转化生长因子β1基因沉默脂肪干细胞移植对盐敏感性高血压大鼠心肌纤维化和心肌细胞的影响研究

摘要

目的 探讨转化生长因子β1(TGF-β1)基因沉默脂肪干细胞(ADSCs)移植对盐敏感性高血压大鼠心肌纤维化和心肌细胞凋亡的影响.方法 本实验于2015年6月-2016年7月在陕西省医学实验动物中心实验室完成.体外培养ADSCs,构建TGF-β1为靶向基因的mRNA和siRNA表达质粒,病毒转染至ADSCs,取第三代ADSCs进行慢病毒转染,转染时分为空白组、对照组、TGF-β1-siRNA转染组.采用Western blot法检测基因转染后第3天和第14天不同基因转染组ADSCs TGF-β1蛋白表达情况.选取80只SPF级雄性Dahl盐敏感大鼠构建盐敏感性高血压大鼠模型,采用随机数字表法分为正盐组、高盐组、ADSCs组及TGF-β1基因沉默组,每组20只.正盐组给予0.3%氯化钠饮食;高盐组给予8%氯化钠饮食;ADSCs组予8%氯化钠饮食6周后尾静脉移植ADSCs,持续3 d;TGF-β1基因沉默组大鼠给予8%氯化钠饮食6周后尾静脉移植TGF-β1基因沉默ADSCs,持续3 d.细胞移植2周后,采用反转录-聚合酶链反应(RT-PCR)检测4组大鼠心肌组织TGF-β1 mRNA表达情况,采用Western blot法检测4组大鼠心肌组织TGF-β1蛋白表达情况,采用超声心动图检测4组大鼠左心室射血分数(LVEF)和缩短分数(FS),并计算左心室质量指数(LVMI),VG染色测算胶原容积积分,HE染色观察心肌细胞形态,荧光显微镜下观察心肌组织中CM-Dil标记的ADSCs分布情况,采用TUNEL法检测4组大鼠心肌细胞凋亡情况.结果 (1)荧光显微镜下显示,CM-Dil标记的ADSCs呈橘红色,标记率为97%.(2)基因转染后第3天和第14天空白组和对照组ADSCs TGF-β1蛋白相对表达量比较,差异无统计学意义(P>0.05),而TGF-β1-siRNA转染组ADSCs TGF-β1蛋白相对表达量低于空白组和对照组(P<0.05).(3)细胞移植2周后,TGF-β1基因沉默组大鼠心肌组织TGF-β1 mRNA、蛋白相对表达量低于正盐组、高盐组及ADSCs组,ADSCs组大鼠心肌组织TGF-β1 mRNA、蛋白相对表达量低于正盐组、高盐组,高盐组大鼠心肌组织TGF-β1 mRNA、蛋白相对表达量高于正盐组(P<0.05).(4)细胞移植2周后,高盐组和ADSCs组大鼠LVEF和FS低于正盐组和TGF-β1基因沉默组,LVMI高于正盐组和TGF-β1基因沉默组(P<0.05);ADSCs组和TGF-β1基因沉默组大鼠LVEF和FS高于高盐组,LVMI低于高盐组(P<0.05);正盐组与TGF-β1基因沉默组大鼠LVEF、FS及LVMI比较,差异无统计学意义(P>0.05).(5)细胞移植2周后,高盐组、ADSCs组和TGF-β1基因沉默组大鼠胶原容积积分高于正盐组,ADSCs组和TGF-β1基因沉默组大鼠胶原容积积分低于高盐组,TGF-β1基因沉默组大鼠胶原容积积分低于ADSCs组(P<0.05).(6)HE染色结果显示,细胞移植2周后高盐组大鼠心肌出现明显纤维化且巨噬细胞浸润明显增多,ADSCs组大鼠心肌纤维化较轻,TGF-β1沉默组大鼠心脏纤维化最轻、巨噬细胞浸润最少.(7)荧光显微镜下显示,细胞移植2周后正盐组和高盐组大鼠心肌组织中未发现CM-Dil标记的ADSCs,TGF-β1基因沉默组大鼠心肌组织中CM-Dil标记的ADSCs多于ADSCs组(P<0.05).(8)细胞移植2周后,正盐组大鼠心肌细胞凋亡率为0,ADSCs组和TGF-β1基因沉默组大鼠心肌细胞凋亡率低于高盐组,TGF-β1基因沉默组大鼠心肌细胞凋亡率低于ADSCs组(P<0.05).结论 TGF-β1基因沉默ADSCs移植能有效减轻盐敏感性高血压大鼠心肌纤维化,减少心肌细胞凋亡.%Objective To investigate the impact of transplantation of TGF-β1 gene silencing adipose-derived stromal cells(ADSCs)on myocardial fibrosis and apoptosis of salt-sensitive hypertensive rats.Methods This experiment was completed in the Central Laboratory for Medical Experimental Animals of Shaanxi Province from June 2015 to July 2016.ADSCs was cultured in vitro,mRNA and siRNA expressed plasmids with TGF-β1 targeted gene were constructed and infected by virus;the third-generation ADSCs were infected by slow virus and divided into blank group,control group and experiment group(with TGF-β1-siRNA transfection).Western blot method was used to detect the protein expression of TGF-β1 of the above three groups after 3 and 14 days of gene transfection.A total of 80 SPF male Dahl salt-sensitive rats were selected to prepare the salt-sensitive hypertensive rats model,and they were divided into A group,B group,C group and D group according to random number table,each of 20 rats.Rats of A group received 0.3% NaCl diet,rats of B group received 8% NaCl diet,rats of C group received caudal vein transplantation of ADSCs after 6 weeks of 8% NaCl diet for 3 days,while rats of D group received caudal vein transplantation of TGF-β1 gene silencing ADSCs after 6 weeks of 8% NaCl diet for 3 days.After 2 weeks of transplantation,T-PCR was used to detect the mRNA expression of TGF-β1 of myocardial tissue,Western Blot method was used to detect the protein expression of TGF-β1 of myocardial tissue,ultrasonic cardiogram was used to detect the LVEF and FS,meanwhile LVMI was calculated,VG staining method was used to observe and calculate the collagen volume fraction,HE staining method was used to observe the myocardial cell morphology,CM-Dil marked ADSCs in myocardial tissue was observed by fluorescence microscope,and TUNEL method was used to detect the apoptosis of myocardial cells.Results (1)Fluorescence microscope found that,CM-Dil marked ADSCs were red,and the marking percentage was 97%.