TNF-α a antisense transfer improves ischemia / reperfusion injury of the hepatic graft

移植肝冷缺血/再灌注损伤是肝移植中尚未解决的问题，其原因是激活的枯否细胞释放炎性细胞因子。我们设计了一种新型的脂质体-日本血凝病毒（HVJ）-阳离子脂质体（HVJ-CL）载体系统，并利用该载体系统，研究了异硫氰酸荧光素（FITC）标记的寡脱氧核苷酸（FITC-ODNs）对枯否细胞的细胞毒性、转染效率以及TNF-α反义核酸对移植肝缺血/再灌注损伤的影响。FITC-ODNs转染后4 h，移植肝中95%以上的ED 2阳性枯否细胞可识别FITC信号。使用以常规HVJ-脂质体（HVJ-L）的HVJ和脂质的量的八分之一施加的HVJ-CL的转移效率（18.5 ± 3.1%）远高于HVJ-L实现的转移效率（10.4 ± 1.2%，p<0.01），而在HVJ-CL和HVJ-L组之间，载体诱导的细胞溶质酶从肝细胞释放到血液循环中没有显著差异。TNF-α反义基因转染可显著抑制肝移植后4 h肝细胞胞浆酶的释放、血清TNF-α水平和枯否细胞TNF-α的免疫组化表达。HVJ-CL法是一种安全有效的将ODNs导入大鼠移植肝Kupffer细胞的方法

Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantation that has been attributed to the release of inflammatory cytokines from activated Kupffer cells. We recently designed a novel vehicle composed of cationic liposomes fused with hemagglutinating virus of Japan (HVJ), namely HVJ-cationic liposomes (HVJ-CL), and using this vector system, we assessed the cytotoxicity, efficiency of Fluorescein isothiocyanate (FITC)-labeled oligodeoxynucleotides (FITC-ODNs) delivery to Kupffer cells and effects of TNF-α antisense delivery against ischemia/reperfusion injury in a rodent hepatic graft. FITC signals were recognized in over 95% of ED2-positive Kupffer cells of transplanted liver 4hrs after FITC-ODNs transfer. The transfer efficiency (18.5 ± 3.1%) using HVJ-CL applied at one-eighth the amount of HVJ and lipids of conventional HVJ-liposomes (HVJ-L) was much higher than that achieved by HVJ-L (10.4 ± 1.2%, p<0.01), while there was no signincant difference in the vector-induced release of cytosolic enzymes from hepatocytes into the blood circulation between HVJ-CL and HVJ-L groups. Treatment with TNF-α antisense transfer significantly suppressed the release of cytosolic enzymes from hepatocytes, the serum levels of TNF-α and immunohistochemical expression of TNF-α by Kupffer cells 4hrs after liver transplantation. This HVJ-CL method provides a safe and effective ODNs delivery to Kupffer cells of hepatic grafts in rats