韓春梅王姍姍高慶華,鄭永富馬夢(mèng)婷張勤
(1.塔里木大學(xué)動(dòng)物科學(xué)學(xué)院,阿拉爾 843300;2.新疆生產(chǎn)建設(shè)兵團(tuán)塔里木畜牧科技重點(diǎn)實(shí)驗(yàn)室,阿拉爾 843300;3.中國(guó)農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,北京100193)
TGF-β1誘導(dǎo)馬鹿茸MSCs軟骨分化及c-myc基因的表達(dá)
韓春梅1,3王姍姍1高慶華1,2鄭永富1馬夢(mèng)婷2張勤3
(1.塔里木大學(xué)動(dòng)物科學(xué)學(xué)院,阿拉爾 843300;2.新疆生產(chǎn)建設(shè)兵團(tuán)塔里木畜牧科技重點(diǎn)實(shí)驗(yàn)室,阿拉爾 843300;3.中國(guó)農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,北京100193)
鹿茸間充質(zhì)干細(xì)胞(MSCs)是維持茸再生與骨化的重要組織,旨在研究鹿茸MSCs的軟骨分化及原癌基因c-myc對(duì)該過程的調(diào)控作用。利用成年塔里木馬鹿生長(zhǎng)60 d的鹿茸第2代間充質(zhì)干細(xì)胞(MSCs,P2),通過TGF-β1(10 ng/mL濃度)刺激,誘導(dǎo)塔里木馬鹿茸間充質(zhì)干細(xì)胞向軟骨分化,采用免疫組化和阿利新藍(lán)染色鑒定誘導(dǎo)結(jié)果,并通過qPCR方法檢測(cè)軟骨分化過程中c-myc基因的表達(dá)變化。結(jié)果顯示,MSCs在誘導(dǎo)后的第9天開始出現(xiàn)細(xì)胞形態(tài)變化,由梭形向多角形轉(zhuǎn)變,原來菊花狀的分布逐漸向鋪路石狀變化,至14 d可觀察到軟骨陷窩,21 d軟骨細(xì)胞基質(zhì)明顯,并開始出現(xiàn)細(xì)胞凋亡。非誘導(dǎo)組28 d細(xì)胞出現(xiàn)凋亡,細(xì)胞內(nèi)發(fā)現(xiàn)空泡。35 d兩組細(xì)胞凋亡明顯,細(xì)胞折光性變差,間隙變大。阿利新藍(lán)染色鑒定,誘導(dǎo)至第14天細(xì)胞基質(zhì)中開始出現(xiàn)大量陽性染色。免疫組化實(shí)驗(yàn)檢測(cè),誘導(dǎo)至21 d的細(xì)胞基質(zhì)中出現(xiàn)棕色Col II陽性反應(yīng)物,隨培養(yǎng)時(shí)間增加顏色加深,并集中分布在細(xì)胞及其周圍基質(zhì)中。在軟骨分化進(jìn)程中,第7、14、21和28天,誘導(dǎo)組c-myc基因表達(dá)與非誘導(dǎo)組相比顯著下調(diào)(P<0.05),但誘導(dǎo)至35 d,誘導(dǎo)組c-myc表達(dá)與非誘導(dǎo)組相比無顯著差異(P>0.05)。在TGF-β1刺激下,塔里木馬鹿茸MSCs可以分化成軟骨,原癌基因c-myc下調(diào)表達(dá)誘導(dǎo)鹿茸MSCs進(jìn)入凋亡狀態(tài)并分化為軟骨細(xì)胞。
馬鹿茸;間充質(zhì)干細(xì)胞;軟骨分化;轉(zhuǎn)化生長(zhǎng)因子β1;c-myc基因
鹿茸是哺乳動(dòng)物唯一能夠割后再生的器官,其割后再生與快速生長(zhǎng)類似腫瘤的特性,吸引了國(guó)內(nèi)外眾多科學(xué)家的興趣。鹿茸間充質(zhì)干細(xì)胞位于茸皮的真皮層下,是一類新的干細(xì)胞。在鹿茸不同生長(zhǎng)期,間充質(zhì)細(xì)胞層的厚度不斷變化,從生長(zhǎng)早期到后期即骨化期,逐漸變薄。一系列研究[1-5]發(fā)現(xiàn)鹿茸的間充質(zhì)細(xì)胞層(MSCs)來源于位于鹿額外脊的骨膜干細(xì)胞,由間充質(zhì)細(xì)胞分化成集落生成單位成纖維細(xì)胞,然后分化成軟骨細(xì)胞進(jìn)而形成骨細(xì)胞,是維持鹿茸的生長(zhǎng)發(fā)育的源泉之一。在鹿茸整個(gè)生長(zhǎng)過程中,不斷發(fā)生著這樣的變化,來實(shí)現(xiàn)鹿茸的生長(zhǎng)[6,7]。但該理論的分子生物學(xué)證據(jù)國(guó)內(nèi)外很少有相關(guān)報(bào)道。
