Caspase-3 Gene Silencing for Inhibiting Apoptosis in Insulinoma Cells and Human Islets
Caspase-3 Gene Silencing for Inhibiting Apoptosis in Insulinoma Cells and Human Islets
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DOI：
10.1021/mp800093f
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发表时间：
2008-11-01
影响因子：
4.9
通讯作者：
Mahato, Ram I.
中科院分区：
文献类型：
--
作者：
Cheng, Guofeng;Zhu, Lin;Mahato, Ram I.
Although islet transplantation has great potential to treat type I diabetes, most islet grafts do not function due to the host immune rejection, nonspecific inflammatory response and poor revascularization.由于caspase-3在移植胰岛细胞的凋亡中起着至关重要的作用，我们使用化学合成的小干扰RNA（siRNA）来沉默胰岛素瘤（INS-1E）细胞和人胰岛中的caspase-3，然后确定caspase-3基因沉默是否可以阻止这些细胞细胞因子诱导的细胞凋亡。用 siRNA 转染 INS-1E 细胞和胰岛，使 caspase-3 转录物分别减少 50-67% 和 50%。此外，转染的胰岛素瘤细胞的凋亡被显着抑制。 Since gene silencing did not last beyond two days, we converted potent siRNA into shRNA and constructed replication deficient adenoviral (Adv) vectors encoding these shRNAs driven by a U6 or H1 promoter. Compared to chemically synthesized siRNA, Adv-caspase-3-ShRNA efficiently transduced islets, showed relatively higher and prolonged levels of gene silencing beyond five days, with higher gene silencing with a U6 promoter, and protected islets from cytokine-induced apoptosis. Finally, return to normoglycemia was achieved at 1 day post-transplantation of Advcaspase-3-shRNA transduced islets under the kidney capsules of streptozotocin induced nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and maintained beyond two weeks. Blood glucose levels returned to >= 325 mg/dL upon removal of the islet graft-bearing kidney at 32 days after transplantation, confirming that transplanted islets were functional.
Although islet transplantation has great potential to treat type I diabetes, most islet grafts do not function due to the host immune rejection, nonspecific inflammatory response and poor revascularization. Since caspase-3 plays a crucial role in apoptosis of transplanted islet cells, we used chemically synthesized small interfering RNAs (siRNAs) to silence caspase-3 in insulinoma (INS-1E) cells and human islets, and then determined whether caspase-3 gene silencing can prevent these cells from cytokine-induced apoptosis. Transfection of INS-1E cells and islets with siRNAs reduced caspase-3 transcripts by 50-67% and 50%, respectively. Additionally, apoptosis in transfected insulinoma cells was markedly inhibited. Since gene silencing did not last beyond two days, we converted potent siRNA into shRNA and constructed replication deficient adenoviral (Adv) vectors encoding these shRNAs driven by a U6 or H1 promoter. Compared to chemically synthesized siRNA, Adv-caspase-3-ShRNA efficiently transduced islets, showed relatively higher and prolonged levels of gene silencing beyond five days, with higher gene silencing with a U6 promoter, and protected islets from cytokine-induced apoptosis. Finally, return to normoglycemia was achieved at 1 day post-transplantation of Advcaspase-3-shRNA transduced islets under the kidney capsules of streptozotocin induced nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and maintained beyond two weeks. Blood glucose levels returned to >= 325 mg/dL upon removal of the islet graft-bearing kidney at 32 days after transplantation, confirming that transplanted islets were functional.