Klotho是一個由990個胺基酸組成的第一型穿膜蛋白 (Type-I membrane protein) 在人類基因組的編碼為KL，Klotho基因會轉譯出一種單通道的穿膜蛋白，此種蛋白會與許多 Fibroblast growth factor 結合並作為其co-receptor 輔助與其相應之受體結合，Klotho與衰老(aging)也有相當程度的關聯性，Klotho 突變的小鼠相較於野生型小鼠壽命是相對較短的，大約只有野生型小鼠壽命的5% 左右，並且有提早出現類似人類衰老相似的徵狀或疾病，例如貧血 (anemia)、動脈硬化 (arteriosclerosis)、骨質酥鬆 (osteoporosis)、皮膚老化……等等，但是目前在各個物種上針對Klotho 在早期發育的方面研究資訊並不充足，目前僅能得知Klotho 缺陷的老鼠在早期未發現任何phenotypes，不過我們推測也許是因為小鼠為哺乳類動物，胚胎早期發育在子宮內不易觀察、亦或是帶嚴重缺陷的無法發育而直接成死胎流產。因此可藉助斑馬魚早期胚胎易於觀察之特性，來觀察Klotho對於早期胚胎發育的影響。
  在先前文獻中Klotho在早期胚胎發育時期的表現位置，可以看到在yolk syncytial layer有訊號表現 (Lin CH, 2017)，另外在神經管的位置亦有Klotho的訊號表現 (Steve M, 2012)。故猜測klotho會影響胚胎早期cell migration以及神經發育。根據實驗結果，利用幾個針對早期胚胎cell migration的marker來做ISH，可以觀差到在knockdown klotho後，胚胎cell migration會有異常。再來透過anti-acetylated tubulin抗染的結果可以看到斑馬魚胚胎的背部neural crest cell及神經突觸有明顯的減少。

Klotho is type-I membrane protein which has 990 amino acids. Klotho protein will binds to many fibroblast growth factor and acts as its co-receptor to bind its corresponding receptor. Klotho is also an aging-related protein, but little is known about the mechanisms by which Klotho controls early embryonic development.
  According to previous study, we can see that Klotho express at yolk syncytial layer (Lin CH, 2017), and the neural tube (Steve M, 2012). So we hypothesize that klotho will effect embryo cell migration and neurodevelopment. In this study, we use morpholino to knockdown Klotho. In our data, whole mount in situ hybridization showed that the expressions of bmp4, gsc, flh and eve1 genes were affected. And the anti-acetylated tubulin antibody IHC showed that dorsal neural crest cell and nerve has significant reduction.

序論---------------------------------------------------01
Klotho 介紹--------------------------------------------01
Klotho 的生理功能---------------------------------------02
Klotho 的動物實驗---------------------------------------04
Klotho 在斑馬魚上的研究---------------------------------06
斑馬魚的 klotho基因表現位置----------------------------07
斑馬魚的神經發育 ---------------------------------------07
斑馬魚的胚胎發育優勢 -----------------------------------08
材料與方法----------------------------------------------11
斑馬魚-------------------------------------------------11
顯微注射-----------------------------------------------12
轉型作用-----------------------------------------------12
大腸桿菌培養-------------------------------------------13
質體抽取-----------------------------------------------13
聚合酶連鎖反應-----------------------------------------14
DNA純化-----------------------------------------------15
合成RNA探針-------------------------------------------16
原位雜交法---------------------------------------------17
RNA萃取-----------------------------------------------19
反轉錄聚合酶連鎖反應-----------------------------------20
即時聚合酶連鎖反應-------------------------------------20
抗體染色----------------------------------------------21
結果---------------------------------------------------24
顯微注射Klotho Morpholino對胚胎造成的影響---------------24
注射不同濃度之Klotho MO後的存活率 ----------------------25
以原位雜交法觀察klotho被抑制後對胚胎發育的影響------------27
注射klotho MO後對於神經發育基因表現量的影響--------------32
注射klotho MO後對神經生長的影響-------------------------34
斑馬魚胚胎各時期神經發育與marker時間比對-----------------36
討論--------------------------------------------------37
Knockdown klotho後造成胚胎發育畸形---------------------37
以原位雜交法觀察注射klotho MO對於胚胎發育的影響 --------37
假設 knockdown klotho會造成胚胎神經發育的異常----------38
以Q-PCR比較Knockdown klotho神經發育相關基因的表現量----38
以Anti-aat免疫螢光染色觀察Knockdown klotho後對於神經發育的影響---------------------------------------------39
參考資料---------------------------------------------40
附錄-------------------------------------------------45

圖表目錄
Fig. 1	Klotho morpholino 與 Klotho 序列比對	P.12
Fig .2	胚胎注射完 klotho MO 後的 phenotypes	P.24
Fig .3	注射各劑量 MO之存活率	P.25
Fig. 4	bmp4在9 hpf時的表現位置	P.29
Fig. 5	eve1在9 hpf時的表現位置	P.29
Fig. 6	gsc在9 hpf時的表現位置	P.30
Fig. 7	flh在9 hpf時的表現位置	P.31
Table. 1	Q-PCR 所使用基因之生理功能	P.32
Fig. 8	Q-PCR 基因表現量之比較	P.33
Fig .9	配胎注射 klotho MO後背部神經元減少	P.34
Fig. 10	神經元數量統計	P.34
Fig. 11	胚胎注射 klotho MO後神經生長較少且較短	P.35
Fig. 12	神經數量統計	P.35
Fig. 13	胚胎各stage 之神經發育	P.36
Fig. 14	實驗中所使用之marker所表現之時間	P.36
Table. 2	Q-PCR 之 primer序列	P.45
Table. 3	注射 klotho MO後的存活率統計	P.45
Table. 4	神經元及神經生長畸形率	P.46
Table. 5	Plasmid 清單	P.46

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