Clinical Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 7, 2006; 12(17): 2762-2766
Published online May 7, 2006. doi: 10.3748/wjg.v12.i17.2762
Expression of angiostatin cDNA in human gallbladder carcinoma cell line GBC-SD and its effect on endothelial proliferation and growth
Ding-Zhong Yang, Jing He, Ji-Cheng Zhang, Zuo-Ren Wang
Ding-Zhong Yang, Department of Surgery, The First Hospital, Xi’an Jiaotong University, Xi’an 710065, Shannxi Province, China
Jing He, Department of Pharmacology, University of Texas Medical Branch, Galveston 77555, Texas State, United States
Ji-Cheng Zhang, Department of Surgery, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China
Zuo-Ren Wang, Department of Surgery, The First Hospital, Xi’an Jiaotong University, Xi’an 710065, Shannxi Province, China
Co-first-authors: Jing He
Correspondence to: Ding-Zhong Yang, Shannxi Provincial Hospital, Xi’an 710065, Shannxi Province, China. doctoryang8@126.com
Telephone: +86-29-85272432
Received: August 5, 2005
Revised: August 12, 2005
Accepted: September 8, 2005
Published online: May 7, 2006
Abstract

AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma.

METHODS: A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR, immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope.

RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). After 14 d of transfection and selection with G418, macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBC-SD cells between groups that were transfected with and without angiostatin. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited.

CONCLUSION: Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth.

Keywords: Angiostatin; Gallbladder carcinoma; Endothelial cell