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World J Gastroenterol 2005;11(19):2932-2940 World Journal of Gastroenterology ISSN 1007-9327 © 2005 The WJG Press and Elsevier Inc. All rights reserved.
• BASIC RESEARCH •
Expressed genes in regenerating rat liver after partial hepatectomy Cun-Shuan Xu, Cui-Fang Chang, Jin-Yun Yuan, Wen-Qiang Li, Hong-Peng Han, Ke-Jin Yang, Li-Feng Zhao, Yu-Chang Li, Hui-Yong Zhang, Salman Rahman, Jing-Bo Zhang Cun-Shuan Xu, Cui-Fang Chang, Jin-Yun Yuan, Hong-Peng Han, Ke-Jin Yang, Li-Feng Zhao, College of Life Science, Henan Normal University, Xinxiang 453007, Henan Province, China Salman Rahman, Homophilia Research Center, London University, London SE17EH, UK Wen-Qiang Li, Yu-Chang Li, Hui-Yong Zhang, Jing-Bo Zhang, Key Laboratory for Cell Differentiation Regulation, Xinxiang 453007, Henan Province, China Supported by the National Natural Science Foundation of China, No. 30270673 Correspondence to: Professor Cun-Shuan Xu, College of Life Science, Henan Normal University, Xinxiang 453007, Henan Province, China.
[email protected] Telephone: +86-373-3326001 Fax: +86-373-3326524 Received: 2004-06-08 Accepted: 2004-08-05
Abstract AIM: To reveal the liver regeneration (LR) and its control as well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers. METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of them showed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressed in 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Subtracted cDNA libraries; Complementary DNA microarray; Liver regeneration; Partial hepatectomy; Cluster analysis Xu CS, Chang CF, Yuan JY, Li WQ, Han HP, Yang KJ, Zhao LF, Li YC, Zhang HY, Rahman S, Zhang JB. Expressed genes in regenerating rat liver after partial hepatectomy. World J Gastroenterol 2005; 11(19): 2932-2940
http://www.wjgnet.com/1007-9327/11/2932.asp
INTRODUCTION In the healthy adult rat liver, most of the hepatocytes lie in G 0 phase, and their cell division index is very low (about one ten thousandth)[1-5]. However, metabolism of hepatocytes is quickly altered after partial hepatectomy (PH) [6-10] . Activation of hepatocytes in G 0 phase occurs about 2 h after PH, and they progress to G 1 phase about 6 h after PH. Then, the cells enter into S phase of cell cycle in 12 h. DNA synthesis occurs in the early 6 h (12-17 h) of S phase, and then DNA is synthesized 18-30 h after PH, which reaches a maximum at 24 h. The G2 phase of cell cycle lies in the subsequent 2-4 h (31-34 h after PH). After that, hepatocytes go on dividing, and the peak of cell division is at 36 h after PH. The next cycle of hepatocytes is in the following 36-66 h after PH[11,12]. The re-differentiation of liver cells and the re-building of regenerated livers are in 72-144 h after PH. Many experiments have confirmed that a cell cycle of hepatocytes lasts for about 30 h, but that of other cells distinguishes from them[13]. Briefly, cells in the residual liver would be activated to proliferate, re-differentiate and rebuild their structure and function after PH. In different phases of liver regeneration (LR), the physiological and biochemical actions of different kinds of cells of the liver are different. The categories and amounts of the expressed genes in them are various [14,15]. To learn the molecular mechanism of LR, it is essential to highlight how many genes are related to it. Therefore, this paper reports that 300 genes have been successfully identified to correlate with LR by combing microarray in combination with suppression subtractive hybridization. MA TERIALS AND METHODS MATERIALS Partial hepatectomy of rats Healthy SD rats weighing 200±20 g were obtained from the Experimental Animal Center of Henan Normal University. Following the method of Hig gins and
Xu CS et al. Expressed genes in regenerating rat liver
Anderson [16], 70% of the rat liver was removed under sterile conditions. Regenerating liver preparation and RNA isolation The regenerating livers of four rats (male:female = 1:1) were taken 2, 4, 8, 12, 16, 24, 36, 48, 72, 96 and 144 h after PH. The livers were rinsed in cold PBS and immersed refrigerator for RNA extraction. Total RNA in a -80 was isolated from frozen livers according to the manual of TRIzol kit of Invitrogen. In brief, 50-100 mg liver was homogenized in 1 mL TRIzol reagent containing phenol and guanidinium isothiocyanate/cationic detergent, followed by phenol-chloroform extraction and isopropyl alcohol precipitation. The quantity and integrity of total RNA were examined by an ultraviolet spectrometer and denaturing formaldehyde agarose electrophoresis by ethidium bromide staining. Subtracted cDNA library construction and screening cDNA subtracted libraries were generated from total RNA by PCR-SelectTM cDNA subtraction kit (Clontech) following the manufacturer’s instructions. Briefly, total RNA was transcripted into double cDNA strands and digested with restriction enzymes, followed by subtracted hybridization with drivers and testers. Finally, differential expression sequence tags were performed to construct subtracted cDNA libraries with suppression PCR. Sequence analysis Base sequence assay of ESTs was carried out according to the current protocols in molecular biology. All sequences were determined on both strands. Comparison analysis of the selected sequences was conducted with the DNAman and the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) GenBank database[17]. cDNA microarray construction cDNA fragments amplified by PCR with nested PCR primer1 and primer2 and purified by NaAc/isopropyl alcohol were spotted onto glass slides (BioStar) with the help of the ProSys-5510A spotting machine. Then the gene chips were ready by hydrating and blocking and drying. A total of 1 152 elements (double spot chip) including 50 control system genes (8 negative control, 12 void control, 30 internal control) and 551 target genes to be studied comprised 8 submatrixes (12*12) occupying 9 mm×18 mm (BioStar). Then the gene chips were ready by hydrating, blocking and drying. Fluorescence-labeled cDNA preparation RNA prepared from rat livers before PH was ready for a reference for all cDNA microarray analyses. Total denatured RNA was reverse transcribed with cy3-conjugated dCTP (control group) and cy5-conjugated dCTP (test group) (Amersham-Pharmacia Biotech) using MMLV reverse transcriptase (Promega) with oligo(dT) primers. After bath incubation for 2 h, labeled buffers I and II were subsequently added to the reaction. The control group and test group were mixed symmetrically and stored avoiding light for application[17].
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Hybridization and scanning for 5-6 h in Glass slices were prehybridized at 42 hybridization buffer containing freshly cooked salmon sperm DNA. The labeled denatured probes were hybridized against cDNA microarray and incubated overnight (16-18 h) at 42 . The slices were then washed twice with 2× SSC containing 0.5% SDS for 5 min at room temperature, then for 10 min, with 0.2× SSC containing 0.5% SDS at 60 and finally with 0.2× SSC at 60 for 10 min. The slices were exposed to a photographer. Hybridized images were scanned by a fluorescence laser-scanning device, GenePix4000A. At least, two hybridizations were performed at each time point. In addition, a semiquantitative inspection of the hybridization results was performed for green signals (downregulation), yellow signals (no obvious regulation), and red signals (upregulation)[17]. Data analysis cy3 and cy5 signal intensities were quantified by GenePixPro 3.0 software. Subsequently, we normalized the obtained numerical data with classical linear regression techniques. In brief, quantified cy3 and cy5 signal intensities were obtained when foreground signal intensities were deducted by background signal intensities and cy5 signal intensities were replaced by 200 when they were
