转化生长因子-β1基因修饰的树突状细胞对重症肌无力大鼠发病的干预

Effects of dendritic cells modified with transforming growth factor β1 gene on the attack of myasthenia gravis in rats

目的探讨转化生长因子(TGF)-β1基因修饰的树突状细胞(DC)对实验性自身免疫性重症肌无力(EAMG)大鼠病情的影响.方法将8周龄的近交系、健康、雌性Lewis大鼠30只应用随机数字表分为6组:正常组、EAMG组、DC治疗组、pcDNA3-TGF-β1-DC治疗组、pcDNA3-DC治疗组和生理盐水对照组.除正常组外,其余均采用乙酰胆碱受体(AChR)蛋白2次免疫的方法诱导EAMG.初次免疫后第5 天上述各组除正常组与EAMG组外分别皮下注射2×106的DC、pcDNA3-TGF-β1-DC、pcDNA3-DC及等体积的生理盐水.观察各组大鼠的临床表现,并于初次免疫后7周进行低频重复电刺激,血清AChR抗体滴度检测及神经肌肉接头(NMJ)处的超微病理学观察.结果 (1)免疫后1周左右,除正常组外各组均有1～2只大鼠出现轻度的肌无力症状,持续2～5 d自行缓解.免疫后5周左右EAMG组、DC治疗组、pcDNA3-DC治疗组及生理盐水对照组大鼠均相继出现肌无力症状,而pcDNA3-TGF-β1-DC治疗组仅1只四肢力量稍差.正常组大鼠始终未见异常.(2) EAMG组、DC治疗组、pcDNA3-DC治疗组及生理盐水对照组大鼠低频电刺激肌电图波幅均有不同程度的衰减,而正常组及pcDNA3-TGF-β1-DC治疗组(分别为3.20±3.70 和5.60±2.70)均无明显衰减;pcDNA3-TGF-β1-DC治疗组大鼠肌电波波幅衰减率明显低于EAMG组(分别为5.60±2.70和16.75±6.13,P<0.05).(3) pcDNA3-TGF-β1-DC治疗组大鼠AChR抗体的水平明显低于EAMG组(分别为0.168±0.039 和0.306±0.034,P<0.01),而DC治疗组、pcDNA3-DC治疗组、生理盐水对照组AChR抗体的水平与EAMG组比较无明显差异(分别为0.276±0.066,0.294±0.082,0.288±0.079和0.306±0.034,P>0.05).(4)除正常组外,其余各组大鼠NMJ处AChR均出现不同程度的丢失.pcDNA3-TGF-β1-DC治疗组AChR丢失减少,突触结构的改变明显减轻,而DC、pcDNA3-DC及等体积的生理盐水治疗对EAMG大鼠的病理学变化无明显作用.结论初发的EAMG大鼠经皮下注射TGF-β1基因修饰的DC,可以有效地阻断EAMG病情的进展.

Objective To study the effects of dendritic cells (DC) modified with transforming growth factor β1 (TGF-β1) gene on experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. Methods 30 female Lewis rats were divided randomly into 6 groups: normal group, EAMG group, DCs treatment group, pcDNA_3-TGF-β1-DCs treatment group, pcDNA_3-DCs treatment group and normal saline group. The rats were immunized with the acetylcholine receptor (AChR) protein extracted from electric organ of Narcine timilei and completed Freund’s adjuvant (CFA) in the experiment groups except normal group. 2×106 pcDNA_3-TGF-β1-DCs/rat were injected subcutaneously into the backs of the rats that had been immunized 5 days earlier with AChR+CFA. The rats in DCs treatment group, pcDNA_3-DCs treatment group and normal saline group were injected in parallel with untreated DCs, pcDNA_3-DCs and normal saline respectively. Then the clinical manifestations were observed everyday. And 7 weeks after the first immunization, repetitive nerve stimulation, detection of acetylcholine receptor antibody (AChRab) and ultrastructural study of neuromuscular junction (NMJ) were performed. Results (1) The mild symptoms were observed on 1 or 2 rats in the experiment groups except normal group after a week, which lasted for 2 to 5 days. After about 5 weeks, the rats in EAMG group, DCs treatment group, pcDNA_3-DCs treatment group and normal saline group presented some symptoms at different degree like myasthenia gravis, and only one of the rats in pcDNA_3-TGF-β1-DCs treatment group presented mildly decreased activity. (2) The significant decrement of repetitive nerve stimulation were found in EAMG group, DCs treatment group, pcDNA_3-DCs treatment group and normal saline group(16.75±6.13, 17.75±7.81, 18.25±8.22 and 16.50±7.14, respectively), but there was no attenuation in pcDNA_3-TGF-β1-DCs treatment group and normal group(3.20±3.70 and 5.60±2.70, respectively). The percentage of decrement in pcDNA_3-TGF-β1-DCs treatment group was lower than that in EAMG group(5.60±2.70 and 16.75±6.13, respectively,P<0.05).(3)The level of AChR-Ab of the rats in pcDNA_3-TGF-β1-DCs treatment group was lower than that in EAMG group significantly (0.168±0.039 and 0.306±0.034, respectively, P<0.01), and no difference among EAMG group, DCs treatment group, pcDNA_3-DCs treatment group and normal saline group were found(0.276±0.066,0.294±0.082, 0.288±0.079 and 0.306±0.034, respectively, P>0.05). (4) The combined AChRs in NMJ of the rats in pcDNA_3-TGF-β1-DCs group were higher than that in EAMG group, and the structure changes of the synapse were relieved.Conclusion It suggests that DCs, transfected with pcDNA_3-TGF-β1, when injected subcutaneously into Lewis rats with incipient EAMG, could inhibit the production of AChR-Ab, relieve the pathologic changes in NMJ and ameliorate the development of EAMG.