【作者】 吴晓蓉；

【导师】 唐罗生； Premkumar Christadoss；

【作者基本信息】 中南大学 ， 临床医学， 2012， 博士

【摘要】 目的：观察E.coli质粒表达重组人乙酰胆碱(human acetylcholine receptor, H-AChR)γ亚单位免疫诱导HLA-DQ8转基因小鼠眼肌型实验性自身免疫性重症肌无力(ocular experimental autoimmune myasthenia gravis, oEAMG)模型的建立并探讨oEAMG模型的特点,明确乙酰胆碱受体抗体(acetylcholine receptor antibody, AChR-Ab)在oEAMG发病机制中的作用和特点。方法：HLA-DQ8转基因小鼠27只,随机平均分成H-AChR γ亚单位免疫组,E. Coli.提取物免疫组,CFA免疫组,用乳化于CFA的重组H-AChR γ亚单位,或E. Coli提取物,或纯CFA分别于0,30,60天免疫。第二次免疫后,每周进行一次小鼠眼部和全身症状评估,小鼠肌肉握力测量,实验结束前进行小鼠前庭眼反射检测。第二次免疫后第15天和45天收集小鼠血清,RIA检测小鼠血清AChR-Ab水平,ELISA检测小鼠血清中IgG、IgG1、IgG2b、IgG2c、IgM水平。结果：H-AChR γ亚单位免疫组9只小鼠中8只(89%)出现眼部症状,6只小鼠有完全上睑下垂症状,9只小鼠中6只(67%)小鼠出现轻度的全身症状(1级),而E. coli免疫组,CFA免疫组无明显眼部表现及全身肌力减退症状,H-AChR γ亚单位免疫组眼部及全身临床症状严重度评分明显高于其它两组(P均<0.001)；第二次免疫后35天起,H-AChR γ亚单位免疫组小鼠肌肉握力与E. coli免疫组,CFA免疫组两组比较具有显著性差异(P均<0.05),H-AChR γ亚单位免疫组小鼠肌肉握力在实验终止前降至最低；与E. coli免疫组,CFA免疫组两组相比,H-AChR γ亚单位免疫组小鼠水平维度的视动反应增益明显下降(P均<0.05)；第二次免疫后15,45天,H-AChRγ亚单位免疫组小鼠血清中AChR-Ab和抗-AChR Ig (IgM、IgG、Ig2b、Ig2c、 IgG1)水平均明显高于E. coli免疫组,CFA免疫组,差别有显著性(P均<0.001)。结论：重组H-AChR γ亚单位能成功诱导小鼠oEAMG模型,AChR-Ab在诱导oEAMG发生和发展的过程中发挥重要作用。目的：探讨重组H-AChRγ亚单位免疫诱导下oEAMG的免疫学发病机制。方法：HLA-DQ8转基因小鼠用乳化于CFA的重组H-AChRγ亚单位,或E. Coli提取物,或纯CFA分别免疫,免疫后第8天后,取淋巴结和脾淋巴细胞体外培养,3H-TdR掺入法检测淋巴细胞增殖反应,收集培养上清液用ELISA检测IL-2,IFN-γ,IL-6,IL-10水平。第3次免疫后30天,流式细胞仪观察各组小鼠脾脏和淋巴结中CD4+CD25+细胞和CD19+细胞百分比变化,免疫荧光法检测3组小鼠眼外肌(extraocular muscle, EOM)和后肢肢体肌(limb muscle, LM)神经肌肉结点处(neuromuscular junction, NMJ) IgG或补体沉积情况,RT-PCR法检H-AChRγ亚单位免疫组和CFA免疫组小鼠EOM和LM的AChR α, γ和ε亚单位mRNA转录水平的变化。结果：H-AChR γ亚单位免疫组小鼠淋巴结和脾淋巴细胞增殖反应显著高于E. Coli提取物或CFA免疫组(P均<0.05),ELISA显示H-AChR γ亚单位免疫组小鼠淋巴细胞体外培养上清液中IL-2, INF-γ水平显著均高于E. coli提取物和CFA免疫组(P<0.05),IL-6,IL-10水平与E. coli提取物和CFA免疫组相比均无显著差异(P均>0.05)。流式细胞结果显示,H-AChR γ亚单位免疫组小鼠淋巴结和脾脏中CD4+CD25+细胞和CD19+细胞百分比显著高于E. coli提取物和CFA免疫组(P均<0.05)。H-AChR γ亚单位免疫组小鼠EOM和LM NMJs处可见大量IgG, C3,和MAC沉积,而E. coli提取物和CFA免疫组小鼠NMJs未见IgG, C3,和MAC沉积(P均<0.001)。与CFA免疫组相比,H-AChR γ亚单位免疫组小鼠EOM和LM的AChR α, γ和ε亚单位mRNA转录水平显著增高,有统计学差异(P均<0.05)。结论：H-AChR γ亚单位免疫能诱导oEAMG和gEAMG的发生与发展,细胞免疫、体液免疫和补体等多种机制参与其发病。目的：观察H-AChR ε亚单位免疫原性在眼肌型重症肌无力发病机理中的作用,进一步探讨AChR亚单位诱导产生的自身免疫反应与oMG和oEAMG的关系。方法：通过PT-PCR检测HLA-DQ8转基因小鼠出生后0,1,2,4周EOM和LM的AChR α, γ和ε亚单位表达水平变化。HLA-DQ8转基因小鼠用乳化于CFA的重组H-AChR ε亚单位,或E. Coli提取物,或纯CFA分别于0,30,60天免疫,第二次免疫后每周进行一次小鼠眼部和全身症状评估,小鼠肌肉握力测量,第二次免疫后第45天收集小鼠血清,RIA检测小鼠血清中AChR-Ab水平,ELISA检测小鼠血清中IgG、IgG1、IgG2b、IgG2c、IgM水平,第3次免疫后30天,免疫荧光法检测小鼠NMJ处IgG或补体沉积情况。结果：EOM的AChR α, γ和ε亚单位的mRNA转录水平随出生后时间的增加无明显改变(P均>0.05),在出生后第0周小鼠LM的中可见AChR γ亚单位的表达,无AChR ε亚单位的表达,出生后第1周LM中,AChR γ亚单位的表达消失,出现AChR ε亚单位的表达,而α亚单位表达无明显变化(P均>0.05)。H-AChR ε亚单位免疫组9只小鼠中3只(33%)有眼部症状,2只(22%)小鼠有轻度的全身症状(1级),而E.coti免疫组,CFA免疫组无明显眼部和全身改变,H-AChR ε亚单位免疫组眼部和全身临床症状严重度评分明显高于其它两组(P均<0.05)。H-AChR ε亚单位免疫组小鼠平均肌力与E-coli免疫组,CFA免疫组两组比较无显著性差异(P均>0.05)。第二次免疫后45天,H-AChR ε亚单位免疫组小鼠血清中AChR-Ab和抗-AChR Ig(IgM、IgG、Ig2b、Ig2c’IgG1)水平均明显高于E.coli免疫组、CFA免疫组(P均<0.01)。有明显眼部和全身症状的H-AChRε亚单位免疫组小鼠EOM和LM NMJs处可见大量IgG,C3,和MAC沉积,而E.coli提取物和CFA免疫组小鼠NMJs基本没有IgG,C3,和MAC沉积(P均<0.01)。结论：HLA-DQ8转基因小鼠对H-AChR ε亚单位免疫诱导的oEAMG的易感性低,可能与AChR ε亚单位与HLA-DQ8分子的交互作用所诱导的自身免疫应答反应较弱有关。更多还原

【Abstract】 Objective:To investigate the ocular experimental autoimmune myasthenia gravis (oEAMG) induced by E. coli plasmid expressing recombinant human acetycholine receptor (H-AChR) γ subunit immunization in HLA-DQ8transgenic mice, and to determine the role and characteristic of acetylcholine receptor antibody (AChR-Ab) in the pathogenesis of oEAMG.Methods:Twenty-seven HLA-DQ8transgenic mice were divided equally into H-AChR γ subunit-immunized, crude E. coli extract-immunized and only CFA-groups randomly. Mice were immunized with AChR y subunit in