Tranchart Hadrien, Gaillard Martin, Diop Papa Saloum, Goulinet Sylvie, Lainas Panagiotis, Dagher Ibrahim
INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France.
INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France.
J Surg Res. 2018 Apr;224:23-32. doi: 10.1016/j.jss.2017.11.060. Epub 2017 Dec 22.
Hepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia.
Our autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure.
Three rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor.
Autotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.
肝细胞移植是治疗遗传性代谢性肝病的一种潜在侵入性较小的肝移植替代方法。我们开发了一种体外基因修饰肝细胞的自体移植方案，该方案结合了慢病毒转导和部分门静脉栓塞预处理肝脏后的移植。我们在家族性高胆固醇血症动物模型渡边兔中研究了这种方法的代谢效率。
我们的自体移植实验方案用于两组兔子（n = 10），分别为实验组和假手术组，分别接受转导的和对照肝细胞。使用重组慢病毒转导从左肝叶分离的肝细胞。在荧光透视控制下栓塞中叶门静脉分支。通过低密度脂蛋白（LDL）内化试验评估LDL受体表达的功能测量。监测胆固醇水平变化。术后20周处死兔子。
每组三只兔子在肝细胞移植后数小时死亡；尸检显示每组两只兔子出现门静脉血栓形成。因此，该方案进行了修改，通过脾内注射移植肝细胞。慢病毒肝细胞转导效率为64.5%。荧光显微镜显示转导肝细胞的Dil-LDL内化。每组七只兔子进行脂质分析。自体移植后四周，实验组总胆固醇水平中位数下降，但未达到统计学意义（8.9[8.0 - 9.8]g/L对6.3[0.5 - 8.3]；P = 0.171）。在实验组中，酶联免疫吸附测定检测到针对人低密度脂蛋白受体的显著抗体表达。
自体移植方案使渡边兔的脂质谱有了非统计学意义的改善。需要进行更多动物的进一步实验来证实或反驳我们的发现。
