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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 毒理學研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32351
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor劉興華(Shing-Hwa Liu)
dc.contributor.authorCheng-Tien Wuen
dc.contributor.author吳鎮天zh_TW
dc.date.accessioned2021-06-13T03:44:19Z-
dc.date.available2012-12-31
dc.date.copyright2011-10-07
dc.date.issued2011
dc.date.submitted2011-07-29
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J Neurosci 30:3933-3946.
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32351-
dc.description.abstract內質網壓力 (Endoplasmic Reticulum Stress, ER stress) 為細胞受到外在壓力時所產生的訊息反應,與很多疾病的產生有關如:神經退化疾病、糖尿病、缺血再灌流反應及急性腎衰竭等等。目前有越來越多的文獻指出內質網壓力訊息會參與在化學物質引起的腎臟傷害過程中,但其作用機轉並不清楚。會造成腎傷害的毒性化學物質很多如:顯影劑(Contrast medium),主要作用在腎臟近曲小管部位,會引發急性腎衰竭使腎臟過濾能力降低,尤其對腎功能已受損的病患(如糖尿病患者、慢性腎臟病變病人或需洗腎的病患)傷害更為明顯。亦如帝鉑注射液 (Cisplatin),本身為廣泛使用的抗癌藥物可以有效的治療睪丸癌、卵巢癌、膀胱癌及頭頸癌等癌症,但對腎臟卻有嚴重的副作用,易造成腎小管細胞凋亡且有產生急性腎衰竭的可能性。這樣的傷害除了透過氧化壓力 (oxidative stress)、細胞循環 (cell cycle) 調控失衡外,許多文獻認為內質網壓力訊息改變可能也參與腎臟傷害。因此本實驗將探討內質網壓力參與在腎臟毒性中的機轉。
實驗結果顯示在細胞實驗中處裡40-60 mg I/ml 的顯影劑後會促進 GRP78, GRP94 表現及Caspase 12 cleaved,同時以GRP78 SiRNA 轉染實驗發現GRP78表現扮演著保護的角色。而在PERK-eIF2α路徑中PERK和eIF2α會被磷酸化,利用eIF2α去磷酸化酶抑制劑salubrinal 則會減少細胞凋亡的產生。雖然salubrinal 在細胞實驗中可以保護顯影劑對近曲小管細胞的傷害。但在動物實驗中是否仍有療效在未來則要進一步加以探討。而在帝鉑注射液 (cisplatin) 的細胞實驗中亦發現salubrinal 可以保護細胞凋亡的生成。但在進行動物實驗後卻意外發現salubrinal 會增加帝鉑注射液所誘發的血中 尿素氮 (BUN) 和血清肌酸酐 (Creatinine) 的比例。腎臟傷害指標kidneyinjury marker (kim-1) 也會增加。病理切片上則發現salubrinal 會使腎小管壞疽細胞及空泡狀細胞增加、刷狀緣掉落、呈扁平狀的情形增,且細胞凋亡螢光染色 (TUNEL, fluorescence stain) 也有增加的情況。在帝鉑注射液造成腎臟傷害的過程中以西方點墨法檢測發現ER stress 相關之peIF2α、ATF4、CHOP、aspase cascade 等訊息有增加的情形。接著以MDA 檢測腎臟中產生脂質過氧化 (lipidperoxidation) 的現象發現ROS 可能扮演著很重要的角 色。此外並非透過CYP2E1 的影響而是透過 NOX4 的表現增加而促進 ROS 的生成,並進一步加強帝鉑注射液造成的腎臟傷害。綜合上述我們發現顯影劑無論在動物或細胞實驗中皆可誘發內質網壓力相關訊息產生。同時在細胞實驗中,以內質網壓力修飾試劑salubrinal 處裡後發現其具有保護腎臟傷害的作用。但在順鉑注射液處裡的動物模式中salubrinal 卻沒有防止細胞凋亡達到保護腎臟的能力。在未來我們還需要再透過修飾不同內質網壓力的調控路徑去減少臨床化學藥物對腎臟傷害的目的。
zh_TW
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Previous issue date: 2011
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dc.description.tableofcontents口試委員審定書 …………………………………………………. I
誌謝 ………………………………………………………………. II
中文摘要 …………………………………………………………. III
Abstract …………………………………………………………… V
List of Abbreviation ………………………………………………VII
I. Introduction …………………………………………………….. 1
1.1 Endoplasmic Reticulum Stress Related Signals and Disorder 1
1.2 Chemicals-Induced Nephrotoxicity and Endoplasmic Reticulum
Stress Signals……………………………………………………. 3
1.2.1 Contrast medium-induced renal injury……………………………... 3
1.2.2 Anti-cancer agent, cisplatin, induced renal toxicity………………… 4
1.2.3 The roles of ER stress protection agents……………………………... 5
1.3 The Aims of This Study………………………………………… 6
II. Methods and Materials ………………………………………… 10
