反义bcl-2寡脱氧核苷酸对BcaCD885细胞内源性bcl-2表达的阻断作用  

INHIBITION OF THE EXPRESSION OF INTRINSICAL BCL-2 PROTEIN BY ANTISENSE OLIGODEOXYNUCLEOTIDES IN BcaCD885 CELLS

作　　者：何永文[1] 毛租彝 高志[2] 王升志[3] 赵德萍 

机构地区：[1]昆明医学院第一附属医院口腔颌面外科,650031 [2]华西医科大学口腔医学院 [3]汕头大学医学院第一附属医院 [4]云南省肿瘤研究所

出　　处：《口腔颌面外科杂志》2000年第3期243-246,共4页Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金资助项目! ( 396 70 788);云南省自然科学基金资项目 !( 1999C0 0 0 7R)

摘　　要：目的　探讨与 bcl-2翻译起始部位碱基互补的反义 bcl-2寡脱氧核苷酸 ( oligodeoxynucleotide,ODN)对 Bca-CD885细胞内源性 bcl-2表达的阻断作用。方法　以脂质体 Lepofectin为载体 ,一过性转染 2 0 μmol/L 反义及正义 bcl-2 ODN于 Bca CD885细胞中 ,FCM-抗体观测转染后 2 4h、3 6h、48h细胞内 bcl-2基因蛋白表达变化 ,RT-PCR技术检测转染后 2 4h bcl-2 m RNA的表达变化。结果　转染反义 bcl-2 ODN后 2 4h、3 6h、48h Bca CD885细胞内 bcl-2基因蛋白表达与对照组相比都明显下降 ( P<0 .0 5 ) ,而正义组与对照组无差异 ( P>0 .0 5 ) ;正反义组 bcl-2m RNA与对照组相比无明显变化 ( P>0 .0 5 )。结论　反义 bcl-2 ODN能在蛋白翻译水平特异性抑制颊癌细胞内源性Objective To study the function of antisense oligodeoxyncleotides which complements to translation initiation site of human bcl 2 mRNA in inhibition of the expression of intrinsical bcl 2 protein in BcaCD885 cells. Method Through lepofectin vector, we transfected 20 μm sense (5′ GGG AAG GAT GGC GCA CGC TG 3′) or antisense (5′ CAG CGT GCG CCA TCC TTC CC 3′) bcl 2 oligodeoxynucleotids into human BcaCD885 cells in 12 hours with 37°C and 5% CO 2 conditions. At 24 h, 36 h and 48 h after transfection, we studied the expession of bcl 2 protein with bcl 2 polyclonal antibody by flow cytometry analysi (FCM). At 24 h after transfection, we studied the expression of bcl 2 mRNA through reverse transcription polymerase chain reaction (RT PCR) technique. Total RNA was extracted from cell sample with TRIzol kit. Primer pairs used fr PCR are: bcl 2 mRNA (385 bp) Forward 5′ ACT TGT GGC CCA GAT AGG CAC CCA G 3′, Reverse 5′ GCA CTT CGC CGA GAT GTC CAG CCA G 3′; β actin mRNA ((154bp) Forward 5′ TCA TCA CCA TTG GCA ATG AG 3′, Reverse 5′ GTG TTG GCG TAC AGG T 3′. PCR samples were run through 1.8% agarose gel and specific amplified lines were analysised with UVP GDS 800 system. Bcl 2 mRNA quantity was the ratio of bcl 2 line density to β actin line density. Results At 24 h, 36 h and 48 h after transfection of antisense bcl 2 oligodeoxynucleotids, there was down regulation of bcl 2 protein in BcaCD885 cells. But the expression of bcl 2 protein did not change after transfection of sense bcl 2 oligo deoxynucleotids. The expression of bcl 2 mRNA was the same in the antisense or sense group with the control group. Conclusion Antisense bcl 2 oligodeoxynucleotids can inhibite intrinsical bcl 2 protein expression in BcaCD885 cells.

关 键 词：反义寡脱氧核苷酸 BCACD885细胞 bcl-2基因 

分 类 号：R739.8[医药卫生—肿瘤]