原癌基因c-myc對(duì)干細(xì)胞的增殖分化起著關(guān)鍵的作用。對(duì)塔里木馬鹿茸c-myc基因的研究發(fā)現(xiàn),不同生長(zhǎng)期鹿茸不同組織中的c-myc基因有明顯時(shí)空表達(dá)變化[8],說明c-myc基因與鹿茸的組織分化和細(xì)胞生長(zhǎng)密切關(guān)聯(lián)。但c-myc在MSCs軟骨分化過程行使何種功能,其表達(dá)是分化的結(jié)果還是原因均無相關(guān)報(bào)道。
轉(zhuǎn)化生長(zhǎng)因子(TGF-β1)是MSCs軟骨分化不可缺少的作用因子[9,10]。Kocamaz等[11]利用10 ng/μL TGF-β1成功誘導(dǎo)了20日齡雞的骨髓間充質(zhì)干細(xì)胞(hMSCs)向軟骨分化。Christopher等[12]用相同劑量的TGF-β1刺激山羊MSCs,3周后RT-PCR檢測(cè)II型膠原蛋白(COL2)和聚集蛋白聚糖的表達(dá)顯著高于非誘導(dǎo)組。Mueller等[13]在人hMSCs軟骨分化的研究中發(fā)現(xiàn),10 ng/μL TGF-β1及其不同配體不但能誘導(dǎo)軟骨形成,同時(shí)還能促使軟骨肥大化。
本研究利用10 ng/mL TGF-β1誘導(dǎo)塔里木馬鹿茸MSCs軟骨分化,確定體外誘導(dǎo)的最佳條件;并在此過程中研究c-myc基因的表達(dá)變化,一方面探討馬鹿茸MSCs體外軟骨的發(fā)育模式;另一方面研究c-myc對(duì)馬鹿茸軟骨分化機(jī)制的影響作用,以期為茸MSCs軟骨分化提供分子依據(jù)。
1.1 材料
1.1.1 實(shí)驗(yàn)材料 生長(zhǎng)60 d的塔里木馬鹿茸體外培養(yǎng)第2代MSCs和軟骨細(xì)胞(P2),由塔里木畜牧科技重點(diǎn)實(shí)驗(yàn)室胚胎工程實(shí)驗(yàn)室提供,HeLa細(xì)胞由華中農(nóng)業(yè)大學(xué)武軍元老師提供。
1.1.2 試劑和儀器 胎牛血清FBS、細(xì)胞培養(yǎng)液DMEM、Ⅱ型膠原酶、胰蛋白酶、TGF-β1均為Sigma公司產(chǎn)品,TRIZOL(Invitrogen);反轉(zhuǎn)錄試劑盒(ThermoFisher),定量PCR試劑盒(Roche),DMSO;CO2加濕培養(yǎng)箱(MCO-15A),eppendorplex熒光定量PCR儀,倒置相差顯微鏡(Nikon);低溫高速離心機(jī)(Jouan);生物顯微鏡(舜宇);血細(xì)胞計(jì)數(shù)板;無菌操作臺(tái)等。阿利新藍(lán)染色試劑盒,羊Col II一抗和免疫組化試劑盒(北京博奧)。
1.2 方法
1.2.1 誘導(dǎo)培養(yǎng)及誘導(dǎo)結(jié)果鑒定 選擇5個(gè)6孔板,每個(gè)孔中加入1 mL 10% FBS-DMEM,約含細(xì)胞1×105個(gè)。設(shè)置其中15孔為對(duì)照組,另15孔為誘導(dǎo)組。誘導(dǎo)組每孔于接種P2細(xì)胞后第3天,細(xì)胞生長(zhǎng)至70%融合時(shí),換用軟骨誘導(dǎo)培養(yǎng)基,即含10 ng/mL TGF-β1的10% FBS-DMEM及1×10-7mol/L地塞米松。每2 d半量換液。換液時(shí)在倒置顯微鏡下觀察拍照記錄細(xì)胞生長(zhǎng)情況。在培養(yǎng)至第7、14、21、28和35天時(shí),每個(gè)時(shí)間點(diǎn)分別收集3個(gè)孔的細(xì)胞,每孔細(xì)胞數(shù)約為4.5×105個(gè),在倒置顯微鏡下觀察細(xì)胞生長(zhǎng)情況,用II型膠原蛋白免疫組化和阿爾新藍(lán)染色鑒定軟骨誘導(dǎo)結(jié)果。
1.2.2 c-myc基因的定量檢測(cè) 對(duì)每個(gè)時(shí)間點(diǎn)收集的細(xì)胞,用Invitrogen公司的TRIZOL Reagent? RNA提取試劑盒方法提取RNA,使用ThermoFisher公司的反轉(zhuǎn)錄試劑盒合成cDNA,1∶10稀釋后待用。根據(jù)GenBank數(shù)據(jù)庫(kù)提供的鹿c-myc基因序列和內(nèi)參基因牛GAPDH基因序列,用Primer 5.0軟件設(shè)計(jì)PCR的引物(表1)。qPCR反應(yīng)條件為:95℃ 5 min;95℃ 10 s,60℃ 30 s,40個(gè)循環(huán)。熔解曲線程序:55-95℃;半定量PCR反應(yīng)條件:95℃,5 min;95℃ 10 s,65℃ 30 s,72℃ 2 min,28個(gè)循環(huán)。
