四环素调控的肿瘤坏死因子相关凋亡诱导配体对胃癌细胞系NCI-N87的杀伤作用

Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87.

作者信息

Wei X C, Wang X J, Chen K, Zhang L, Liang Y, Lin X L

机构信息

Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xue Yuan Road, Haidian District, Beijing 100083, China.

出版信息

World J Gastroenterol. 2001 Aug;7(4):559-62. doi: 10.3748/wjg.v7.i4.559.

DOI:10.3748/wjg.v7.i4.559

PMID:11819829

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4688673/

Abstract

AIM

To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells.

METHODS

The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction.

RESULTS

The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium.

CONCLUSION

With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.

摘要

目的

将人TRAIL（肿瘤坏死因子相关凋亡诱导配体）的cDNA片段克隆到四环素调控基因表达系统RevTet-On系统中，将表达载体转导至胃癌细胞系NCI-N87，并检测体外可控表达TRAIL对胃癌细胞的影响。

方法

将TRAIL全长cDNA插入到四环素反应元件（TRE）控制的载体中，获得质粒pRevTRE-TRAIL，将其转染至包装细胞系PT67。此外，将载体pRev-Tet On和pRevTRE也分别转染至PT67。经潮霉素和G418筛选后，测定病毒滴度。收集含有逆转录病毒载体的培养基，用于转导胃癌细胞系NCI-N87。建立了所得细胞系NCI-N87-Tet On TRE-TRAIL和对照细胞系NCI-N87 Tet On-TRE。用强力霉素（一种四环素类似物）孵育细胞，诱导细胞系中TRAIL的表达。诱导后分析对胃癌细胞的杀伤作用。

结果

构建了重组质粒pRev-TRE-TRAIL。经潮霉素或G418筛选后，获得了生产细胞系PT67-TRE、PT67-TRE-TRAIL和PT67-Tet On，滴度约为10(8)CFU.L(-1)。用这些细胞系的逆转录病毒载体转导NCI-N87细胞，建立了稳定细胞系NCI-N87-Tet-On TRE-TRAIL（NN3T）和对照细胞系NCI-N87-Tet-On-TRE（NN2T）。所选细胞系的生长曲线与野生型NCI-N87相同。加入强力霉素后，试验组细胞死亡明显（29%-77%），而对照和野生型细胞系未观察到差异。加入试验组培养基后，人白血病细胞系Jurkat被激活直至死亡（83%），表明试验细胞向培养基中分泌了活性TRAIL蛋白。