摘要
目的研究重组腺相关病毒(AAV)载体介导胰岛素样生长因子I(IGFI)在体外神经元表达的情况,及其对高糖诱导神经元凋亡的保护作用。方法利用分子克隆技术将大鼠IGFI基因克隆到pSNAV2.0质粒上,构建包含重组质粒pSNAV2.0-IGFI的杂合型重组AAV载体rAAV2/1-IGFI。将人神经母细胞瘤SH—SY5Y细胞分为正常组(A组)、无血清组(B组)、无血清高糖组(C组),无血清高糖+rAAV2/1-IGFI组(D组),其中A组不做任何处理;B组使用无血清培养基培养;C组用含100mmol/LD.葡萄糖的无血清培养基培养;D组则先用rAAV2/1-IGFI病毒载体感染后再用含100mmol/LD.葡萄糖的无血清培养基处理。干预24h后应用RT—PCR和Western免疫印迹检测各组IGFI基因和蛋白的表达情况;应用Hoechst 33342荧光染色法、AnnexinV—FITC/PI双染法流式细胞仪检测细胞凋亡。观察rAAV2/1-IGFI感染后对高糖诱导SH—SY5Y细胞凋亡的保护作用。结果成功构建rAAV2/1-IGFI重组AAV载体。感染SH-SY5Y细胞后,RT—PCR显示SHSY5Y能表达大鼠IGFI基因;Western免疫印迹检测发现D组IGFI蛋白的表达水平(0.44±0.04)显著高于其他3组(A组0.29±0.02,B组0.17±0.02,C组0.08±0.02,均P〈0.05)。rAAV2/1-IGFI能明显降低高糖诱导细胞凋亡的凋亡率:Hoechst33342荧光染色检测总凋亡率分别为A组(2.71±1.03)%,B组(9.17±1.72)%,C组(25.63±1.81)%,D组(14.50±2.27)%,各组间差异均有统计学意义(P〈0.05);流式细胞仪分析结果表明Annexin V-FITC^*/PI^-的早期凋亡细胞加上Annexin V—FITC^*/PI^*的晚期凋亡细胞的总细胞凋亡率A组为(5.01±1.17)%,B组为(9.87±1.38)%,C组为(27.56±2.25)%,D组为(17.34±2.08)%,各组间差异均有统计学意义(P〈0.05)。结论rAAV2/1-IGFI感染体外神经元SH—SY5Y细胞能
Objective To study the expression of recombinant adeno- associated virus (AAV) mediated insulin-like growth factor I (IGF I ) in cultured neurons and the protection of recombinant AAV against neuron apoptosis induced by high-level glucose. Methods The rat IGF I gene was transferred into pSNAV2.0 using molecular cloning technique to construct a heterozygotic recombinant AAV vector rAAV2/I- IGF I including recombinant plasmid (pSNAV2.0-IGF I ). Human neuroblastoma cells (SH-SYSY) was allocated into normal group (Group A, no intervention), serum-free group (Group B, cultured in serum-free medium), serum-free high-level glucose group (Group C, cultured in serum-free medium containing 100 mmol/L D-glucose), and serum-free high-level glucose + rAAV2/1-IGF I group (Group D, infected with rAAV2/1- IGF I and then cultured in serumfree medium containing 100 mmol/L D- glucose). After intervention for 24 h, the expression of IGF I was detected by RT-PCR and Western blotting, and the cell apoptosis was determined by Hoechst33342 fluorescence staining, AnnexinV-FITC/PI dual staining and flow cytometry. With these tests, protection of rAAV2/1-IGF I against apoptosis of SH-SY5Y cells induced by high-level glucose was evaluated. Results rAAV2/1-IGF I was constructed successfully. RT-PCR revealed the expression of IGF I mRNA in SH-SY5Y cells after infection with rAAV2/1 -IGF I. Western blotting demonstrated remarkably elevated expression of IGF I protein in Group D (0.44±0.04) as compared with other three groups (Group A 0.29±0.02, Group B 0.17±0.02, Group C 0.08±0.02, all P〈0.05). rAAV2/1- IGF I was shown to significantly reduce the rate of cell apoptosis. By Hoechst33342 fluorescent staining, the total apoptosis rates were (2.71±1.03)% in Group A, (9.17±1.72)% in Group B, (25.63±1.81)% in Group C and (14.50±2.27)% in Group D. There were significant differences among four groups (P〈0.05). By AnnexinV-FITC/PI flow cytometry, the total apoptosis rates (earl
出处
《中华生物医学工程杂志》
CAS
2009年第5期330-335,共6页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金(30860297)
广东省自然科学基金(06022679)
关键词
胰岛素样生长因子I
神经元
细胞凋亡
腺相关病毒
高糖
Insulin-like growth factor I
Neurons
Apoptosis
Adeno-associated virus
High-level glucose