改进原位循环灌流法分离小鼠肝细胞研究

Improved in situcirculatory perfusion method of separating mice hepatocytes

【目的】建立小鼠肝细胞分离原位循环灌流方法,为肝脏细胞蛋白质组研究提供技术技持。【方法】参考Seglen胶原酶两步原位灌流法,对其进行改进,建立原位循环灌流分离小鼠肝细胞方法,对小鼠肝脏进行原位循环灌流,离体消化肝脏组织,差速离心获得纯化小鼠肝细胞;台盼蓝拒染试验检测肝细胞活性,常规血球计数法计算肝细胞得率;利用显微形态观察、HE、PAS及免疫细胞化学等检测方法鉴定所获得的肝细胞纯度;用含体积分数20%FBS的RPMI-1640细胞营养液对原代小鼠肝细胞进行培养。【结果】建立了原位循环灌流分离小鼠肝细胞的方法,采用该方法可从每只小鼠肝脏中成功分选到细胞活性大于90%、纯度在95%以上的(4.2×107)～(6.5×107)个肝细胞。原代分离的小鼠肝细胞培养4h后,大多数细胞可贴壁,12h后贴壁完全,细胞呈伸张状态,该原代肝细胞可持续培养7d以上,且细胞形态状况良好。【结论】成功建立了小鼠肝脏原位循环灌流试验体系,获得了纯度和活性均较高的小鼠肝细胞,分选所得的小鼠肝细胞完全满足构建肝细胞蛋白质组表达谱试验的需要。

【Objective】 The study was to establish in situ circulation perfusion method of separation mice hepatocytes,providing technical support for liver cell preteomics research.【Method】 By referencing and improving Seglen＇s two-steps in situ circulatory perfusion method,mice liver in situ perfusion method was established.After perfusion,the liver was cut and digested in vitro.Then the hepatocytes were purified by differential centrifugal methods.Trypan blue dye test was taken to detect hepatocytes activity,and the yield of hepatocytes with routine blood cell was calculated by counting method;The purity of hepatocytes was detected by microscopic morphology,HE,PAS and ICH tests.Purified hepatocytes was cultured in RPMI-1640 nutrients containing 20% FBS.【Result】 An improved in situ circulatory perfusion method of separation mice hepatocytes was established.Taking this method,4.2×107-6.5×107cells was obtained with above 95% cells purity and 90% cell viability from per mice liver.After cultured for 4 hours,most of hepatocytes sticked to the plate wall and were in the state of stretching in 12 hours culturing.Harvested hepatocytes can be cultivated continuously for about one week.【Conclusion】 Mice liver in situ circulation perfusion system was successfully established.Enough mice hepatocytes with high cells purity and cell activity were harvested.These hepatocytes can completely meet proteomics research.