磁性壳聚糖纳米介导eNOS基因转染受损平滑肌细胞初探

Magnetic chitosan nanoparticle-mediated eNOS gene transfer into ballon-injured vascular smooth muscle cells

目的:探讨磁性壳聚糖(后简称磁聚糖)纳米载体对血管平滑肌细胞的毒性及介导内皮型一氧化氮合酶(eNOS)基因对受损血管平滑肌细胞的转染效率、表达及对细胞增生的影响。方法:原位共沉淀法制备磁聚糖并耦合eNOS质粒;MTT法检测磁聚糖对血管平滑肌细胞生长的毒性;分别以重组腺病毒表达载体(AdCMV-eNOS)、磁聚糖-eNOS和磁聚糖空载体在外加磁场下转染受损血管平滑肌细胞,免疫荧光染色及RT-PCR测定eNOS表达,流式细胞术检测转染效率及血管平滑肌细胞周期和凋亡情况。结果:在浓度低于20 mg.ml-1时,磁聚糖对血管平滑肌细胞的生长无明显毒副作用;磁聚糖-eNOS组转染效率[(58.7±3.6)%]较AdCMV-eNOS组[(78.1±2.5)%]低,但两组均检测到eNOS mRNA和蛋白表达,且两组受损血管平滑肌细胞G0/G1期细胞较磁聚糖空载体组明显增多(P<0.01),S/G2-M期细胞减少(P<0.01),3组的细胞凋亡无显著差异(P>0.05)。结论:正常浓度的磁聚糖对血管平滑肌细胞生长无抑制作用,该载体能成功介导eNOS基因的转染,延缓受损后血管平滑肌细胞增生周期。

Objective： To investigate the toxicity and transfection efficiency of magnetic chitosan nanoparticle as a vestor mediated eNOS gene delivery to vascular smooth muscle cells（VSMC） in vitro,and then the gene expression and effect on ballon-injured VSMC proliferation were also detected.Methods： Prepare chitosan coated magnetic nanoparticles by the in-situ co-precipitation reaction and couple eNOS plasmid.The cytotoxicity to VSMC of magnetic chitosan was evaluated by MTT.In the apposite magnetic field,recombinant adenovirus carried eNOS（AdCMV-eNOS）,magnetic chitosan-eNOS complex and magnetic chitosan alone were transfected respectively into the cultured ballon-injured VSMC.The expression of the eNOS gene was detected by immunofluorescence and RT-PCR,and the VSMC generation cycle and apoptosis were examined by flow cytometer.Results： Below the concentration of 20 mg·ml^-1,magnetic chitosan nanoparticle had little toxic effect on VSMC proliferation.Though the transfection efficiency in magnetic chitosan-eNOS complex group[（58.7±3.6）%] was lower than that of AdCMV-eNOS group[（78.1±2.5）%],the expressions of eNOS gene in VSMC were detected in both groups by immunofluorescence and RT-PCR.By flow cytometer VSMC in G0/G1 stage were detected more in both groups abovementioned than the magnetic chitosan empty vestor group after gene transfer,while cells in S/G2-M were less.And no significant differences were found in apoptosis among the three groups.Conclusion： Normal concentration of magnetic chitosan nanoparticle vector has no inhibition on VSMC proliferation.It can efficiently mediate gene transfection into VSMC in vivo and delays the growth cycle of VSMC.