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Copyright ©The Author(s) 1999. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 15, 1999; 5(5): 443-444
Published online Oct 15, 1999. doi: 10.3748/wjg.v5.i5.443
The effect of retinoic acid on Ito cell proliferation and content of DNA and RNA
Ze-Li Gao, Xiao-Hong Gu, Department of Gastroenterology, Yangpu Central Hospital, Shanghai 200092, China
Ding-Guo Li, Han-Ming Lu, Department of Gastroenterology, Xin Hua Hospital, Shanghai Second Medical University, Shanghai 200090, China
Ze-Li Gao, male, born on 1961-05-30 in Huanren County, Liaoning Province, graduated from the Harbin Medical University in 1983, got the doctor degree in Shanghai Second Medical University in 1997, now working in the Department of Gastroenterology, Yangpu Central Hospital, Shanghai, having 11 papers published.
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Ze-Li Gao, Department of Gastroenterology, Yangpu Central Hospital, 130 Boyang Road, Shanghai 200090, China.
Telephone: +86·21·58615242
Received: April 15, 1999
Revised: August 20, 1999
Accepted: September 24, 1999
Published online: October 15, 1999

Key Words: liver fibrosis, retinoic acid, Ito cell, cell culture, microspectrophotometer, DNA, RNA


The development of liver fibrosis is due to an imbalance between synthesis and degradation of extracellular matrix. Recent studies have shown that Ito cells which are located along the sinuses of liver, can store and metabolize Vit A, and are the main cells that produce collagen, among which type I, III and IV and laminin, account for 80%-95% of the total hepatic collagen[1]. Ito cells in the course of proliferation and synthesis of collagen showed a reduction of Vit A contents and retinoic acid receptors[2]. This study was designed to investigate the effect of retinoic acid on Ito cell proliferation and the contents of DNA/RNA and to analyse further its mechanism or pathways.


DMEM medium (Gibco Compony); all trans-retinoic acid (RA), retinol palmitate (RP) (Sigma Company); 3H-TdR (Shanghai Nuclear Energy Research Institute); Solution A: 0.1% Triton X-100, 0.08N HCl, 0.15N NaCl; solution B: 120 μM acridine orange (AO), 1 mM EDTA, 0.15N NaCl; vidas microspectr ophotometer analysis system.

3H-TdR incorporation test

The isolation and culture of Ito cells were done by the method as published before[3]. Rat Ito cells of 5-d primary culture were suspended on DMEM me dium containing 20% bovine serum. The number of the cells was adjusted to 1 × 105/mL. One mL of the cell suspension was put into each well of the 24-well culture plate, which contained serial concentrations of RA/RP. The control wells contained no RA/RP. After incubating for 24 h, 8 μci-3H-TdR was added to each well and incubated for another 48 h. The cells were collected on the F49 filter paper, fixed with 4 mL 10% trichloro-acetic acid and dehydrated with 4 mL alcohol. The dried filter papers were put on the bottom of the scinti bottle which contained 5 mL scintillating solution for counting the pulse per minute (CPM). The degree of DNA replication was indicated by CPM/well. We set three wells for each sample.

DNA/RNA content analysis

Ito cell culture and treatment were handled with the same procedure as above and the difference was as follows: drug treatment with RA/RP at 10-4 mol/L, small cover glass was put into each well of the plate to bear the growing cells. After the culture medium was discarded, the Ito cells on the small cover g lass were fixed for 30 min with 70% alcohol and washed with PBS solution. Solution A (0.4 mL) was added into each well, the preparation was set onice bath for reacting 15 seconds, 1.2 mL solution B was then used for 8 min staining. The small cover glass was put on a glass slide, fluorescent microscopic examination was made immediately with the inciting light wave of 488 nm. DNA was examined with a screened filter at a wave-length of 530 nm, with green in stain, whereas RNA examined with a filter at 610 nm showed a red fluorescence. Fifty cells were examined in each group with fluorescent microspectrophotometer-30 (FMSP-30); the fluorescence intensity was converted into grey scale value, corresponding to the relative aver age DNA/RNA contents of Ito cells.

