Basic research on xenotransplantation u sing genetic engineering and its future application in clinic meidicine

异种器官移植被认为是解决供体器官短缺的一种有前途的替代方法。本研究旨在为异种器官移植的临床应用提供基础知识。我们研究了(1)超急性排斥反应和(2)细胞反应的机制，包括延迟排斥反应和免疫耐受，并尝试了成功的异种移植的新策略。结果如下：(1)对大鼠的Crry基因和豚鼠的DAF基因进行了分析。通过将受体的补体抑制蛋白遗传插入到供体器官中，可以抑制补体的激活。对α 1,3半乳糖基转移酶（α 1,3 GT）在小鼠和猪体内的主要异种抗原α 1,3半乳糖基（α Gal）抗原进行了遗传分析。我们现在正在生产具有α 1,3 GT功能的敲除小鼠……更多的是在猪中敲除的靶向载体。由于我们也阐明了猪α 1,3 GT的几个剪接变体，我们现在正在研究这些变体之间的功能差异。由于α 1,2 focusyl transferase （α 1,2 FT）具有竞争性抑制α 1,3 GT的功能，因此转染α 1,2 FT基因后，猪培养细胞中α Gal抗原的表达降低，我们正在建立α 1,2 FT转基因猪。然而，由于α 1,2 FT基因转染降低了内皮细胞唾液酰化，抑制了管的形成能力，因此需要进一步的实验来修饰细胞表面的碳水化合物抗原。在豚鼠与大鼠的联合实验中，凝血和补体系统的抑制被证明是抑制超急性排斥反应的重要机制。(2)通过抑制供体抗原呈递细胞上的粘附分子获得供体特异性免疫耐受。胰岛微囊移植皮下引导大网膜的方法是可行的。确定了腺病毒载体转染供体器官的最佳条件。同时移植骨髓细胞或逆转录病毒载体移植供体MHC基因有利于诱导免疫耐受。少

Xenotransplantation is considered to be promising as an alternative method to solve the shortage of donor organs. The purpose of this research is to get the basic knowledge for clinical application of xenotransplantation. We investigated the mechanisms of (1) hyperacute rejection and (2) cellular response including delayd rejection and immunological tolerance, and attempted the new strategy for successful xenotransplantation. The obtained results are as follows,(1) Crry gene in rats and decay accelerating factor (DAF) gene in guinea pigs were analyzed. The experimental model, which shows that complement activation can be suppressed when complement inhibitory proteins of recipient are genetically inserted into donor organ, was established. Genetic analysis of alpha 1,3 galactosyl transferase (alpha 1,3 GT), which forms main xenoantigens, namely alpha 1,3 galactosyl (alpha Gal) antigens, wes performed in mice and pigs. We are now producing knock out mice of alpha 1,3 GT function and cons … More tructing a targeting vector for knock out in pigs. Since we also elucidated several splicing variants in porcine alpha 1,3 GT,we are now examining the functional difference between these variants. As gene transfection of alpha 1,2 fucosyl transferase (alpha 1,2 FT), which is capable of competitive inhibition of alpha 1,3 GT,caused the decreased expression of alpha Gal antigens in pig cultured cells, we are establishing alpha 1,2 FT transgenic pigs. However, as decreased sialylation in cultured endothelial cells by alpha 1,2 FT gene transfer inhibited the capacity of tube formation, further experiment will be necessary for modification of carbohydrate antigens in cell surface. In guinea pig to rat combination, inhibition of coagulation as well as complement system was proved to be important to suppress hyperacute rejection.(2) Donor specific immunological tolerance was found to be acquired by inhibition of adhesion molecules on donor antigen presenting cells in rat to mouse combination.The method of microcapsulated islets transplantation into subcutaneously guided greater omentum was proved to be useful. The optimal condition of adenovirus vector for gene transfer to donor organs was determined. The simultaneous transplantation of bone marrow cells or gene transfer of donor MHC using retrovirus vector was beneficial for induction of immunological tolerance. Less