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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳建春,陳玉怜 | |
dc.contributor.author | Chih-Kai Liao | en |
dc.contributor.author | 廖智凱 | zh_TW |
dc.date.accessioned | 2021-06-13T03:30:01Z | - |
dc.date.available | 2014-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-29 | |
dc.identifier.citation | 第1章:
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32062 | - |
dc.description.abstract | 星狀神經膠細胞具有維持腦部恆定性的功能,過度的發炎反應與星狀神經膠細胞間隙接合功能的改變皆可導致腦部的損傷與神經退化疾病的發生。本研究以脂多醣刺激初級培養的新生大鼠星狀神經膠細胞,探討發炎反應對星狀神經膠細胞間隙接合的影響。脂多醣以濃度與時間依賴的方式降低星狀神經膠細胞間隙接合蛋白Cx43的表現量,卻不影響細胞的存活度與Cx43的磷酸化狀態。脂多醣也造成Cx43的免疫染色在細胞相鄰的位置減弱,以及抑制細胞的間隙接合功能。脂多醣刺激星狀神經膠細胞促使其類鐸受體TLR4蛋白質的含量上升2至3倍,當TLR4被干擾RNA抑制之後可以回復Cx43蛋白質的表現,顯示脂多醣藉由活化TLR4下游的訊息傳遞途徑來抑制Cx43的表現。此外,脂多醣也會抑制脂筏凹陷蛋白caveolin-3的表現,且處理的時間愈長其表現量下降的趨勢愈明顯。利用專一性caveolin-3干擾RNA轉染星狀神經膠細胞抑制內生性caveolin-3的表現,Cx43的表現量與細胞間隙接合功能也明顯地受到抑制。以脂多醣刺激星狀神經膠細胞1小時之後,細胞內磷酸化ERK的表現量上升,而在刺激6小時之後其iNOS蛋白質的表現也隨著增加。以iNOS cofactor抑制劑1400W處理星狀神經膠細胞,可以防止脂多醣抑制Cx43與caveolin-3的表現。然而ERK抑制劑PD98059與脂多醣共同處理3小時,卻不影響Cx43與caveolin-3的表現。另一方面,星狀神經膠細胞以脂多醣刺激1小時後造成磷酸化JNK的表現量上升,且在刺激8小時後其轉錄因子NF-κB蛋白質的含量也增加。預先以JNK抑制劑SP600125處理可以避免脂多醣所引起Cx43蛋白質含量減少的情況,但以抑制劑BAY11-7082阻斷NF-κB轉位至細胞核卻不能回復Cx43蛋白質的表現。細胞若預先以SP600125抑制JNK的磷酸化,則可以防止脂多醣造成細胞間隙接合的抑制作用,以及細胞膜上Cx43免疫螢光染色的減弱。利用cycloheximide抑制新生蛋白質的合成途徑,會加速脂多醣對Cx43蛋白質的降解作用。當加入蛋白酶體抑制劑MG132或lactacystin,都可以阻止脂多醣造成Cx43降解的現象;若以leupeptin抑制溶酶體的途徑則無法得到相同的效果。此外,以脂多醣刺激6至8小時也會促使星狀神經膠細胞內Cx43與泛素的交互作用,若抑制JNK的活化則可防止脂多醣造成Cx43的泛素化。總結上述研究:脂多醣經由類鐸受體TLR4 (1)促使iNOS表現量上升並產生NO、(2)調降caveolin-3的表現以抑制Cx43的表現、(3)活化下游的JNK訊息傳遞路徑、(4)經由泛素-蛋白酶體路徑降解Cx43的蛋白質而減少Cx43在細胞膜上的分佈,進而抑制星狀神經膠細胞間隙接合的功能。 | zh_TW |
dc.description.abstract | Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell-cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2- to 3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18-24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. We next examined the short-term effects of LPS on astrocytes. Short-term treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 24 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin, but not using the lysosome inhibitor leupeptin. Immunoprecipitation analyses revealed an increased association of Cx43 with ubiquitin in astrocytes after LPS stimulation for 6 or 8 h and this effect was prevented by SP600125. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway. Short-term LPS stimulation leads to Cx43 degradation by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:30:01Z (GMT). No. of bitstreams: 1 ntu-100-D95446006-1.pdf: 8420993 bytes, checksum: 0ca3bb2de0dc53d18b80ffa2a924d29a (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 口試委員會審定書........................................ I
致謝 .................................................. II 中文摘要.............................................. III Abstract................................................ V 英文縮寫與全名對照表 ................................. VII 目錄 .................................................. IX 圖目錄 ................................................ XI 第1章:前言 ............................................ 1 1.1 星狀神經膠細胞 ..................................... 1 1.2 間隙接合與connexin ................................. 2 1.3 間隙接合蛋白Cx43 的組裝、運送與分解 ................ 3 1.4 脂筏與caveolin ..................................... 4 1.5 Cx43 與caveolin 的關係.............................. 5 1.6 脂多醣、類鐸受體、iNOS 與NO ........................ 6 1.7 文獻回顧 ........................................... 8 1.8 研究目的 ........................................... 8 1.9 參考文獻 .......................................... 10 第2章:脂多醣對大鼠星狀神經膠細胞間隙接合功能之影響 ... 15 2.1 序論 .............................................. 15 2.2 實驗材料與方法 .................................... 17 2.3 結果 .............................................. 25 2.4 討論 .............................................. 28 2.5 參考文獻 .......................................... 31 圖與圖片說明 .......................................... 36 第3章:脂筏凹陷蛋白caveolin-3 在脂多醣影響星狀神經膠細胞 Cx43的表現與間隙接合的功能所扮演的角色 ................ 53 3.1 序論 .............................................. 53 3.2 實驗材料與方法 .................................... 55 3.3 結果 .............................................. 62 3.4 討論 .............................................. 64 3.5 參考文獻 .......................................... 67 圖與圖片說明 .......................................... 70 第4章:脂多醣對星狀神經膠細胞間隙接合蛋白的降解作用 ... 81 4.1 序論 .............................................. 81 4.2 實驗材料與方法 .................................... 84 4.3 結果 .............................................. 90 4.4 討論 .............................................. 94 4.5 參考文獻 .......................................... 98 圖與圖片說明 ......................................... 102 第5章:結論及未來展望 ................................ 121 5.1 結論 ............................................. 121 5.2 未來展望 ......................................... 121 5.3 參考文獻 ......................................... 123 模式圖 ............................................... 124 發表文獻 ............................................. 125 | |
dc.language.iso | zh-TW | |
dc.title | 脂多醣對大鼠星狀神經膠細胞間隙接合功能調控之探討 | zh_TW |
dc.title | Effects of Lipopolysaccharide on Gap Junctional Intercellular Communication in Rat Primary Astrocytes | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 王淑美,王懷詩,卓貴美 | |
dc.subject.keyword | 星狀神經膠細胞,脂多醣,類鐸受體,間隙接合,脂筏凹陷蛋白,iNOS,泛素,蛋白酶,體, | zh_TW |
dc.subject.keyword | Astrocytes,Lipopolysaccharide,Toll-like receptor,Cx43,Caveolin-3,iNOS,Ubiquitin,Proteasome, | en |
dc.relation.page | 125 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-29 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 解剖學暨生物細胞學研究所 | zh_TW |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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