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

异种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。本研究的目的是为异种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(包括延迟排斥反应和免疫耐受)的机制，并尝试了成功异种移植的新策略。主要结果如下：(1)对大鼠CRY基因和豚鼠衰变加速因子(DAF)基因进行了分析。建立了将受体补体抑制蛋白基因插入供体器官可抑制补体活化的实验模型。对形成主要异种抗原的α-1，3-半乳糖基转移酶(α-1，3-GT)的遗传分析，即α-1，3-半乳糖(α-Gal)抗原，在小鼠和猪身上进行。我们现在正在生产具有α1，3 GT功能和CONS…的基因敲除小鼠更多的是为猪的基因敲除提供靶向载体。由于我们还阐明了猪α1，3 GT中的几个剪接变体，我们现在正在研究这些变体之间的功能差异。由于α-1，2-岩藻糖基转移酶(α-1，2-FT)能竞争性抑制α-1，3-GT，导致猪培养细胞中α-Gal抗原表达下降，因此我们建立了α-1，2-FT转基因猪。然而，由于α-1，2-FT基因转移降低了培养内皮细胞的唾液酸化，抑制了管状细胞的形成能力，因此还需要进一步的实验来修饰细胞表面的糖类抗原。在豚鼠到大鼠的联合移植中，抑制凝血和补体系统是抑制超急性排斥反应的重要因素。(2)在大鼠到小鼠的联合移植中，通过抑制供体抗原提呈细胞上的黏附分子，获得了供体特异性免疫耐受。确定了腺病毒载体向供体器官转导基因的最佳条件。同时进行骨髓细胞移植或逆转录病毒载体介导的供体MHC基因转移有利于诱导免疫耐受。较少

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