摘要
目的构建含人酪氨酸激酶受体B(tyrosine kinase receptorB,TrkB)基因的重组慢病毒质粒,建立稳定过表达Trk B的肾癌细胞株786-O并观察TrkB过表达后细胞增殖能力变化。方法扩增TrkB编码序列(coding sequence,CDS),并与pLV-EGFP(2A)puro载体连接。将连接产物导入HEK293V细胞包装成病毒颗粒并感染肾癌细胞株786-O。Western blotting检测TrkB蛋白表达。MTS法和克隆形成实验检测786-O增殖能力变化。结果重组慢病毒质粒pLV-EGFP(2A)puro-h TrkB构建成功,感染靶细胞后获得稳定过表达Trk B的肾癌细胞株786-O。重组质粒组TrkB蛋白表达显著升高。MTS实验中,重组质粒组490 nm吸光值明显降低(P<0.05);克隆形成实验中,重组质粒组克隆形成数明显低于空载体组(P<0.05)。结论稳定过表达TrkB可能抑制肾癌细胞786-O细胞增殖。
Objective To build the lentiviral expression vector of p LV-EGFP（2A） puro-TrkB（Tyrosine kinase receptor B）, establish the renal cell carcinoma cell line with stable overexpression of TrkB and observe the effect of TrkB on the proliferation of 786-O. Methods Coding sequence（CDS） of TrkB was amplified and combined with p LV-EGFP（2A） puro, then the connected product was transfected into human embryonic kidney HEK293 V cell. Western blotting was used to detect TrkB protein level. The effect of TrkB on the proliferation of 786-O was detected by MTS assay and clone formation assay. Results p LV-EGFP（2A） puro-TrkB lentiviral expression vector was successfully constructed and overexpression of renal cell carcinoma cell line 786-O was achieved after transfecting with target cells. The expression of TrkB significantly up-regulated protein level compared with empty vector group. MTS assay showed that the absorbance in recombinant vector group decreased significantly after transfection（P〈0.05） and cell clone formation assay showed that cell colony numbers in recombinant vector group also decreased significantly compared with the empty vector group（P〉0.05）. Conclusion The overexpression of TrkB may significantly decrease cell proliferation of 786-O cell line.
出处
《解放军医学院学报》
CAS
2016年第6期617-620,624,共5页
Academic Journal of Chinese PLA Medical School
基金
国家高技术研究发展计划(863计划)(2014AA020607)~~
关键词
酪氨酸激酶受体B
肾细胞癌
增殖
tyrosine kinase receptor B
renal cell carcinoma
proliferation
