奈米二氧化鈦對痲瘋樹懸浮培養細胞蛋白質的影響

Nano particles-TiO2 specific proteomic profiling of Jatropha curcas suspension cells

陳津甚(Chin-Shen Chen)

指導教授 ： 周映孜

國立屏東科技大學/農學院/生物科技研究所/碩士(2016年)

摘要

中文摘要
學號:M10318012
論文名稱: 奈米二氧化鈦對痲瘋樹懸浮培養細胞蛋白質的影響
總頁數:65
學校名稱:國立屏東科技大學 系別:生物科技系
畢業時間及摘要別:一百零四學年度第二學期碩士學位論文摘要
指導教授:周映孜 研究生:陳津甚
論文摘要內容:
奈米二氧化鈦(nanoparticle-TiO2，NP-TiO2)粒子廣泛被應用生活上，奈米粒子微小容易懸浮於空氣中或汙染水源，可能會產生一些急性毒性，但它們對生物體影響仍是未知。特別是，奈米粒子在自然條件及其所產生的生態效應仍然知之甚少。文獻指出生質柴油的油料作物麻瘋樹可以吸收汙水中的奈米顆粒(奈米汙染物)，發展生質作物的附加價值，可降低替代能源的生產成本。本論文利用痲瘋樹組織培養懸浮細胞，分別測定NP-TiO2對細胞活性、生長量、蛋白質體變化、細胞抗氧化活性、脂肪酸變化等探討NP-TiO2對麻瘋樹細胞生理造成的影響。分別以光照、黑暗、100 mg/L TiO2處理光照和黑暗等四種處理條件處理兩天，收集100 mg/L處理組懸浮培養液0，6，12，24，48 hr後測OD600發現NP-TiO2處裡不會抑制細胞生長，但從光照、黑暗、100 mg/L TiO2處理光照和黑暗懸浮培養細胞中萃取蛋白質以二維電泳分析，分析後比對蛋白質點有所差異，將差異表現的蛋白質透過液相質譜-串聯式質譜分析及資料庫比對，在黑暗處理組明顯增加累積量的蛋白是Major allergen Pru ar和nucleoside diphosphate kinase B。major allergen Pru ar是與植物病原相關聯，在光照和黑暗處理條件下皆有，但在黑暗處理下較多，是一個脂質轉移蛋白(lipid-transfer proteins, LTPs)。Nucleoside diphosphate kinase B (NDPKB) 只在黑暗處理中被比對到，這是最初發現光敏色素相互作用蛋白。100 mg/L NP-TiO2處理光照/黑暗皆比對到peroxidase A2-like但在光照處理組有過量表現，是ㄧ個過氧化酵素。因為在蛋白鑑定中比對到過氧化酵素的累積，以WST-1 kit測定過氧化酶活性，結果顯示光照和100 mg/L NP-TiO2處理光照相較於黑暗組都有較高的過氧化酶活性。由NP-TiO2產生的光觸媒特性在本實驗中證實會造成痲瘋樹細胞過氧化逆境，導致過氧化酶過度表現，但不至於影響細胞生長。經奈米二氧化碳處理之後光照或是黑暗組C18:3的脂肪酸沒有表現，C18:0、C20:0和C24:0飽和脂肪酸被降解了，C18:2(ω-6)在奈米二氧化鈦黑暗處理組沒有被表達。

關鍵字:痲瘋樹、奈米二氧化鈦、蛋白質、脂肪酸、液相質譜分析、氣相質譜分析

關鍵字

痲瘋樹 ； 奈米二氧化鈦 ； 蛋白質 ； 脂肪酸 ； 液相質譜分析 ； 氣相質譜分析

並列摘要

Abstract
Student ID:M10318012
Title of thesis: Nano particles -TiO2 specific proteomic profiling of Jatropha curcas suspension cells
Total:65
Name of institute: Graduate Instituted of Biotechnology
Graduate date: July, 2016
Degree Conferred: Master
Name of students: Chin-Shen Chen
Adviser : Dr. Ying-Tzy Jou
The contents of abstract in this thesis:
Nano titanium dioxide (Nanoparticle-TiO2, NP-TiO2) particles are widely used in modern life. Nanoparticles are easily pollution into the water and air. It may have some acute toxicity, but effect on the organism is still unknow a lot, and the ecological effect in nanoparticles in natural conditions, and the resulting still poorly understood. Literature indicates that biodiesel Jatropha curcas, an oil crops can absorb nanoparticles (nano-pollutants) in the sewage. The added value of developing biomass crops can reduce the cost of alternative energy. In this thesis, we used Jatropha tissue suspension culture cells as the martial. NP-TiO2 treated on Jatropha cells and measured the cell activity, cell growth; proteomics, cellular antioxidant activity, and fatty acid investigate. The NP-TiO2 effects of NP-TiO2 on Jatropha cells caused the physiological. We treated the Jatropha cell for two days, and change the light, dark, and 100 mg/L TiO2, four kinds of treatment conditions. The results show that NP-TiO2 does not inhibit cell growth. But the light, dark, 100 mg /L TiO2 process light and dark suspension culture cell to extracted the proteins in a two-dimensional electrophoresis assay. The difference in performance analysis database of proteins by the LC-MS, the major allergen Pru ar and Nucleoside diphosphate kinase B (NDPKB) were found in dark treatment. Major allergen Pru ar was associated with plant pathogenic. In the light and dark treatment conditions, but more accumulated in the dark treatment, that is a lipid transfer protein (lipid-transfer proteins, LTPs). NDPKB only found in the dark. This is the first discovery phytochrome interacting protein. In 100mg /L NP-TiO2 treated light/dark were found peroxidase A2-like, it is a peroxidase, but both NP-TiO2 and light treatment were overexpression. In comparison did the identify protein correlated with peroxidase, the peroxide enzyme activity were measured in WST-1 kit, results showed that light and 100 mg/L NP-TiO2 light treated had a higher than dark peroxide enzyme activity. In this experiment shown the NP-TiO2 produced photocatalytic properties cause Jatropha cell peroxide stress. Cause overexpression peroxidase, but does not affect cell growth. After the NP-TiO2 treated light or dark change the C18: 3 fatty acids did not show. C18: 0, C20: 0 and C24: 0 saturated fatty acids are degraded, C18: 2 (ω-6) is not expressed in TiO2 dark treatment. The NP-TiO2 were change the protein accumulation, ROS related enzyme activity, and fatty acid species, although the phenotype and cell viability did not change, but inside the cell were shift a lot to tolerance the new type stress NP-TiO2.

Keywords: Jatropha curcas, Nanoparticle titanium dioxide (NP-TiO2), protein, fatty acids, liquid chromatography-mass spectrometry, Gas chromatography-mass

並列關鍵字

Jatropha curcas ； Nanoparticle titanium dioxide ； protein ； fatty acids ； liquid chromatography-mass spectrometry ； Gas chromatography-mass

參考文獻

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