胃癌细胞中端粒位置效应研究

目的研究插入端粒片段的荧光素酶报道质粒在胃癌细胞的转染及它对相邻荧光素酶报道基因表达的影响,探讨胃癌细胞中端粒位置效应的存在.方法将pTET-OFF调节质粒和携带荧光素酶报道基因的pTRE2-Hyg-Luciferase反应质粒共转染胃癌细胞SGC7901,经强力霉素(deoxycyline)诱导24h后检测报道基因活性,观察强力霉素诱导的抑制反应.然后将插入有端粒片段的荧光素酶报道质粒pBX质粒和对照质粒pBX-nfl(no teloemere fragment)分别与pTET-OFF调节质粒共转染细胞,在强力霉素诱导下,检测报道基因活性的改变.结果共转染pTET-OFF和pTRE-Hyg-Luciferase质粒的SGC7901细胞,在强力霉素作用下荧光素酶报道基因的活性逐渐降低.强力霉素诱导分别共转染有pTET-OFF和pBX质粒或pTET-OFF和pBX-nfl的细胞时,前者荧光素酶活性较后者降低4倍.结论转染TET-OFF基因表达调控系统的胃癌细胞SGC7901受强力霉素的诱导抑制;转染入胃癌细胞中的端粒片段降低了存在它附近荧光素酶报道基因的表达水平.

Telomere position effect in gastric cancer cell SGC7901

Objective To determine the possibility of telomere seeding in human gastric cancer cell SGC7901 and the influence of telomere on the expression of luciferase reporter gene nearby and to explore the position effect of telomere in gastric cancer cell. Methods The TET-OFF regulatory system was prepared and then transfected to gastric cancer cell SGC7901 by Lipofectamine TM 2000. With doxycycline added, the activity of the fluorescence caused by the expression of the target gene (the luciferase gene) with TET-OFF regulatory system was analyzed. The luciferase reporter plasmid in which telomere fragment pBX was inserted and the control plasmid pBX-ntf (no teloemere fragment) were co-transfected to cell 7901 with pTET-OFF regulate plasmid respectively and the luciferase activity was determined with doxycycline's induction. Results With the inducing of Dox, the activity of the fluorescence caused by the expression of the luciferase gene was inhibited. Treated with deoxycyline, there was a 4-fold decrease in the activity of the fluorescence in the cells infected with pBX and pTET-OFF as compared with that in the cells infected with pBX-ntf and pTET-OFF. Conclusion With the help of Lipofect2000, the gastric cancer cell SGC7901 can be transfected by TET-OFF regulatory system successfully. Dox could repress luciferase gene expression. Telomere seeding in gastric cancer cell SGC7901 could decrease the expression of luciferase reporter gene.