CD4^(+)T细胞在心力衰竭小鼠模型中的变化及细胞因子释放的研究

Changes of CD4^(+)T cells and release of cytokines in a mouse model of heart failure

目的:建立小鼠心力衰竭(HF)模型,应用超声影像学、病理学和分子生物学技术综合评价疾病发展过程中CD4^(+)T细胞和炎症因子变化。方法:30只10周龄SPF级C57BL/6J小鼠行外周负荷法制备HF模型,然后随机分为造模后2周组(n=10)、造模后4周组(n=10),另外10只设为假手术组,假手术组仅打开胸腔暴露主动脉但不进行结扎。采用小鼠心脏超声评价心功能;Masson染色评价心肌纤维化程度;免疫组化染色观察Ⅰ型胶原阳性面积及CD4^(+)T细胞数量变化;ELISA检测小鼠心肌组织IFN-γ、IL-2、IL-4、IL-6和IL-10水平;Western blot检测T-bet、GATA-3、FoxP3水平。结果:心脏超声结果显示,与假手术比较,造模2周、4周后LVESD和LVEDD增大(P<0.05),而LVFS、LVEF下降(P<0.05),与造模后2周相比,造模4周后LVFS、LVEF降低(P<0.05),但LVESD和LVEDD并没有进一步增大(P>0.05)。病理染色结果显示,与假手术组相比,造模2周、4周后小鼠心肌组织纤维化和Ⅰ型胶原阳性面积显著增加(P<0.05),与造模后2周相比,造模后4周心肌组织纤维化和Ⅰ型胶原阳性面积显著增加(P<0.05)。免疫组化结果显示,与假手术组相比,造模2周、4周后CD4^(+)T淋巴细胞显著增多(P<0.05);与造模后2周相比,造模后4周CD4^(+)T淋巴细胞显著增多(P<0.05)。炎症因子水平比较:与假手术组相比,造模后2周、4周心肌中IFN-γ、IL-2水平显著升高(P<0.05),IL-4、IL-6、IL-10水平显著降低(P<0.05);与造模后2周相比,造模后4周小鼠心肌组织炎症因子IFN-γ、IL-2水平显著升高(P<0.05),IL-4、IL-6、IL-10水平显著降低(P<0.05)。Western blot结果显示,与假手术组相比,造模后2周、4周心肌T-bet水平显著升高(P<0.05),GATA-3和FoxP3水平降低(P<0.05);与造模后2周相比,造模后4周T-bet显著升高(P<0.05),GATA-3,FoxP3降低(P<0.05)。结论:随着HF的发生发展,CD4^(+)T淋巴细胞亚群数量增加并发生比例失衡,导致炎症因子水平改变,从而引起心室重构加重HF的病理表现。

Objective:To comprehensively evaluate the changes of CD4^(+)T cells and inflammatory factors during development of heart failure(HF)in mouse model by ultrasound imaging,pathology and molecular biology techniques.Methods:Thirty 10-weekold SPF C57BL/6J mice were subjected to peripheral overload method to prepare HF models,and mice were then randomly divided into 2 weeks after modeling group(n=10)and 4 weeks after modeling group(n=10),the other 10 mice were assigned to sham operation group.Thoracic cavity was only opened to expose the aorta but not ligated in sham group.Cardiac ultrasound was used to evaluate cardiac function;Masson staining was used to evaluate the degree of myocardial fibrosis;immunohistochemical staining was used to observe positive area of typeⅠcollagen and change of CD4^(+)T cell number;ELISA was used to detect IFN-γ,IL-2,IL-4,IL-6 and IL-10 levels in mouse myocardial tissues;Western blot was used to detect T-bet,GATA-3 and FoxP3 levels.Results:Ultrasound of the heart showed that compared with sham group,LVESD and LVEDD increased significantly at 2,4 weeks after modeling(P<0.05),while LVFS and LVEF decreased significantly(P<0.05),compared with 2 weeks modeling,LVFS and LVEF were significantly reduced at 4 weeks after modeling(P<0.05),while LVESD and LVEDD did not increase further(P>0.05).Pathological staining results showed that compared with sham group,fibrotic and typeⅠcollagen positive area were increased significantly at 2,4 weeks after modeling(P<0.05);compared with 2 weeks after modeling,myocardial fibrosis and typeⅠcollagen positive area were increased significantly at 4 weeks after modeling(P<0.05).Results of immunohistochemistry showed that CD4+T lymphocytes in myocardial tissue of mice were increased significantly at 2 and 4 weeks after modeling(P<0.05);compared with 2 weeks after modeling,CD4^(+)T lymphocytes were in‐creased significantly at 4 weeks after modeling(P<0.05).Comparision of levels of inflammatory factors:compared with sham group,levels of inflammatory factors IFN-γand IL-2 were significantly increased(P<0.05),while levels of IL-4,IL-6 and IL-10 were signifi‐cantly decreased at 2 and 4 weeks after modeling(P<0.05);compared with 2 weeks after modeling,levels of IFN-γ and IL-2 were sig‐nificantly increased(P<0.05),while levels of IL-4,IL-6 and IL-10 were significantly decreased(P<0.05);Western blot results showed that T-bet level was increased significantly at 2 and 4 weeks after modeling(P<0.05),while levels of GATA-3 and FoxP 3 were decreased(P<0.05);compared with 2 weeks after modeling,T-bet level was increased significantly(P<0.05),while levels of GATA-3 and FoxP 3 were decreased at 4 weeks after modeling(P<0.05).Conclusion:With the onset and development of HF,the number of CD4^(+)T lymphocytes are increased and the proportion is unbalanced,which leads to changes in levels of inflammatory factors and causes ventricular remodeling to aggravate the pathological manifestations of HF.