Basic Research
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 21, 2005; 11(23): 3549-3553
Published online Jun 21, 2005. doi: 10.3748/wjg.v11.i23.3549
Effects of adrenomedullin gene overexpression on biological behavior of hepatic stellate cells
Yi Wang, Jin-Sheng Zhang, Guang-Cun Huang, Qi Cheng, Zhong-Hua Zhao
Yi Wang, Jin-Sheng Zhang, Guang-Cun Huang, Qi Cheng, Zhong-Hua Zhao, Department of Pathology, Medical Center, Fudan University, Shanghai 200032, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Professor Jin-Sheng Zhang, Department of Pathology, Medical Center, Fudan University, Shanghai 200032, China. jszhang44@shmu.edu.cn
Telephone: +86-21-54237049
Received: May 27, 2004
Revised: May 28, 2004
Accepted: August 30, 2004
Published online: June 21, 2005
Abstract

AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection.

METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2’-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types I and III. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot.

RESULTS: Two cell clones (A-2, A-8) transfected with the AM gene expressed higher levels of AM mRNA (non-transfected group: 0.86±0.11, empty vector group: 1.01±0.11, A-2 clone group: 1.44±0.08 and A-8 clone group: 1.36±0.05) and protein (12.31±0.17, 12.35±0.12, 12.56±0.06 and 12.62±0.07) (P<0.05). AM gene overexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P<0.05) and expression of procollagen type I (0.58±0.1, 0.48±0.11, 0.3±0.06 and 0.31±0.07 at mRNA level) (0.27±0.07, 0.3±0.06, 0.14±0.05 and 0.13±0.05 at protein level) (P<0.05) and procollagen type III (0.17±0.04, 0.15±0.03, 0.1±0.02 and 0.09±0.02 at mRNA level) (0.22±0.04, 0.2±0.03, 0.11±0.04 and 0.13±0.03 at protein level) (P<0.05). Compared with cells non-transfected (TIMP2: 2.77±0.03, MMP-2: 0.5±0.04, MMP-1: 0.49±0.07) and transfected with empty vector (TIMP2: 2.79±0.04, MMP-2: 0.48±0.03, MMP-1: 0.45±0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7±0.02 and A-8 clone group: 2.71±0.02) (P<0.05) and MMP-2(A-2 clone group: 0.15±0.05 and A-8 clone group: 0.13±0.04) (P<0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68±0.06 and A-8 clone group: 0.81±0.09) (P<0.05).

CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.

Keywords: Adrenomedullin; Hepatic stellate cell; Procollagen type I and III