猪肝细胞异种移植过程中移植肝细胞凋亡

The apoptosis of transplanted hepatocytes during porcine hepatocytes xenotransplantation

目的探讨猪肝细胞异种移植后移植肝细胞凋亡情况。方法采用原位胶原酶循环灌注法分离中国实验用小型猪肝细胞。D氨基半乳糖腹腔内注射制作大鼠急性肝衰竭模型，48h时将培养24h的猪肝细胞悬液2ml（含5．0×10^7个肝细胞）移植到急性肝衰竭大鼠腹腔内，观察移植后ALF大鼠2周存活率和肝功能变化；收集移植后不同时段的移植肝细胞观察其病理变化，应用Annexin V-FITC凋亡检测试剂盒结合流式细胞仪检测移植肝细胞的凋亡率、坏死率及存活率。结果2周存活率√UF大鼠为18．75％，移植组为56．25％（P〈0．05）。注药后48h肝功能明显恶化，移植后肝功能明显改善。移植肝细胞在腹腔内存活时间约3～5d。肝细胞体外培养24h已有凋亡出现。与培养24h时相比，移植后24h，移植肝细胞凋亡率明显上升，活率明显下降（P〈0．05），坏死率无明显上升（P〉0．05）；与移植后24h相比，移植后48h，移植肝细胞凋亡率进一步上升，活率进一步下降（P均〈0．05），坏死率仍没有明显变化（P〉0．05）；移植后72h，移植肝细胞摄亡率明显下降，与体外培养24h相比，差异无显著性（P〉0.05），与移植48h相比，移植肝细胞活率无明显变化（P〉0．05），移植肝细胞坏死率明显上升（P〈0.05）。结论猪肝细胞异种腹腔内移植时，移植肝细胞仅能存活3～5d，早期免疫排斥以肝细胞凋亡为主，3d后则以肝细胞坏死为主。

Objective To evaluate the apoptosis of transplanted hepatocytes during porcine hepatocytes intraperitoneal xenotransplantation. Methods Recirculating collagenase perfusion method was used to isolate hepatocytes of Chinese experimentmini-pigs and the isolated porcine hepocytes suspensions were cultured for 24 h. 2 ml free porcine hepatocytes suspensions （ 5. 0 ×10^7 cells） were transplanted into abdominal cavity of SD rats at 48 h after D-gal introperitonear injection （1.2 g/kg）. The two-week survival rate and the hepatic functions were examined. The pathological changes of transplanted hepatocytes were observed. The rate of apoptosis, viability and necrosis of the transplanted hepatocytes was measured by Annexin V-FITC apoptosis detecting kit and flow cytometry. Results There was a significant difference in two-week survival rate between acute liver failure（ALF） group （56.25%） and transplantation group （18. 75%） （P〈0. 05）. The hepatic functions were damaged significantly and recovered after hepatocyte transplantation. The survival time of the transplanted porcine hepatocytes was from 3 to 5 days after D-gal injection. The apoptosis of hepatocytes occurred after culture for 24 h in vitro. Compared with culture of 24 h, the apoptosis rate of the transplanted hepatocytes increased and the viability of the transplanted hepatocytes decreased significantly （P〈 0. 05）. The necrosis rate of the transplanted hepatocytes changed unsignificantly （P〈0. 05）. Compared with transplantation for 24 h, the rate of apoptosis increased and the viability of the transplanted hepatocytes decreased continuously at 48 h after transplantation （P〈0.05）. The necrosis rate of the transplanted hepatocytes changed unsignificantly （P〈0. 05）. The apoptosis rate of the transplanted hepatocytes decreased at 72 h after transplantation. Compared with transplantation for 48 h, the viability of the transplanted hepatocytes changed unsignificantly （P〈0. 05） and the necrosis rate of the transplanted hepatocytes increased significantly （P〈0. 05）. Conclusion The transplanted hepa- tocytes can only survive for 3 to 5 days by using free porcine hepatocytes intraperitoneal xenotransplantation. The apoptosis is the main way in early immune rejection of the transplanted hepatocytes, and necrosis three days later.