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（transfor-ming 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 MPC 30 -DEA70 ( PC poly-mer) , 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 la-beled 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 ①The AFs were spindle-shaped, polygonal or irregular , with large nucleuses and clear outline .② According to confocal microscopy , most MPC30 -DEA70/AS-ODN complexes were found around the nucleus .③ According to flow cytometry , the transfection efficiency and fluorescent intensity of the complexes were significantly enhanced with the increasing of N /P ratio.④Western blot a-nalysis showed that the expression of TGF -β1 was remarkably decreased with the rising of N/P value (P<0.05).Con-clusions TGF-β1-AS-ODN can be trasfected by MPC 30 -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 .
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