(2)No statistically significant differences of relative expression quantity of TGF-β1 protein of ADSCs was found between blank group and control after 3 or 14 days of gene transfection(P>0.05),while relative expression quantity of TGF-β1 protein of ADSCs of experiment group was statistically significantly lower than that of blank group and control group,respectively(P<0.05).(3)After 2 weeks of transplantation,relative expression quantity of TGF-β1 mRNA and TGF-β1 protein of myocardial tissue of D group was statistically significantly lower than that of A group,B group and C group,respectively,relative expression quantity of TGF-β1 mRNA and TGF-β1 protein of myocardial tissue of C group was statistically significantly lower than that of A group and B group,respectively,while relative expression quantity of TGF-β1 mRNA and TGF-β1 protein of myocardial tissue of B group was statistically significantly higher than that of A group(P<0.05).(4)After 2 weeks of transplantation,LVEF and FS of B group and C group were statistically significantly lower than those of A group and D group,while LVMI of B group and C group was statistically significantly higher than that of A group and D group,respectively(P<0.05);LVEF and FS of C group were statistically significantly higher than those of B group,while LVMI of C group was statistically significantly lower than that of control group(P<0.05);no statistically significant differences of LVEF,FS or LVMI was found between A group and D group(P>0.05).After 2 weeks of transplantation,collagen volume fraction of B group,C group and D group was statistically significantly higher than that of A group,respectively,while collagen volume fraction of C group and D group was statistically significantly lower than that of B group,respectively,collagen volume fraction of D group was statistically significantly lower than that of C group(P<0.05).(6)After 2 weeks of transplantation,HE staining results showed that,myocardial tissue of B group occurred obvious fibrosis and microphages infiltration,myocardial tissue of C group occurred mild fibrosis,while myocardial tissue of C group occurred a little fibrosis and microphages infiltration.(7)After 2 weeks of transplantation,no CM-Dil marked ADSCs of A group or B group was found by fluorescence microscope,while CM-Dil marked ADSCs of D group was statistically significantly more than that of C group(P<0.05).(8)After 2 weeks of transplantation,myocardial cell apoptosis rate of A group was 0,myocardial cell apoptosis rate of C group and D group was statistically significantly lower than that of B group,while myocardial cell apoptosis rate of D group was statistically significantly lower than that of C group(P<0.05).Conclusion Transplantation of TGF-β1 gene silencing ADSCs can effectively relive the myocardial fibrosis and reduce the myocardial cell apoptosis of salt-sensitive hypertensive rats.