9% NaCl; n = 6) For bile duct ligation (BDL), 8-12-week-old mice

9% NaCl; n = 6). For bile duct ligation (BDL), 8-12-week-old mice underwent BDL or sham surgery as previously described.13 WT and Tg animals received 100 μg/g/day of GCV (IP) diluted in 0.9% NaCl (or 0.9% NaCl alone for sham animals), beginning the day after surgery, for 11 consecutive days. At least 5 animals were treated per BDL group (n = 5; sham+GCV: n = 3; sham+saline: n = 2). The murine HSC line, JS1, has been

previously described,14 the mouse hepatocyte cell line, AML12, was purchased from ATCC (Manassas, VA), and the immortalized EC line, TSEC, was kindly donated by Vijay Shah, M.D.15 Mouse HSCs were isolated by in situ perfusion of livers with collagenase and pronase as well as Percoll gradient centrifugation. Primary hepatocytes were isolated by in situ perfusion with collagenase, followed BGB324 ic50 by differential centrifugation. Histological liver analysis was performed by an expert pathologist (I.F.) using a score from 0-3 for both centrilobular and parenchymal necrosis, according to the following: 0 = none; BVD-523 1 = isolated hepatocytes; 2 = groups of hepatocytes; 3 = bridging. Ballooning of hepatocytes was scored as follows: 0 = none; 1 = mild; 2 = moderate; 3 = severe. For each mouse, 10 fields at ×100 magnification were analyzed, and

the average was calculated for each mouse. Unless otherwise stated, data represent mean ± standard error of the mean. Statistical analysis was performed by SPSS software (version 17; SPSS, Inc., Chicago, IL). Significance was calculated by the Student t test or, when appropriate, by analysis of variance. Differences were considered significant if P < 0.05. Reverse transcriptase polymerase chain reaction (PCR) was performed on messenger RNA (mRNA) extracted from whole liver and HSCs isolated

from both WT and GFAP-HSV-Tk (Tg) mice. Only Tg samples consistently expressed the HSV-Tk transcript for up to 7 days in primary culture (Supporting Fig. 2D). HSV-Tk expression was absent from both WT and Tg primary hepatocytes (data not shown). In initial studies, we first established a dose-dependent toxicity curve for GCV in established murine cell lines and then applied the same concentrations to primary HSCs isolated from WT and Tg mice. Both immortalized mouse stellate cells (JS1) and MCE公司 hepatocytes (AML12) were incubated with incremental GCV concentrations for 3 days. GCV-mediated toxicity unrelated to HSV-Tk gene expression was analyzed by assessing 3H-thymidine incorporation as well as alamarBlue assay. Cell death was determined by staining with trypan blue and determining the percentage of viable cells. Using this approach, GCV doses higher than 10 μM were toxic in cell lines (Supporting Fig. 2A-C), and subsequent experiments in primary cells therefore used 5 μM of GCV, which avoided nonspecific toxicity. Next, primary HSCs from both WT and Tg mice were cultured for 5 days with GCV (5 and 500 μM) or saline.

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