骨髓间充质干细胞治疗实验性自身免疫性重症肌无力的作用机制探讨

Treatment of experimental autoimmune myasthenia gravis with bone marrow stromal stem cells in rats

目的探讨骨髓间充质干细胞(BMSCs)治疗实验性自身免疫性重症肌无力(EAMG)的作用机制。方法将Lewis大鼠随机分为移植组、模型组和正常对照组,每组各10只。移植组、模型组大鼠腹腔注射单克隆抗体35(m Ab35)建立EAMG模型,正常对照组大鼠腹腔注射等量0.9%氯化钠溶液。分离培养BMSCs并作鉴定后,调整密度至1.5x106/600μl PBS,移植组尾静脉注射细胞悬液600μl,模型组和正常对照组均予600μl PBS。采用双盲法、免疫荧光检测、ELISA和real-time RT-PCR分别测定3组大鼠Lennon临床评分、腓肠肌乙酰胆碱受体(AChR)数量变化、血清乙酰胆碱受体抗体(AChR-Ab)含量和腓肠肌AChR、基质金属蛋白酶9(MMP-9)m RNA表达。结果移植组Lennon临床评分、血清AChR-Ab含量、腓肠肌AChR m RNA表达均低于模型组(P<0.05)。正常对照组大鼠神经肌肉接头处可见明显红色荧光,移植组可见断断续续、较为微弱的荧光,模型组未见红色荧光。模型组大鼠腓肠肌MMP-9 mRNA表达量明显高于移植组(P<0.05)。结论 BMSCs能缓解EAMG大鼠的临床症状,抑制MMP-9 mRNA表达可能是其作用机制。

Objective To investigate the effects of bone marrow stromal stem cells （BMSCs） in treatment of experimental autoimmune myasthenia gravis （EAMG） in rats. Methods Thirty Lewis rats were divided randomly into the transplantation group, the model control group and the normal control group with 10 rats in each group. EAMG model was induced by intraperitoneal injection of mAb-35 in transplantation group and model control group. BMSCs were isolated, cultured and identified from healthy Lewis rats. Rats in transplantation group were injected with 1.5 × 10^6 BMSCs diluted in 600ml PBS, and the same volume of PBS was injected in the model control and the normal control group. The symptoms of rats were observed, the titers of plasma AChR-Ab were detected by ELISA, the AChR changes in gastrocnemius were examined by immunofluorescence technique, and the expressions of AChR and MMP-9 mRNA were detected by real-time RT-PCR in all groups. Results The clinical score, the titers of AChR-Ab, and the expression of AChR mRNA in the transplantation group were significantly lower than those in the model control group. There were no significant differences in above variates between transplantation and the normal control group. The immunofluorescence assay showed EAMG group had no red fluorescence, while the transplantation group had intermittently weak fluorescence. The expression of MMP-9 mRNA was higher in the model control group. However, there was no significant difference between transplantation group and normal control group. Conclusion BMSCs can ameliorate clinical manifestations in EAMG rats, which may be associated with inhibition of MMP-9 expression.