Schmidt Peter, Goto Masafumi, Le Mauff Brigitte, Anegon Ignacio, Korsgren Olle
Department of Clinical Immunology, Uppsala University, Uppsala, Sweden.
Transplantation. 2003 Mar 15;75(5):697-702. doi: 10.1097/01.TP.0000053249.39753.D6.
Protection against complement activation may reduce acute islet damage in pig-to-human islet xenotransplantation. Expression of the human complement regulatory proteins decay-accelerating factor (DAF, CD55) or CD59 was induced on intact adult porcine islets (APIs) by adenoviral transduction. The functional capacity of the transgenes was examined in vitro after exposure to fresh human serum.
Intact APIs were transduced with adenoviral vectors Ad.hDAF or Ad.hCD59 or a control vector. After 3 days, the islets were trypsin dissociated to a single-cell suspension. A cytotoxicity assay was performed in which the islet cells were incubated with human complement active AB serum. Flow cytometry and immunohistochemistry were used to evaluate transgene expression.
APIs could be transduced to express hDAF or hCD59. Flow cytometry analysis of islet single cells revealed that only a fraction of the cells expressed the transgene; immunohistochemical staining of transduced islets demonstrated that mainly cells located in the periphery of the islets were expressing the protein. Cells from nontransduced islets or islets expressing the control protein were sensitive to lysis in human sera (66+/-4.0% and 73+/-3.7% cytotoxicity, respectively). Single cells from islets transduced with hDAF and hCD59 were partially protected from lysis. Islet cells expressing hCD59 were slightly less sensitive to lysis (33+/-3.3%) than cells expressing hDAF (45+/-3.5%).
These data show that intact pig islets can be transduced to express human regulators of complement activation on the surface and that pig islet cells expressing hDAF or hCD59 are less sensitive to complement-mediated lysis.
防止补体激活可能会减少猪到人胰岛异种移植中的急性胰岛损伤。通过腺病毒转导在完整的成年猪胰岛（API）上诱导人补体调节蛋白衰变加速因子（DAF，CD55）或CD59的表达。在体外暴露于新鲜人血清后检测转基因的功能能力。
用腺病毒载体Ad.hDAF或Ad.hCD59或对照载体转导完整的API。3天后，将胰岛用胰蛋白酶解离成单细胞悬液。进行细胞毒性试验，将胰岛细胞与人补体活性AB血清一起孵育。采用流式细胞术和免疫组织化学评估转基因表达。
API可被转导以表达hDAF或hCD59。胰岛单细胞的流式细胞术分析显示只有一部分细胞表达转基因；转导胰岛的免疫组织化学染色表明主要是位于胰岛周边的细胞表达该蛋白。来自未转导胰岛或表达对照蛋白的胰岛的细胞对人血清中的裂解敏感（细胞毒性分别为66±4.0%和73±3.7%）。用hDAF和hCD59转导的胰岛单细胞受到部分保护而免于裂解。表达hCD59的胰岛细胞对裂解的敏感性略低于表达hDAF的细胞（33±3.3%）（45±3.5%）。
这些数据表明完整的猪胰岛可以被转导以在表面表达人补体激活调节因子，并且表达hDAF或hCD59的猪胰岛细胞对补体介导的裂解不太敏感。
