Narang Ajit S, Cheng Kun, Henry James, Zhang Chunxiang, Sabek Omaima, Fraga Daniel, Kotb Malak, Gaber A Osama, Mahato Ram I
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 26 South Dunlap Street, Feurt Building RM 406, Memphis, Tennessee 38163, USA.
Pharm Res. 2004 Jan;21(1):15-25. doi: 10.1023/b:pham.0000012147.52900.b8.
Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival.
We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry.
After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 microg DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation.
These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.
胰岛移植受限于胰岛移植失败，原因包括血管再生不良、宿主免疫排斥和非特异性炎症反应。人血管内皮生长因子（hVEGF）基因传递可能会促进胰岛血管再生和存活。
我们评估了编码hVEGF和增强型绿色荧光蛋白（EGFP）的双顺反子质粒（pCMS-EGFP-hVEGF）的基因表达。在体外和体内评估了胰岛的葡萄糖反应性，并通过免疫组织化学评估了胰岛移植中的血管再生情况。
转染后，hVEGF和EGFP在Jurkat细胞中的表达水平与原始单顺反子质粒相当，但在用双顺反子质粒转染的胰岛中观察到hVEGF表达更高且持续时间更长，这可能是启动子强度和缺氧反应差异的结果。3:1 w/w复合物在每2000个胰岛5微克DNA的剂量下对胰岛几乎没有毒性。在葡萄糖刺激下，转染胰岛的胰岛素释放以及移植到小鼠肾包膜下的胰岛在葡萄糖刺激后的分泌随时间增加。使用小鼠抗人胰岛素、小鼠抗人血管性血友病因子和大鼠抗小鼠CD31抗体对移植胰岛进行免疫组织化学染色表明，胰岛具有功能且有新血管形成。
这些发现表明，胰岛短暂表达hVEGF基因可能会促进胰岛血管再生并延长移植后胰岛的存活时间。
