参考文献:1. Efrat, S, Russ, HA (2012) Making beta cells from adult tissues. Trends Endocrinol Metab 23: pp. 278-285 blank" title="It opens in new window">CrossRef <br> 2. Parnaud, G, Hammar, E, Ribaux, P, Donath, MY, Berney, T, Halban, PA (2009) Signaling pathways implicated in the stimulation of beta-cell proliferation by extracellular matrix. Mol Endocrinol 23: pp. 1264-1271 blank" title="It opens in new window">CrossRef <br> 3. Halban, PA, German, MS, Kahn, SE, Weir, GC (2010) Current status of islet cell replacement and regeneration therapy. J Clin Endocrinol Metab 95: pp. 1034-1043 blank" title="It opens in new window">CrossRef <br> 4. Yang, SN, Shi, Y, Yang, G, Li, Y, Yu, J, Berggren, PO (2014) Ionic mechanisms in pancreatic beta cell signaling. Cell Mol Life Sci 釁? pp. 釁<br> 5. Hughes, A, Jessup, C, Drogemuller, C, Mohanasundaram, D, Milner, C, Rojas, D, Russ, GR, Coates, PT (2010) Gene therapy to improve pancreatic islet transplantation for Type 1 diabetes mellitus. Curr Diabetes Rev 6: pp. 274-284 blank" title="It opens in new window">CrossRef <br> 6. Shapiro, AM (2011) Strategies toward single-donor islets of Langerhans transplantation. Curr Opin Organ Transplant 16: pp. 627-631 b013e32834cfb84" target="_blank" title="It opens in new window">CrossRef <br> 7. Li, F, Mahato, RI (2011) RNA interference for improving the outcome of islet transplantation. Adv Drug Deliv Rev 63: pp. 47-68 blank" title="It opens in new window">CrossRef <br> 8. Jiao, Y, Le Lay, J, Yu, M, Naji, A, Kaestner, KH (2014) Elevated mouse hepatic betatrophin expression does not increase human beta-cell replication in the transplant setting. Diabetes 63: pp. 1283-1288 b13-1435" target="_blank" title="It opens in new window">CrossRef <br> 9. Bernal-Mizrachi, E, Kulkarni, RN, Scott, DK, Mauvais-Jarvis, F, Stewart, AF, Garcia-Ocana, A (2014) Human beta-cell proliferation and intracellular signaling part 2: still driving in the dark without a road map. Diabetes 63: pp. 819-831 b13-1146" target="_blank" title="It opens in new window">CrossRef <br> 10. Fiaschi-Taesch, NM, Salim, F, Kleinberger, J, Troxell, R, Cozar-Castellano, I, Selk, K, Cherok, E, Takane, KK, Scott, DK, Stewart, AF (2010) Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6. Diabetes 59: pp. 1926-1936 b09-1776" target="_blank" title="It opens in new window">CrossRef <br> 11. Mulligan, G, Jacks, T (1998) The retinoblastoma gene family: cousins with overlapping interests. Trends Genet 14: pp. 223-229 blank" title="It opens in new window">CrossRef <br> 12. Lefebvre, B, Vandewalle, B, Longue, J, Moerman, E, Lukowiak, B, Gmyr, V, Maedler, K, Kerr-conte, J, Pattou, F (2010) Efficient gene delivery and silencing of mouse and human pancreatic islets. BMC Biotechnol 10: pp. 28 blank" title="It opens in new window">CrossRef <br> 13. Barbu, AR, Bodin, B, Welsh, M, Jansson, L, Welsh, N (2006) A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans. Diabetologia 49: pp. 2388-2391 blank" title="It opens in new window">CrossRef <br> 14. Csete, ME, Afra, R, Mullen, Y, Drazan, KE, Benhamou, PY, Shaked, A (1994) Adenoviral-mediated gene transfer to pancreatic islets does not alter islet function. Transplant Proc 26: pp. 756-757 <br> 15. Giannoukakis, N, Mi, Z, Gambotto, A, Eramo, A, Ricordi, C, Trucco, M, Robbins, P (1999) Infection of intact human islets by a lentiviral vector. Gene Ther 6: pp. 1545-1551 blank" title="It opens in new window">CrossRef <br> 16. Rehman, KK, Wang, Z, Bottino, R, Balamurugan, AN, Trucco, M, Li, J, Xiao, X, Robbins, PD (2005) Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors. Gene Ther 12: pp. 1313-1323 blank" title="It opens in new window">CrossRef <br> 17. Takahashi, R, Ishihara, H, Takahashi, K, Tamura, A, Yamaguchi, S, Yamada, T, Katagiri, H, Oka, Y (2007) Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors. J Mol Endocrinol 38: pp. 127-136 blank" title="It opens in new window">CrossRef <br> 18. Harris, SL, Levine, AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24: pp. 2899-2908 blank" title="It opens in new window">CrossRef <br> 19. Dalboge, LS, Almholt, DL, Neerup, TS, Vassiliadis, E, Vrang, N, Pedersen, L, Fosgerau, K, Jelsing, J (2013) Characterisation of age-dependent beta cell dynamics in the male db/db mice. PLoS One 8: pp. e82813 blank" title="It opens in new window">CrossRef <br> 20. Efrat, S (2013) Recent progress in generation of human surrogate beta cells. Curr Opin Endocrinol Diabetes Obes 20: pp. 259-264 <br> 21. Scharp, D, Arulmoli, G, Taeidi, P, Albert, E, Avakian, A, Sunshine, H, Kucera, C, McRaney, D, Bondurant, D, Haight, B, Magaldi, M (2013) Survival and Function of Human Islets Cultured for 4 Weeks Post-Processing. International Pancreas and Islet Transplantation Association: 9/27/2013; Monterey California. <br> 22. Corey, DR (2007) Chemical modification: the key to clinical application of RNA interference?. J Clin Invest 117: pp. 3615-3622 blank" title="It opens in new window">CrossRef <br> 23. Kenski, DM, Butora, G, Willingham, AT, Cooper, AJ, Fu, W, Qi, N, Soriano, F, Davies, IW, Flanagan, WM (2012) siRNA-optimized modifications for enhanced In Vivo activity. Mol Ther Nucleic Acids 1: pp. e5 blank" title="It opens in new window">CrossRef <br>
Background Human pancreatic islet structure poses challenges to investigations that require specific modulation of gene expression. Yet dissociation of islets into individual cells destroys cellular interactions important to islet physiology. Approaches that improve transient targeting of gene expression in intact human islets are needed in order to effectively perturb intracellular pathways to achieve biological effects in the most relevant tissue contexts. Results Electroporation of intact human cadaveric islets resulted in robust and specific suppression of gene expression. Two genes were simultaneously suppressed by 80% from baseline levels. When multiple (up to 5) genes were simultaneously targeted, effective suppression of 3 of 5 genes occurred. Enzymatic pretreatment of islets was not required. Simultaneous targeting of RB and p53 pathway members resulted in cell cycle reentry as measured by EDU incorporation in 10% of islet nuclei. Conclusions At least three genes can be effectively suppressed simultaneously in cultured intact human pancreatic islets without disruption of islet architecture or overt alterations in function. This enabled the effective modulation of two central growth control pathways resulting in the phenotypic outcome of cell cycle reentry in postmitotic islet cells. Transient exposure to multiple siRNAs is an effective approach to modify islets for study with the potential to aid clinical applications.