磷酸胆碱聚合物载TGF—β1反义寡核苷酸转染血管外膜成纤维细胞对TGF—β1的影响  

Effects of phosphorylcholine polymer carried antisense oligonucleotides on TGF - β1 expression in rat vascular adventitial fibroblasts

作　　者：李菲[1] 王志荣[2] 张卓琦[2] 程明月[2] 张超群[2] 

机构地区：[1]徐州医学院研究生学院2010级,江苏徐州221004 [2]徐州医学院附属医院心内科,江苏徐州221002

出　　处：《徐州医学院学报》2014年第4期211-214,共4页Acta Academiae Medicinae Xuzhou

基　　金：国家自然科学基金（30800219）

摘　　要：目的观察新型阳离子磷酸胆碱聚合物MPC30-DEA70能否有效地转染针对转化生长因子β1（transforming growth factor-β1，TGF-β1）的反义寡核苷酸（AS—ODN）进入到血管外膜成纤维细胞中及转染后对血管外膜成纤维细胞内TGF-β1表达的影响，为MPC30-DEA70，聚合物作为药物基因治疗载体在心血管疾病中的运用奠定基础。方法组织贴块法培养大鼠胸主动脉外膜成纤维细胞；应用共聚焦激光扫描显微镜（CLSM）观察MPC30-DEA70／TGF-β1AS-ODN（FAM标记）在细胞内的分布和定位；流式细胞仪（FCM）检测二者的细胞转染效率、荧光强度；免疫印迹实验（Western blot）检测TGF-β1细胞内表达。结果①原代培养的细胞呈纺锤形，多角形或者不规则形，细胞核较大，数量1～2个不等，轮廓清楚，核仁大而明显，呈椭圆形；②激光共聚焦显微镜观察到FAM标记的基因复合物多数分布在细胞核周围；③流式结果显示转染效率及荧光强度随N／P比增加而明显增大；④Western blot结果显示随着N／P比值的增大，TGF-β1的蛋白表达明显下降（P〈0．05）。结论MPC30-DEA70，能够携带TGF-β1-AS—ODN进入离体培养的大鼠胸主动脉外膜成纤维细胞，并能下调TGF—β1蛋白的表达。Objective To observe whether AS - ODN of TGF - β1 can be transfected by MPC30 - DEA70 （ PC polymer） , a novel cationic phosphorylcholine polymer into rat vascular adventitial fibroblasts and to investigate the expression of TGF- β1 after transfection. Methods The primary culture of rat vascular adventitial fibroblasts was performed. Then, confocal microscopy were used to detect the distribution and location of the complex of TGF - β1 AS - ODN labeled by carboxyfluorescein （FAM） in the cells. The transfection efficiency and fluorescent intensity were detected by flow cytometry. The expression of TGF - β1 was examined by western blot. Results （1） The AFs were spindle - shaped, polygonal or irregular, with large nucleuses and clear outline. （2） According to eonfoeal microscopy, most MPC30 - DEA70/AS- ODN complexes were found around the nucleus. （3） According to flow cytometry, the transfeetion efficiency and fluorescent intensity of the complexes were significantly enhanced with the increasing of N/P ratio. （4） Western blot analysis showed that the expression of TGF - β1 was remarkably decreased with the rising of N/P value （ P 〈 0.05 ）. Conclusions TGF - β1 - AS - ODN can be trasfected by MPC30 - DEA70 into rat vascular adventitial fibroblasts in vitro and lower the expression of TGF - β1. This study may lay foundation for the use of the PC polymer as a non - viral transgenic vector in cardiovascular diseases.

关 键 词：磷酸胆碱聚合物 反义寡核苷酸 转化生长因子Β1 非病毒基因载体 

分 类 号：R541.42[医药卫生—心血管疾病] Q782[医药卫生—内科学]