树突状细胞在大鼠再生肝中分布及数量变化

Distribution and Quantity Changes of Dentritic Cells in Rat Regenerating Liver

探讨树突状细胞在大鼠再生肝中分布及数量变化,为深入研究其在肝再生进程中的作用奠定基础。制作大鼠2/3肝切除模型(PH),用两步灌流法分散肝脏细胞,用Percoll密度梯度离心和ox62免疫磁珠分离相结合方法分离树突状细胞(DC),用免疫组织化学方法定性、定位CD86和CD103在再生肝(RL)、分散的肝脏细胞及分离的树突状细胞中分布,用蛋白免疫印迹方法定量树突状细胞的CD86和CD103,用RT-PCR定量分离的树突状细胞CD86和CD103的mRNA。结果表明,0h再生肝树突状细胞分布于肝血窦、中央静脉、胆管周围,12h在远离中央静脉和胆管的部位出现,24h呈弥散状分布,168h分布与对照相同;从肝切除后0、2、6、12、24、30、36、72、120、168h等10个时间点的大鼠再生肝中收获的树突状细胞平均数分别为每只大鼠1.55、1.59、1.87、2.46、1.49、2.73、3.87、6.04、6.52、8.40百万个,CD86和CD103阳性细胞数目、细胞活性均在95%以上。上述结果说明在肝再生进程中树突状细胞的分布由肝血窦、中央静脉、胆管周围逐渐向全肝弥散,24h弥散达到高峰,之后回落,至168h分布与对照相同,但其数量随肝再生进程而增加。

To explore the distribution and quantity changes of dentritic cells in rat regenerating liver and lay the foundation for researching their roles deeply in the process of regenerating liver, we make rat 2/3 hepateetomy model according to the method of Higgins et al, scatter liver cells by two-step perfusion, and isolate and purify dendritic cells by the way of percoll density gradient centrifugation and ox62 immunomagnetic beads. Immunocytochemistry method is used to qualitify and localize CD86 and CD103 cells in hepatic tissue, disperse hepatic cells and purify dendritic cells. CD86 and CD103 are quantified by Western blot while their mRNA levels are quantified by RT-PCR. The results show that dendritic ceils distribute in the hepatic sinusoids and round central veins and bile ducts at 0 h, far from central veins and bile ducts at 12 h. At 24 h they diffuse in the whole liver. Their distribution at 168 h is similar to that at 0 h. The average numbers of dendritic ceils are obtained from rat regenerating liver at 0, 2, 6, 12, 24, 30, 36, 72, 120 and 168 h post partial hepatectomy are 1.55×10^6, 1.59×10^6 , 1.87 ×10^6, 2.46 ×10^6, 1.49 ×10^6, 2.73 ×10^6, 3.87 ×10^6, 6.04 ×10^6, 6. 52 ×10^6 and 8.40 ×10^6 respectively. The percentage of CD86 and CD103-postive cells and viability of dendritic cells are both more than 95%. These results indicate that the distribution of dendritic cells gradually disperse from the hepatic sinusoids and regions round central veins and bile ducts to the whole liver. They reach peak at 24 h, and then fall back to the same level as 0 h at 168 h. The number of purified dendritic cells increase in a time-dependent manner.