小鼠uroplakinⅡ启动子在人细胞中的作用

Effect of mouse uroplakin Ⅱ promoter on human bladder cancer cell line

目的 研究小鼠UroplakinⅡ (UPⅡ )启动子在人细胞中的作用。方法 采用半定量RT PCR方法 ,检测人类膀胱移行细胞癌细胞系 (BIU 87)、肾癌细胞系 (GRC 1)、脐静脉血管内皮细胞系(EC)、肺癌细胞系 (A549)和皮肤成纤维细胞系 (Hs2 7)中UPⅡ基因mRNA的表达情况 ;同时构建pEGFP UPⅡ、pGL UPⅡ重组质粒 ,应用脂质体转染技术 ,将重组质粒转染入BIU 87、GRC 1、EC、A549和Hs2 7;利用共聚焦显微镜、流式细胞仪和微板检测仪观察细胞表达绿色荧光蛋白 (GFP)和荧光素酶(Luc)的情况。结果 UPⅡ基因mRNA在BIU 87中有较高的表达 ,而在其他组织细胞中表达极低。BIU 87表达GFP较多 (5～ 10 /HP) ,亮度较强 ,流式细胞仪测定表达量为 10 .1% ;而GRC 1、EC细胞表达GFP极少 (0～ 2 /HP) ,亮度暗 ,流式细胞仪测定表达量分别为 0 %和 1.8%。Luc在BIU 87中的表达量是其他组织细胞的 1.8～ 8.2倍 ,差异有显著性 (P <0 .0 1)。结论 UPⅡ特异性的表达在膀胱癌细胞中。

Objective To study the effect of gene expression of mouse uroplakinⅡ (UPⅡ) promoter on human bladder cell cancer cell line. Methods The mRNA expression of different cell lines was quantified by RT-PCR. Green fluorescent protein (G F P) and luciferase (Luc) were used as reporter genes. The plasmids carrying UPⅡ or GFP were constructed and transfected into human cell lines of bladder transit ional cell cancer (BIU-87), kindey cancer (GRC-1), vascular endothelium (EC), lung ca ncer cell line (A549) and skin fibroblast cell line (Hs27). GFP activity of cell s was detected by confocual microscopy and flow cytometry (FCM). Luciferase valu e was measured by luminometer (microplate) and luciferase to β-galactosidase r atios (L/G values) were used for evaluating transfection efficiency. Results RT-PC R showed high expression level of UPⅡ mRNA in bladder cancer cell line BIU-87, whereas low level or no expression in nonbladder cancer cell lines. The activity of GFP in bladder cancer (BIU-87) cell was higher than that in the other cell l ines (5～10/HP versus 0～2/HP), with 4.34% positive cells in BIU-87 detecte d by FC M, but no positive cell was found in the other cell lines. L/G values indicated that the luciferase expression in human bladder cancer cells transfected with mo use UPⅡ promoter was 1.8～8.2-fold as high as that in the nonbladder cell line s. Conclusion Mouse UPⅡ promoter gene can be expressed in a tissue-specific fashi on in human urinary bladder cancer. It is capable of initiating transcription of reporter genes in human bladder cancer cell line.