肝细胞体外培养及其在药物毒理学研究中的应用

目的对肝细胞体外培养及其在毒理学中的应用进行综述。方法查阅相关文献,归纳体外肝细胞模型制作及分类,总结肝细胞模型的应用方向、评价指标。结果与结论肝细胞体外培养方法成熟,在药物毒理学研究中的应用广泛,是未来药物毒理学研究的一大方向。

体外肝细胞三维培养的研究进展

肝细胞体外培养如何能长时间维持肝细胞分化表型及功能活力，是目前生物人工肝研究关键问题之一。体外培养细胞功能改善的一个重要原则就是模拟体内细胞环境。由此，众多研究学者提出体外肝细胞三维培养是解决上述问题的有效途径。近年来，国内外在该领域取得了较大的进展，使体外肝细胞培养功能活力、细胞分化表型维持时间都大大延长。本文就近年来体外肝细胞三维培养的研究进展、存在问题及展望做一简要综述。

基于3D体外培养模型的化学物质肝毒性预测研究进展

精准预测化学物质肝毒性对保护人类生命健康安全具有重要意义。为了避免动物实验固有的物种间差异性和局限性,开发和利用与人源肝脏生理功能直接相关的体外模型至关重要。三维(3D)体外细胞培养模型相比于二维(2D)模型能更好地保留肝细胞代谢功能,再现肝脏内多种细胞相互作用的复杂环境,是体外模拟肝脏生理功能的一大进步,并初步在药物毒性评估方面获得应用的同时,也被引入到环境毒理学领域用于预测环境化学物质的肝毒性。本文介绍了目前常用3D体外细胞培养模型的制备方法,综述了其在环境化学物质(纳米材料、持久性有机污染物和新型有机污染物等)肝毒性预测方面的应用现状,最后探讨了3D肝细胞体外培养模型在有害结局路径指导下开展肝毒性预测的研究与应用前景。

乙型肝炎体外实验模型的方法学研究

目的本研究拟在原有体外模型,如体外鸭原代肝细胞培养模型、大鼠原代肝细胞培养和HBV基因转染人肝癌细胞系-2215细胞培养模型的基础上,对相关实验方法和内容进行改进,以便为防治乙肝药物的应用研究提供比较理想的实验方法。方法根据乙型肝炎的病症和乙肝病毒感染的特点,分别以嗜肝DNA病毒感染鸭的原代肝细胞、HBV基因转染人肝癌细胞系-2215细胞和CCl4熏染大鼠原代肝细胞体外培养试验,观察肝原代细胞培养后的存活状态、DHBVDNA和ALT的水平。结果受染后的鸭、2215细胞和鼠原代肝细胞存活时间较未染细胞明显缩短,DHBVDNA和ALT的水平显著升高。结论改进后的乙肝鸭原代肝细胞培养、CCl4熏染大鼠原代肝细胞培养和人肝癌2215细胞体外培养模型稳定,重复性好,适用于防治乙型肝炎有效药物的筛选和研究。

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepato- cytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxan- induced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/ progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/ progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.

Hepatoprotective evaluation of Anogeissus latifolia:In vitro and in vivo studies

AIM： To evaluate the hepatoprotective activity of a hydroalcoholic extract of the bark of Anogeissus latifolia; in vitro in primary rat hepatocyte monolayer culture and in vivo in the liver of Wistar rats intoxicated by carbon tetrachloride （CCl4）. METHODS： In the in vitro study, a primary hepatocyte monolayer culture was treated with CCh and extract of Anogeissus latifolia. Hepatoprotective activity was demonstrated in the CCh damaged primary monolayer culture. In the in vivo study, the hepatoprotective activity of a hydroalcoholic extract ofAnogeissus latifolia was analyzed in liver injured CCh-treated rats. Biochemical parameters including serum transaminases [aspartate aminotransferase （AST） and alanine aminotransferase （ALT）] and alkaline phosphatase （ALP） in serum wereanalyzed. The biochemical findings were supplemented with histopathological examination of rat liver sections. RESULTS： In vitro： primary hepatocyte monolayer cultures were treated with CCh and extract of Anogeissus latifolia. A protective activity could be demonstrated in the CCh damaged primary monolayer cultUre. In vivo： Hydroalcoholic extract of Anogeissus latifolia （300 mg/kg） was found to have protective activity in rats with CCh-induced liver damage as judged from serum marker enzyme activity. CONCLUSION： The above findings lead to the conclusion that the hydroalcoholic extract of Anogeissus latifol/a is hepatoprotective. Hence, we suggest that the inclusion of this plant in the management of liver disorders is justified.

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

In vitro incubation of cytokine-induced killer cells from patients with and without hepatitis B virus and a cell subset analysis

Objective The aim of the study was to explore the difference between immune cell subsets during the incubation of cytokine-induced kill cells （CIKs） from patients with and without hepatitis B virus （HBV）. Methods Peripheral blood samples were extracted from 50 tumor patients, and were divided into two groups according to the presence or absence of HBV. The proliferation rate and activity of CIK cells were examined based on counts on days 1, 5, 7, 9, 11, 13, and 15 of culture. Additionally, the CD3＋, CD4＋, CD8＋, CD3＋CD8＋, C＋）3＋CD4＋, and CD3＋CD56＋ T cell populations were analyzed by flow cytometry on days 5, 7, 10, 13, and 15 of culture. Results Proliferation over a 15-day period was higher in the HBV-positive group than in the negative group （280-fold vs. 180-fold increase, respectively）, but there was no significant difference between the two groups at each time point. The frequencies of CD3＋, CD8＋ T, CD3＋CD8＋, and CD3＋CD56＋T cells increased over time, while those of CD4＋ and CD3＋CD4＋ T cells decreased over time, and these changes were greater in the positive group than in the negative group. The differences in CD8＋ T cells and CD3＋CD4＋ T cells between the two groups were significant （P 〈 0.05）. Conclusion The proliferative capacity of CIK cells was higher for patients in the HBV-positive group than those in the HBV-negative group, and immune cell subsets were more favorable in the HBV-positive group than the neaative arouD.