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重组人乙酰胆碱受体γ亚单位蛋白诱导HLA-DQ8转基因小鼠实验性自身免疫性眼肌型重症肌无力可能性研究 |
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| 重组人乙酰胆碱受体γ亚单位蛋白诱导HLA-DQ8转基因小鼠实验性自身免疫性眼肌型重症肌无力可能性研究 |
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作者：吴晓蓉,唐… 文章来源：中南大学湘雅二医院,410011 点击数：1713 更新时间：2007/4/29 23:15:39 
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| 目的 利用HLA－DQ8转基因小鼠建立一个全新的实验性自身免疫性眼肌型重症肌无力(oEAMG)模型,并探讨oEAMG免疫学发病机制。方法 HLA－DQ8转基因小鼠18只，随机平均分成对照组和实验组，实验组小鼠分别用乳化于完全弗氏佐剂的E.coli质粒表达重组人乙酰胆碱受体(H-AChR)γ单位20微克于0天免疫,乳化于不完全弗氏佐剂的重组H-AChR γ亚单位于30天和60天强化免疫,对照组用PBS代替H-AChR γ亚单位进行初次和强化免疫.眼部症状根据单眼或双眼的上睑下垂程度评估.全身肌力改变使用握力器评估.第二次免疫后第15天和45天收集小鼠血清,放射免疫分析法(RIA)和酶联免疫吸附实验(ELISA)检测小鼠血清中抗AChR抗体水平,并用逆转录聚合酶链式反应（RT-PCR）检测各组小鼠眼外肌中AChR α、 γ和ε亚单位mRNA表达变化。结果 （1）实验组小鼠单眼或双眼均出现不同程度的上睑下垂症状，眼部症状评分明显高于对照组（P<0.001），实验组oEAMG发病率为89%，明显高于对照组（P<0.001）；（2）第二次免疫后第6周开始，实验组小鼠肌肉握力明显降低，差异有显著性（第6、7周P值分别等于0.009、0.0191）；（3）第二次免疫后第15、45天，RIA结果显示实验组血清中抗－AchR抗体水平均明显高于对照组（P<0.01）,ELISA结果显示实验组血清中抗－AchR抗体(IgM、IgG、Ig2b、Ig2c、IgG1)水平均明显高于对照组（P均<0.01）；（4）与对照组相比，RT-PCR结果显示实验组AChR α、γ和ε亚单位mRNA表达明显增强（P均<0.01）。结论 重组H-AChR γ 亚单位免疫后能诱发HLADQ8转基因小鼠发生oEAMG。抗－AChR抗体在oEAMG发病机理中起重要作用，参与oEAMG的发生与发展。重组H-AChR γ亚单位的免疫原性诱导的自身免疫应答破坏眼外肌AChR，启动AChR基因转录的上调机制,免疫后小鼠眼外肌（EMOs）AChR α、γ和ε亚单位基因转录增加。这个全新的oEAMG模型能成为研究人类眼肌型重症肌无力（oMG）和全身型重症肌无力（gMG）发病机制和临床疗效评价的有效手段。
Objective: To establish a novel model of experimental autoimmune ocular myasthenia gravis (oEAMG) in HLA–DQ8 transgenic mice and study the immunologic pathogenesis of oEAMG..
Methods: Eighteen HLA-DQ8 transgenic mice were randomly divided equally into control group, experimental group. The mice in experimental group were immunized with 20ug of E. coli plasmid expressing recombinant human acetycholine receptor gamma (AChR γ) subunit emulsified in CFA on day 0 and in IFA on days 30 and 60, while mice in control group were injected with PBS emulsified in CFA or IFA. Ocular symptom was evaluated by documentation of partial or full closure of one or both eyes and generalized muscle weakness was measured by grip strength machine. Sera were collected on days 15，45 after the second immunization. The seurum anti-AChR antibody levels were tested by RIA and ELISA. The mRNA expression of mouse AChR α, fetal γ and ε subunit proteins was quantitated in the extra-ocular muscles of each group by RT-PCR.
Results:（1）Compared with the control group, the mice from experimental group exhibited partial or full closure of one or both eyelids, the scores of ocular symptom in experimental group were higher than that in control group（P<0.001）,and the oEAMG incidence between experimental（89%）and control group are significantly different. (P<0.001)；（2）The 6th and 7th week after the 2nd immunization, experimental group mice had higher average grip strengths,as compare to control group mice(all P value <0.05); （3）The serum anti-AChR antibodies levels were significantly higher in experimental group mice as compared to control group mice (p<0.01). ELISA detected the serum anti-AChRIgM、IgG、Ig2b、Ig2c、IgG1 Antibodies levels were significantly higher（all P value <0.01) in experimental group mice as compared to control group mice. （4）Compared with the control group , the mRNA expression of AChR subunits( α，γ，ε) were increased in immunized mice.
Conclusions: Our findings suggest that oEAMG can be induced in HLA-DQ8 transgenic mice by immunizing with recombinant H-AChR γ subunit. Anti-AChR antibodies played a crucial role in the pathogenesis and were involved in the development of oEAMG in transgenic mice. The H-AChR γ subunit induced the autoimmune response to mouse AChR which led to destruction of AChR, and triggered the up-regulation of AChR genetic transcription, the mRNA expression of AChR subunits( α，γ，ε) were increased in AChR-immunized mice. This new model of oEAMG could be a valuable tool to study the pathogenesis of ocular MG(oMG) and generalized MG (gMG) in humans and further for clinical therapeutic analysis.
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