You Position: Home > Paper

Protection of adeno-associated virus mediated insnlin-like growth factor Ⅰ against neuron apoptosis induced by high-level glucose

( views:277, downloads:16 )
Author:
No author available
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
5
DOI:
10.3760/cma.j.issn.1674-1927.2009.05.002
Key Word:
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

Abstract: 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 rAAV2/1-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- 1GF Ⅰand then cultured in serum- free 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 Hoeehst33342 fluorescent staining, the total apoptosis rates were (2.71±1.03)% in Group A, (9.17±1:1.72)% 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). ByAnnexinV-FITC/PI flow cytometry, the total apoptosis rates (early phase AnnexinV-FITC~+/PI~- and late phase AnnexinV-FITC~+/PI~+ apoptosis cells) were (5.01±1.17)% in Group A, (9.87±1.38)% in Group B, (27.56± 2.25)% in Group C and (17.34±2.08)% in Group D. There were significant differences among four groups (P<0.05). Conclusion Infection with rAAV2/1-IGF Ⅰ may significantly increase IGF Ⅰ expression in SH-SY5Y cells and substantially ameliorate neuron apoptosis induced by high-level glucose.

WanfangData CO.,Ltd All Rights Reserved
About WanfangData | Contact US
Healthcare Department, Fuxing Road NO.15, Haidian District Beijing, 100038 P.R.China
Tel:+86-010-58882616 Fax:+86-010-58882615 Email:yiyao@wanfangdata.com.cn