摘要
目的评价包载抗凋亡基因bcl-xL的纳米微粒对缺氧大鼠心肌细胞的保护作用。方法从人肝脏组织获取bcl-xLcDNA(基因全长710 bp),连接至真核表达载体pTargeTTM上;用超声双乳化蒸发法制备纳米微粒;大鼠心肌细胞分为未转染组、脂质体转染组和纳米转染组共3组, 每组8孔;缺氧培养(5％CO2+95％N2)48 h后,采用逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学染色法半定量检测bcl-xL mRNA及蛋白表达,末端DNA转移酶dUTP缺口末端标记法 (TUNNEL)法检测细胞凋亡指数。结果制备的纳米微粒大小为200-260 nm,基因包封率为 89．26％;与未转染组比较,纳米转染组和脂质体转染组bcl-xL mRNA和蛋白表达水平均升高(P< 0．01);与另两组比较,纳米转染组心肌细胞凋亡指数(14．063±3．396)％显著下降(P<0．05)。结论成功制备包载bcl-xL基因的纳米微粒,外源性bcl-xL能够提高心肌细胞对缺氧的耐受性,纳米微粒具有低细胞毒性特点,是一种较理想的基因载体。
Objective To investigate the effect of nanosphere containing bcl-xL gene on tolerance of rat cardiac myocytes to hypoxia in vitro. Methods bcl-xL cDNA （710 bp） was cloned from human liver tissues by RT-PCR and cloned into pTargeT^TM. The nanospheres were prepared by the solvent evaporation technique. Cardiac myocytes in vitro were divided into three groups： nanosphere-transferred, lipofectamine 2000-transferred and control group. Forty-eight h after hypoxic culture （5 % CO2 ＋ 95 % N2）, bcl-xL mRNA and its protein expression in all three groups was detected by RT-PCR and immunochemistry respectively. Apoptosis index was measured by TUNEL. Results The diameters of nanospheres were in the range of 200 260 nm, and were well packaged and stable. In Lipofectamine 2000-transferred group and nanosphere-transferred group, the expression of bcl-xL mRNA and protein in cardiac myocytes was significantly increased as compared with control group （P 〈 0.01 ）. In nanospher-transferred group, apoptosis index was markedly decreased to （14. 063 ± 3. 396）% as compared with other groups （P 〈 0.05）. Conclusion We acquired nanosphere containing bcl-xL gcne successfully. The exogenous bcl-xL can enhance tolerance of rat cardiac myocytes to hypoxia. Nanoparticle is an optimal gene vector.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第4期576-578,共3页
Chinese Journal of Experimental Surgery
基金
卫生部科研基金资助项目(981093)
关键词
基因
纳米
基因载体
心肌细胞
Gene
Nanoparticle
Gene vector
Cardiac myocytes
