Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro
- 주제(키워드) Biomedical Sciences
- 발행기관 아주대학교
- 지도교수 Gwang Lee
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024236
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, I assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 µg/µl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 µg/µl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamine metabolism related enzymes, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles.
more목차
A. INTRODUCTION……………………………………………………………………….................1
B. RESULTS
1) Clathrin-mediated endocytosis related genes are altered in MNPs@SiO2(RITC) treated HEK293 cells………………………………………………..........8
2) MNPs@SiO2(RITC) disturb the expression pattern of proteasome related genes in HEK 293 cells………………………………………………………………………………….10
3) MNPs@SiO2(RITC) facilitate formation of inclusion bodies in Synphilin-1 stably over-expressed 293 (Synph-293) cells……………………………………………….13
4) MNPs@SiO2(RITC) cause disturbance in proteasome activity of primary neuronal cells………………………………………………………………………………………...........17
5) MNPs@SiO2(RITC) facilitate the formation of inclusion bodies in SH-SY5Y cells and primary neuronal cells…………………………………………………………………….19
6) Nanoparticles induce mild Endoplasmic Reticulum (ER) stress in HEK293 cells………….......................................................................................................25
7) MNPs@SiO2(RITC) induce ROS generation and cell death in primary neurons…...…....................................................................................................27
8) MNPs@SiO2(RITC) alter polyamine metabolism in SH-SY5Y cells………...31
C. DISCUSSION………………………………………………………………………….................33
D. MATERIALS AND METHODS
a) Cell culture………………………………………………………………………………..............39
b) Primary neuronal culture………………………………………………………………….......39
c) MNPs@SiO2(RITC) and silica nanoparticles……………………………………………41
d) RNA purification………………………………………………………………………...............41
e) Quantitative real-time PCR (qPCR) and reverse transcription PCR (RT-PCR)……….........................................................................................................42
f) Proteasome activity assay……………………………………………………………….......47
g) MTS assay………………………………………………………………………………...............48
h) Immunocytochemistry…………………………………………………………………...........48
i) ROS measurement in neuronal cells……………………………………………………....51
j) Western blotting…………………………………………………………………………............51
k) Statistical analysis………………………………………………………………………...........52
E. REFERENCES…………………………………………………………………………................53
