反义基因治疗对H9C2心肌细胞β_1受体表达的影响  

Effects of β_1 adrenoceptor antisense oligonucleotide on β_1 adrenoceptor mRNA and protein in H9C2 myocardial cells

作　　者：林纯莹[1] 陈泗林[1] 梁远红[1] 周燕[2] 余细勇[1] 

机构地区：[1]广东省人民医院广东省心血管病研究所,广州510100 [2]武汉大学人民医院急诊科,武汉000000

出　　处：《岭南心血管病杂志》2008年第2期140-143,共4页South China Journal of Cardiovascular Diseases

基　　金：中国博士后科学基金(20060400780);广东省自然科学基金(A2007022);广东省医学科研基金(A2007022)

摘　　要：目的观察阳离子脂质体介导的β1受体反义基因对H9C2心肌细胞β1受体mRNA及受体蛋白表达的影响,寻求最佳转染比率。方法H9C-2心肌细胞,用去甲肾上腺素(norepinephrine,NE)刺激24h后转染β1受体反义寡核苷酸(β1 antisense oligonucleotide,β1-AS-ODN),脂质体与β1-AS-ODN的混合摩尔比为2.0、2.5和3.0,分别转染18h、24h和36h;在倒置荧光显微镜下观察心肌细胞的形态;用免疫印迹技术测定心肌细胞β1受体蛋白的时间表达曲线。实验分为4组:NE组、NE加β1受体反向寡核苷酸(β1 inverse oligonucleotides,β1-IN-ODN)组、NE加β1-AS-ODN组、心肌细胞组,用半定量逆转录多聚酶链反应检测心肌细胞β1受体mRNA的表达。结果β1-AS-ODN分布在心肌细胞的胞浆及细胞核中,转染24h效率达70%,此后平缓降低;NE组的心肌细胞β1受体蛋白显著升高;与NE组相比,脂质体与β1-AS-ODN的混合摩尔比2.0、2.5,和3.0的心肌细胞β1受体蛋白显著降低(P<0.05,P<0.01,P<0.05);与心肌细胞组比较,NE组和β1-IN-ODN组的心肌细胞β1受体mRNA和受体蛋白显著升高(P<0.01);与NE组相比,NE加AS-ODN组的心肌细胞β1受体mRNA和受体蛋白显著降低(P<0.01)。结论阳离子脂质体与β1-AS-ODN比率为2.5转染24h效果最好,其机制可能是通过激活核糖核酸酶H降解靶mRNA,在转录和翻译水平抑制目的基因表达。Objectives To evaluate the effects of β1 receptor antisense oligodeoxynucleotides （β1-AS-ODN） by delivery with the different charge ratios lipofectamin on β1 adrenoceptor mRNA and protein in H9C2 myocardial cells. Methods H9C-2 myocardial cells were stimulated by norepinephrine （NE） for 24 hours. The β1-AS-ODN was delivered with different ratio of the lipofectamin, then the complexes were transformed for 18 hours, 24 hours, 36 hours.β1-AS-ODN labeled with FAM were measured by fluorescence microscopy. The study was divided into 4 groups： NE group, myocardial cell group （normal group）, β1-AS-ODN group and [31-IN-ODNs group. β1AR mRNA level was measured by RT-PCR, β1AR protein level was measured by Western blot. Results It showed that the β1-AS-ODN at 24 hours （70%） was taken up into H9C-2 myocardial cells remaining up to 36 hours. The maximum drop of β1AR protein was greater at ratio 2.5 than at ratio 2.0 and 3.0. Compared with the NE group, the β1AR mRNA and protein in β1-AS-ODN group were significantly decreased （P〈 0.01）. Conclusions β1-AS-ODN could transfect cultured myocardial cells triumphally. The best ratio of the lipofectamin/β1-AS-ODN was 2,5 for 24 hours. The inhibition of β1-AR expression probably occurs in transcriptional and translational steps.

关 键 词：Β1受体 反义寡核苷酸 心肌细胞 

分 类 号：R541.61[医药卫生—心血管疾病]