Objective To establish a proliferating model of hepatic oval cells (HOCs) with adult Wistar rats, isolate and culture in vitro the HOCs, and to approach the possibility of inducing the HOCs differentiated into hepatocytes. Methods Rats were fed with 0.07% wt/wt ethionine. On day 8, 2/3 partial hepatectomy (2/3 PH) was performed. Isolate, harvest and purify HOCs by type Ⅳ collagenase perfusion with semi in situ 2-step method and Percoll density gradient method. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to complete Williams' medium E (WE) to culture the HOCs. HOCs was induced differentiation with HGF, oncostatin m (OSM) and fibroblast growth factor-4 (FGF4) into hepatocytes. Results The concentration of cell after purification was about 1.34?105/ml. Most cells were small, about 1/6~1/3 the size of normal hepatocyte. Ovoid, elliptical or polygonal in shap, the nucleus-cytoplasm ratio was relatively large, and clone-like proliferation appeared 2 weeks later. LSCM revealed positive expression of Thy-1 and C-kit in cytoplasm and membrane of HOCs. ICC showed AFP in cytoplasm of HOCs. Stimulated by inducing system, the shape of HOCs changed gradually. The volume enlarged and cells lost their adherence ability. ICC indicated apparent positive stain of cytoplasm Alb 14 days after differentiated induction, and the positive ratio increased along with the extension of induction duration. Cytochemical tests indicated brown or black sediment with G-6-P staining and red particles with PAS staining, respectively. Conclusion The proliferation model of rats' HOCs was established after ethionine feeding and 2/3 PH. HOCs can be obtained with type IV collagenase perfusion and Percoll density gradient isolation and purification. Clone proliferation can be achieved through culturing HOCs in vitro. Under certain conditions, HOCs can be induced and differentiated into certain typical hepatocyte phenotype.

Objective Identification of colorectal cancer specific antigens from cDNA phage expression library by recombinant expression cloning (SEREX). Methods The cDNA phage expression library derived from colorectal cancer tissue was constructed using the SMART (Switching Mechanism at 5'end of RNA Transcript) techniques. The cDNA phage expression library was screened with SEREX (serological identification of antigen by recombinant cDNA expression libraries), the positive clones encoding antigenic genes were obtained after immunoscreening, and the nucleotide sequences of cDNA inserts were determined and analyzed with BLST software in GenBank, and the antigenic gene function was analyzed by bioinformatics. Results The cDNA phage expression library derived from colorectal cancer tissues was successfully constructed. The primary library consisted of 2.39?10 6 recombinants, and the recombinant rate was more than 97.5%. The titer of the amplified cDNA phage expression library was 4.1?10 10pfu/ml, and the size of inserted cDNA varied from 0.5 to 4.0kb. Sixteen positive clones encoding antigenic genes were obtained after immunoscreening, and results showed that 16 reactive clones were derived from 12 different genes. Ten of 12 genes were highly homologous to the genes known in GenBank, such as IFITM1, CD24, Survivin, KLK6, et al. Two expressed sequence tags (ESTs) were not found in GenBank. The antigenic genes included structural gene, regulation gene and metabolizing gene. Conclusion The tumor-associated antigen genes and the two ESTs were obtained by SEREX were worthy of further study on their structures and functions.

Objective To determine the effects of angiotensin Ⅱ (Ang Ⅱ) on NF-κB DNA binding activity in alveolar macrophage. Method Human alveolar macrophages were isolated and made homogeneous from alveo-lar lavage fluid, and cuhtured in DMEM. Alvcolar macrophages were treated with AugⅡ (10-6M) for 15 min, 30 min, 60 min and 120 min, respectively. Moreover, alveolar macmphages were pretreated with irbesartan (AngⅡ type 1 receptor blocker) for Ⅰ hour before stimulated with Angiotensin Ⅱ for Ⅰ hour. Electrophoretic gel mobility shift assay (EMSA) was used to detect NF-κB DNA binding activity. The protein expression of IκBα was examined by Western blot. Expressions of TNF-α and ICAM-1 mRNA were detected by using RT-PCR. Results EMSA re-vealed that there was a increase in up-regulation of NF-κB DNA binding activity after alveolar macrophages were treated with Ang Ⅱ for 15 rain and peaked at 60 min. Irbesartan treatment reduced DNA binding activity. Com-pared with control group, the protein expression of IκBα decreased in Ang Ⅱ treatment group(0.29±0.11, P= 0.013), and Irbesartan treatment significantly increased protein expression of IκBα(0.83±0.12, P=0.001). The expressions of TNF-α and ICAM-1 mRNA were up-regulated by AngⅡ in comparison with the control group (TNF-α:1.13±0.17 vs. 0.42±0.099; ICAM-1 0.55±0.08 vs. 0.16±0.050, P=0.003). Irbesartan inhibited the expressions of TNF-α (0.77±0.15 vs 1.13±0.17, P=0.02; ICAM-1(0.32±0.07 vs 0.55±0.08, P =0.001). Conclusions Ang Ⅱ is capable to stimulate NF-κB signal pathway in alveolar macrophages.

