摘要
背景:大量动物实验证明转化生长因子β1基因是目前肺纤维化的研究热点和重要的药物作用靶点。目的:探讨转化生长因子β1基因沉默内皮祖细胞移植对大鼠肺纤维化的影响。方法:(1)构建靶向转化生长因子β1的siRNA质粒,转染内皮祖细胞,分为空白组、对照组及转化生长因子β1基因沉默组,Western blot检测转染第3天和第28天转化生长因子β1表达;(2)80只Wistar大鼠(北京维通利华动物实验技术有限公司提供)分为正常对照组、肺纤维化模型组、内皮祖细胞组、转化生长因子β1沉默内皮祖细胞组,后3组大鼠气管内分别一次性注入0.3m L博莱霉素溶液(5mg/kg)以制备大鼠肺纤维化病理模型,造模后24 h,正常对照组和肺纤维化模型组大鼠尾静脉注射20μL生理盐水,内皮祖细胞组及转化生长因子β1沉默组大鼠尾静脉注射20μL细胞浓度为3×106 L-1的内皮祖细胞悬液或转化生长因子β1沉默内皮祖细胞悬液;(3)移植28d后,ELISA测定大鼠支气管肺泡灌洗液中白细胞介素10、白细胞介素6以及肿瘤坏死因子α水平,苏木精-伊红染色观察肺组织病理变化,RT-PCR、Western blot检测肺组织中转化生长因子β1及Smad-2、Smad-7的表达。结果与结论:(1)在转染后第3天和第28天,转化生长因子β1基因沉默组的转化生长因子β1蛋白相对表达水平低于空白组和对照组,差异有显著性意义(P <0.05);(2)内皮祖细胞组较肺纤维化模型组病理变化减轻,肺泡间隔厚度低,转化生长因子β1沉默内皮祖细胞组病理变化进一步减轻;(3)内皮祖细胞组白细胞介素10、白细胞介素6以及肿瘤坏死因子α水平较肺纤维化模型组降低(P <0.05),转化生长因子β1沉默内皮祖细胞组进一步降低,但与内皮祖细胞组比较,差异无显著性意义(P> 0.05);(4)与肺纤维化模型组相比,内皮祖细胞组和转化生长因子β1沉默组大鼠肺组织中转化生长因子β1和Smad-2的基因和蛋白表达水平明显降低(P <0.05),而Smad-7表达水平明显升高(P <0.05)。转化生长因子β1沉默内皮祖细胞组上述3种基因和蛋白表达水平均优于内皮祖细胞组(P <0.05);(5)结果表明,转化生长因子β1沉默内皮祖细胞移植对肺纤维化大鼠有保护作用,可能通过降低Smad-2及增强Smad-7表达,发挥抑制肺纤维化作用。
BACKGROUND:Abundant animal experiments have shown that transforming growth factorβ1(TGF-β1)gene is a hotspot of pulmonary fibrosis and an important target for drugs.OBJECTIVE:To explore the therapeutic effect of the transplantation of endothelial progenitor cells with RNA interference targeting TGF-β1 in the rats with pulmonary fibrosis.METHODS:(1)The siRNA plasmid directed to TGF-β1 was constructed and transfected into endothelial progenitor cells.It was divided into blank group,control group and TGF-β1-siRNA transfection group.The expression of TGF-β1 at 3 and 28 days after transfection was detected by western blot assay.(2)Eighty Wistar rats provided by Beijing Vital River Laboratory Animal Technology Co.Ltd.were divided into control,model,endothelial progenitor cell,and TGF-β1 silent groups.The rat trachea in the latter three groups was injected with 0.3 mL of bleomycin(5 mg/kg)to establish the rat model of pulmonary fibrosis.At 24 hours after modeling,the control and model groups received the injection of 20μL of normal saline via caudal vein,and the endothelial progenitor cell,and TGF-β1 silent groups subjected to the injection of 20μL 3×106/L endothelial progenitor cell suspension and TGF-β1 silent endothelial progenitor cell suspension,respectively.(3)After 28 days of transplantation,the levels of interleukin-10,interleukin-6,and tumor necrosis factor-αin the rat bronchoalveolar lavage fluid were detected by ELISA.The pathological changes of lung tissue were observed by hematoxylin-eosin staining.Expression levels of TGF-β1,Smad-2,and Smad-7 were detected by RT-PCR,and western blot assay.RESULTS AND CONCLUSION:(1)The protein expression of TGF-β1 in the TGF-β1-siRNA transfection group was significantly lower than that in the blank and control groups at 3 and 28 days after transfection(P<0.05).(2)Compared with the model group,the pulmonary fibrosis was relieved in the endothelial progenitor cell group with reduced alveolar interval thickness and further attenuated in the TGF-β1 silent group.(3)The levels of interleukin-10,interleukin-6,and tumor necrosis factor-αin the endothelial progenitor cell group were significantly lower than those in the model group(P<0.05).These factor levels in the TGF-β1 silent group were lower than those in the endothelial progenitor cell group(P>0.05).(4)Compared with the model group,the mRNA and protein expression levels of TGF-β1 and Smad-2 in the endothelial progenitor cell and TGF-β1 silent groups were significantly decreased,the levels of Smad-7 were significantly increased(both P<0.05).All above levels in the TGF-β1 silent group were significantly superior to those in the endothelial progenitor cell group(P<0.05).(5)To conclude,TGF-β1 silent endothelial progenitor cell transplantation has a protective effect against pulmonary fibrosis in rats and probably inhibits pulmonary fibrosis by reducing Smad-2 and enhancing Smad-7 expression.
作者
彭秋凤
高静珍
叶奎
邢爱民
Peng Qiufeng;Gao Jingzhen;Ye Kui;Xing Aimin(Second Department of Pneumology,Tianjin 4th Central Hospital,Tianjin 300140,China;Department of Vascular Surgery,Tianjin 4th Central Hospital,Tianjin 300140,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第1期90-95,共6页
Chinese Journal of Tissue Engineering Research
