大鼠肝卵圆细胞的增殖模型建立和体外分离的实验研究

发布时间：2018-08-29 16:45

【摘要】：肝干细胞在理论研究和临床应用都有重要意义。一方面，，肝干细胞可能参与肝脏严重损伤后的再生与修复，因此，肝干细胞的增殖与分化及其机理的研究，有助于阐明肝脏的发育机制。另一方面，肝干细胞具有强大的增殖能力，并能分化为肝细胞，这将为肝病的细胞移植、组织工程和基因治疗带来新的希望。肝组织是否存在肝干细胞的问题一直在争论，由于肝干细胞缺乏特异性表面标记物，给肝干细胞的分离和鉴定带来很大困难。成年肝组织的肝干细胞数量很少，且处于休眠期，要获取足量且存活率高的肝干细胞，必须建立良好的激活模型。 目的 建立大鼠肝卵圆细胞增殖模型，进一步进行肝干细胞的分离、纯化和鉴定。 方法 选择81只体重200±20g的雄性Wistar大鼠，随机分为对照组和模型组。采用2-AAF+2／3肝切除方法(2-acetylaminofluorene+two thirds partial hepatectomy，2-AAF+2／3PH)建立肝卵圆细胞增殖模型。使用不同剂量的2—AAF通过胃管灌喂，5天后行肝2／3切除，术后第二天各组按各自剂量继续灌喂，并于术后第4h、4d、8d、12d、16d不同的时间点取肝组织，光镜及电镜下观察肝干细胞增殖情况，并行免疫组织化学染色。利用胶原酶原位灌注及Percoll密度梯度离心分离大鼠肝卵圆细胞，并把分离细胞置于37℃5％ CO_2孵箱中培养，细胞在倒置显微镜、电镜下观察细胞特点，免疫组织化学染色鉴定肝卵
[Abstract]:Liver stem cells are of great significance in both theoretical research and clinical application. On the one hand, liver stem cells may be involved in the regeneration and repair of the liver after severe injury. Therefore, the study of the proliferation and differentiation of hepatic stem cells and its mechanism will help to clarify the mechanism of liver development. On the other hand, liver stem cells have the ability to proliferate and differentiate into hepatocytes, which will bring new hope for cell transplantation, tissue engineering and gene therapy of liver disease. The question of whether liver stem cells exist in liver tissue has always been debated. Due to the lack of specific surface markers of liver stem cells, it is difficult to isolate and identify liver stem cells. The number of liver stem cells in adult liver tissue is very small and it is in dormancy stage. To obtain enough liver stem cells with high survival rate, a good activation model must be established. Objective to establish rat liver oval cell proliferation model and further isolate, purify and identify hepatic stem cells. Methods 81 male Wistar rats weighing 200 卤20 g were randomly divided into control group and model group. Liver oval cell proliferation model was established by 2-AAF 2 / 3 hepatectomy (2-acetylaminofluorene two thirds partial hepatectomy,2-AAF 2/3PH). After 5 days of intragastric administration of 2-AAF, 2 / 3 hepatectomy was performed in each group, and the liver tissues were taken at different time points at 4 h, 4 d, 8 d, 12 d and 16 d after operation. The proliferation of hepatic stem cells was observed under light microscope and electron microscope, and immunohistochemical staining was used. Rat hepatic oval cells were isolated by in situ perfusion of collagenase and Percoll density gradient centrifugation. The isolated cells were cultured in 5% CO_2 incubator at 37 鈩

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