李明洋，劉乙蒙，劉 超，李淑玲，欒治東*

(遼寧醫(yī)學(xué)院 基礎(chǔ)醫(yī)學(xué)院 1.發(fā)育生物學(xué)教研室；2.組胚教研室；3.生物化學(xué)教研室, 遼寧 錦州 121000)

研究論文

Ubc9對(duì)雞胚神經(jīng)嵴細(xì)胞發(fā)育的影響

李明洋1，劉乙蒙3，劉 超1，李淑玲2，欒治東1*

(遼寧醫(yī)學(xué)院 基礎(chǔ)醫(yī)學(xué)院 1.發(fā)育生物學(xué)教研室；2.組胚教研室；3.生物化學(xué)教研室, 遼寧 錦州 121000)

目的探索Ubc9和SUMO通路對(duì)雞胚神經(jīng)嵴細(xì)胞發(fā)育的影響。方法1)采集雞胚，通過(guò)免疫熒光實(shí)驗(yàn)和原位雜交實(shí)驗(yàn)來(lái)檢測(cè)SUMO結(jié)合酶Ubc9及神經(jīng)嵴細(xì)胞標(biāo)志基因的表達(dá)情況。2)設(shè)置對(duì)照組(非特異性嗎啉代處理)和Ubc9-嗎啉代組來(lái)處理各組8期雞胚，隨后通過(guò)原位雜交實(shí)驗(yàn)檢測(cè)Snail2，Sox9和FoxD3等神經(jīng)嵴細(xì)胞晚期標(biāo)志基因的表達(dá)情況。結(jié)果1)Ubc9與標(biāo)志基因Pax7共表達(dá)于4、6和8期雞胚神經(jīng)嵴細(xì)胞前體。2)與對(duì)照組相比，Ubc9-嗎啉代組雞胚單側(cè)上Snail2(P<0.05)，Sox9(P<0.01)和FoxD3(P<0.05)的mRNA水平均有下降。結(jié)論Ubc9表達(dá)于4、6和8期雞胚的神經(jīng)脊細(xì)胞前體，雞胚神經(jīng)嵴細(xì)胞的形成需要SUMO通路參與。

雞胚；神經(jīng)嵴細(xì)胞；Ubc9；SUMO通路

神經(jīng)嵴細(xì)胞(neural crest cell, NCC)起源于脊椎動(dòng)物背部神經(jīng)管，為多潛能的干細(xì)胞并具有高度遷移性，能夠分化產(chǎn)生多種組織，包括頭部的大部分骨骼和肌肉、 外周神經(jīng)系統(tǒng)的神經(jīng)元和膠質(zhì)細(xì)胞及皮膚黑色素細(xì)胞等[1]。NCC發(fā)育異常會(huì)導(dǎo)致缺陷病。研究表明，許多蛋白參與NCC形成和遷移，如NCC早期標(biāo)志基因Pax7[2]以及晚期調(diào)控因子如Snail2、Sox9和FoxD3[3]等。

小泛素相關(guān)修飾蛋白(small ubiquitin-related modifier，SUMO)化是一種廣泛存在的蛋白質(zhì)翻譯后修飾形式[4]，參與核質(zhì)轉(zhuǎn)運(yùn)，細(xì)胞周期調(diào)控，信號(hào)傳導(dǎo)和轉(zhuǎn)錄活性調(diào)控等[5]。本研究將觀察SUMO化中唯一的E2結(jié)合酶-Ubc9[6]在早期雞胚中的表達(dá)情況，隨后檢測(cè)Ubc9敲除后雞胚的NCC晚期標(biāo)志基因的表達(dá)水平，探索SUMO通路對(duì)雞胚NCC發(fā)育的影響。

1 材料與方法

1.1 試劑

受精雞蛋(遼寧醫(yī)學(xué)院動(dòng)物實(shí)驗(yàn)中心)；嗎啉代 (Gene Tools)；抗體:anti-DIG, anti-rabbit alexa fluor 448(invtrogen)。

1.2 雞胚的制備

受精雞蛋于37 ℃孵育箱中孵育到4(18 h)、6(24 h)和8(28 h)期[7]。用無(wú)菌注射器抽取部分蛋清后敲開(kāi)蛋殼，濾紙覆蓋雞胚，再沿濾紙邊剪下雞胚，于4%多聚甲醛中固定2 h。

