人pre-miR-15a真核表达载体的构建及其对Raji细胞生长的抑制作用  

Construction of eukaryotic expression vector of pre-miR-15a and its inhibitory effect on Raji cells proliferation

作　　者：谌琴[1] 何冬梅[1] 

机构地区：[1]暨南大学医学院血液病研究所,广州510632

出　　处：《第三军医大学学报》2009年第23期2334-2337,共4页Journal of Third Military Medical University

基　　金：国务院侨办重点学科建设基金(51205002)~~

摘　　要：目的构建人pre-miR-15a真核表达载体,并研究其对Raji细胞的生长抑制作用。方法将pre-miR-15a与目标载体(PGCSIL-GFP)定向连接,转化细菌感受态细胞,PCR鉴定阳性克隆,并测序。利用脂质体法将该载体转染Raji细胞,实验分为空白对照组、阴性对照质粒组和pre-miR-15a组(n=5)。RT-PCR检测Bcl-2mRNA表达,间接免疫荧光法检测Bcl-2蛋白表达,台盼蓝细胞计数法检测细胞增殖活性。结果PCR阳性克隆鉴定及测序结果均与目的序列一致。倒置荧光显微镜下见绿色荧光表达;RT-PCR法示各组间Bcl-2mRNA表达差异无显著性(P>0.05);间接免疫荧光法示pre-miR-15a组的Bcl-2蛋白表达量较空白对照组和阴性对照质粒组显著降低(P<0.05);台盼蓝拒染法示pre-miR-15a组Raji细胞生长受抑。结论本实验成功构建了per-miR-15a真核表达载体,且pre-miR-15a可以抑制Raji细胞生长。Objective To construct an eukaryotic expression vector of pre-miR-15a, and to investigate the inhibitory effect of pre-miR-15a to Raji cells. Methods The pGCSIL-GFP vector encoding pre-miR-15a nucleotides was transfected into the bacterial competent cells, and then confirmed by PCR and sequencing analysis. The identified vector was transfected into Raji cells with oligofeetamine 2000. The cells were divided into 3 groups, blank, negative control and pre-miR-15a group. Semi-quantitative RT-PCR was used to detect the expression of Bcl-2 mRNA, and immunofluorescence indirect for Bcl-2 protein expression. The growth of Raji cells was assayed by trypan blue dye exclusion method. Results PCR and sequences analysis indicated that the recombinant clones was identical with target sequences. Many green fluorescent cells were observed under fluorescent microscopy. The levels of Bcl-2 mRNA at every group had no obviously difference. Bcl-2 protein expression was obviously decreased at pre-miR-15a group compared with the other groups. Trypan blue dye exclusion method showed the cell growth was inhibited at 48 h and 72 h post-transfection. Conclusion We successfully construct the eukaryotic expression vector of pre-miR-15a, and it can inhibit the growth of Raji cells.

关 键 词：pre-miR-15a RAJI细胞 淋巴瘤 

分 类 号：R394-33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]