Chen Zhi-hong, Li Tang, Luo Bing, Zhu Wei, Sun Ruo-peng
Department of Peadiatrics, Shandong University Qilu Hospital, Jinan 250012, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2007 Jun;21(2):188-90.
To construct the recombinant adenovirus containing rat insulin-like growth factor 1 (rIGF-1), and then infect to rat islet beta cells-RINm5F cells with the virus to investigate the role of rIGF-1 to streptozotocin-induced cell impairment in vitro.
Recombinant adenovirus encoding rIGF-1 was constructed, and then infect to RINm5F cells. rIGF-1 protein was detected by Western blot analysis and ELISA method. Then streptozotocin was used to induce RINm5F cells impairment. The levels of nitric oxide were detected in cells culture supernatants. The cells function was evaluated by glucose-stimulated insulin production. The apoptosis was analyzed by flow cytometry. Thiaoollyl blue viability assay was applied to exam the number of viable cells.
The recombined adenovirus-rIGF-1 was constructed successfully and its titer was about 4.0x10(8) pfu/ml. The rIGF-1 was expressed in the RINm5F cells and cell culture supernatants. rIGF-1 expression could inhibit islet cells apoptosis, significantly decrease the level of NO induced by streptozotocin and significantly increase insulin secretion and cells viability.
These results suggested that the high concentration of rIGF-1 in cultured islets may be beneficial in maintaining islet beta cells function and protecting islet beta cells from apoptosis-mediated factors. The apoptosis induced by STZ may be NO-dependent.
构建含大鼠胰岛素样生长因子1（rIGF-1）的重组腺病毒，并用该病毒感染大鼠胰岛β细胞-RINm5F细胞，以研究rIGF-1对链脲佐菌素诱导的体外细胞损伤的作用。
构建编码rIGF-1的重组腺病毒，然后感染RINm5F细胞。通过蛋白质免疫印迹分析和酶联免疫吸附测定法检测rIGF-1蛋白。然后用链脲佐菌素诱导RINm5F细胞损伤。检测细胞培养上清液中一氧化氮的水平。通过葡萄糖刺激的胰岛素分泌来评估细胞功能。通过流式细胞术分析细胞凋亡情况。应用噻唑蓝活力测定法检测活细胞数量。
成功构建重组腺病毒-rIGF-1，其滴度约为4.0×10(8) pfu/ml。rIGF-1在RINm5F细胞和细胞培养上清液中表达。rIGF-1的表达可抑制胰岛细胞凋亡，显著降低链脲佐菌素诱导的一氧化氮水平，并显著增加胰岛素分泌和细胞活力。
这些结果表明，培养的胰岛中高浓度的rIGF-1可能有利于维持胰岛β细胞功能，并保护胰岛β细胞免受凋亡介导因子的影响。链脲佐菌素诱导的细胞凋亡可能依赖于一氧化氮。