1.3 原位雜交

雞胚先經(jīng)4%多聚甲醛固定，然后經(jīng)過(guò)梯度濃度的甲醛處理，再用100%甲醇洗兩次。65 ℃預(yù)雜交1 h，80 ℃預(yù)熱探針5 min,再65 ℃孵育過(guò)夜，用含有2% 勃林格試劑(Boehringer)和滅活的胎牛血清的三羥甲基氨基甲烷-鹽酸緩沖液室溫封閉2 h，anti-DIG經(jīng)1∶1 000稀釋，4 ℃搖床過(guò)夜，堿性磷酸酶顯色[8]。

1.4 免疫熒光及成像

免疫熒光實(shí)驗(yàn)按照標(biāo)準(zhǔn)步驟[9]。簡(jiǎn)要概括，將胚胎固定在4 %多聚甲醛中，用磷酸鹽緩沖液 (含有2 g/L牛血清白蛋白,0.1% Tween)潤(rùn)洗(3×60 min)。一抗孵育，4 ℃過(guò)夜。特異性熒光二抗經(jīng)1∶2 000稀釋，顯色。用Nikon體視顯微鏡(Spot SE鏡頭和Nikon Eclipse 80i顯微鏡的軟件)成像。

1.5 電穿孔法敲除Ubc9

將嗎啉代(Mo)置于3%蔗糖溶液使其濃度為0.48 μmol/L，添加1 μg DNA (pCIG)作為載體和提供綠熒光，單側(cè)電穿孔法[10]轉(zhuǎn)染入固定在濾紙環(huán)中的4期雞胚。雞胚孵育[11]過(guò)夜，隨后用于原位雜交和免疫熒光分析。

1.6 統(tǒng)計(jì)學(xué)分析

所得實(shí)驗(yàn)數(shù)據(jù)采用四格表的Fisher確切概率法分析，統(tǒng)計(jì)學(xué)處理由SPSS 17.0統(tǒng)計(jì)軟件完成。

2 結(jié)果

2.1 Ubc9在不同發(fā)育時(shí)期雞胚的表達(dá)

4～6期雞胚中Ubc9 mRNA廣泛而分散地表達(dá)(圖1A，B)，有逐漸向神經(jīng)褶匯聚的趨勢(shì)，在8期可見(jiàn)Ubc9在神經(jīng)褶表達(dá)明顯增強(qiáng)(圖1C)，并且8期3個(gè)切片處的原位雜交檢測(cè)也顯示Ubc9在此處表達(dá)增強(qiáng)(圖1D)。

A～B.Ubc9 expression in chick embryos at st 4(18 hours)and 6(24 hours)；C.Ubc9 expression in chick embryos at st 8(28 hours)；D.sections at the indicated level of the st 8 embryo

圖1原位雜交檢測(cè)Ubc9在早期(4,6,8期)雞胚中的表達(dá)
Fig1Ubc9expression(insituhybridization)inchickembryosatearlystages(4,6,8st)

2.2 Ubc9與 Pax7在雞胚神經(jīng)褶上的共定位情況

NCC早期標(biāo)志基因Pax7(圖2A) 與Ubc9(圖2B)共表達(dá)于神經(jīng)褶(圖2C，D)，即神經(jīng)脊細(xì)胞前體。

2.3敲除8期雞胚的Ubc9后，神經(jīng)嵴細(xì)胞無(wú)法形成

對(duì)照組(圖3A,B,C)經(jīng)5堿基錯(cuò)配的對(duì)照-Mo(非特異性)處理后， 9個(gè)雞胚出現(xiàn)了1個(gè)Snail2(A)的異常表達(dá)(1/9),Sox9(B,1/10),FoxD3(C,1/8)，近似無(wú)影響。而實(shí)驗(yàn)組(圖3 D,E,F)經(jīng)Ubc9-Mo處理后，與對(duì)照組相比，Snail2(D,7/9),Sox9(E,5/6)和FoxD3(F,7/10)的mRNA表達(dá)明顯減少(P<0.05,P<0.01和P<0.05) (表 1)。

3 討論

本研究首先用原位雜交法檢測(cè)了Ubc9的mRNA的表達(dá)情況，發(fā)現(xiàn)Ubc9在8期雞胚的神經(jīng)褶即NCC的前體表達(dá)，同時(shí)，免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)NCC早期標(biāo)志基因Pax7[2]與Ubc9共表達(dá)于8期雞胚神經(jīng)褶，即神經(jīng)嵴前體細(xì)胞所在位置。這些結(jié)果提示，Ubc9可能在NCC形成過(guò)程中起作用。

A.Pax7 expression at NC precursors of the neural fold；B.Ubc9 expression at NC precursors of the neural fold；C.Ubc9 are coexpressed with Pax7 in NC precursors of the neural fold of st 8 embryos；D.Framed regions of merged images at higher magnification;Ubc9(green) and Pax7(red) in C,D

