Modification of donor MHC antigens with ribozyme RNA transfer to prevent rejection of graft liver

在我们的第一项研究中，我们检测了将核酶RNA插入同种异体移植物中是否可以下调目标抗原的表达并防止移植器官的排斥反应。然而，目前，即使在体外，也未观察到核酶RNA插入供体血管内皮细胞后供体MHC I类抗原表达的显著下调。因此，对这些细胞的免疫反应没有明显降低。可能是核酶RNA的结构不适合这些靶抗原。现在我们正在开发新的核糖酶RNA，以提供有效的基因治疗。在我们的第二项研究中，我们研究了70%肝切除术后大鼠再生肝组织和离体肝细胞中血管生成素(Ang)-1、Ang-2、Tie-2和血管内皮生长因子（VEGF）的表达。分别用增殖细胞核抗原（PCNA）和TUNEL染色评价窦状内皮细胞（SECs）的增殖和凋亡情况。结果：VEGF rRNA表达升高，在肝切除术后72 h达到峰值，随后逐渐降低。肝切除术前，血管内皮生长因子（VEGF） mRNA水平可检测到，并在96 h时缓慢升高，达到峰值。同时，血管内皮生长因子（VEGF） mRNA在肝切除术前几乎未检测到，但在120 h时显著升高。在离体细胞中，VEGF mRNA仅在肝细胞中表达，而血管内皮生长因子（VEGF） mRNA在窦状细胞中表达，主要在肝星状细胞（hsc）中表达。SECs的PCNA标记指数缓慢升高，在72 h时达到峰值，而在120 h时检测到凋亡的SECs。结论：肝细胞分泌的VEGF可能在SEC增殖中起关键作用，其次是Ang-1升高，可能促进了窦状动脉成熟。因此，在缺乏VEGF的情况下，主要从hsc中释放的Ang-2可能会导致多余的SECs凋亡，从而停止再生过程。

In our first study, we examined whether insertion of ribozyme RNA into the allograft could down-regulate the expression of the target antigens and prevent the rejection of transplanted organs. However, at present, significant down-regulation of donor MHC class I antigen expression on the donor vascular endothelial cells was not observed after insertion of ribozyme RNA even in vitro. Therefore, immunological response to these cells was not significantly reduced. Probably, structure of ribozyme RNA was not suitable for these target antigens. Now we are developing new ribozyme RNA to provide for efficient gene therapy.In our second study, we investigated the expression of angiopoietin (Ang)-1, Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in both regenerating liver tissue and isolated liver cells after 70% hepatectomy in rats. Proliferation and apoptosis of sinusoidal endothelial cells (SECs) were also evaluated with proliferating cell nuclear antigen (PCNA) and TUNEL staining, respectively. Results : Expression of VEGF rRNA increased, with a peak at 72 h after hepatectomy, decreasing thereafter. Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but increased remarkably at 120 h. In isolated cells, VEGF mRNA expression was limited to hepatocytes, but Ang-2 mRNA was found in sinusoidal cells, mainly in hepatic stellate cells (HSCs). The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected at 120 h. Conclusions : VEGF secreted by hepatocytes may play a key role in SEC proliferation, followed by increased Ang-1, presumably promoting sinusoidal maturation. Thereafter, Ang-2 mainly released from HSCs in the absence of VEGF may contribute to apoptosis of superfluous SECs for cessation of regenerative process.