Lee B W, Kim M-H, Chae H Y, Hwang H J, Kang D, Ihm S H
Department of Internal Medicine, Hallym University College of Medicine, Chuncheon, Korea.
Transplant Proc. 2013 Mar;45(2):591-6. doi: 10.1016/j.transproceed.2012.10.040.
The efficient transfer of genes into intact islets is difficult since islets exist as clusters of differentiated cells with little replication potential. Cell proliferation in response to growth factors is known to be accompanied by loosening of cell-to-cell contacts and increasing paracellular permeability. In this study, we investigated whether gene delivery into intact islet cells was facilitated by modulating β-cell proliferation.
Isolated rat islets were pretreated with glucagon-like peptide (GLP)-1 or human growth hormone for 24 hours, or with 300 mg/dL of glucose for 48 hours before transduction with a suboptimal dose of recombinant adenoviral vector expressing green fluorescent protein (GFP) and β-galactosidase (multiplicity of infection of 25). Transduction efficiency was assessed by measuring β-galactosidase activity and GFP expression using enzyme-linked immunosorbent assay, flow cytometry, and fluorescence microscopy. The numbers of 7-aminoactinomycin D-positive dead cells and 5-ethynyl-2-deoxyuridine (EdU)-positive proliferating cells were also monitored using flow cytometry and fluorescence microscopy.
The transduction efficiency of rat islet cells by a suboptimal dose of viral vector was significantly improved by GLP-1 pretreatment, accompanied by enhanced cell viability and cell proliferation. An increased GFP expression in islet cells after GLP-1 pretreatment was observed among the increased numbers of EdU-positive proliferating cells.
Pretreatment of rat islets with GLP-1 enhanced the transduction efficiency of an adenoviral vector, reducing viral dose burden while improving islet cell viability. From a therapeutic standpoint, genetic modification of pancreatic islets combined with GLP-1 pretreatment may be a promising option for ex vivo gene therapy prior to islet transplantation.
由于胰岛是以几乎没有复制潜能的分化细胞簇形式存在，因此将基因有效导入完整胰岛很困难。已知细胞对生长因子的增殖反应伴随着细胞间接触的松动和细胞旁通透性的增加。在本研究中，我们调查了通过调节β细胞增殖是否有助于将基因导入完整的胰岛细胞。
分离的大鼠胰岛在用表达绿色荧光蛋白（GFP）和β-半乳糖苷酶的重组腺病毒载体（感染复数为25）进行次优剂量转导之前，先用胰高血糖素样肽（GLP）-1或人生长激素预处理24小时，或用300mg/dL葡萄糖预处理48小时。通过酶联免疫吸附测定、流式细胞术和荧光显微镜测量β-半乳糖苷酶活性和GFP表达来评估转导效率。还使用流式细胞术和荧光显微镜监测7-氨基放线菌素D阳性死细胞和5-乙炔基-2'-脱氧尿苷（EdU）阳性增殖细胞的数量。
GLP-1预处理显著提高了次优剂量病毒载体对大鼠胰岛细胞的转导效率，同时增强了细胞活力和细胞增殖。在EdU阳性增殖细胞数量增加的情况下，观察到GLP-1预处理后胰岛细胞中GFP表达增加。
用GLP-1预处理大鼠胰岛可提高腺病毒载体的转导效率，降低病毒剂量负担，同时提高胰岛细胞活力。从治疗角度来看，胰腺胰岛的基因修饰与GLP-1预处理相结合可能是胰岛移植前体外基因治疗的一个有前景的选择。
