CAJ | 학술논문

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。
목적：관찰신형양리자린산담감취합물 MPC30-DEA70능부유효지전염침대전화생장인자β1（transfor-ming growth factor-β1，TGF-β1）적반의과핵감산（AS-ODN）진입도혈관외막성섬유세포중급전염후대혈관외막성섬유세포내TGF-β1표체적영향，위MPC30-DEA70취합물작위약물기인치료재체재심혈관질병중적운용전정기출。방법조직첩괴법배양대서흉주동맥외막성섬유세포；응용공취초격광소묘현미경（CLSM）관찰MPC30-DEA70/TGF-β1AS-ODN（ FAM표기）재세포내적분포화정위；류식세포의（ FCM）검측이자적세포전염효솔﹑형광강도；면역인적실험（Western blot）검측TGF-β1세포내표체。결과①원대배양적세포정방추형，다각형혹자불규칙형，세포핵교대，수량1～2개불등，륜곽청초，핵인대이명현，정타원형；②격광공취초현미경관찰도FAM표기적기인복합물다수분포재세포핵주위；③류식결과현시전염효솔급형광강도수N/P비증가이명현증대；④Western blot결과현시수착N/P비치적증대，TGF-β1적단백표체명현하강（ P＜0．05）。결론 MPC30-DEA70능구휴대TGF-β1-AS-ODN진입리체배양적대서흉주동맥외막성섬유세포，병능하조TGF-β1단백적표체。
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 .