Establishment of the method for gene therapy of β cell regeneration by non-invasive gene delivery via pancreatic duct

本研究旨在为糖尿病尤其是β细胞缺乏型糖尿病的治疗提供新的思路。我们报道了EGF家族的β细胞素和肝素结合EGF样生长因子（HB-EGF）在人胰腺，特别是胎儿胰腺中大量表达，重组人β细胞素具有诱导非β胰腺外分泌细胞系分化为β细胞的能力。为了证实β细胞素在体内的这种作用，我们接下来证明了重组人β细胞素改善了由四氧嘧啶的选择性灌注诱导的糖尿病小鼠模型中的葡萄糖耐受不良。这些结果提示β细胞素和HB-EGF可能在胰腺中起生长和/或分化因子的作用，基于这些证据，我们拟通过内镜逆行胰管注射的非侵入性方法，发展新的β细胞再生基因治疗。我们尝试从导管上皮细胞中诱导β细胞新生， ...更多信息 通过在小鼠中注射含有LacZ或推定的β细胞分化因子如β细胞素和肝素结合EGF样生长因子的基因的腺病毒载体，我们成功地诱导LacZ主要在导管细胞表达的腺病毒从胰管口在十二指肠导管注射没有任何严重的副作用，表明这种再生治疗糖尿病的方法适用于大型动物，包括人类。作为下一步，我们尝试使用腺病毒载体在胰管细胞中诱导Betacellulin和HB-EGF基因。这两种基因都能在导管细胞中表达，并观察到导管内分泌细胞包括β细胞的新生，但这些基因产物的表达水平似乎不足以显著提高胰岛素含量。通过内镜逆行胰管注射腺病毒载体，提高β细胞分化因子基因的表达水平，为糖尿病的基因治疗提供新的途径。少

This research was undertaken to establish the new therapy for diabetes mellitus especially for βcell-depleted type of diabetes. We reported that Betacellulin and Heparin-binding EGF-like growth factor (HB-EGF), both of which belong to EGF family, are expressed abundantly in human pancreas, especially developing fetal pancreas, and that recombinant human betacellulin, has a potency to induce differentiation of βcell from non-β pancreatic exocrine cell line. To confirm this effect of betacellulin in vivo, we next demonstrated that recombinant human betacellulin ameliorated glucose intolerance in diabetic mice model induced by selective perfusion of alloxan. These results indicate that betacellulin and HB-EGF may act as a growth and/or differentiation factor in the pancreas.Based on these evidence, we intend to develop the new gene therapy for βcell regeneration by non-invasive method of endoscopic retrograde injection into pancreatic duct. We tried to induce βcell neogenesis from duct ce … More lls by injecting adenovirus vector containing LacZ or genes of putative β cell differentiation factors such as Betacellulin and Heparin-binding EGF-like growth factor in mice. We succeeded in inducing the expression of LacZ mainly in duct cells by injecting LacZ-containing adenovirus into duct from the orifice of pancreatic duct in the duodenum without any serious side effect, suggesting that this method of regeneration therapy for diabetes is applicable to larger animals including human. As a next step, we tried to induce Betacellulin and HB-EGF genes in pancreatic duct cells using adenovirus vector. Both genes could be expressed in duct cells, and neogenesis of endocrine cells including β cells from ducts was observed, but expression levels of these gene products appeared to be not enough to elevate the insulin content significantly. It is necessary to improve the expression level of such genes to develop the new gene therapy for diabetes by the method of endoscopic retrograde injection of adenovirus vectors containing such genes of β cell differentiation factors into pancreatic duct. Less