2.1 Contrast medium-induced nephropathy……………………… 10
2.1.1 Cell culture and chemical treatment…………………………………. 10
2.1.2 Cell viability assay…………………………………………………….. 10
2.1.3 Acridine orange/ethidium bromide dual staining…………………… 10
2.1.4 Hochest 33258 staining………………………………………………… 11
2.1.5 Immunoblotting analysis……………………………………………… 11
2.1.6 GRP78 SiRNA Transfection………………………………………….. 12
2.1.7 Contrast medium-induced renal toxicity in rat……………………… 12
2.2 Salubrinal Enhances Cispaltin-induced nephropathy………. 13
2.2.1 Cisplatin-induced acute renal injury in mice………………………. 13
2.2.2 Real-time RT-PCR…………………………………………..……….. 14
2.2.3 Histological assessment of renal injury……………………………... 15
2.2.4 TUNEL staining for apoptosis……………………………..………... 15
2.2.5 Caspase 3 activity assay……………………………..……………….. 15
2.2.6 Immunoblotting analysis…………………………..………………… 16
2.2.7 Tissue oxidative injury assessment: Malondialdehyde (MDA) assay 16
2.2.8 Statistical analysis…………………………..……………………….. 17
III. Results………………………………………………………… 18
3.1 Contrast medium induced ER stress response in the tubular cells
…………………………………………………………..…… 18
3.1.1. CM induces cell death in the renal tubular cell line NRK52E….. 18
3.1.2 CM induces the ER stress response……………..………………… 18
3.1.3 PERK/elF2α activation rescued CM-induced tubular cell apoptosis 19
3.1.4 Effect of GRP78 knockdown on the CM-induced apoptosis…….. 20
3.1.5 CM induces ER stress response in the animal model……………. 20
3.2 Salubrinal mediated cisplatin-induced renal injury..……... 21
3.2.1 Salubrinal prevents the cisplatin-induced renal injury in the
renal tubular cells…………………………..……………………… 21
3.2.2 Salubrinal enhances the cisplatin-induced renal injury in animal
model…………………………..…………………………..………… 21
3.2.3 Salubrinal enhances cisplatin-induced renal tubular cell apoptosis 22
3.2.4 Salubrinal enhances cisplatin-induced oxidative stress in the kidneys 23
IV. Discussion …………………………………………………… 25
4.1 The ER stress involves in the contrast medium, Urographin 25
4.2 The ER stress effects in the chemical reagent, cispltin……. 27
V. Summary and Perspective …………………………………… 33
Reference …………………………………………………………. 34
Appendix …………………………………………………………. 75
1. List of Publication: …………………………………………… 75
Figure 3-1. Effects of urografin (CM, 20–60 mg I/ml) on cell viability and
apoptosis in renal tubular cells. ……………..………………….. 47
Figure 3-2. Effects of urografin on necrosis in renal tubular cells. ..……….. 49
Figure 3-3. Effects of urografin on ER stress expressions in renal tubular cells. 50
Figure 3-4. Urografin-induced ER stress signals PERK/eIF2α in renal
Tubular cells. ……………………………………………………. 51
Figure 3-5. Effects of iopromide on ER stress expressions in renal
tubular cells. ……………………………..……………….……… 52
Figure 3-6. Salubrinal decreased urografin-induced apoptosis was detected
