目的 观察结缔组织生长因子(connective tissue growth factor,CTGF)在病毒性心肌炎( viral myocarditis,VMC)小鼠心肌组织中的表达情况.方法 实验选取BALB/C小鼠100只,分为对照组、VMC组各50只.应用柯萨奇B3病毒(coxsackie virus B3,CVB3)感染小鼠建立VMC模型.在CVB3感染后第4、7、14、21天各随机抽取8只采集心脏标本,行HE染色测定心肌病理积分,应用Masson染色法观察小鼠心肌胶原纤维分布,测量胶原容积分数(collagen volume fraction,CVF),检测血清CK-MB水平,应用免疫组织化学方法检测转化因子β1(transforming growth factor-β1,TGF-β1)、CTGF蛋白在小鼠心肌中的分布；应用RT-PCR方法检测小鼠心肌组织TGF-β1、CTGF mRNA的相对表达量；并作相关性分析.结果 (1)VMC组小鼠CK-MB水平于第7天达高峰,后逐渐下降(各时间点分别为455.45±37.95,606.95±35.64,573.62±42.90,308.60±20.49),其中第4、7、14天时高于对照组,差异有统计学意义(t值分别为6.144,12.558,11.182,P均＜0.01)；(2)VMC组小鼠第14、21天时CVF明显高于对照组(CVF值分别为8.22±1.95,9.46±1.87,t值分别为4.486,5.552,P均＜0.01)；(3)免疫组织化学法检测VMC组小鼠各时间点心肌CTGF蛋白(各时间点分别为171.50±10.25,141.70±10.863,110.35±11.051,81.05±10.190)及TGF-β1蛋白(184.90±11.480,150.25±9.915,103.50±10.455,84.15 ±9.848)阳性表达均较对照组高(P均＜0.01)；(4)VMC小鼠心肌组织中CTGF mRNA(0.4728±0.0328,0.5750±0.0439,0.6228±0.0458,0.7265±0.0469)及TGF-β1mRNA(0.5757 ±0.0426,0.6922 ±0.0408,0.7447±0.0510,0.8513±0.0505)表达随病毒感染时间延长而增强(P均＜0.01)；(5)VMC小鼠心肌组织中CTGF的表达与TGF-β1的表达呈显著正相关(r=0.987,P＜0.01)；VMC小鼠心肌组织中CTGF的表达与CVF密切相关(r=-0.901,P＜0.01),但CTGF的表达早于心肌纤维化的出现.结论 CTGF的表达伴随VMC的心肌纤维化程度而增加,提示CTGF的异常表达可能参与了VMC中心肌纤维化的发展过程.
目的 觀察結締組織生長因子(connective tissue growth factor,CTGF)在病毒性心肌炎( viral myocarditis,VMC)小鼠心肌組織中的錶達情況.方法 實驗選取BALB/C小鼠100隻,分為對照組、VMC組各50隻.應用柯薩奇B3病毒(coxsackie virus B3,CVB3)感染小鼠建立VMC模型.在CVB3感染後第4、7、14、21天各隨機抽取8隻採集心髒標本,行HE染色測定心肌病理積分,應用Masson染色法觀察小鼠心肌膠原纖維分佈,測量膠原容積分數(collagen volume fraction,CVF),檢測血清CK-MB水平,應用免疫組織化學方法檢測轉化因子β1(transforming growth factor-β1,TGF-β1)、CTGF蛋白在小鼠心肌中的分佈；應用RT-PCR方法檢測小鼠心肌組織TGF-β1、CTGF mRNA的相對錶達量；併作相關性分析.結果 (1)VMC組小鼠CK-MB水平于第7天達高峰,後逐漸下降(各時間點分彆為455.45±37.95,606.95±35.64,573.62±42.90,308.60±20.49),其中第4、7、14天時高于對照組,差異有統計學意義(t值分彆為6.144,12.558,11.182,P均＜0.01)；(2)VMC組小鼠第14、21天時CVF明顯高于對照組(CVF值分彆為8.22±1.95,9.46±1.87,t值分彆為4.486,5.552,P均＜0.01)；(3)免疫組織化學法檢測VMC組小鼠各時間點心肌CTGF蛋白(各時間點分彆為171.50±10.25,141.70±10.863,110.35±11.051,81.05±10.190)及TGF-β1蛋白(184.90±11.480,150.25±9.915,103.50±10.455,84.15 ±9.848)暘性錶達均較對照組高(P均＜0.01)；(4)VMC小鼠心肌組織中CTGF mRNA(0.4728±0.0328,0.5750±0.0439,0.6228±0.0458,0.7265±0.0469)及TGF-β1mRNA(0.5757 ±0.0426,0.6922 ±0.0408,0.7447±0.0510,0.8513±0.0505)錶達隨病毒感染時間延長而增彊(P均＜0.01)；(5)VMC小鼠心肌組織中CTGF的錶達與TGF-β1的錶達呈顯著正相關(r=0.987,P＜0.01)；VMC小鼠心肌組織中CTGF的錶達與CVF密切相關(r=-0.901,P＜0.01),但CTGF的錶達早于心肌纖維化的齣現.結論 CTGF的錶達伴隨VMC的心肌纖維化程度而增加,提示CTGF的異常錶達可能參與瞭VMC中心肌纖維化的髮展過程.
