稳定表达淋巴细胞趋化因子的大鼠系膜细胞载体的构建  

Stable Expression of Lymphotactin Gene in Rat Mesangial Cells

作　　者：朱春芳[1] 徐荣左[1] 欧周罗[1] 

机构地区：[1]复旦大学上海医学院生物化学与分子生物学系,上海200032

出　　处：《复旦学报（医学版）》2003年第6期565-568,共4页Fudan University Journal of Medical Sciences

摘　　要：目的 探讨以肾小球系膜细胞作为高表达大鼠淋巴细胞趋化因子(Ltn)的载体的可行性,从而为深入研究淋巴细胞趋化因子的作用和体内活性创造条件。方法 采用RT-PCR法获得大鼠的Ltn的编码区全长的cDNA序列;应用基因重组技术和限制性内切酶酶切法构建并鉴定pcDNA 3.1/Ltn真核表达载体;用Lipofec-tAMINE PLUS脂质体转染法将构建的质粒及空质粒瞬时转染于SKOV3细胞;用RT-PCR和ELISA法检测LtnmRNA及蛋白质水平的表达,进而用pcDNA 3.1/Lac Z和pcDNA 3.1/Ltn稳定转染原代培养的WKY大鼠肾小球系膜细胞,用潮霉素B筛选获得稳定表达报告基因Lac Z和大鼠Ltn的细胞克隆。结果 测序结果显示质粒载体中正确地插入了大鼠Ltn的编码基因;RT-PCR显示在瞬时转染的SKOV3细胞和稳定转染的4个系膜细胞克隆中Ltn mRNA表达增高;ELISA证实细胞培养上清中Ltn的蛋白质浓度较对照组明显升高。此外,X-gal染色显示获得了2个稳定表达β-半乳糖苷酶基因的系膜细胞克隆。结论 本研究成功地构建了真核表达质粒载体pcDNA 3.1/hygro/Ltn,获得了稳定表达大鼠Ltn的肾小球系膜细胞克隆,并在mRNA及蛋白质水平上证实了Ltn的表达,为进一步研究Ltn的体内活性奠定了基础。Purpose: To set up a stable vector, rat glomerular mesangial cell, to express lymphotactin gene. Methods: We amplified the rat lymphotactin(Ltn) cDNA by RT-PCR for constructing the plasmid pcDNA 3. 1/Ltn. Lipofectin method was used to transfect pcDNA 3. 1/Ltn into SKOV3 cells instantaneously. RT-PCR and ELISA were applied to detect Ltn mRNA and protein. Then pcDNA 3. 1/Ltn and pcDNA 3. 1/Lac Z were transfected into WKY rat glomerular mesangial cells and Hygromycin B was scanned. Results: The recombinant eukaryotic expression plasmid, pcDNA 3. 1/Ltn was successfully constructed. RT-PCR and ELISA showed 4 cell clones positively expressed Ltn. X-gal staining showed 2 cell clones positively expressed Lac Z. Conclusions: Lymphotactin gene was stably expressed in rat mesangial cells which established the base for further function research of Lymphotactin in kidney diseases in vivo.

关 键 词：淋巴细胞趋化因子 大鼠 载体 肾小球系膜细胞 基因转染 肾小球疾病 

分 类 号：R692.6[医药卫生—泌尿科学] R-33[医药卫生—外科学]