臺灣博碩士論文加值系統

自分泌移動因子受器(AMFR)為分子量78 kDa的醣蛋白，因此又稱為gp78。
AMFR為多功能的蛋白質，當位在細胞表面時，會做為細胞激素的受器，透過與配體的結合來刺激細胞的移行。位在內質網膜上時，則扮演E3汎素蛋白接合酵素( E3 ubiquitn ligase)的角色， 協助內質網膜上的蛋白質降解作用。AMFR在臨床研究上，常做為癌症預後的指標，但目前對於AMFR在胚胎發育的功能與機制尚不明瞭。因此我們選擇斑馬魚做為研究的動物，主要分析在斑馬魚中amfr在胚胎發育早期的功能及作用機制為何。利用全覆式定位雜交來偵測amfr於胚胎發育早期的基因表現，在胚胎受精後3.3小時，amfr表現於卵黃融合層中(yolk syncytial layer, YSL)中，一直到原腸晚期都可觀察到amfr表現於YSL上，由於YSL在早期胚胎發育扮演重要的角色，因此推測amfr與YSL的功能上具有關聯性。利用反義股morpholino RNA抑制斑馬魚胚胎amfr基因的表現，造成缺陷表現型的產生。除了在原腸時期的外包(epiboly)有遲緩的情形外，在器官發育也有圍心腔腫大、腦發育不全、肌肉畸型、尾部彎曲以及卵黃囊延伸萎縮等現象。隨著注射劑量的提升，缺陷表現型的比例也會隨之升高。接著研究外包的遲緩是否會影響三胚層的形成，造成晚期發育時器官的各種缺陷。藉由全覆式定位雜交分析中內胚層(gsc)、中胚層(ntl)及內胚層(sox32)的標記(marker)，觀察amfr基因表現被抑制的胚胎發育情形。結果顯示amfr基因表現被抑制的胚胎，會導致中內胚層、中胚層及內胚層細胞的移行遲緩。進一步探討造成外包遲緩的機制為何，利用微小管/小管素分析套組進行分析，經過紫杉醇處理與amfr基因表現被抑制的胚胎，發現微小管的含量明顯高於野生型的胚胎，小管素則無明顯的差異性。再將胚胎進行免疫組織染色，經過紫杉醇處理過的胚胎，發現有緻密的束狀卵黃細胞微小管的形成，而受到amfr基因表現抑制的胚胎也有相似的情形。我們的結果顯示，amfr在斑馬魚早期胚胎發育，表現於YSL且對於原腸期外包的進行及三胚層的生成皆有重要性，並且具有促進微小管解聚合的能力。

Autocrine motility factor receptor (AMFR) is a glycoprotein originally named gp78. AMFR/gp78 plays a dual role as a cytokine receptor on the cell surface or as an E3 ubiqui¬tin ligase involved in endoplasmic reticulum associated degradation (ERAD). AMFR has important clinical implication, which has prognostic value in clinical cancer research. However, the exact function of AMFR in embryo¬nic development remains un¬clear. Using in situ hybridization to detect amfr mRNA in zebrafish embryos, we found that amfr mRNA was localized in blastomere and yolk syncytial layer (YSL) at mid-blastula stage. The amfr expression patten is correlated with the development of YSL from mid-blastula to late gastrulation. YSL appears to play an important role in the formation of 3 germ layers, therefore we pro¬pose that amfr is essential to the function of YSL. Antisense morpholino knock¬down of amfr pro¬duced defective phenotypes and caused epibolic delay in zebrafish embryos. Defective phenotypes of amfr knockdown embryos include bent tail, pericar¬dial edema, malformed muscles, cerebral hypoplasia and abnormal yolk cell extension. The percentage of defective phenotypes increased as the dosage of amfr antisense morpholino injection increased. We found that amfr morphant with epib¬oly delay also exhibited abnormalities in mesoendodermal, mesodermal and endo¬dermal cell migration. To study the mechanism of epiboly delay caused by amfr knockdown, we analyzed the effect on microtubule by assessing microtubule and tubulin contents in amfr knockdown em¬bryos. Our analyses indicated that the con¬tent of microtubule increase in embryos treated with microtubule stabiliz¬ing agent taxol and in amfr knockdown embryos. Immunohistochemical analysis also showed that both amfr knockdown and taxol treatment produced dense and bundles of microtubule in yolk cytoplasmic layer. These results suggest that amfr knock¬down inhibits the depolymertization of yolk cell microtubule. We propose that amfr expressed in the YSL functions in the epibolic movement and formation of three germ layers.

