Uromodulin基因启动子在真核细胞中表达的特异性及活性

Tissue-specific and transcriptional activity of uromodulin promoter

目的 观察Uromodulin基因启动子在各种真核细胞中表达的特异性及活性.方法 对含Uromodulin基因启动子的真核表达质粒pEGFP-URO进行测序鉴定,利用脂质体转染技术,将质粒转染人近端肾小管上皮细胞株（HK-2）、人肾癌细胞株（ACHN）、人膀胱癌细胞株（T24）、大鼠系膜细胞株（HBZY-1）,用荧光显微镜和流式细胞仪观察绿色荧光蛋白（EGFP）的表达.经激发光照射后发出绿色荧光的为阳性细胞,并和pEGFP-N3进行对照研究.结果 转染24 h后HK-2、ACHN阳性率较高（7～12/HP）,流式检测结果为13.2%和11.4%;而T24、HBZY-1阳性率较低（0～1/HP）,流式检测结果为0%和0.8%.pEGFP-N3在各组细胞中均有较高表达,其阳性率分别为23.08%（HK-2）、19.24%（ACHN）、26.14%（T24）和18.03%（HBZY-1）.结论 小鼠Uromodulin基因启动子在HK-2及ACHN中具有特异性的启动活性,其活性约为CMV的50%.

Objective To investigate tissue-specific and transcriptional activity of Uromodulin （URO） gene promoter in cell lines. Methods Enhanced green fluorescent protein （EGFP） was used as reporter to construct the expression vector pEGFP-URO and transfected into cell lines of HK-2, ACHN, T24 and HBZY-1.Positive cells were observed as a green fluorescence under excitation light. EGFP activity of the cells was detected by fluorescence microscopy and flow cytometry （ FCM ） , and compared to the vector pEGFP-N3.Results The activity of EGFP in HK-2 and ACHN cell lines was stronger than that in the other cell lines （5-10/HP versus 0-1/HP）, with positive rate being 13.2% and 11.4% in HK-2 and ACHN cells respectively, but no positive cells were found in the other cell lines. The vector pEGFP-N3 highly expressed EGFP in all cell lines, with positive rate being 23.08% （HK-2） and 19.24% （ACHN） respectively. Conclusion Mouse URO gene promoter can be expressed in a tissue-specific way in human HK-2 and ACHN cells. The URO promoter transcriptional capacity is half of CMV promoter.