大鼠原代肝细胞的标记和移植方法

Method of label and transplant for primary hepatocyte of rat

探讨CM-DiI标记大鼠原代肝细胞的最佳浓度、细胞的移植量和移植途径.采用原位胶原酶灌注和密度梯度离心法分离、纯化大鼠肝细胞,用不同浓度CM-DiI进行标记,筛选出CM-DiI的最佳标记浓度,分别从门静脉、尾静脉移植到2/3肝切除0h大鼠,对细胞移植36h后的肝组织石蜡切片和冰冻切片分别进行免疫组织化学分析和荧光观察.结果显示,4μmol·mL-1浓度的CM-DiI和肝细胞孵育5min,细胞标记率高达95%.每克大鼠残肝移植0.8mL的细胞悬液(107个·mL-1)和0.2,0.4,0.6mL移植后检测到的标记细胞数目相比有显著性差异(P<0.01),经肝门静脉移植后可以检测到的标记细胞数比尾静脉多(P<0.01).CM-DiI标记大鼠原代肝细胞的最佳浓度为4μmol·mL-1,标记细胞的最佳移植量为每克大鼠残肝移植0.8mL的细胞悬液(107个·mL-1)、最佳移植途径为经肝门静脉移植.

The optimum concentration of CM-DiI incubated with primary hepatocyte of rat,optimum transplant volume and route of labeled primary hepatocyte are studied.The rat hepatocytes are isolated and purified by collagenase and density gradient centrifugation,and labeled with different concentration of CM-DiI,and then transplanted on rat which are previously suffered 2/3partial hepatectomy 0hby liver portal vein and tail vein,respectively.The livers are removed after transplanting hepatocyte 36h,frozen section and paraffin section are prepared and analyzed by immunohistochemistry and fluorescence observation,respectively.The results show that hepatocyte can be labeled to 95%after incubating with CM-DiI 4μmol·mL-1 for 5min,labeled hepatocytes which are observed on frozen section transplanted by0.8mL per keilogram remnant liver weight are more than that of 0.2,0.4and 0.6mL（P0.01）,and labeled hepatocytes transplanted by liver portal vein are more than that of tail vein（P 0.01）.Hepatocytes incubated with CM-DiI 4μmol·mL-1 for 5min can be labeled to 95%,and 0.8mL labeled hepatocytes per keilogram remnant liver weight transplanted by liver portal vein can get preferable label and transplantation efficiency.