臺灣博碩士論文加值系統

Muscleblind-like (MBNL)是一群參與調控特定組織所需差異性剪輯的蛋白家族。在果蠅中，muscleblind蛋白參與了肌肉及感光受器的末端分化調控。在人類及小鼠中都具有3種MBNL的paralogs。先前我們實驗室已在斑馬魚選殖出其3種mbnl基因(zmbnl1-3)以研究其在斑馬魚早期發育所扮演的角色。在本篇論文主要集中於zmbnl3的相關研究。藉由pre-mRNA的差異性剪輯，zmbnl3至少可轉譯出5種不同的蛋白質異構物。zmbnl3除了含有MBNL典型的CCCH zinc finger domains，也含有其他特殊的保留區域(如LEV box, NGR box, Ser/Thr-rich domain)。zmbnl3廣泛表現於成魚組織，但不同的isoforms在各組織的表現量不盡相同。在胚胎發育時期，zmbnl3在24hpf之後才漸漸出現。利用全胚體原位雜交技術觀察(WISH)到zmbnl3在胚胎中呈現廣泛性表現。利用antisense morpholino knockdown zmbnl3，並以不同marker gene(wnt1, tnnt2, anxa5)進行WISH，及分析其可能的下游基因clc-1 and tnnt2，並沒有觀察到與WT明顯的差異。但藉由alcian blue staining發現 zmbnl3 morphants其咽喉鰓弓的軟骨發育有異常的情況，且在48hpf之後，myoD表現量較WT下降。另一方面，顯微注射zmbnl3 cRNA發現其異常表型主要為體軸彎曲及體節縮短。隨著施打劑量增加，亦發現畸形率有dose-dependent的情況。利用頭部及肌肉發育相關marker(wnt1, pax2.1, myoD, myogenin)進行WISH，發現過量表達zmbnl3的胚胎腦部發育受到影響且體節排列嚴重異常。但以RT-PCR分析其可能下游基因(clc-1, myoD, myogenin,)並沒有發現改變。將zmbnl3過量表達於C2C12細胞，可發現zmbnl3會抑制肌肉細胞的分化(如:缺乏融合的多核肌纖維細胞、mef2a 剪輯形式停留在分化前及MHC蛋白表現量下降)。由Dual-luciferase reporter assaye更發現了在斑馬魚胚胎中，zmbnl3可以降低myoD promoter的活性。综合以上結果，我們推測zmbnl3藉由MyoD-dependent途徑來干擾肌肉細胞進行分化。

Muscleblind-like (MBNL) is a family of proteins that participate in regulation of tissue-specific alternative splicing. In Drosophila, the muscleblind protein was shown to regulate terminal differentiation of photoreceptors and muscles. Three MBNL paralogs have been identified in humans and mice. Previously, we cloned three mbnl genes (zmbnl1 – 3) in zebrafish in order to study their function during fish development. In this study, we focus on zmbnl3. Alternative splicing of the zmbnl3 primary transcripts gives rise to at least 5 protein isoforms. In addition to the characteristic CCCH zinc finger domains, several structural motifs (like LEV box, NGR box, Ser/Thr-rich domain) are also found conserved in zmbnl3. Zmbnl3 is expressed in most adult tissues although the expression of specific spliceforms varies. During embryogenesis, zmbnl3 transcripts are hardly detected until 24hpf. Whole-mount in situ hybridization (WISH) reveals that zmbnl3 expression in the embryo is more ubiquitous rather than specific. zmbnl3 morphants of antisense morpholono knockdown were examined by WISH analysis of several marker genes (wnt1, tnnt2, anxa5), as well as by RT-PCR analysis of the splicing patterns of its suspected targets, clc-1 and tnnt2. The morphants did not show clear difference from WT embryos. However, alcian blue staining revealed overt defects in the pharyngeal arches of zmbnl3 morphants, and myoD expression was also decreased. On the other hand, microinjection of zmbnl3 cRNA into the embryos resulted in defective embryos with crooked body axes and short somites. The defective rate of the injected embryos increased in a dose-dependent manner. WISH analysis of several marker genes (wnt1, pax2.1, myoD, myogenin) revealed that embryos overexpressed with zmbnl3 had disorganized somite formation and abnormal brain development. RT-PCR analysis indicated that the splicing patterns of clc-1 and expression levels of myoD and myogenin were not changed. When introduced into C2C12 cells, zmbnl3 inhibited cell differentiation as judged by lack of cell fusion, change of mef2a splicing pattern and reduction of MHC expression. Dual-luciferase reporter assay further revealed that zmbnl3 down-regulated myoD promoter activity in fish embryos. These data suggest that zmbnl3 may interfere with muscle differentiation through the MyoD-dependent pathway.