3H-TdR incorporation test

Lower densities of RA/RP (10-6 mol/L) had no effect on the 3H-TdR incorporation of Ito cell. With higher densities 3H-TdR incorporation of Ito cell was inhibited as compared with the control (P < 0.05)(Table 1).

Table 1 3H-TdR incorporation test.
GroupDosage (mol/L)(CPM ± s)/well
Control2866.4 ± 253.2
Test groups
RA10-62657.6 ± 104.8
10-51279.4 ± 236.4a
10-4182.2 ± 67.6a
RP10-62598.4 ± 76.4
10-51182.6 ± 154.4a
10-4897.2 ± 82.6a
DNA/RNA content analysis

The results showed that RA/RP at 10-4 mol/L could reduce DNA/RNA- contents of Ito cells as compared control (P < 0.01) (Table 2).

Table 2 DNA, RNA content analysis.
GroupDosage (mol/L)DNARNA
Control101.98 ± 21.5889.38 ± 22.03
RA10-461.79 ± 18.31a56.31 ± 14.72a
RP10-446.85 ± 11.52a49.20 ± 10.12a

Ito cells which have the characteristics of fibroblasts cell and myofibroblasts are the main collagen-producing cells in the liver. Bamard H[4] and Seifert WF[5] reported that RA could reduce the deposition of types I and III collagen in the CCl4-induced liver fibrosis of the rat through its inhibitory effect on the transformation of Ito cell to myofibroblasts.

Our study showed that RA/RP could inhibit 3H-TdR incorporation of rat Ito cells and reduced the DNA/RNA contents of rat Ito cells. Our previous study indicated that RA could restore retinoic acid receptor content and increase cAMP content in the primary culture of rat Ito cells[6]. The form of biologic effect of RA was similar to that of thyroxin[7], i.e. they both act on nuclear receptor resulting in a change of the second messenger and regulating the gene expression of RAR and collagen in the Ito cells. RA may be expected to be an effective antihepatofibrotic agent.


Edited by Ma JY

1.  Ramadori G. The stellate cell (Ito-cell, fat-storing cell, lipocyte, perisinusoidal cell) of the liver. New insights into pathophysiology of an intriguing cell. Virchows Arch B Cell Pathol Incl Mol Pathol. 1991;61:147-158.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Gao ZL, Li DG, Lu HM. The retinoic aci content of Ito cell, the expression of retinoic acid receptor and liver fibrosis. Zhonghua Xiaohua Zazhi. 1993;13:295-296.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Gao ZL, Liu SR, Li DG, Lu HM. The isolation and culture of rat ito cell and the effect of retinoic acid on the proliferation of Ito cell. Shanghai Dier Yikedaxue Xuebao. 1995;15:93-95.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Davis BH, Kramer RT, Davidson NO. Retinoic acid modulates rat Ito cell proliferation, collagen, and transforming growth factor beta production. J Clin Invest. 1990;86:2062-2070.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 115]  [Cited by in F6Publishing: 121]  [Article Influence: 3.6]  [Reference Citation Analysis (0)]
5.  Seifert WF, Bosma A, Hendriks HF, van Leeuwen RE, van Thiel-de Ruiter GC, Seifert-Bock I, Knook DL, Brouwer A. Beta-carotene (provitamin A) decreases the severity of CCl4-induced hepatic inflammation and fibrosis in rats. Liver. 1995;15:1-8.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 33]  [Cited by in F6Publishing: 36]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
6.  Gao ZL, Li DG, Lu HM. The effect of retinoic acid on Ito cell retinoic acid receptor and cAMP content. Weichangbingxue He Ganbingxue Zazhi. 1995;4:20.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Weiner FR, Blaner WS, Czaja MJ, Shah A, Geerts A. Ito cell expression of a nuclear retinoic acid receptor. Hepatology. 1992;15:336-342.  [PubMed]  [DOI]  [Cited in This Article: ]