高表达重组人组织激肽释放酶7基因的前列腺癌单克隆细胞株的构建

目的：建立稳定高表达重组人组织激肽释放酶7基因（ KLK7）的前列腺癌细胞株DU145，为前列腺癌发生机制的研究奠定基础。方法 PCR法扩增KLK7 cDNA，并克隆到真核表达载体pcDNA3．1上；经过限制性内切酶酶切、DNA测序验证正确后，以脂质体法转染人前列腺癌DU145细胞；通过G418筛选，每个单克隆细胞扩大培养，建立稳定转染KLK7的DU145单克隆细胞株。采用Western blot 法检测DU145细胞株KLK7表达。结果酶切鉴定及DNA 测序分析显示 pcDNA3．1-KLK7真核表达载体成功构建；转染后，成功获得稳定高表达KLK7的DU145单克隆细胞株。 Western blot 结果显示，pcDNA3．1-KLK7转染的DU145细胞KLK7表达量明显高于转染空载体组细胞（P＜0．01）。结论 pcDNA3．1-KLK7真核表达载体的建立及稳定高表达KLK7的前列腺癌单克隆细胞株的建立，为研究KLK7在前列腺癌发生发展中的作用提供了条件。

Establishment of a human prostatic carcinoma cell line stably overexpressing Kallikrein 7

Objective To establish a human prostatic carcinoma cell line stably overexpressing Kallikrein 7 （KLK7） and provide foundation for prostate cancer pathogenesis .Methods KLK7 cDNA was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1.Then it was confirmed by restriction endonuclease and DNA sequencing test , and the correct vectors were transfected into prostate cell line DU 145 with lipofectamine .The survival cell clones treated by G418 were amplified.The KLK7 expression of cell line DU145 was detected by Western blotting in each cell clone .Re-sults After restriction endonuclease analysis and DNA sequencing , the pcDNA3.1-KLK7 eukaryotic expression vector was successfully constructed .The stably transfected DU145 cell line highly expressing KLK7 was successfully established after being transfected .The results of Western blotting showed the expression of KLK 7 protein was significantly higher in stable transfectants with the KLK7 vector than that in the cells with empty vector （P＆lt;0.01）.Conclusion The construc-tion of the eukaryotic expression vector pcDNA 3.1-KLK7 and the establishment of stable prostatic cell line overexpressing KLK7 provide basis for further studies on the function KLK 7 in the prostate pathogenesis .