Callewaert H, Gysemans C, Cardozo A K, Elsner M, Tiedge M, Eizirik D L, Mathieu C
Laboratory of Experimental Medicine and Endocrinology (LEGENDO), UZ Gasthuisberg O&N, Katholieke Universiteit Leuven, Leuven, Belgium.
Cell Transplant. 2007;16(5):527-37. doi: 10.3727/000000007783464948.
Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors-improving beta-cell resistance against immune attack-is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 +/- 4.1% at MOI 50 in whole islets to 80.0 +/- 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive beta-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of beta-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.
胰岛移植是1型糖尿病一种很有前景的治疗方法，但需要长期免疫抑制是其广泛应用的主要障碍。通过慢病毒载体在体外导入药物——提高β细胞对免疫攻击的抵抗力——是一条值得探索的途径。本研究的目的是调查在病毒感染前将胰岛解离成单个细胞并在移植前重新聚集是否能提高病毒转导效率且无细胞毒性。该程序将LV-pWPT-CMV-EGFP构建体的转导效率从在MOI为50时全胰岛的11.2±4.1%提高到在MOI为5时的80.0±2.8%。活力（通过Hoechst/PI测量）和功能（通过葡萄糖刺激测量）仍保持较高水平。移植后，转染后的假胰岛聚集体在90多天内仍保持EGFP阳性，且EGFP的表达主要与胰岛素阳性的β细胞共定位。在体外或体内均未观察到对免疫攻击的易感性增加。这些数据表明，在慢病毒转染前将胰岛分散并在移植前重新聚集是一种在体外和体内将感兴趣的基因导入胰岛用于移植目的的高效方法，但与未处理的胰岛相比，使用分散的细胞聚集体使血糖正常化所需的β细胞数量高出八倍多。在胰岛细胞中实现稳定和强转基因表达所付出的高昂代价肯定是重要的细胞损失。