目錄
中文摘要-------------------------------------------- p.01
英文摘要-------------------------------------------- p.03
序論
Muscleblind protein 基本結構---------------------- p.6
Muscleblind之基因表現及蛋白質功能--------------- p.9
Muscleblind與末端肌肉細胞分化 ------------------ p.12
Muscleblind與疾病 ------------------------------ p.15
動機 ----------------------------------------------- p.17
材料與方法
斑馬魚的飼育與胚胎操作 -------------------------- p.18
1.斑馬魚(Danio rerio的飼養) -------------------- p.18
2.胚胎取得與保存 ----------------------------- p.19
斑馬魚zmbnl3基因之選殖及建構 -------------------- p.20
1.生物資訊序列比對 --------------------------- p.20
2. RACE-PCR --------------------------------- p.21
3. Total RNA萃取 ----------------------------- p.23
4. RT-PCR ------------------------------------ p.24
5.不同物種同源性胺基酸序列比對 --------------- p.25
全胚體原位雜交技術 ---------------------------- p.25
顯微注射 -------------------------------------- p.31
1.morpholino注射 --------------------------- p.31
2.cRNA注射 ------------------------------- p.32
全胚體軟骨染色 -------------------------------- p.34
抗體製備 -------------------------------------- p.35
1.構築重組pET-29b(+)-zmbnl3C基因表現質體 ---- p.35
2. 6xHis-tagged zmbnl3C重組蛋白的表現與純化 --- p.36
3. 6xHis-tagged zmbnl3C重組蛋白之免疫注射 ----- p.39
細胞的培養 -------------------------------------- p.40
微脂粒之基因轉染 -------------------------------- p.41
蛋白質的表現 ------------------------------------ p.42
1.total protein萃取 ---------------------------- p.42
2.蛋白質之定量 ------------------------------ p.43
3.SDS-PAGE --------------------------------- p.44
4.西方點墨分析 ------------------------------ p.45
Dual-Luciferase Reporter Assay --------------------- p.47
結果
一、zmbnl3之選殖和序列分析 ---------------------- p.50
二、zmbnl3基因的表現 --------------------------- p.51
三、顯微注射antisense morpholino， zmbnl3 knock down 後的表
型觀察 -------------------------------------- p.52
四、zmbnl3沒有直接參與Clc-1及Tnnt2的splicing調節- p.53
五、zmbnl3 knock down的胚胎在midbrain hindbrain boundary、
心臟和魚鰾並無發現發育上的異常---------------- p.54
六、zmbnl3 knock down的胚胎有頭部咽喉鰓弓(pharyngeal arch)
之軟骨構造發育上的異常 ---------------------- p.55
七、zmbnl3 knock down對 myoD表現量的影響 ------- p.55
八、zmbnl3過量表達後之表型觀察 ------------------ p.56
九、過量表達zmbnl3對於肌肉發育的相關marker及Clc-1 splicing
的影響 ---------------------------------------- p.57
十、過量表達zmbnl3造成胚胎體節不正常排列 ------- p.58
十一、過量表達zmbnl3造成胚胎腦部發育上的異常 --- p.59
十二、zmbnl3會抑制C2C12細胞分化 --------------- p.59
十三、zmbnl3能抑制myoD promoter的表現 ---------- p.61
十四、zmbnl3的抗體製備 -------------------------- p.62
討論
一、zmbnl3基因序列及特殊功能區在脊椎動物中具有高度保
留性 ---------------------------------------- p.64
二、zmbnl3基因與脊椎動物發育時期表現之差異性 ---- p.65
三、zmbnl3 loss/gain of function之討論 --------------- p.67
四、zmbnl3與myoD promoter之關係 ---------------- p.70
圖表 ------------------------------------------------ p.72
