腺病毒介导的白细胞介素10基因转移对胰岛β细胞凋亡及胰岛素释放的影响

目的 了解鼠白细胞介素10(mIL-10)基因转移对RINm5F胰岛β细胞凋亡及胰岛素释放功能的影响,以探讨其对1型糖尿病的潜在治疗价值.方法 携带有IL-10基因的重组腺病毒载体Ad-mIL-10感染RINm5F细胞(Ad-mIL-10组),并设置重组腺病毒载体(Ad-eGFP)组及未感染组为对照,通过RT-PCR、Western印迹及酶联免疫吸附试验(ELISA)分别检测IL-10基因和蛋白表达,四甲基偶氮唑盐(MTT)法分析细胞活力,放射免疫法行胰岛素释放实验.检测加入白细胞介素1-β(IL-1β)诱导细胞破坏后,Ad-mIL-10对培养上清中一氧化氮(NO)、一氧化氮合酶(NOS)水平的影响,Hoechst33258核染色分析细胞凋亡,流式细胞仪检测细胞表面Fas表达.结果 RINm5F细胞中检测到mIL-10基因及蛋白的有效表达,培养液中町测得IL-10蛋白.IL-10重组腺病毒感染胰岛β细胞后仍具有在高糖浓度下刺激胰岛素分泌的功能.将IL-1β作用于细胞后,Ad-mIL-10组NO、NOS水平低于未感染组及Ad-eGFP组[NO水平(nmol/106 cells):52.9±3.2比227.3±26.4、235.1±28.6,均P＜0.05;NOS水平(U/106 cells):9.3±1.2比29.8 ±2.5、30.5±2.8,均P＜0.05].Fas表达阳性细胞数明显低于未感染组及Ad-eGFP组(24.6%±1.0%比33.3%±5.1%、32.6%±1.1%,均P＜0.05);细胞凋亡率也低于其他两组(9.4%±1.1%比19.2%±2.2%、20.6%±2.3%,均P＜0.05).结论 携有mIL-10基因的重组腺病毒可在RINm5F细胞有效表达,通过抑制IL-1β诱导的Fas表达而起到抗凋亡作用,胰岛细胞生理功能不受影响.

Effects of adenovirus-mediated interleukin-10 gene transfer on apoptosis and insulin secretion function of beta cell

Objective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. Methods The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose ( 16. 7mmol/L) for a 1 -hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant After induction of IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Results Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-lβ [ NO level ( nmol/106 cells): 52. 9 ± 3. 2 vs 227. 3 ± 26. 4, 235. 1 ±28.6, both P＜0.05; NOS level (U/106 cells): 9. 3 ±1.2 vs 29. 8 ±2.5, 30.5 ±2.8, both P ＜ 0. 05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1β. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24. 6% ± 1.0% vs 33. 3% ± 5. 1% , 32. 6% ±1.1%) (P＜0.05).The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4%±1.1% vs 19.2%±2.2%,20.6%±2.3%,P＜0.05).Conclusions IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function,protecting islet cells from IL-1β-mediated apoptosis and promoting islet cells survival.It is a potential therapy for type 1 diabetes mellitus.