CAJ | 학술논문

目的：探讨新型阳离子磷酸胆碱聚合物MPC30-DEA70载AT1受体的反义寡核苷酸(AS-ODN)对离体培养的大鼠心肌成纤维细胞 AT1受体及Ⅰ型胶原蛋白表达的影响。方法：MTT 法检测 MPC与心肌成纤维细胞的细胞相容性；应用激光扫描共聚焦显微镜(CLAM)检测复合物在细胞内的定位，流式细胞仪(FCM)检测转染效率及荧光强度， Western blot检测AT1受体蛋白、Ⅰ型胶原蛋白的表达。结果：MPC浓度＞40μg/mL时才出现明显细胞毒性，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，少量分布于细胞核内。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。Western blot结果显示，转染复合物后各组细胞AT1受体及Ⅰ型胶原蛋白的表达也相应减少。结论：MPC30-DEA70在安全浓度范围内就能够携带AT1R-ASODN以较高的转染率进入离体培养的大鼠心肌成纤维细胞，并成功靶向抑制AT1R及Ⅰ型胶原蛋白的表达。从而可以推断阳离子磷酸胆碱聚合物可以有效负载反义寡核苷酸(AS-ODN)，靶向性抑制基因的表达，为新型非病毒性基因载体的研究提供了一定的理论依据。
목적：탐토신형양리자린산담감취합물MPC30-DEA70재AT1수체적반의과핵감산(AS-ODN)대리체배양적대서심기성섬유세포 AT1수체급Ⅰ형효원단백표체적영향。방법：MTT 법검측 MPC여심기성섬유세포적세포상용성；응용격광소묘공취초현미경(CLAM)검측복합물재세포내적정위，류식세포의(FCM)검측전염효솔급형광강도， Western blot검측AT1수체단백、Ⅰ형효원단백적표체。결과：MPC농도＞40μg/mL시재출현명현세포독성，격광공취초현미경관찰도FAM표기적기인복합물다수분포재세포핵주위，소량분포우세포핵내。류식결과현시전염효솔급형광강도수N/P비증가이명현증대。Western blot결과현시，전염복합물후각조세포AT1수체급Ⅰ형효원단백적표체야상응감소。결론：MPC30-DEA70재안전농도범위내취능구휴대AT1R-ASODN이교고적전염솔진입리체배양적대서심기성섬유세포，병성공파향억제AT1R급Ⅰ형효원단백적표체。종이가이추단양리자린산담감취합물가이유효부재반의과핵감산(AS-ODN)，파향성억제기인적표체，위신형비병독성기인재체적연구제공료일정적이론의거。
Objective:To investigate the impact of novel cationic phosphorylcholine polymer of MPC30-DEA70 load AT1 receptor of antisense oligonucleotide(AS-ODN) on the expression of AT1 receptor and type I collagen in cultured rat myocardial. Methods:MTT assay for detection of MPC and cardiac fibroblasts cell compatibility;application of laser scanning confocal microscope(CLAM) to detect complex in the cell,flow cytometry(FCM) detected transfection efficiency and fluorescence intensity, the expression of Western blot detection of AT1 receptor protein and collagen I.Results:When the concentration of MPC>40 μg/ml cytotoxicity will obviously appeared,under the confocal laser scanning microscopy,most of gene complexes marked by FAM distributed in nucleus around,a small amount of them distributed in the nucleus.Flow cytometry analysis revealed that with N/P ratio increased the efficiency of transfection and fluorescence intensity increased obviously.The Western blot results showed that expression of intracellular AT1 receptor and type I collagen was reduced accordingly after transfection complexes.Conclusion:MPC30-DEA70 can carry AT1-R AS-ODN in cultured rat cardiac fibroblasts with high transfection rate in the safe concentration range,and inhibit the expression of AT1R and type I collagen.So then we can infer that cationic phosphorylcholine polymer can effectively load antisense oligonucleotide(AS-ODN),targeted inhibition of gene expression.This research will provide certain theoretical basis for study on the novel non viral gene vector.