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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
dc.contributor.author | Han-Hsuan Chou | en |
dc.contributor.author | 周漢軒 | zh_TW |
dc.date.accessioned | 2021-06-16T13:02:16Z | - |
dc.date.available | 2015-09-24 | |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61399 | - |
dc.description.abstract | 個體在受到B型肝炎病毒(HBV)感染後成為慢性帶原者的機率和受感染時的年紀有高度相關性。若是在一歲以前受到感染,該個體變成慢性帶原者的機率高達九成;若是在一到五歲之間受到HBV感染,其帶原率降到三成;若是成人受到感染,其帶原率降到只有5%以下。對於這個現象的正確發生原因目前還不清楚。
在前驅實驗中,我們使用尾靜脈動力注射(HDI)將10微克的HBV表現質體從尾靜脈注射進小鼠體內,作為一個體內肝細胞轉染HBV的小鼠動物模式,結果發現十二周大的小鼠清除血清中表面抗原的速度很顯著的比六周大的小鼠快。在許多前人的研究中,腸道菌被認為影響了許多部份的免疫系統發展,例如腸道周邊淋巴組織、腸道lamina propria淋巴球數量以及細胞激素濃度等。基於以上的知識及觀察,我提出我的假說:C3H/HeN小鼠的腸道菌在出生後六周到十二周這段期間'訓練'了小鼠的免疫系統,使其足夠成熟而可以在十二周時對HBV產生免疫反應。 為了測試我的假說,我將C3H/HeN小鼠分成三組:'Old1'組喝的是正常的飲用水,而'OA1'組在五周大到十二周大這段時間喝的是加了抗生素的飲用水[Ampicillin (1g/L), neomycin (1g/L), vancomycin (0.5g/L), metronidazole (1g/L)]。在抗生素水給予結束後,我讓'OA1'組小鼠飲用正常飲用水兩天以去除腸道中殘留的抗生素,接著和'Old1'組一起用HDI注射10微克的HBV表現質體溶在百分之八體重重量的PBS中。另一個''Young1'組則是飲用正常的飲用水,在六周大時用HDI注射10微克的HBV表現質體溶在百分之八體重重量的PBS中。每一週的體重及飲用水的重量都有被記錄以監測小鼠的健康狀況,且糞便及血清也都是每週採集。 實驗結果顯示,'Old1'組在HDI後七週內就將血清中的表面抗原完全清除,並且產生比其他兩組顯著的更高的表面抗體,並且他們也有最低的e抗原陽性率及最高的e抗體陽性率。“Young1”組的小鼠在HDI七周後還有90%的表面抗原陽性率,並且他們也有最高的e抗原陽性率及最低的e抗體及表面抗體陽性率。”OA1”組的小鼠在HDI七週後表面抗原陽性率還有六成,表面抗體的值介於兩組之間,但比較靠近”Young1”組。e抗原及e抗體的陽性率則都介於其餘兩組之間。雖然”OA1”組的小鼠對於HBV的免疫反應介於其他兩組之間,其組內的變異卻很大。有一群表現得像”Old1”而另外一群表現得像”Young1”組。另外我也用定量聚合酶連鎖反應(Quantitative PCR)測量了血清中HBV-DNA的濃度,結果顯示在HDI後八週之內”Young1”組的值都最高,其次是”OA1”組,最低的是”Old1”組。 另一方面,我也針對16s rRNA中的V3區域來量化腸道菌的變化。”Old1”和”Young1”組的腸道中真細菌的量在整個實驗過程中都維持穩定。在”OA1”組中,腸道中真細菌的量在抗生素開始給予一週後就降到同時間”Old1”組的量的1%,而在抗生素停止給予之後,腸道菌的量在幾周內就快速的增加。我也用時象溫度凝膠梯度電泳(TTGE)來利用V3區域中的GC含量來分析腸道菌相。實驗結果顯示C3H/HeN小鼠的腸道菌相大約在出生後八到九週達到平衡。除此之外,在同一個組的個體之間雖然比較暗的片段有一些個體變異,主要比較亮的主要片段大致相同。腸道菌相在抗生素開始給予後的兩週內減少並且達到一個新的平衡菌相,在抗生素停止給予後,腸道菌相在五周內都還呈現動態。再重複的實驗中,我也得到相似的結果。 以上的實驗結果顯示腸道菌的菌相及菌量確實受到了抗生素的影響,且這些改變也很可能是造成”OA1”組清除表面抗原顯著的比”Old1”組慢的原因。我們還需要更多資料才能分析出造成此現象的關鍵菌種,以及它所影響的主要免疫因子。 | zh_TW |
dc.description.abstract | The chance of becoming a chronic carrier of hepatitis B virus (HBV) strongly related to the age of exposure to the virus. For those who acquire the infection perinatally or < 1 year old, the chance of becoming chronic carrier is over 90%; the chance drops to 30% for children of 1 -5 years old; and the incidence is lower than 5% when infected during adulthood. The reason for this phenomenon is not yet clear.
