结缔组织生长因子在病毒性心肌炎心肌组织中的变化

Experimental study on expression of connective tissue growth factor in viral myocarditis in mice

目的观察结缔组织生长因子（connectivetissuegrowthfactor，CTGF）在病毒性心肌炎（viralmyocarditis，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（coxsackievirusB3，CVB，）感染小鼠建立VMC模型。在CVB，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（collagenvolumefraction，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（transforminggrowthfactor-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、CTGFmRNA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，￡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CTGFmRNA（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。

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.