목적 관찰결체조직생장인자(connective tissue growth factor,CTGF)재병독성심기염( viral myocarditis,VMC)소서심기조직중적표체정황.방법 실험선취BALB/C소서100지,분위대조조、VMC조각50지.응용가살기B3병독(coxsackie virus B3,CVB3)감염소서건립VMC모형.재CVB3감염후제4、7、14、21천각수궤추취8지채집심장표본,행HE염색측정심기병리적분,응용Masson염색법관찰소서심기효원섬유분포,측량효원용적분수(collagen volume fraction,CVF),검측혈청CK-MB수평,응용면역조직화학방법검측전화인자β1(transforming growth factor-β1,TGF-β1)、CTGF단백재소서심기중적분포；응용RT-PCR방법검측소서심기조직TGF-β1、CTGF mRNA적상대표체량；병작상관성분석.결과 (1)VMC조소서CK-MB수평우제7천체고봉,후축점하강(각시간점분별위455.45±37.95,606.95±35.64,573.62±42.90,308.60±20.49),기중제4、7、14천시고우대조조,차이유통계학의의(t치분별위6.144,12.558,11.182,P균＜0.01)；(2)VMC조소서제14、21천시CVF명현고우대조조(CVF치분별위8.22±1.95,9.46±1.87,t치분별위4.486,5.552,P균＜0.01)；(3)면역조직화학법검측VMC조소서각시간점심기CTGF단백(각시간점분별위171.50±10.25,141.70±10.863,110.35±11.051,81.05±10.190)급TGF-β1단백(184.90±11.480,150.25±9.915,103.50±10.455,84.15 ±9.848)양성표체균교대조조고(P균＜0.01)；(4)VMC소서심기조직중CTGF mRNA(0.4728±0.0328,0.5750±0.0439,0.6228±0.0458,0.7265±0.0469)급TGF-β1mRNA(0.5757 ±0.0426,0.6922 ±0.0408,0.7447±0.0510,0.8513±0.0505)표체수병독감염시간연장이증강(P균＜0.01)；(5)VMC소서심기조직중CTGF적표체여TGF-β1적표체정현저정상관(r=0.987,P＜0.01)；VMC소서심기조직중CTGF적표체여CVF밀절상관(r=-0.901,P＜0.01),단CTGF적표체조우심기섬유화적출현.결론 CTGF적표체반수VMC적심기섬유화정도이증가,제시CTGF적이상표체가능삼여료VMC중심기섬유화적발전과정.
Objective To study the expression of connective tissue growth factor (CTGF) in the myocardial tissue of mice with viral myocarditis ( VMC ).Method Balb/c mice were infected with coxsackie virus B3 (CVB3) to establish VMC model.The mice were divided into control group ( n =50) and VMC group (n =50).on days 4,7,14 and 21 after infection,heart specimens of 8 mice were randomly taken and examined after HE staining for myocardial necrosis and cellular infiltration.The area of positive Masson stained myocardium collagen fibers was measured,and collagen volume fraction (CVF) was measured.Then the level of serum creatine phosphokinase-MB (CKMB) was determined.The levels of CTGF and TGF-β1 were detected by streptavidin peroxidase immunoperoxidase technique.Expression of CTGF and TGF-β1 were detected with reverse transcription-polymerase chain reaction (RT-PCR).At the same time,the correlations were analyzed.Result ( 1 ) The level of CKMB peaked on day 7,and decreased afterwards (455.45 ± 37.95,606.95 ± 35.64,573.62 ± 42.90,308.60 ± 20.49,respectively,4-21 d points),in which 4,7,14 d points,there was significant difference compared with control group(t =6.144,12.558,11.182,respectively,P ＜0.01 ).(2) CVF increased significantly on day 14 (8.22±1.95,t=4.486,P＜0.01) and day 21 (9.46±1.87,t =4.486,P＜0.01) in VMC group.(3)Measured by streptavidin peroxidase immunoperoxidase technique,the levels of CTGF ( 171.50 ± 10.25,141.70 ± 10.863,110.35 ± 11.051,81.05 ± 10.190,respectively,4-21 d points) and TGF-β1 ( 184.90 ±11.480,150.25 ±9.915,103.50± 10.455,84.15 ±9.848,respectively,4-21 d points) increased after day4 in VMC ( P ＜ 0.01 ).( 4 ) Measured by RT-PCR,the expression of CTGF mRNA and TGF-β1 increased in VMC group,and the increase was enhanced with the disease development ( P ＜ 0.01 ).(5)The expression of CTGF and TGF-β1 was positively linearly correlated ( r =0.987,P ＜ 0.01 ),the expression of CTGF was negatively correlated with CVF ( r =- 0.901,P ＜ 0.01 ),but the expression of CTGF was detected earlier than myocardial fibrosis.Conclusion The increase of CTGF expression was associated with the severity of myocardial fibrosis in VMC.These results suggest that abnormal expression of CTGF may take part in the development of fibrosis in VMC.
