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Graduate School of Ajou University Department of Biomedical Sciences 4. Theses(Ph.D)

Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

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dc.contributor.advisorGwang Lee-
dc.contributor.authorPHUKAN, GEETIKA-
dc.date.accessioned2019-10-21T07:30:06Z-
dc.date.available2019-10-21T07:30:06Z-
dc.date.issued2017-02-
dc.identifier.other24236-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/19020-
dc.description학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2017. 2-
dc.description.tableofcontentsA. 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-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titleSilica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2017. 2-
dc.description.degreeDoctoral-
dc.identifier.localId770678-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000024236-
dc.subject.keywordBiomedical Sciences-
dc.description.alternativeAbstractThe 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.-
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