结缔组织生长因子单克隆抗体对小鼠慢性结肠炎肠壁纤维化的作用

The effect of connective tissue growth factor monoclonal antibody on intestinal fibrosis in mice with chronic colitis

目的研究结缔组织生长因子(CTGF)单克隆抗体对葡聚糖硫酸钠(DSS)诱导下小鼠慢性结肠炎肠壁纤维化的作用。方法采用自由饮用葡聚糖硫酸钠(DSS)诱导的小鼠慢性结肠炎模型;Balb/c雌性小鼠25只,随机分为5组,每组5只。其中1组为空白对照组,饮用蒸馏水36天,另外4组自由饮用2.5%DSS溶液4天,饮用蒸馏水5天,循环重复至第36天,同时从第一天起这4组分别用安慰剂(非特异性人IgG 20 mg/kg)及不同浓度CTGF单抗(10 mg/kg、20 mg/kg、40 mg/kg)腹腔注射,每周2次。每天观察各组小鼠的临床表现、粪便性状、体重、粪便隐血情况,予以疾病活动指数(disease activity index,DAI)评分,于第36天取小鼠结肠,采用HE染色进行组织病理学评分,masson染色及α-平滑肌肌动蛋白(α-SMA)免疫组化染色检测胶原及α-SMA含量,采用实时荧光定量PCR(RT-PCR)检测小鼠结肠组织中α-SMA、Ⅰ型前胶原(PC-Ⅰ)、CTGF mRNA的表达水平。结果安慰剂组与空白组相比,DAI评分、病理学评分、胶原含量及α-SMA含量显著上升(P=0.0005,P=0.0133,P<0.0001,P=0.0207),α-SMA、PC-Ⅰ、CTGF mRNA的表达水平显著提高(P=0.0086,P=0.0109,P=0.0046)。与安慰剂组相比,10 mg/kg、40 mg/kg单抗组DAI评分显著降低(P=0.0111,P=0.0011)。20 mg/kg单抗组较安慰剂组病理学评分显著降低(P=0.0216),不同浓度单抗组间病理学评分差异无统计学意义(P=0.4004)。与安慰剂组相比,10 mg/kg、20 mg/kg及40 mg/kg单抗组胶原含量显著降低(P=0.0011,P=0.0006,P=0.0002),不同浓度组间比较差异无统计学意义(P=0.3762)。与安慰剂组相比,40 mg/kg单抗组α-SMA含量显著降低(P=0.0281),不同浓度组间比较差异无统计学意义(P=0.0766)。10 mg/kg单抗组PC-Ⅰ、CTGF mRNA的表达水平较安慰剂组显著减少(P=0.0421,P=0.0296),20 mg/kg单抗组α-SMA、PC-Ⅰ、CTGF mRNA的表达水平较安慰剂组显著减少(P=0.0425,P=0.0233,P=0.0107),40 mg/kg单抗组α-SMA、PC-Ⅰ、CTGF mRNA的表达水平较安慰剂组显著减少(P=0.0087,P=0.0031,P=0.0024),40 mg/kg单抗组α-SMA mRNA的表达水平较10 mg/kg单抗组显著减少(P=0.0434)。结论肠道慢性炎症伴随纤维化的形成,CTGF单抗可以减少α-SMA、PC-Ⅰ、CTGF mRNA的表达,降低慢性结肠炎小鼠肠壁的胶原、α-SMA含量,抑制肠壁纤维化。

Objective To research the function of connective tissue growth factor(CTGF)monoclonal antibody(mAb)on dextran sodium sulfate(DSS)induced intestinal wall fibrosis in mice with chronic colitis.Methods Mouse chronic colitis model was induced by free drinking dextran sulfate sodium(DSS).Twenty-five Balb/c female mice were randomly divided into 5 groups with 5 mice in each group.One group was the blank control group which drinking distilled water for 36 days,and the other four groups drank 2.5%DSS solution for 4 days and distilled water for 5 days,and the cycle was repeated until the 36th day.At the same time,the four groups were given placebo(20 mg/kg non-specific human IgG)and different concentrations of CTGF monoclonal antibody(10 mg/kg,20 mg/kg,40 mg/kg)by intraperitoneal injection twice a week from the first day.The Clinical manifestations,feces characteristics,body weight,feces occult blood of mice were observed every day,and the disease activity index(DAI)score was given.On the 36th day,the colons of mice were obtained for histopathological scoring with HE staining.Collagen andα-smooth muscle actin(α-SMA)contents were detected by Masson staining andα-SMA immunohistochemical staining,and the expression levels ofα-SMA,procollagen typeⅠ(PC-Ⅰ)and CTGF mRNA in colonic tissues of mice were detected by real-time fluorescence quantitative PCR(RT-PCR).Results Compared with the blank group,the DAI score,pathological score,collagen content andα-SMA content in the placebo group were significantly increased(P=0.0005,P=0.0133,P<0.0001,P=0.0207),and the mRNA expression levels ofα-SMA,PC-Ⅰand CTGF were significantly increased(P=0.0086,P=0.0109,P=0.0046).Compared with the placebo group,the DAI scores of the 10 mg/kg and 40 mg/kg mAb groups were significantly lower(P=0.0111,P=0.0011).The pathological scores of the 20 mg/kg mAb group were significantly lower than those of the placebo group(P=0.0216),and there was no significant difference among the groups with different concentrations of mAb(P=0.4004).Compared with placebo group,collagen content in 10 mg/kg,20 mg/kg and 40 mg/kg mAb groups was significantly decreased(P=0.0011,P=0.0006,P=0.0002),and there was no statistical difference among different concentration groups(P=0.3762).Compared with placebo group,α-SMA content in 40 mg/kg mAb group was significantly decreased(P=0.0281),but there was no significant difference among different concentration groups(P=0.0766).The expression levels of PC-Ⅰand CTGF mRNA in the 10 mg/kg mAb group were significantly lower than those in the placebo group(P=0.0421,P=0.0296).The expression levels ofα-SMA,PC-Ⅰand CTGF mRNA in the 20 mg/kg mAb group were significantly lower than those in the placebo group(P=0.0425,P=0.0233,P=0.0107).The expression levels ofα-SMA,PC-Ⅰand CTGF mRNA in the 40 mg/kg mAb group were significantly lower than those in the placebo group(P=0.0087,P=0.0031,P=0.0024).The expression level ofα-SMA mRNA in the 40 mg/kg mAb group was significantly lower than that in the 10 mg/kg mAb group(P=0.0434).Conclusion Chronic intestinal inflammation is accompanied by the formation of fibrosis.Anti-CTGF mAb can reduce the mRNA expression ofα-SMA,PC-Ⅰand CTGF,reduce the content of collagen andα-SMA in the intestinal wall of mice with chronic colitis,and inhibit the fibrosis of the intestinal wall.