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研究生:王淞民
研究生(外文):Sung-Min Wang
論文名稱:從人類皮膚微生物總體中鑑定溶解磷酸鈣的細菌
論文名稱(外文):Identification of Calcium Phosphate-degrading Bacteria from Human Skin Microbiome.
指導教授:黃俊銘黃俊銘引用關係
指導教授(外文):Chun-Ming Huang
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:52
中文關鍵詞:微生物總體皮膚磷酸鈣發酵短鏈脂肪酸
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磷酸鈣在人體上過的累積會導致不同類型的疾病產生,像是結石以及皮膚搔癢等。我們的研究主要是研究皮膚微生物對於磷酸鈣結石的影響,我們利用含有豐富磷酸鈣的培養基從人類皮膚上鑑定出一株具有降解磷酸鈣的液化澱粉芽孢桿菌,細菌可以利用葡萄糖進行發酵反應產生短鏈脂肪酸,進而降解磷酸鈣。秀麗隱桿線蟲已經被證實是研究許多生物過程一個非常好的動物模型,在此我們用它做為模型來探討液化澱粉芽孢桿菌的發酵液對於磷酸鈣的分解,透過餵食已用Alizarin red S (ARS) 染色後的磷酸鈣給線蟲,並加入發酵液與線蟲共同培養。我們發現發酵液 (包含短鏈脂肪酸) 可以有效的降解線蟲體內的磷酸鈣,其中醋酸的降解活性已經在小鼠中獲得證實。在本實驗中我們鑑定出一株能夠做為益生菌的液化澱粉芽孢桿菌,此外該菌株的發酵液能夠有效的降解磷酸鈣。
The accumulation of calcium phosphate stones in humans is a modern common disease of civilization, and contributes to many different types of stones-associated diseases. Our research has been mainly aimed to study the probiotic effects of skin bacteria on the degradation of calcium phosphate stones. We have identified a Bacillus amyloliquefaciens bacterium from the human skin by selecting from calcium phosphate rich agar plates in conjunction with 16S rRNA sequencing. The bacteria can ferment the glucose to short-chain fatty acids (SCFAs) which efficiently dissolve the calcium phosphate. Caenorhabditis elegans (C. elegans), proven to be an excellent model for the study of many biological processes, has been used as one of models to study the degradation of calcium phosphate by fermentation media of Bacillus amyloliquefaciens. C. elegans was fed with Alizarin red S-stained calcium phosphate particles along with/without the fermentation media. We found that the fermentation media containing SCFAs can degrade calcium phosphate in C. elegans. The degradation activity of acetic acid, one of SCFAs, was verified in mice. The novelty in our study includes the identification of a Bacillus amyloliquefaciens as a probiotic bacterium. Furthermore, the fermentation media of this bacteria can effectively degrade calcium phosphate.
目錄
圖目錄 vi
表目錄 vii
第壹章、 緒論 1.
1. 研究背景 1.
1.1 介紹 1.
1.2 研究動機 2.
1.3 泌尿系統結石的分類 3.
1.4 泌尿系統結石的形成假說 4.
1.5 泌尿系統結石的臨床症狀及治療 4.
2. 人類微生物總體 5.
3. 秀麗隱桿線蟲 (Caenorhabditis elegans) 6.
3.1 介紹 6.
3.2 生命週期 6.
4. 液化澱粉芽孢桿菌 (Bacillus amyloliquefaciens) 7.
第貳章、 研究目的及架構 8.
1. 研究目的 8.
2. 研究架構 8.
第參章、 材料與方法 10.
1. 使用儀器 10.
2. 使用藥品 11.
3. 實驗方法 13.
3.1 線蟲的來源及繼代培養 13.
3.1.1 線蟲的來源 13.
3.1.2 NGM 培養基之製備 13.
3.1.3 S. Basal Solution之製備 13.
3.1.4 線蟲飼料之製備 14.
3.1.5 Freezing Solution之製備 14.
3.1.6 線蟲的繼代培養 14.
3.1.7 線蟲的保存 15.
3.2 液化澱粉芽孢桿菌之培養 15.
3.2.1 液化澱粉芽孢桿菌的篩選 15.
3.2.2 液化澱粉芽孢桿菌之培養 16.
3.2.2 菌種的保存 16.
3.2.4 抽取菌種DNA 16.
3.2.5 DNA瓊脂醣膠體電泳 17.
3.3 磷酸鈣樣品之製備 18.
3.3.1 磷酸鈣溶液 18.
3.4 發酵實驗 18.
3.4.1 分光光度計測定 19.
3.4.2 Phenol Red酸檢指示劑 19.
3.4.3 菌種培養 19.
3.4.2 培養液之製備 20.
4. Dynamic Light Scattering (DLS)儀器測定 20.
5. Gas Chromatography-Mass Spectrophotometer (GC-MS-MS) 20.
6. 利用線蟲作為模型來觀察發酵液對於體內磷酸鈣的影響 21.
7. 利用小鼠作為模型來觀察醋酸對於背部皮下磷酸鈣的影響 21.
第肆章、 結果與討論 23.
1. 16S rRNA定序之結果 23.
2. 發酵實驗之結果 23.
3. 利用GC-MS-MS測定發酵液之內容物 24.
4. 發酵液對於磷酸鈣粒子大小的影響 24.
5. 發酵液對於線蟲體內的磷酸鈣之影響 24.
6. 醋酸對於小鼠背部皮下的磷酸鈣之影響 25.
7. 統整實驗結果 26.
第伍章、 結論與未來展望 27.
1. 菌種的使用及後續開發研究 27.
2. 發酵液與單一短鏈脂肪酸的比較 27.
3. 動物模型的選擇 28.
4. 篩選方式的多樣化 28.
5. 利用不同的碳源進行發酵 28.
6. 草酸鈣結石 28.
7. 活菌的運用性 29.
8. 結論 29.
參考文獻 31.

圖目錄
圖1. 為篩選液化澱粉芽孢桿菌所使用之方式及基因序列 33.
圖2. 利用液化澱粉芽孢桿菌 (Bacillus amyloliquefaciens)進行發酵實驗之結果 34.
圖3. 利用GC-MS-MS測定發酵液之內容物 35.
圖4. 將磷酸鈣溶液與不同條件的發酵液反應後的粒徑大小 38.
圖5. 利用線蟲作為模型來觀察發酵液對於體內磷酸鈣的影響 40.
圖6. 醋酸對於小鼠背部皮下的磷酸鈣之影響 41.

表目錄
表1. 聚合酶連鎖反應所使用之條件 42.

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