參考文獻 -------------------------------------------- p.93
附圖 ------------------------------------------------ p.98
附表 ------------------------------------------------ p.108
圖與表次
圖一.經選殖、定序及BLAST分析，建構 zmbnl3的基因結構- p.72
圖二.將zmbnl1和zmbnl2, zmbnl3和其他物種muscleblind家族的分
子演化樹(phylogenetic tree)關係圖。----------------- p.73
圖三.將zmbnl3和其他物種mbnl3家族胺基酸序列比對圖。-- p.74
圖四.利用RT-PCR分析zmbnl3在成魚組織的分布情況及胚胎時期的
表現。----------------------------------------- p.75
圖五.利用全胚體原位雜交技術(whole-mount in situ hybridization)觀
察zmbnl3在胚胎發育時間和空間上的表現。---------- p.76
圖六.zmbnl3 morphants於24 hpf至120 hpf時期的表型。---- p.77
圖七.利用RT-PCR分析zmbnl3 knockdown之後並沒有對clc-1及tnnt2
的splicing剪輯上的產生異常。--------------------- p.78
圖八. zmbnl3 knock down morphants在midbrain hindbrain boundary、
心臟和魚膘並無發現明顯發育的異常。-------------- p.79
圖九. zmbnl3 morphants部分有頭部咽喉鰓弓(pharyngeal arch)之軟骨
構造發育上的異常。------------------------------- p.80
圖十.利用RT-PCR分析zmbnl3 knockdown morphants之myoD表現。
----------------------------------- p.81
圖十一.利用RT-PCR分析overexpression zmbnl3B及zmbnl3C之後，
發現對於肌肉發育相關marker(myoD、myogenin)及調控clc-1
的splicing並無明顯影響。----------------------- p.82
圖十二.過量表達zmbnl3造成胚胎發育上體節不正常排列。-- p.83
圖十三.過量表達zmbnl3造成胚胎體節不正常排列。-------- p.84
圖十四.過量表達zmbnl3造成胚胎腦部發育上的異常。------ p.85
圖十五.利用C2C12肌肉纖維母細胞進行pCS2+/ pCS2+-zmbnl3B和
pEGFP-N3 co-transfection實驗觀察zmbnl3B對於C2C12細 胞分化型態的影響。---------------------------- p.86
圖十六.利用RT-PCR及西方點墨法分析zmbnl3B對於C2C12細胞分
化指標基因表現的影響(co-transfection實驗)。------ p.87
圖十七.利用抗生素篩選使C2C12肌肉纖維母細胞大量表達進行
zmbnl3B-pEGFP-N3以觀察zmbnl3B對於C2C12細胞分化型
態的影響。------------------------------------- p.88
圖十八.利用RT-PCR及西方點墨法分析zmbnl3B對於C2C12細胞分
化指標基因表現的影響(enrich實驗)。------------- p.89
圖十九.zmbnl3能抑制myoD promoter的表現。------------ p.90
表一. zmbnl3 knockdown morphants 表型統計表。---------- p.91
表二. zmbnl3 ovexpression morphants 表型統計表。--------- p.92
附圖一.建構zmbnl3基因結構時所使用的TA-cloning質體，pGEM-T
Easy vector (promega)。--------------------------- p.98
附圖二.zmbnl3 cRNA製作時所用之質體pCS2+。----------- p.99
附圖三.蛋白純化所利用之質體pET-29b(+)。-------------- p.100
附圖四.建立最佳誘導帶有pET-29b(+)-zmbnl3C質體之大腸桿菌株
BL21表現zmbnl3C-6xHis重組蛋白質之條件。------- p.101
附圖五-a. zmbnl3C-6xHis重組蛋白純化結果。-------------- p.102
附圖五-b. zmbnl3C-6xHis重組蛋白純化結果。------------- p.103
附圖五-c. 利用西方點墨法檢測所得之蛋白純化結果帶有
zmbnl3C-6xHis之重組蛋白。------------------- p.104
附圖六. 利用西方點墨法檢測兔子血清帶有Anti-zmbnl3C-6xHis之抗
體。------------------------------------------ p.105
附圖七.建構pEGFP-N3-zmbnl3B質體以進行大部分表達(enrich)
zmbnl3B-EGPF蛋白對於C2C12肌肉纖維母細胞分化的影響。
-------------------------- p.106
附圖八.Dual-Luciferase reporter assay實驗所用質體map圖。-- p.107
附表一. RT-PCR所用之primer列表 (zmbnl3)-------------- p.108
附表二.RT-PCR所用之primer列表 (zebrafish)------------ p.109
附表三. RT-PCR所用之primer列表 (mouse – C2C12)------ p.110
附表四.全胚體原位雜交所使用之probe列表 -------------- p.110

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