by flow cytometry. .……………………………..……………….. 53
Figure 3-7. Salubrinal decreased urografin-induced apoptosis was detected by
Hoechst staining. …………………………………..…………….... 55
Figure 3-8. Effects of GRP78 RNA interference on urografin-induced apoptosis of
renal tubular cells. …………………………………..…………….. 57
Figure 3-9. Effects of salubrinal on apoptosis and expressions of GRP78 and
phospho-eIF2α in GRP78-siRNA–transfected renal tubular cells. 58
Figure 3-10. Effects of urographin in the rats. ………………………………. 60
Figure 3-11.Effects of salubrinal on apoptosis in the renal tubular cells. … 62
Figure 3-12. Schematic representation of the experimental procedure. ….. 64
Figure 3-13. Enhancement of salubrinal on cisplatin-induced injury. ……. 65
Figure 3-14. Enhancement of salubrinal on renal histomorphological
changes in cisplatin-treated mice.…………………………….. 66
Figure 3-15. Enhancement of salubrinal on apoptosis and caspase 3
activity induction in the kidneys of cisplatin-treated mice. …. 67
Figure 3-16. Enhancement of salubrinal on the induction of ER stress-related
XI
molecules in the kidneys of cisplatin-treated mice. ……………… 68
Figure 3-17. Enhancement of salubrinal on the induction of oxidative stress
in the kidneys of cisplatin-treated mice. …...………………… 70
Figure 3-18. The enhancement of salubrinal on cisplatin-induced increased
expressions of phospho-eIF2α, CHOP, cleaved caspase 9, cleaved caspase
12, and Bax, and decreased expression of Bcl-2 in the kidneys could be
reversed by NAC. ………………………………………………….. 72
Figure 3-19. Antioxidant NAC reverses the enhancement of salubrinal on
cisplatin-induced increased serum BUN and creatinine levels and renal
caspase 3 activity in mice. …………………………………………. 74
dc.language.isoen
dc.subject氧化壓力zh_TW
dc.subject薩伯&#63965zh_TW
dc.subject顯影劑誘發腎臟病變zh_TW
dc.subject內質網壓力zh_TW
dc.subject急性腎衰竭zh_TW
dc.subject顯影劑zh_TW
dc.subject帝鉑注射液zh_TW
dc.subjectAKIen
dc.subjectEndoplasmic Reticulum Stressen
dc.subjectContreat mediumen
dc.subjectCisplatinen
dc.subjectSalubrinaen
dc.subjectNephrotoxicityen
dc.subjectAcute kidney injuryen
dc.title內質網壓力在化學物誘導之腎臟毒性的角色探討zh_TW
dc.titleThe Role of Endoplasmic Reticulum Stress in the Chemicals-Induced Nephrotoxicityen
dc.typeThesis
dc.date.schoolyear99-2
dc.description.degree博士
dc.contributor.oralexamcommittee蕭水銀(Shoei-Yn Lin-Shiau),楊榮森(Rong-Sen Yang),林石化(Shih-Hua Lin),姜至剛(Chih-Kang Chiang)
dc.subject.keyword內質網壓力,顯影劑,帝鉑注射液,氧化壓力,薩伯&#63965,&#63837,顯影劑誘發腎臟病變,急性腎衰竭,zh_TW
dc.subject.keywordEndoplasmic Reticulum Stress,Contreat medium,Cisplatin,Salubrina,Nephrotoxicity,Acute kidney injury,AKI,en
dc.relation.page75
dc.rights.note有償授權
dc.date.accepted2011-07-29
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept毒理學研究所zh_TW
顯示於系所單位:毒理學研究所

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