圖2免疫熒光顯示Ubc9和Pax7共定位于雞胚8期的神經(jīng)嵴細(xì)胞前體(神經(jīng)褶)

Fig2Ubc9proteinsarecoexpressed(Immunofluorescence)withPax7inNCprecursorsoftheneuralfoldofst8embryos

A～C.unilateral electroporation of 5 mismatch control morpholinos have no effect on the expression of NC markersSnail2,Sox9 andFoxD3； D～F.Ubc9-morpholino downregulates the expression ofSnail2,Sox9 andFoxD3; right panels show higher-magnification of framed regions, arrows point to untreated internal control signal and brackets demarcate treated areas; insets show the localization of morpholinos (green) beforeinsituhybridization

圖3 嗎啉代法敲除Ubc9導(dǎo)致了Snail2(D), Sox9(E),和FoxD3(F)表達(dá)的下降Fig 3 Unilateral Ubc9-morpholino downregulates the expression of Snail2(D), Sox9(E), and FoxD3(F)

*P<0.05,**P<0.01 compared with control group.

Ubc9是SUMO化唯一的E2結(jié)合酶。SUMO化反應(yīng)包括3個(gè)步驟：活化，結(jié)合和連接，涉及多個(gè)酶的級(jí)聯(lián)反應(yīng)：E1活化酶、E2結(jié)合酶以及E3連接酶，其中Ubc9，參與SUMO化中結(jié)合和連接兩步[12]，阻斷了Ubc9的表達(dá)，即阻斷了SUMO通路[4]。

敲除小鼠SUMO1會(huì)導(dǎo)致唇裂等缺陷病[13]的發(fā)生，說(shuō)明NCC形成或遷移需要SUMO通路的參與，并且，一些NCC相關(guān)轉(zhuǎn)錄因子如Sox9等可被SUMO化修飾[14- 15]。但是NCC形成和遷移是否需要Ubc9或需要SUMO通路的參與，目前沒(méi)有直接證據(jù)。本研究通過(guò)嗎啉代敲除法降低了雞胚Ubc9的mRNA表達(dá)水平，發(fā)現(xiàn)NCC晚期標(biāo)志基因Snail2,Sox9和FoxD3的表達(dá)明顯下降或不表達(dá)，說(shuō)明NCC形成需要Ubc9 和SUMO通路的參與。

綜上所述，Ubc9表達(dá)于早期雞胚神經(jīng)嵴細(xì)胞前體，NCC形成需要SUMO通路的參與。本研究為神經(jīng)嵴細(xì)胞的進(jìn)一步探索提供了新的思路，而Ubc9和SUMO通路如何影響神經(jīng)嵴細(xì)胞形成及機(jī)制仍有待研究。

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The effect of Ubc9 on neural crest cell development of chicken embryo

LI Ming-yang1, LIU Yi-meng3, LIU Chao1, Li Shu-ling2, LUAN Zhi-dong1*

(1.Dept. of Developmental Biology; 2.Dept. of Histology and Embryology; 3.Dept. of Biochemical andMolecular Biology，Liaoning Medical College.Jinzhou 121001，China)

ObjectiveTo examine the effect of Ubc9 and SUMO pathway on neural crest cell development of chicken embryo.Methods1)Chicken embryos were collected,Insituhybridization and Immunofluorescence were used to observe the expression of SUMO-conjugating enzyme-Ubc9 and early markers of neural crest. 2)In the control group, 5-mismatch Ubc9-control morpholino (5MM-Mo) was used to treat chick embryos; In the Ubc9-Mo group, sequence-specific Morpholinos(Mo) was used to reduce Ubc9 level in chicken embryos, then the mRNA of neural crest markers(Snail2，Sox9andFoxD3) was examined.Results1)Ubc9 was coexpressed with Pax7 in neural crest precursors of the neural fold at 4,6 and 8 stage of early chicken embryos. 2)Compared to the control group,Ubc9-Mo treatment led to a reduce of mRNA level of neural crest markers,Snail2(P<0.05)，Sox9 (P<0.01)andFoxD3(P<0.05).ConclusionsSUMO-conjugating enzyme, Ubc9 expression start at neural fold at 4,6 and 8 stage of early chicken embryo, Ubc9 and SUMO pathway are required for neural crest cell development of chicken embryo.

chicken embryo；neural crest cell; Ubc9；SUMO pathway

2014- 04- 10

2014- 06- 20

*通信作者(correspondingauthor)：zhidongluan@lnmu.edu.cn

1001-6325(2014)08-1079-04

R 392.11

A