丙种球蛋白和维生素C对实验性自身免疫性心肌炎作用的研究

Effect of intravenous immunoglobulin and vitamin C on progression of experimental autoimmune myocarditis in mice

目的:探讨丙种球蛋白(IVIG)和不同剂量维生素C(VitC)对实验性自身免疫性心肌炎(EAM)的保护作用。方法:Balb/c小鼠52只,随机分为6组:空白组不予任何处理;其余小鼠分别于第1天和第7天在双测腹股沟皮下注射充分乳化的猪心肌肌凝蛋白,剂量为每次每只100μg;从免疫的当天开始给每只小鼠腹腔注射所给药物。小剂量VitC组:150mg/kg·d-1VitC;大剂量VitC组:300 mg/kg·d-1VitC;IVIG组:1g/kg.d-1IVIG;IVIG+VitC组:1 g/kg.d-1IVIG和150mg/kg·d-1VitC;对照组:与干预组等量的生理盐水。小鼠于第21天给予称重,麻醉后处死,并摘眼球取血,用Elisa法测定血清肿瘤坏死因子α(TNF-α)水平;取心脏、脾脏和肾脏分别称重,得心脏、脾脏和肾脏与体重之比(C/W、S/W、R/W);脾脏在肉眼观察后作病理切片和H-E染色。心脏分为三部分:一部分作病理切片和H-E染色,一部分作冰冻切片,在荧光显微镜下用直接免疫荧光法测定心肌组织中沉积的IgG水平,一部分作电镜检查。结果:(1)心脏的病理改变:大、小剂量VitC组心肌炎性细胞的浸润及心包钙化有所减轻;IVIG组和IVIG+VitC组心肌偶有淋巴细胞浸润,无心包钙化。(2)脾脏的病理改变:除空白组外,其余脾脏均明显增大,IVIG组和IVIG+VitC组更是明显,镜检表现为红髓充血和白髓增生。(3)心脏、脾脏、肾脏与体重之比:各干预组C/W明显低于对照组;各干预组和对照组S/W明显高于空白组,IVIG组和IVIG+VitC组S/W明显高于大、小剂量VitC组;R/W各组无差异。(4)TNF-α水平:大、小剂量VitC组的TNF-α水平略低于对照组,IVIG组和IVIG+VitC组的TNF-α水平明显低于对照组。(5)心肌的免疫荧光检测:对照组比较粗的荧光条带主要集中在心肌间质,大、小剂量VitC组荧光的密度和强度有所降低,而IVIG组和IVIG+VitC组的荧光条带增宽,在心肌间质中呈宽大的条索状分布,亮度明显增强。(6)心肌的电镜检测:对照组心肌肌丝排列紊乱,肌节断裂严重,线粒体肥大,并出现空泡变性;大、小剂量VitC组病变比对照组有所减轻;IVIG组和IVIG+VitC组肌丝排列紊乱明显减轻,肌节断裂比对照组减少,线粒体基本正常。结论:IVIG和VitC对EAM都有一定的保护作用,可以减轻心脏的病理改变,抑制TNF-α的产生;但IVIG或IVIG+VitC作用更为明显,并可刺激机体的免疫反应,增加心肌中IgG的沉积。

Objective： To evaluate the effect of intravenous immunoglobulin （IVIG） and vitamin C on the progression of experimental autoimmune myocarditis （EAM）. Methods： Fifty-two Balb/c mice were randomized into six groups： The blank group received no treatment, the remaining 5 groups were immunized with 100μg emulsified porcine myosin at d 1 and d 7. Different agents were injected from d 1, SVitC group： 150 mg/kg · d^-1 vitamin C; LVitC group： 300 mg/kg· d^-1 vitamin C; IVIG group： 1 g/kg · d^-1 IVIG; IVIG＋VitC group： 1 g/kg· d^-1 IVIG and 150 mg/kg· d^-1 vitamin C; The control group same volume of normal saline. All mice were sacrificed at d 21, and serum TNF-α levels were detected with enzyme linked immunosorbent assay （ELISA）. The ratio of heart to body weight （C/W）, spleen to body weight （S/W） and kidney to body weigh （R/W） were calculated. The spleens and heart were examined pathologically and/or immunohistochemically. Results： Compared with those of control group, inflammatory cells infiltration in the myocardium and calcification in the pericardiume in SVitC and LVitC groups were extenuated. There were inflammatory cells infiltrating in the myocardium sparely and no calcification in the pericardium in IVIG and IVIG ＋VitC groups. The size of spleens enlarged especially in IVIG and IVIG＋VitC groups. White and red pulps of spleens were hyperplastic microscopically. The C/W of treatment groups decreased significantly compared with that of control group. The S/W of therapy groups and control group was significantly higher than that of blank group; and the S/W Of IVIG and IVIG ＋ VitC groups was significantly higher than that of SVitC and LVitC groups. The R/W in each groups had no significant difference. The TNF-α level in SVitC and LVitC groups was a little lower than that in control group; TNF-α level in IVIG and IVIG＋VitC groups was significantly lower than that of control group. Wide fluorescence stripe was found along extracellular matrix surrounding the damaged cardiomyocytes of control group. Both density and intensity of fluorescence in SVitC and LVitC groups were lower than those of control group. There were much wider fluorescence stripe and strengthened intensity in IVIG and IVIG ＋ VitC groups. The myofilaments were in wild disorder and sarcomere had severe breakage in control group. Moreover, chondriosome hypertrophy and vacuolar degeneration were found. The damage lessened in SVitC and LVitC groups. Both myofilaments and sarcomeres in IVIG and IVIG ＋ VitC groups were almost normal, and the chondriosome was normal. Conclusion： IVIG and vitamin C have some protective and therapeutic effect on the progression of EAM by decreasing pathological damage of myocardium and depressing TNF-α production, and IVIG combined with vitamin C is more effective.