短发卡RNA对黑素瘤B16F10细胞BAG-1基因表达的抑制作用  

Silencing BAG-1 gene by eukaryotic expression vector containing short hairpin RNA in mouse melanoma B16F10 cells

作　　者：宋亚丽[2] 陈浩[1] 刘毅[1] 陈佳[1] 薛燕宁[1] 曾学思[1] 孙建方[1] 

机构地区：[1]中国医学科学院皮肤病研究所病理科,南京210042 [2]山东省立医院皮肤科,250021

出　　处：《中华皮肤科杂志》2008年第9期594-597,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（30672474） 志谢山东省立医院张莉、张捷、马春燕、牟文丽、田飞等医师

摘　　要：目的构建针对小鼠BAG-1（Bcl-2-associated athanogene 1）基因的短发卡RNA（shRNA）真核表达质粒，探讨其对小鼠黑素瘤B16F10细胞BAG-1基因表达的抑制作用。方法以小鼠BAG-1 mRNA编码区作为RNA干扰靶点，构建shRNA真核表达质粒pRNAT—U6．1／Neo—BAG-1，应用lipofectamin^TM2000转染小鼠黑素瘤B16F10细胞，设阴性对照组（转染阴性对照质粒Neg）和空白对照组（未转染），转染后48h开始Gg18筛选。转染1个月后采用RT—PCR、蛋白质印迹检测BAG-1基因的表达。结果酶切及测序结果证实，重组质粒pRNAT—U6．1／Neo—BAG-1构建成功。质粒在B16F10细胞的转染效率为20％-30％。阴性对照组细胞BAG-1基因表达与空白对照组无差异，shRNA质粒处理组细胞BAG-1基因表达较空白对照组显著下降（P〈0．05），表达抑制率在mRNA和蛋白水平分别为77％±4％和62％±2％。结论针对小鼠BAG—1基因的shRNA真核表达质粒pRNAT—U6．I／Neo—BAG-1能高效特异地抑制小鼠黑素瘤B16F10细胞BAG-1基因的表达。Objective To construct the eukaryotic expression plasmids of short hairpin RNA （shRNA） specific for mouse Bcl-2-associated athanogene 1 （BAG-1） and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16F10 cells. Methods Plasmids named pRNAT-U6.1/Neo-BAG-1, were designed and constructed to target the mouse BAG-1 mRNA coding region. LipofectaminTM 2000 was used to transfect plasmids into B16F10 cells. Negative plasmid-transfected and untransfected B16F10 cells served as negative and blank controls respectively. Forty-eight hours following Wansfection, G418 was used to select the resistant cells. The mRNA and protein expression of BAG-1 gene was measured by reverse transcription-PCR and Western blot respectively about 1 month after the transfection. Results The eukaryotic expression plasmids, pRNAT-U6.1/Neo-BAG-1, were constructed, and verified by restriction enzyme digestion and DNA sequencing. The transfection rate in B16F10 cells was 20% -30%. Compared with the blank control, the mRNA and protein expression of BAG-1 in B16F10 cells was significantly inhibited by BAG-1 shRNA （both P 〈 0.05 ）, and the inhibition rates were （77±4）% and （62 ±2）%, respectively. Conclusions These results indicate that the eukaryotic expression vectors containing shRNA against BAG-1 gene, pRNAT-U6.1/Neo-BAG-1, are successfully constructed, and can significantly inhibit the expression of BAG-1 gene in mouse melanoma B16F10 cells.

关 键 词：黑色素瘤 RNA 小分子干扰 基因 BAG-1 

分 类 号：R739.5[医药卫生—肿瘤]