一种同时分离培养大鼠肝细胞及肝枯否细胞技术的建立

Establishment of a Technique to Isolate and Culture of Rat Hepatocytes and Kupffer Cells Simultaneously

目的:建立简便、经济的同步分离培养肝细胞及kupffer细胞的方法。方法:采用肝脏原位灌洗结合离体胶原酶灌注消化的方法获得总细胞悬液,差速离心分离肝细胞及肝非实质细胞,经多次低速离心可分离肝细胞,经percoll密度梯度离心以及选择性贴壁法得到纯化的kupffer细胞。台盼蓝染色鉴定细胞活力。使用倒置相差显微镜、HE染色、PAS染色及白蛋白免疫组织化学染色对培养肝细胞的形态及功能进行检测。使用光学显微镜、荧光显微镜及CD68免疫荧光染色鉴定分离的kupffer细胞。结果:体外成功的同步分离培养了肝细胞及kupffer细胞,肝细胞产率为1.37±0.53×108/大鼠,kupffer得率为3.45±0.41×106/g肝脏。细胞存活率及纯度都可达90%。肝细胞培养24 h后呈典型肝细胞形态,7天后仍具有糖原合成和白蛋白合成能力。贴壁后的kupffer细胞呈典型的星型或三角形,且其标志分子CD68免疫荧光染色阳性。结论:应用改良的原位灌注方法可以很好的同时分离具有活性及功能的肝细胞和kupffer细胞。

Objective： To establish a simple and reliable technique to isolate and culture the rat hepatocytes and kupffer cells simultaneously.Methods： To obtain liver cell suspension,the liver in vivo was perfused at first,then the liver with collagenase in vitro were perfused and digested.The filtrated cell suspension was separated into liver parenchymal and non-parenchymal cells using differential centrifugation.Then hepatocytes were harvested by repeatedly low speed centrifugation,kupffer cells were purified by density gradient centrifugation with percoll and further enriched by selective attachment.The viability of these cells was determined by Typan blue exclusion staining.Inverted phase contrast microscope,HE staining,periodic acid-schiff（PAS） staining and immunohistochemical staining of albumin were used to examine the morphological characteristics and function of the hepatocytes.Kupffer cells were identificated by light microscope,fluorescent microscope and immunofluorescence staining of CD68.Results： Hepatocytes and kupffer cells were isolated synchronously and cultured successfully in vitro.The yield of hepatocytes was about 1.37±0.53×108 per rat and 3.45±0.41×106 kupffer cells were harvested per gram liver.The viability and purity of these cells were 90%.Hepatocytes presented its typical cell morphology after 24 hours of cultivation and still remained functions of glycogen and albumin synthesis after day 7.The isolated kupffer cells showed polymorphism with typical polygon-like and star-like shapes and exhibited positive result of CD68.Conclusion： Hepatocytes and kupffer cells with high viability and function can be separated by the modified perfusion technique.