催產素受體於斑馬魚離子調節機制中扮演之角色

Roles of isotocin receptors in zebrafish ion regulation mechanisms

甘馨惠(Hsin-Hui Kan)

指導教授 ： 黃鵬鵬

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02406

摘要

過去研究指出神經荷爾蒙催產素(oxytocin)在哺乳類腎臟參與離子及水分調節機制，且其同源基因isotocin 已被證實在魚類離子調節中扮演重要角色。然而，在催產素調節表皮細胞分化進而影響魚類離子和滲透壓的這個模式中，其受體所扮演的角色尚未被瞭解。本實驗目的為利用分子生物學、生化及生理學的方法，研究催產素受體在斑馬魚離子運輸及滲透壓平衡所扮演的角色為何。
　　以催產素反義核酸抑制催產素蛋白質的合成後，表皮幹細胞、離子細胞(富含鈉鉀氫離子幫浦細胞與富含氫離子幫浦細胞)和表皮細胞數量都顯著地減少。此外，以注射cRNA 方式過量表現催產素基因，發現表皮幹細胞數量也隨之增加。此實驗證實催產素藉由調控幹細胞進而影響離子細胞的分化。為瞭解催產素與其受體的關係，抑制催產素基因後，發現其第一型受體mRNA 表現量會顯著下降，但第二型受體mRNA 表現量會顯著上升；此實驗說明催產素第一、二型受體作用
相反。將催產素受體用其反義核酸分別抑制其基因表現後，發現第一型受體的基因被抑制時，表皮幹細胞、離子細胞與表皮細胞數量都顯著上升；但在催產素第二型受體的基因被抑制後，所有表皮細胞數量的結果都與第一型受體被抑制的結果相反。且注射cRNA 方式過量表現催產素第一、二型受體，其幹細胞數量都可得到與抑制基因表現相反的結果。這些實驗證實催產素第一、二型受體藉由調控幹細胞進而影響離子細胞的分化，且兩者作用相反。
　　為了更進一步探討這兩型受體間的關係，從共同注射實驗發現同時抑制兩個受體的功能會使幹細胞、離子細胞、表皮細胞和黏液細胞數量與控制組沒有顯著差異；同時抑制催產素第一型及過量表現第二型受體都會使幹細胞數量上升；在同時抑制第二型及過量表現第一型受體時，幹細胞數量會減少。這些實驗更進一步證實，這兩型催產素受體在魚類上調控離子細胞的功能互相牽制。
　　黏液細胞數量在催產素基因被抑制時上升，但在此二受體分別被抑制時下降。這個結果顯示黏液細胞的分化不同於目前已知的幹細胞－離子細胞途徑，推測有其他因子調控黏液細胞的分化。
　　綜上所述，催產素及其受體在斑馬魚表皮細胞及離子細胞功能發展上扮演重要角色。催產素第一型受體對表皮細胞分化為負向調控、催產素第二型受體為正向調控；他們的作用完全相反，藉由這樣的迴饋機制影響離子細胞及表皮細胞的數量，進而調節硬骨魚類體內的離子及滲透壓平衡。本研究提供一個新平台瞭解催產素第一、二型受體在魚類表皮細胞的發育所扮演的角色。

關鍵字

催產素 ； 催產素受體 ； 離子調控 ； 迴饋機制 ； 細胞分化

並列摘要

The mammalian oxytocin homolog in teleost fish, isotocin (itnp), was previously shown to play a major role on the proliferation of epidermal stem cells. However, the involvement of isotocin receptor in the isotocin control pathways of fish ionic and osmotic regulation through the epidermal ionocytes is unknown. In the present study, the roles of isotocin-isotocin neurophysin receptor (itnpr) axis in the epidermal cells development were elucidated using zebrafish as the animal model.
Loss of function assay using morpholino oligos showed that itnp morphants significantly decreased epidermal stem cells (p63-positive cell), matured ionocytes (Na+-K+-ATPase-rich cells: NaRC and H+-ATPase-rich cells: HRC), and keratinocytes numbers significantly decreased. In addition, itnp cRNA over-expression increased epidermal stem cells number. These show profound effects of itnp in epidermal
development through the stem cell. Using quantitative PCR, itnpr1 mRNA decreased significantly in the itnp morphants cDNA templates, but itnpr2 increased. This indicates
itnpr1 and 2 have opposite functions. As confirmed by the knockdown, itnpr1 morphants showed significant increase in stem cells, matured ionocytes and keratinocytes number, whereas loss of itnpr2 has reverse effects. Gain of function
showed that itnpr1 cRNA and itnpr2 cRNA-injected embryos reversed the results in MO injection alone with stem cells results. These data demonstrated that itnpr1 and 2 play opposite roles in fish epidermal development.
To further understand the function of these receptors, series of co-injection experiments were performed. Double knockdown of itnpr1 and r2 showed no significant
effect to the control morphants. The density of stem cell increased in itnpr1-MO plus r2-cRNA but decreased in itnpr2-MO plus r1-cRNA-injected embryos. These data further demonstrated that these two receptors play opposite role and restrict each others actions.
In case of mucous cells, itnp morphants showed increased in density, but decreased in both of receptors morphants. These may show that other factors influences mucous cell development independent from the known stem cell lineage pathway.
Altogether, itnp-itnprs system shows to play an important role in zebrafish epidermal cell development. The effect of itnpr1 (negative) is in contrast to itnpr2
(positive) on regulating epidermal cells number. These opposite roles could be a feedback regulation, which is a physiological need to facilitate proper epidermal cell
development and maintain the iono/osmo balance in zebrafish. Hence, this study provides new knowledge and platform to better understand the specific roles of itnpr1
and r2 in fish epidermal development.

並列關鍵字

oxytocin ； isotocin ； oxytocin receptor ； isotocin receptor. ionoregulation ； feedback regulation ； cell proliferation

參考文獻

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