BACKGROUND: Hepatic oval cells (HOCs) possess the potential of self-renewal, replication, and clone, proliferation and differentiation into mature hepatocytes under a certain condition. HOCs can be used as biomaterial for constructing biological artificial liver in vitro, employed for in vivo transplantation, as well as for tissue engineering as seed cells. HOCs can be widely used for improving clinical treatment of liver diseases. OBJECTIVE: To establish adult Wistar rat models of HOC proliferation, to perform/n vitro isolation and culture of HOCs, and to study the possibility of induction and differentiation of HOCs into hepatucytes. DESIGN: Observational study. SETTING: Institute of Gastroenterology, Nanfang Hospital, Southern Medical University. MATERIALS: Experiments were performed at the Laboratory of Institute of Gastroenterology of Nanfang Hospital from December 2003 to February 2006. Thirty-six healthy male Wistar rats aged 3-4 months (150-200 g) were provided by Experimental Animal Center of Southern Medical University. METHODS: Male Wistar rats were orally fed with ethionine received two-thirds partial hepatectomy (2/3 PH). HOCs were harvested and purified by two-steps perfusion and Percoll density gradient centrifugation, and then cultured in vitro and induced with hepatocyte growth factor (HGF), oncostatin M (OSM) and fibroblast growth factor-4 (FGF4). MAIN OUTCOME MEASURES: Identification and differentiation of HOCs. RESULTS: The concentration of HOCs was about 1.34×108 L-1 in each rat model after in vitro isolation. These cells were round, oval or polygon, about 1/6 1/3 the size of normal hepatocytes. The nucleus-cytoplasm ratio was relatively large. After 2 weeks, clone-like proliferation of HOCs could be observed. Laser scanning confocal microscopy indicated positive expression of stem cells markers Thy-1 and C-kit in cytoplasm and membrane of HOCs. Immunocytochemistry demonstrated positive stem cells marker alpha fetoprotein (AFP) in cytoplasm of HOCs. HOCs can stably passage and its shape gradually changed after inducing with HGF, OSM and FGF4. HOC volume became larger and HOCs lost their ability of sticking to the wall of culture flask. Apparent positive stain of cytoplasm albumin (Alb) was detected 14 days after induction, and the positive ratio increased along with the extension of inducing duration. Results of cytochemistry indicated a brown or black deposit after glucose-6-phosphotase (G-6-P) staining and red particles after periodic acid-Schiff (PAS) staining. CONCLUSION: Adult Wistar rat models of HOC proliferation are replicated by ethionine feeding combined with 2/3 PH. HOCs can be obtained through collagenase perfusion and Percoll density gradient centrifugation. Rat HOCs can be passaged and cultured in vitro. Under a certain condition, HOCs can be induced and differentiated into hepatocytes.

Objective The protective effect of oleanolic acid on acute cholestatic liver injury in rats. Methods Thirty male SD rats were randomly divided into 3 groups ( n= 10 per group): sham group, bile duct ligated (BDL) group, and bile duct ligated with oleanolic acid (BDL+OA ) group. After 7 days, liver samples in all rats were collected. Expressions of bile acids pump and nuclear receptors at mRNA and protein levels were detected by RT- qPCR and Western blotting. Results At mRNA level, the expression of Mrp4 and Oatp1 expression in the BDL and BDL+OA groups were increased as compared with that in the sham group. The expression of Mrp4 increased 1.8 times in the BDL group and increased 2.3 times in the BDL+OA group (P<0.05), but the expression of Oatp1 was not statistically significant; AhR was increased 1.7-fold in the BDL group and 2.8 times in the BDL+OA group, Nrf2 was increased 1.5-fold in the BDL group and 2.1 times in the BDL+OA group with statistically significant difference. At the protein level, in the BDL group, Mrp4 expression increased 1.3 times, Oatp1 expression increased 1.5 times, AhR expression increased 1.3 times, Nrf2 expression increased 1.4 fold; in the BDL+OA group, Mrp4 expression increased 1.8 fold, AhR expression increased 1.9 fold, with statistical significance between the two groups. Oatp1 expression increased 1.4-fold in the BDL+OA group as compared with the BDL group showing no statistical significance. Conclusions Oleanolic acid stimulates the hepatic expression of bile acids pump Mrp4 associated with the activation of nuclear receptors AhR and Nrf2 in acute bile ductligated rats.