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.docVIP

编号： 刊用形式： 人pre-miR-15a 谌 琴，何冬梅 (暨南大学医学院血液病研究所, 广州 510632) 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。RT-PCR检测Bcl-2 mRNA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-2 mRNA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。 关键词：pre-miR-15a；Raji细胞；淋巴瘤 中图法分类号： 文献标识码：A Construction of Eukaryotic Expression Vector of pre-miR-15a and the inhibitory effect of pre-miR-15a CHEN Qin, HE Dong-mei (Institute of Hematology,Medical Collge, Jinan University, Guangzhou 510632, China Abstract：objective To construct eukaryotic expression vector of pre-miR-15a, and to study the inhibitory effect of pre-miR-15a to Raji cells. Methods The pGCSIL-GFP vector which was Lineating with pre-miR-15a nucleotides was transfected into the bacterial competent cell, and confirmed by PCR and sequence analysis. Transfecting this vector into Raji cells with oligofectamine 2000, and the experiment was classed into three groups: blank, negative control and pre-miR-15a group. Semi-quantitative RT-PCR detected the expression level of Bcl-2 mRNA; Bcl-2 protein expression was detected by indirect immunofluorescence; The growth inhibitory effect of Raji cells was measured by trypan blue dye exclusion method. Results the consequences of recombinant clones by PCR and sequences analysis were both coinsided 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 levels obviously decreased at pre-miR-15a group compared with the other groups. Trypan blue dye exclusion method showed the cell growth was inhibited at 48h and 72 h post-transfection. Conclusion We