转人B7-H3基因鳞癌细胞株的建立及其检测

目的探讨转入B7-H3基因鳞癌细胞株的建立及其检测。方法采用脂质体介导法将已建好的真核表达质粒pEGFP-C1-B7-H3导入人鳞癌细胞Tca8113中（Tca8113/B7-H3）,RT-PCR检测B7-H3在该细胞中的表达,Western blot检测其蛋白的表达。经G418筛选后,用有限稀释法建立稳定高表达的带有B7-H3基因的Tca8113鳞癌细胞株。结果RT-PCR方法检测到目的基因B7-H3的一个215bp大小的cDNA扩增产物;DAB显色,裂解的Tca8113/B7-H3基因转染细胞膜蛋白中相对分子质量约65～86ku大小的特异性条带。转染B7-H3的Tca8113细胞,经过细胞传代培养后仍然能够检测到高表达的B7-H3基因,经过有限稀释法建立了Tca8113/B7-H3细胞株。结论成功建立了Tca8113/B7-H3细胞株,为以后的体内外试验提供了很好的平台,为近一步研究其抗肿瘤作用奠定了基础。

Construction and detectionof the cell line of Tca8113/hB7-H3

Objective To construct and detect the cell line of Tca8113/hB7-H3. Methods The eukaryotic expression vector pEGFP-C1-B7-H3 was transfected into human squamous cell carcinoma cell line Tca8113 by lipofectamine 2000. The expression of GFP-B7-H3 protein and B7-H3 was detected by Western blot and RT-PCR respectively. Then the transfected cells were selected in medium containing G418 and termed as Tca8113/hB7-H3, through definite dilution method, to construct the cell line of Tca8113/hB7-H3. Results A 215 bp fragment of RT- PCR product was detected by using the B7-H3 specific primers. DAB showed that a differential belt which was consisted of about 65 - 86 ku protein molecule. Tca8113/hB7-H3 cells expressed B7-H3 protein with highest cell surface level by detecting passage cells. Through definite dilution method, we constructed the cell line of Tca8113/ hB7-H3. Conclusions The cell line of Tca8113/hB7-H3 has be constructed and cancer vaccines have been made too. This gives a good level to research cancer in vivo or vitro and lays a base of making further study on its antitumour function.