Rapsyn对正常及实验性自身免疫性重症肌无力小鼠乙酰胆碱受体的作用

目的: 探讨突触后膜乙酰胆碱受体缔合蛋白(rapsyn)对正常小鼠及实验性自身免疫性重症肌无力(EAMG)小鼠肌肉组织内乙酰胆碱受体(AChR)的作用。方法: 将扩增的pcDNA-rapsyn质粒注入小鼠左后肢,右后肢相同部位注射等量生理盐水,2周后将实验小鼠随机分为E组和C组,E组经腹腔注射AchR单克隆抗体35 (mAb35)诱导EAMG动物模型,C组经腹腔注射相同剂量的生理盐水,模型诱导48 h后处死实验动物分离双侧后肢肌肉,注射pcDNA-rapsyn质粒的左后肢肌肉分别为LE组和LC组,注射生理盐水的右后肢肌肉分别为RE组和RC组。免疫荧光染色法观察突触后膜AChR与运动终板的结合情况,RT-PCR和Western blotting法检测AChRα mRNA和蛋白表达情况。结果: 结果表明,LC组与RC组相比AChRα蛋白表达量增多(P<0.05),LE组与RE组相比AChRα蛋白表达量增多(P<0.05); LC组与RC组相比AChRα mRNA表达变化无统计学意义(P>0.05),LE组与RE组相比AChRα mRNA表达有明显降低(P<0.01)。结论: 在肌肉组织内上调rapsyn蛋白的表达对正常及EAMG小鼠AChR受体发挥保护性作用。

Effect of rapsyn on acetylcholine receptor in normal and experimental autoimmune myasthenia gravis mice

AIM:To investigate the effect of acetylcholine receptor-associated protein at synapse(rapsyn) on acetylcholine receptor(AChR) in normal mice and mice with experimental autoimmune myasthenia gravis(EAMG). METHODS:The left hind limb of each mouse was injected with pcDNA-rapsyn at 8-10 sites equally spread over the muscle,and the right hind limb was injected with the same volume of 0.9%NaCl.Two weeks after electropermeabilization, 36 mice were divided into 2 groups:the mice in group E were intraperitoneally injected with 0.5 mL 40-fold concentrated cell culture supernatant of mAb35,and the mice in group C were intraperitoneally injected with the same volume of 0.9%NaC1.The animals were killed 48 h after injection.The tibialis anterior muscles and calf muscles of bilateral hind limbs were isolated.The muscles of left hind limb received rapsyn plasmid were LE group and LC group,and the muscles of right hind limb muscles pcDNA-received 0.9%NaC1 were RE group and RC group.The expression of AChR and rapsyn at the mouse muscle endplate was detected by immunofluorescence staining.The expression of AChRa at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively.RESULTS;The protein expression of AChRαwas higher in LC group than that in RC group(P < 0.05 ).The protein expression of AChRot was higher in LE group than that in RE group(P < 0.05 ).No statistical difference of the AChRa mRNA expression between LC group and RC group was observed(P > 0.05 ).The mRNA expression of AChRa was significantly lower in LE group than that in RE group(P <0.01).CONCLUSION;Protein overexpression of rapsyn by plasmid transfection protects the AChRs in the muscles of both nomal and EAMG mice.