was detected by cyto-metric bead assay,and the pathomorphological changes in skin and liver tissues were observed. Spleen lymphocytes were sep-arated and treated with TCE,trichloroethanol（ TCOH）and concanavalin A（ ConA）at final concentrations of 13. 14,29. 88 and 5. 00 mg/L respectively,and then the levels of spleen lymphocyte proliferation（SLP）were detected by 5-ethynyl-2’-deoxyuridine incorporation method. Results The established transgenic mice could normally express HLA-B＊1301 pro-tein. The interaction effect among mice type,TCE exposure and treatment time in interleukin（ IL）-6 concentration was statistically significant（P〈0. 01）. The interaction effect between mice type and TCE exposure in IL-4 concentrations was statistically significant（P〈0. 05）. Both in wild type and transgenic mice,IL-6 concentrations of one-and two-week TCE groups were significantly higher than those of one-and two-week control groups of the same kind mice 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/L;P〈0. 01]. In transgenic mice,IL-4 concentrations of one- and two-week TCE groups were significantly higher than those of one- and two-week control groups respectively[ 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]. IL-6 and IL-4 concentrations of two-week TCE group of transgenic mice were significantly lower than those of two-week TCE group of wild type respectively[（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 were significantly lower than those of one-week TCE group of transgenic mice respectively[（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]. After treated with ConA in vitro,the interaction effect among mice type,TCE exposure and treatment time in SLP level was statistically significant（P〈0. 05）. After treated with TCE and TCOH in vitro respec-tively,none of the main effect and interaction effect of SLP level on mice type,TCE exposure and treatment time had sta-tistically significant（P〈0. 05）. After exposure to TCE in vivo for two weeks,one of the four wild-typed mice showed mild granulocyte and lymphocyte infiltration in the back skin without cellular edema in the liver,while one of the four transgenic mice was more serious in this kind of skin pathologic changes obviously with cellular edema in the liver. Conclusion HLA-B＊1301 transgenic mice was established with expression of HLA-B＊1301 protein for the first time. Compared with wild type mice,the transgenic mice are more susceptible to immunotoxicity of TCE. Exposure to TCE in vivo may lead to immune enhancement in the first week and immunosuppression in the second week in the transgenic mice.
出处
《中国职业医学》
CAS
北大核心
2014年第5期481-488,共8页
China Occupational Medicine
基金
国家自然科学基金(81172641)
