BACILLUS AMYLOLIQUEFACIENS生長在高GABA含量稻米刺激膠原蛋白合成以及減緩磷酸三鈣誘導產生的皮膚搔癢

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姓名

Enkhtsetseg Tserenkhuu(Enkhtsetseg Tserenkhuu) 查詢紙本館藏

畢業系所

生物醫學工程研究所

論文名稱

BACILLUS AMYLOLIQUEFACIENS生長在高GABA含量稻米刺激膠原蛋白合成以及減緩磷酸三鈣誘導產生的皮膚搔癢
(GABA-RICH RICES COLONIZED WITH BACILLUS AMYLOLIQUEFACIENS STIMULATE COLLAGEN SYNTHESIS AND ATTENUATE TRICALCIUM PHOSPHATEINDUCED SKIN ITCHING)

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摘要(中)

皮膚是人體最大也是最明顯的器官。瘙癢的皮膚，也稱為瘙癢，是一種刺激性和不舒服的感覺，常常伴隨著想要抓與刮皮膚的慾望以增強感受。慢性瘙癢會誘發發炎反應，持續造成白細胞介素(IL-6)的產生，進而導致表皮增生。另一方面，皺紋的形成是老化的一部分，與膠原蛋白降解有著關聯性。由成纖維細胞產生的第一型膠原(Col1)是人類皮膚中最主要的膠原類型。γ-氨基丁酸(GABA)是中樞神經系統中眾所周知的抑制性神經傳遞物質。近期的研究報導了GABA對傷口癒合的顯著影響及其作為免疫調節劑的功能。根據研究指出，植物生長促進內生細菌可以刺激植物的生長，並促進植物獲取營養、共生性能和脅迫耐受性增加的能力。

在這項研究內容，我們將水稻穀粒與解澱粉芽孢桿菌Bacillusamyloliquefaciens (BA)，一種非致病性土壤細菌，一起培養。我們的研究結果顯示，與BA細菌一同培養的水稻穀粒並沒有改變根和芽的長度，但GABA的含量卻有顯著的增加。經1mM和10mM GABA處理過的NIH 3T3細胞，其細胞中的Col1的蛋白質含量有明顯的提升。此外，GABA處理過的NIH 3T3細胞中的Col1 mRNA轉錄物的表現量會以以劑量依賴性的趨勢增加。

GABAB3-siRNA對GABA受體亞單位β-3 (GABAB3)的抑制顯著降低了NIH 3T3細胞中的Col1含量，說明了GABAB3介導了GABA誘導的Col1產生的過程。真皮內注射磷酸三鈣進入ICR小鼠背部皮膚能夠引起皮膚瘙癢。局部應用GABA或高GABA含量(BA菌共同培養生長)之稻米增加了Col1的產生，並減緩了磷酸三鈣誘導的瘙癢和IL-6含量之增加。

摘要(英)

Skin is the largest and most visible organ in human body. Itchy skin, also known as pruritus, is an irritating and an uncomfortable sensation that associated with the desire to scratch for enhancement the feeling. Chronic itching induces inflammation and sustains production of interleukin (IL)-6, which can lead to epidermal hyperplasia. On the other hand, the formation of wrinkles is part of the aging associated with degradation of the collagen. Type I collagen (Col1) produced by fibroblasts is the main type of the collagen in the human skin. Gamma-aminobutyric acid (GABA) is well known as an inhibitory neurotransmitter in the central nervous system. Recent studies have reported the significant effect of GABA on wound healing and its function as an immune-modulator. It is reported that plant growth-promoting endophytic bacteria can stimulate the growth, nutrient acquisition, symbiotic performance and stress tolerance of plants.
Here, we incubated rice grains with Bacillus amyloliquefaciens (BA), a non-pathogenic soil bacterium. Our results demonstrated that incubation of rice grains with BA bacteria did not change the lengths of roots and sprouts, but significantly increased the content of GABA. Treatment of 1mM and 10mM GABA significantly up-regulated the protein level of Col1 in NIH 3T3 cells. Furthermore, the expression of Col1 mRNA transcript was increased in NIH 3T3 cells when cells were treated with GABA in a dose-dependent manner.
Inhibition of GABA receptor subunit beta-3 (GABAB3) by GABAB3-siRNA considerably reduced the Col1 in NIH 3T3 cells, indicating the GABAB3 mediated the GABA-induced Col1 production. Intra-dermal injection of tri-calcium phosphate (Ca3(PO4)2) into the dorsal back skin of ICR mice caused itching. Topical application of GABA or GABA-rich (BA-colonized) rice onto mouse skin augmented the production of Col1 and attenuated the tri-calcium phosphate-induced decreases in itching and the level of IL-6.

關鍵字(中)

★ 液化澱粉芽孢桿菌
★ 搔癢
★ γ-氨基丁酸
★ 第一型膠原蛋白
★ 白細胞介素

關鍵字(英)

★ Bacillus amyloliquefaciens
★ GABA
★ IL-6
★ Itch
★ Type 1 collagen

論文目次

Contents
Chinese abstract i
English abstract ii
Acknowledgement iii
Contents iv
List of Figures iv
List of Tables vii
List of Abbreviations viii
Chapter 1: Introduction 1
1.1 Skin structure 1
1.2 Skin wrinkle 2
1.3 Collagen 4
1.4 Itching process 7
1.5 Gamma amino butyric acid 9
1.6 Nutrition of Rice 12
1.7 Bacillus amyloliquefaciens 13
Chapter 2: Materials and methods 16
2.1 Materials 16
2.2 Inoculation and bacterial culture 17
2.3 Cultivation of Rice 17
2.4 Determination of GABA content in Rice 17
2.5 Cell culture 19
2.6 Cell counting and treatment 20
2.7 Animal experiment 20
2.8 Protein extraction and measurement 21
2.8.1 Whole cell protein extraction from cell culture 21
2.8.2 Whole protein extraction from mice skin 21
2.8.3 Total protein concentration 21
2.9 Immunoblotting analysis 22
2.9.1 Poly-Acrylamide Gel Electrophoresis 22
2.9.2 Western blotting 23
2.9.3 Immunostaining 24
iv
2.9.4 Band densitometry 24
2.10 Elisa of Interleukin-6 25
2.11 Statistical analysis 25
Chapter 3: Results and discussion 26
3.1 Enhancement of the Col1 in NIH 3T3 cell by GABA compound 26
3.2 Knockdown 27
3.3 Enhancement of the Col1 in ICR mice by GABA compound 28
3.4 Rice sprout and root growth rate of with and without BA bacteria 29
3.5 GABA concentration of Rice 30
3.6 Collagen 1 expression of mice skin treated by GABA rich Rice 31
3.7 Mice skin scratching number treated by GABA compound and GABA rich Rice 32
3.8 IL-6 level of mice skin treated by GABA compound and GABA rich
Rice 33
Chapter 4: Conclusion and Future work 34
References 36

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指導教授

黃俊銘(Eric.Chun-Ming Huang)

審核日期

2019-1-15

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