摘要
用美国FDA批准使用的生物可降解材料聚乳酸聚乙醇酸共聚物(PLGA)为载体材料,制备载p27kip1基因的纳米粒子.用激光光散射法测定纳米粒子的平均粒径为288.9nm,粒径呈窄分布,扫描电镜观察纳米粒子表面光滑.DNA含量为3%.包封效率为86%.观察p27kip1基因纳米粒子的体外释放情况,发现DNA体外最初释放速度较大,约1周后释放速度开始减缓,可维持平稳释放15天以上.体外转染大鼠血管平滑肌细胞,用流式细胞仪检测到p27kip1基因纳米粒子对细胞周期进程的抑制作用.建立自体静脉移植大鼠模型,随机分成三组进行试验:p27kip1基因纳米粒子组,空白纳米粒子组,单纯静脉移植组.分别于给药后3天、7天、14天、28天取材,HE染色及Verhoeff染色检测内膜厚度,蛋白质印迹检测P27抑癌基因蛋白的表达,免疫组化SABC法检测移植静脉内膜PCNA、E2F表达.动物模型试验中转基因组内膜平均厚度较其他组明显减少(P<0.01);转基因组PCNA的表达较其他组明显降低(P<0.01);转基因组E2F的表达7￣14天较其他组显著降低(P<0.01);对照组及单纯静脉移植组之间均无明显差异.纳米粒子作为p27kip1基因载体能够有效抑制自体静脉移植后内膜平滑肌细胞的增殖.
A therapeutic gene-p27kipl gene were encapsulated into poly （lactic-co-glycolic acid） （PLGA） NPs by an emulsification/solvent evaporation technique, and NP size distribution was assessed by submicro laser defractometer. The particle morphology was observed by scanning electron microscopy. The diameter of p27kiplgene-NPs was around 288.9 nm with very narrow size distribution, and p27kipl Nps showed good spherical shape with smooth uniform surface. The p27kipl loading in NPs was about 3%. Encapsulation efficiency of gene was about 86%. In vitro gene release from the NPs was performed in TE buffer at 37℃ under rotation （130 r/min） utilizing double-chamber diffusion cells on a shake stander, the result showed that gene release from the NPs lasted above two weeks. P27kipl gene NPs was transduced in smooth muscle cell in vitro and cell cycle was tested by flowcytometry. Vein grafting model was established in 120 rats by transplanting internal branch of jugular vein to carotid artery. The rats were randomly divided into three groups： 1）The p27kipl gene NPs treated grafting group; 2） Control group （treated with empty NPs without p27kipl gene）; 3） Shame grafting group. The grafted veins were harvested at 3d, 7d, 14d and 28d respectively after the operation. Intimal hyperplasic （IH） was observed by morphologic evaluation. The expression of PCNA, E2F were detected by immunohistochemistry and analyzed by computer digitizing system. The p27kipl gene transfection mediated by NPs complex enabled to produce protein expression of p27kipl gene （P 〈 0.05） and significantly inhibits hyperplasia of the grafted vein especially for 7-28 days in p27 group （P 〈 0.01）. Immunohistochemical analysis of PCNA indicated decreased positive cell in the p27kipl group compared with the control group at 7-28 d （P 〈 0.01）. The expression of E2F was decreased in p27 group at 7-14 d （P 〈 0.01）. There is no significant difference between control group and grafting group in expression of E2F and PCNA
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2005年第12期1192-1198,共7页
Progress In Biochemistry and Biophysics