In a pilot study using hydrodynamic injection (HDI) to transiently transfect HBV into mouse hepatocytes, we found that rate of serum HBsAg clearance in C3H/HeN mice of 12-week-old was significantly faster than the mice of 6-week-old, which resembles the HBV infection in humans. It has been shown that many aspects of innate and adaptive immunities are influenced by gut- microbiota, such as gut-associated lymphoid structure, the number of special lymphocytes in lamina propria of gut, and the cytokine secretions. We hypothesized that the different outcomes of HBV transfection are due to different immunities of C3H/HeN at 6- and 12-week old, which, in turn, are influenced by their gut-microflora compositions. To test this hypothesis, I divided C3H/HeN mice into three groups: the “Old1” group who had normal autoclaved drinking water throughout the experiment; “OA1” group who received antibiotics cocktail [Ampicillin (1g/L), neomycin (1g/L), vancomycin (0.5g/L), and metronidazole (1g/L)] in drinking water from 5 to 12 weeks old. At the end of antibiotics treatment, 'OA1' group received two days of normal water to remove residual antibiotics in the gastrointestinal tract. And both “Old1” and “OA1” received HDI of 10ug pAAV/HBV1.2 in 8% body weight of PBS at the same time. Additional group “Young1” had normal drinking water and received HDI of 10ug pAAV/HBV1.2 in 8% body weight of PBS at 6 weeks old as a control. Body weight and the amount of drinking water consumption were recorded weekly to monitor the health condition of each group and stool and serum sample were collected for further examinations. All “Old1” mice cleared serum HBsAg 7 weeks after HDI, and they produced significantly higher anti-HBs than the other two groups. They also showed the lowest HBeAg positive rate and the highest anti-HBe positive rate among the three groups. Serum HBsAg was still positive in 90% of “Young1” mice 7 weeks after HDI. They also had the highest HBeAg positive rate and the lowest positive rates of anti-HBe and anti-HBs. The “OA1” mice had 60% HBsAg persistent rate 7 weeks after HDI, and their anti-HBs level lies between groups “Old1” and “Young1” but closer to the latter. They also showed intermediate HBeAg positive rate and anti-HBe positive rate in the three groups. It is noted that “OA1” mice showed a large within-group variance. One subgroup of them performed like group “Old1”, while the other subgroup performed like group “Young1”. I also measured the amount of HBV-DNA in the serum using quantitative PCR, and “Young1” mice had the highest value among three groups in all time points within 8 weeks after HDI, followed by “OA1”, and “Old1” mice, respectively. The V3 region of bacterial 16s rRNA was targeted to quantify the changes of gut microbiota. The amount of eubacteria remained stable during the experimental course in groups “Old1” and “Young1”. In group “OA1”, the amount of gut eubacteria showed 100-fold reduction one week after the beginning of antibiotic treatment. After the cessation of antibiotics, the amount of gut microbiota increased dramatically in few weeks. TTGE (Temporal-temperature gradient gel electrophoresis) was conducted to investigate the gut microbiota profiles according to their GC content of V3 region. The result showed that the gut microbiota of C3H/HeN mice reached equilibrium state in approximately 8 to 9 weeks in “Old1” mice. While some minor band differences existed, the major bands were approximately the same among individuals. In “OA1” group, the gut microbiota decreased after the beginning of antibiotic treatment and reached a new equilibrium in two weeks. After the cessation of antibiotics, the gut microbiota profile changed dynamically even after 5 weeks. Similar results were obtained in a repeated experiment. These data showed that the amounts and patterns of gut bacteria were affected by antibiotic treatment. It is likely that these changes contributed to the delayed clearance of serum HBsAg in group “OA1” compared to group “Old1”. More data will be needed to get more insight on the key microbiota that resulted in different serum HBsAg clearance rate, and the immune factors that were responsible. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T13:02:16Z (GMT). No. of bitstreams: 1 ntu-102-R99445119-1.pdf: 2796062 bytes, checksum: 40df446e337fdd8c6257a01e187b3d02 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 口試委員會審定書……………………………………………………………………………………………………i
誌謝…………………………………………………………………………………………………………………………ii 中文摘要…………………………………………………………………………………………………………………iii Abstract…………………………………………………………………………………v Index….……………………………………………………………………………………………………………….viii 圖次及表次………………………………………………………………………………………………………………x Chapter I:Introduction 1.1 Introduction to HBV ……………………………………………………….....1 1.2 The natural infection history of HBV………………………………………..2 1.3 Current research models of HBV ……………………………………………3 1.4 The effect of gut microbiota on immune system………………………….....4 1.5 The effect of antibiotics on the shaping of gut microbiota……………….....7 1.6 Hypothesis and experiment goal……………………………………………..8 Chapter II:Material and Method 2.1 Animals……………………………………………………………………….10 2.2 Experimental design…………………………………………………………10 2.3 Hydrodynamic injection and serum collection…………………………….10 2.4 PCR and Temporal-temperature gel electrophoresis (TTGE) …………...12 2.5 Real-time (RT)-PCR…………………………………………………………13 2.6 Northern blotting…………………………………………………………….13 2.7 Enzyme-linked immunosorbent spot…………………………………….....14 2.8 Data analysis…………………………………………………………………15 Chapter III:Results 3.1 Body weight change and water consumption after antibiotics treatment.15 3.2 Effects of antibiotics on the amount of stool eubacteria……………….…16 3.3 Effects of antibiotics on serum HBsAg clearance and Anti-HBs production …………………………………………………………………………..……16 3.4 Effects of antibiotics on serum HBeAg clearance rate and Anti-HBe……… production…………………………………………………………………...17 3.5 HBV-DNA in serum ………………………………………………………..18 3.6 TTGE results 3.6.1 Establishment of mouse gut microbiota profile by age…………..19 3.6.2 Effect of antibiotics on gut microbiota profile……………………19 3.6.3 Variation in gut microbiota profile between individuals and……… between cages within group…………………………………….….20 3.6.4 Effect of hydrodynamic injection of gut microbiota profile……..21 3.7 Northern blot for HBV transcript in liver and ELISPOT for activated…… T-cells in spleen………………………………………………………………21 Chapter IV:Discussion 4.1 Antibiotics cocktail treatment caused dramatic effect to the immune……… response against HBV in mice. …………………………………………………..22 4.2 Antibiotics cocktail treatment shifted the gut microbiota profile of mice....24 4.3 The body weight decreased dramatically after the consumption of antibiotic cocktail. ………………………………………………………………………..…..25 4.4 Modified hypothesis. ……………………………………………………….…26 References……………………………………………………………………………...28 Figures…………………………………………………………………………….…...38 Tables…………………………………………………………………………….…….65 | |
dc.language.iso | en | |
dc.title | 腸道菌對於B 型肝炎病毒免疫感受性之影響 | zh_TW |
dc.title | Age-dependent Clearance of HBV: The Role of Gut Microbiota | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王弘毅(Hurng-Yi Wang),楊宏志(Hung-Chih),陶秘華(Mi-Hua Tao) | |
dc.subject.keyword | B 型肝炎病毒,腸道菌,免疫,抗生素,尾靜脈動力注射, | zh_TW |
dc.subject.keyword | Hepatitis B virus,gut microbiota,immune system,antibiotics,hydrodynamic injection, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-06 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-102-1.pdf 目前未授權公開取用 | 2.73 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。