Efficient amplifying and culturing method for biliary epithelial cells

Abstract

The invention discloses an efficient amplifying and culturing method for biliary epithelial cells in rabbit livers and belongs to the technical field of biological medicines. The efficient amplifying and culturing method comprises the following steps of: (1) separating, purifying and culturing the epithelial cells; (2) detecting cell growth lines; and (3) identifying the epithelial cells. The efficient amplifying and culturing method for the biliary epithelial cells, disclosed by the invention, has the advantages as follows: through a multi-step separating method, biliary epithelial cells with higher purities are obtained and a long-time multiplication culturing system for the biliary epithelial cells is established; a fact that the multiplication of the biliary epithelial cells in the livers can be obviously promoted through an Rho kinase inhibitor Y-27632 is first found; and the efficient amplifying and culturing method for the biliary epithelial cells, disclosed by the invention, has the advantages of simpler operation and the like, is suitable for promotion and application and can provide a platform for researches on a plurality of disease mechanisms including biliary atresia, primary biliary cirrhosis and the like.

Description

technical field [0001] The invention relates to a high-efficiency expansion and culture method of rabbit intrahepatic bile duct epithelial cells, belonging to the technical field of biomedicine. Background technique [0002] Abnormal pathology of biliary epithelial cells can lead to a variety of diseases including biliary atresia and primary biliary cirrhosis. One of the ideal methods to study the pathogenesis of these diseases is to establish an expandable culture system of biliary epithelial cells in vitro. Using different separation and purification methods, researchers have successively isolated and cultured bile duct epithelial cells from human and rodent bile duct tissues. So far, there has been no research report on the isolation and culture of rabbit bile duct epithelial cells. Rabbit is a commonly used experimental animal, and it is economical and convenient to obtain materials. The physiological and biochemical indicators of rabbit liver have many similar character...

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Problems solved by technology

[0003] At present, the successful culture methods of normal intrahepatic bile duct epithelial cells reported at home and abroad mainly include tissue block culture, density gradient centrifugation and cell sorting, etc., but these methods have the disadvantages of low cell purity and the primary bile duct epithelial cells cultured in vitro The proliferative ability is weak, and long-term expandable culture cannot be carried out, and the research materials often need to involve the whole liver tissue, and the liver tissue is consumed. For example, cell sorting is an effective method to obtain high-purity bile duct epithelial cells, but Cell sorting requires special sorting equipment such as flow cytometry, and the single cells obtained by sorting are not very suitable for the growth of bile duct epithelial cells, and there is currently a lack of specific surface markers for bile duct epithelial cells. The development and application of sorting technology; density gradient centrifugation can purify bile duct epithelial cells to a certain extent, but it is also inevitable to mix other types of cells such as hepatic stellate cells and fibroblast-like cells, so the cell purity is not high; tissue block The advantage of the culture method is that the biological characteristics of the bile duct epithelial cells in the body are preserved to the greatest extent, so it also has good growth activity, but this method can also promote the mixed growth of fibroblasts, hepatocytes and bile duct epithelial cells. low; there are very few normal intrahepatic bile duct epithelial cell lines available at home and abroad, their properties are unstable, and they are prone to malignant transformation; although there have been some research reports on the isolation and culture of bile duct epithelial cells, the research results show that most of the existing The culture system cannot maintain the proliferation of cells for a long time, even with the addition of cytokines such as EGF and HGF, it can only prolong the survival time of cells appropriately, and the effect is not very significant

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