Title

中草藥純物質ABL-007蛋白質體學研究之驗證

Translated Titles

Verification of Proteomic Data for ABL-007 Treated Astrocytes

Authors

吳盛發

Key Words

西方點墨法 ; 細胞萃取液 ; 細胞膜循環 ; 低密度膽固存受體 ; 胰島素生長因子結合蛋白2 ; 星狀膠細胞 ; Western blotting ; conditioned medium ; exsomes ; LDL-R ; IGFBP-2 ; Astrocyte

PublicationName

朝陽科技大學應用化學系生化科技碩博士班學位論文

Volume or Term/Year and Month of Publication

2017年

Academic Degree Category

碩士

Advisor

李孟真

Content Language

繁體中文

Chinese Abstract

李老師實驗室先前與辜韋智老師合作探討ABL-007純化合物對於星狀膠質細胞(Astrocyte CTX TNA2)影響其分泌物蛋白質的產生。此論文在於用西方點墨法驗證在蛋白質體學中得到的結論,並近一步與富含ABL-007的中草藥粗萃物進行比較其藥效。方法為培養星狀膠質細胞,添加/無添加10μM的ABL-007處理24小時候,收集並濃縮細胞培養液 (conditioned medium);透過 LC-MS/MS 進行分析其蛋白質體。同樣處理的樣品以西方點墨法檢查,是否量跟蛋白質體學的結論相同。 接著入加中草藥粗萃物進行比較並使用西方墨點法得到的數據驗證 LC-MS/MS的結果。細胞培養液 及細胞萃取液 (cell lysate)都進行檢驗。在LC-MS/MS的初步結果得知ABL-007純化合物會導致星狀膠質細胞的細胞培養液中LDL- Receptor (LDL-R)及 Insulin-like growth factor binding protein-2 (IGFBP-2)分泌量下降。同樣處理的樣品以西方點墨法檢查發現LDL-R及IGFBP-2有下降趨勢,而在使用中草藥粗萃物處理並使用西方墨點法驗證,得到LDL-R及IGFBP-2 分泌量上升。若細胞萃取液檢驗則看到ABL-007純化合物及中草藥粗萃物處理都會導致LDL-R及IGFBP-2 分泌量下降,以上數據可以得到在體外實驗中ABL-007會導致LDL-R及IGFBP-2分泌量下降。粗萃物的效果則是導致LDL-R及IGFBP-2分泌量上升,這些蛋白質會參與到細胞膜循環、膽固醇利用和胰島素抗阻等,但是他們影響到的功能需要再更進一步的驗證。 關鍵字:西方點墨法、星狀膠質細胞、細胞萃取液、低密度膽固醇受體、第二型胰島素生長因子結合蛋白、細胞膜循環。

English Abstract

Background: Professor Lee previously worked with Professor Ku to explore the effect of ABL-007 pure compound on the production of secretory proteins in astrocyte cell line CTXTNA2. My study is to use Western blot to verify the conclusion drawn from the proteomic data, as well as to compare effects of single compounds versus ABL-007 rich crude extract from plant origin. Material and methods: Cultured astrocytic cell line was treated with and without addition of 10 uM of ABL-007 for 24 hours, and the conditioned medium was collected and concentrated with flow through column. The protein was analyzed by LC-MS/MS. The remaining sample was checked for protein level of interesting clones with Western blots. Both conditioned medium and the cell lysate were tested. Proteomic data from the LC/MS/MS methods showed that ABL-007 pure compounds decreased the level of LDL-Receptor (LDL-R) and insulin-like growth factor binding protein-2 (IGFBP-2) in the conditioned medium. The Western blot demonstrated that in the conditioned medium the LDL-R and IGFBP-2 were down-regulated. We have prepared a crude extract from the plant which contains ABL-007. The cells were treated with diluted crude extract so that the concentration of ABL-007 was 7 uM. The treatment of crude extract results in the increased level of LDL-R and IGFBP-2 in the conditioned medium. When the cell lysates were tested, the LDL-R and IGFBP-2 reduced. Keywords: Western blotting, astrocyte, conditioned medium, LDL-R, IGFBP-2, exosomes.

Topic Category 基礎與應用科學 > 化學
理工學院 > 應用化學系生化科技碩博士班
生物農學 > 生物科學
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