目錄 …………………………………………………………………………… i

表目錄 ………………………………………………………………………… iv

圖目錄 ………………………………………………………………………… v

中文摘要 ……………………………………………………………………… 1

英文摘要 ……………………………………………………………………… 2


壹、 前言……………………………………………………………………… 4

一、AMFR/gp78蛋白質結構………………………………………………… 4

二、AMFR的配體…………………………………………………………… 4

三、AMFR於細胞中的功能…………………………………………………… 5

1. 細胞膜上的功能 ……………………………………………………… 5

2. 內質網膜上的功能…………………………………………………… 6

四、AMFR/AMF於惡性種瘤的相關性……………………………………… 8

五、AMFR在早期小鼠胚胎的表現………………………………………… 10

六、斑馬魚amfr在早期胚胎發育的基因功能……………………………… 10

1. 斑馬魚作為遺傳及發育的動物模式………………………………… 10

2. 斑馬魚amfr於早期胚胎發育的表現………………………………… 11

七、斑馬魚的卵黃融合層(Yolk syncytial layer, YSL) ……………………… 11

八、外包(Epiboly)相關機制…………………………………………………… 13

1. 微小管(Microtubule) ………………………………………………… 13

2. 緊密接合(Tight junction)……………………………………………… 14

3. 細胞移行(Cell migration)……………………………………………… 15

九、實驗目的………………………………………………………………… 15

貳、實驗材料與方法…………………………………………………………… 17

一、實驗動物：斑馬魚………………………………………………………… 17

二、核醣核酸探針的製備……………………………………………………… 17

1. 粹取總核醣核酸(Total RNA extraction)……………………………… 17

2. RT反應成cDNA……………………………………………………… 18

3. PCR反應……………………………………………………………… 18

4. 試管內轉錄反應(in vitro transcription)製作RNA探針……………… 19

三、全覆式定位雜交…………………………………………………………… 19

四、基因表現抑制實驗………………………………………………………… 21

1. 顯微注射(Microinjection)……………………………………………… 21

2. amfr反義股及控制股反義股的製備………………………………… 22

五、胎胎外包百分比的測量…………………………………………………… 23

六、微小管(Microtubulin)含量的分析………………………………………… 23

七、胚胎微小管免疫組織染色(Immunohistochemstry)……………………… 24

八、顯微鏡……………………………………………………………………… 26

參、實驗結果………………………………………………………………… 27

一、斑馬魚amfr基因表現於卵黃融合層…………………………………… 27

二、利用amfr morpholino抑制斑馬魚中amfr基因的表現………………… 27

1. 抑制斑馬魚胚胎amfr基因的表現，產生了缺陷表現型…………… 27

2. 提升amfr morpholino的注射劑量會增加缺陷表現型的百分比…… 27

3. 抑制amfr 基因表現產生三種缺陷表現型，Type I、Type II及
Type III………………………………………………………………… 28

4. amfr缺陷表現型大多為Type II以及Type III………………………… 29

5. 抑制amfr基因表現導致原腸期外包顯著遲緩……………………… 29

三、抑制amfr基因的表現影響了三胚層的形成…………………………… 30

1. 抑制amfr的基因表現會對原腸期中內胚層(mesoendoderm)細胞的移行造成影響………………………………………………………… 30

2. 抑制amfr的基因表現會對中胚層細胞的移行造成影響…………… 31

3. 抑制amfr的基因表現造成外包的遲緩，同時也會使內胚層細胞移行遲緩………………………………………………………………… 31

四、抑制amfr表現會影響YSN及微小管穩定性………………………… 32

1. 抑制amfr基因表現會抑制微小管的解聚合能力…………………… 32

2. 利用免疫組織染色觀察胚胎原腸晚期的微小管…………………… 33

3. 抑制amfr的基因表現會使卵黃細胞產生緻密的束狀微小管……… 33

肆、討論……………………………………………………………………… 35

伍、參考文獻………………………………………………………………… 40

表目錄

Table 1. Sequences of antisense morpholinos……………………………… 48

Table 2. Phenotypic analysis of amfr knockdown morphants……………… 49

Table 3. Embryos show significant decrease in epiboly percentage amfr knockdown morphants……………………………………………… 50

Table 4. Embryos treated with taxol and nocodazole displayed epiboly delay 51

Table 5. Quantitative measurement of the relative amount of microtubule present in the pellets of microtubule/tubulin assay………………… 52




圖目錄

Fig. 1. Whole mount in situ hybridization showing amfr mRNA expression in zebrafish early development……………………………………… 53

Fig. 2. Gene structure and antisense morpholinos of zebrafish amfr
(BC057411) ………………………………………………………… 54

Fig. 3. Morphological phenotypes of amfr knockdown embryos…………… 55

Fig. 4. amfr knockdown caused defective morphant phenotypes in a dosage dependent manner…………………………………………………… 56

Fig. 5. amfr knockdown caused epiboly delay in late gastrulation stage…… 57

Fig. 6. Three types of amfr knockdown morphant…………………………… 58

Fig. 7. Defective rates of three types of amfr knockdown morphants with different dosages……………………………………………………… 59

Fig. 8. amfr knockdown caused epiboly delay in a dose dependent manner… 60

Fig. 9. Zebrafish amfr morphants exhibit delay in the migration of mesoendodermal cells………………………………………………… 61

Fig.10. Zebrafish amfr morphants exhibit delay in the migration of mesodermal cells……………………………………………………… 62

Fig.11. Zebrafish amfr morphants exhibit delay in the migration of endodermal cell……………………………………………………… 63

Fig.12. Microtubule/tubulin assay reveals the increase of microtubule in amfr knockdown embryos………………………………………………… 64

Fig.13. Western blotting data of microtubule/tubulin assay quantified by density detection software from three separate experiments………… 65


Fig.14. Organization of yolk cell microtubules in wild type embryos stained by immunohistochemistry…………………………………………… 66

Fig.15. Knockdown of amfr increases the formation of dense and bundles of microtubules in yolk cell……………………………………………… 67

Supplementary Fig. S1………………………………………………………… 68

Supplementary Fig. S2………………………………………………………… 69

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