CFA emulsion,20μg of crude E. coli extract in CFA or CFA only on day0,30and60. After the2nd immunization, the gMG and oMG scores and were evaluated weekly, generalized muscle weakness was also measured by grip strength machine weekly. The study of vestibulo-ocular reflexes was performed before termination, serum samples from individual mice were collected on day15,45after the2nd immunization, and the serum anti-AChR antibody levels were tested by RIA and serum anti-AChR Ig (Ig G、IgG1、IgG2b、IgG2c、IgM) isotype levels were tested by ELISA.Results:As opposed to the crude E. coli extract-and CFA-immunized mice with no ocular or generalized symptoms,8of9γ subunit-immunized mice (89%) had ocular symptoms,6of9mice with H-AChR γ subunit-immunization had complete ptosis in one or both eyes at termination, Mild generalized muscle weakness (grade1) was observed in6of9H-AChR γ subunit-immunized mice at termination. The scores of ocular and generalized symptoms of the γ subunit-immunized group were significantly higher than those in other two groups (all P<0.001). The difference among the three groups in grip strengths attained statistical significance starting on day35after the2nd H-AChR γ subunit immunization (all p<0.05) and reached the lowest level at termination. The horizontal optokinetic response gains were significantly lower in H-AChR γ subunit immunization group mice as compared to other two group mice(all P<0.05). On day15and45after the2nd immunization, the levels of anti-AChR Abs and anti-AChR Ig (Ig G、IgG1、IgG2b、 IgG2c、IgM) isotype levels were significantly higher in the sera of H-AChR γ subunit-immunized mice as compared to other two groups (all P<0.001)Conclusions:oEAMG can be generated in HLA-DQ8transgenic mice by H-AChR γ subunit immunization, and AChR-Abs played a key role in the pathogenesis of oEAMG. Objective:To explore the immunologic pathogenesis of oEAMG induced by recombinant H-AChR γ subunit immunization.Methods:DQ8mice were immunized as described above with20μg of AChR γ subunit,20μg of crude E. coli extract or CFA only. Seven days following y subunit immunization, mice were euthanized and draining lymph node cells (LNC) and spleen lymphocytes were cultured in vitro. The lymphocyte proliferation was estimated by the3H-TdR incorporation, and the supernatant were collected to observe the IL-2, IFN-y, IL-6, IL-10production by ELISA. The day30after the3rd immunization, The percentages of CD4+CD25+and CD19+cell in LNC and spleen lymphocytes of the mice from H-AChR γ subunit-immunized, crude E. coli extract-immunized and CFA-groups were detected by flow cytometry. IgG, C3and membrane attack complex (MAC) deposits at the NMJ from LM and EOM samples of the mice were detected by immunofluorescence assay. The levels of AChR α, γ and s subunit mRNA expression of LM and EOM samples of the mice were evaluated by RT-PCR.Results:LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice showed significantly increased proliferative responses as compared to E. coli extract-immunized and CFA-immunized mice (all P<0.05). LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice exhibited significantly enhanced IFN-γ and IL-2as compared to those of the other two groups (all P<0.05). There were no significant differences in IL-6, IL-10levels among three groups (all P>0.05). The percentages of CD4+CD25+and CD19+cell in LNC and spleen lymphocytes of the mice from H-AChR γ subunit-immunized were significantly higher than those in other two groups (all P<0.05). H-AChR γ subunit-immunized mice had abundant amounts of C3, IgG and MAC deposits in extraocular and limb muscle NMJs, whereas these deposits could hardly be detected on the NMJs of crude E. coli extract and CFA immunized mice (all P<0.001). The EOM and limb muscle samples from H-AChR γ subunit-immunized mice demonstrated significantly increased mRNA levels for AChR α, γ and ε subunit(all P<0.05)Conclusions:oEAMG and gEAMG could be triggered by autoimmunity to the recombinant H-AChR γ subunit. Multiple mechanisms including cellular Immunity, humoral immunity and complement are involved in the pathogenesis of oEAMG. Objective:To study the role of H-AChR ε immunity in the pathogenesis of oEAMG, and to further explore the relationship between the autoimmune response induced by AChR subunit and oEAMG.Methods:Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the levels of AChR α,γ andesubunit genes mRNA expression in the extra-ocular and limb muscles of HLA-DQ8transgenic mice of Ow,1w,2w,4w of their development. HLA-DQ8transgenic mice were immunized with AChR ε subunit in CFA emulsion,20μg of crude E. coli extract in CFA or CFA only on day0,30and60. Mice After the2nd immunization, the gMG and oMG scores and were evaluated weekly, and grip strength machine was also used to measure the generalized muscle weakness weekly, serum samples from individual mice were collected on day45after the2nd immunization, and the serum anti-AChR antibody levels were tested by RIA and serum anti-AChR Ig (Ig G、IgG1、IgG2b、IgG2c、IgM) isotype levels were tested by ELISA. IgG, C3and membrane attack complex (MAC) deposits at the NMJ from LM and EOM samples of the mice were detected by immunofluorescence assay before termination.Results:There were no significant differences in AChR α,γ and ε subunit expression of EOM samples from HLA-DQ8mice at different stages of ontogeny (P>0.05). At Ow after birth, the AChR Y subunit was detected whereas AChR ε subunit was not detected in the LM samples. The AChR ε subunit mRNA was initially detected at1w, the beginning of AChR ε subunit expression was coincident with the beginning of disappearance in AChR Y subunit expression. There were no significant differences in AChR a subunit expression of LM samples at different stages (P>0.05). As opposed to the crude E. coli extract-and CFA-immunized mice with no ocular or generalized symptoms,3of9ε subunit-immunized mice (33%) had ocular symptoms, Mild generalized muscle weakness (grade1) was observed in2of9H-AChR ε subunit-immunized mice at termination (P<0.05). The scores of ocular and generalized symptoms of the s subunit-immunized group were significantly higher than those in other two groups (all P<0.05). There were no significant differences in grip strengths among three groups (all P>0.05). On day45after the2nd immunization, the levels of anti-AChR Abs and anti-AChR Ig (Ig G、IgG、IgG2b、IgG2c、IgM) isotype levels were significantly higher in the sera of H-AChR ε subunit-immunized mice as compared to other two groups (all P<0.01). H-AChR ε subunit-immunized mice with prominent ocular symptoms had abundant amounts of C3, IgG and MAC deposits in extraocular and limb muscle NMJs, whereas these deposits could hardly be detected on the NMJs of crude E. coli extract and CFA immunized mice (all P <0.01).Conclusions:HLA-DQ8transgenic mice showed lower susceptibility to H-AChR ε subunit immunization, which can be explained by the weaker autoimmune response induced by the specific interaction between AChR ε subunit and HLA-DQ8molecular.更多还原