表1 qPCR檢測(cè)用引物
為比較c-myc軟骨分化過程的基因表達(dá),提取培養(yǎng)至第7天的P2代軟骨細(xì)胞和HeLa細(xì)胞的RNA,反轉(zhuǎn)錄后備用。
1.2.3 統(tǒng)計(jì)學(xué)處理 基因表達(dá)倍增值用2-△△Ct法計(jì)算,利用SPSS17.0 軟件包對(duì)對(duì)照組和誘導(dǎo)組的基因表達(dá)差異進(jìn)行顯著性檢驗(yàn),P <0.05有統(tǒng)計(jì)學(xué)意義。
2.1 誘導(dǎo)結(jié)果
倒置顯微鏡下觀察:誘導(dǎo)組培養(yǎng)至第9天后開始出現(xiàn)細(xì)胞形態(tài)變化。一些細(xì)胞的形態(tài)開始從梭形變成圓形、多角形,隨培養(yǎng)時(shí)間的延長(zhǎng),更多的細(xì)胞形態(tài)發(fā)生類似變化。原本菊花狀的細(xì)胞分布逐漸向鋪路石狀變化。至14 d可觀察到軟骨陷窩,培養(yǎng)至21 d軟骨細(xì)胞基質(zhì)明顯,同時(shí)發(fā)現(xiàn)凋亡細(xì)胞??瞻捉M培養(yǎng)至28 d細(xì)胞出現(xiàn)凋亡,細(xì)胞內(nèi)發(fā)現(xiàn)空泡。35 d兩組細(xì)胞凋亡明顯,凋亡細(xì)胞體積縮小,形態(tài)近似圓形,凸起,中心呈空泡狀,細(xì)胞折光性變差,間隙變大(圖1)。
阿利新藍(lán)染色發(fā)現(xiàn),誘導(dǎo)至第14天細(xì)胞基質(zhì)中出現(xiàn)大量藍(lán)色陽性染色,即聚集蛋白聚糖的軟骨細(xì)胞表面標(biāo)志,隨培養(yǎng)時(shí)間延長(zhǎng)著色面積加大(圖2)。免疫組化實(shí)驗(yàn)發(fā)現(xiàn)誘導(dǎo)至21 d,細(xì)胞基質(zhì)出現(xiàn)棕色Col II陽性反應(yīng)物,隨培養(yǎng)時(shí)間顏色加深集中分布在細(xì)胞內(nèi)及其周圍基質(zhì)中(圖3)。對(duì)照組細(xì)胞無相應(yīng)反應(yīng)變化。
2.2 c-myc基因表達(dá)定量檢測(cè)
通過qPCR定量檢測(cè)誘導(dǎo)不同時(shí)間點(diǎn)c-myc基因的表達(dá)結(jié)果(圖4)發(fā)現(xiàn),第14、21和28天,誘導(dǎo)組c-myc基因的相對(duì)表達(dá)量(倍增值)分別為0.323、0.224和0.319,而對(duì)照組的分別為0.574、0.72和0.457,兩組間差異顯著(P<0.05);但35 d兩組間沒有顯著差異(P>0.05)。第14-35天4個(gè)時(shí)間點(diǎn)誘導(dǎo)組的基因表達(dá)量與P2代軟骨細(xì)胞中的表達(dá)量(倍增值為0.222)比較沒有顯著差異(P>0.05),但與第7天對(duì)照組的表達(dá)量相比均有顯著差異(P<0.05)。對(duì)照組中,第7、14和21天3個(gè)時(shí)間點(diǎn)的基因表達(dá)量無顯著差異(P>0.05),但顯著高于第28和35天的表達(dá)量(P<0.05)。
定量PCR未檢測(cè)到HeLa細(xì)胞的c-myc的表達(dá)量,盡管通過半定量PCR結(jié)果,HeLa細(xì)胞的內(nèi)參基因與目的基因均有表達(dá),且表達(dá)量相似(圖5)。
Wang等[13]通過在人MSCs軟骨分化的培養(yǎng)基質(zhì)中添加血清和TGF-β1誘導(dǎo)劑對(duì)培養(yǎng)細(xì)胞凋亡的影響實(shí)驗(yàn),發(fā)現(xiàn)培養(yǎng)基中加入血清組在培養(yǎng)的第7天細(xì)胞數(shù)與細(xì)胞活力均明顯下降,用TUNEL法染色檢測(cè)培養(yǎng)21 d的細(xì)胞,確定加血清培養(yǎng)組細(xì)胞凋亡比對(duì)照組顯著。而加10 ng/mL TGF-β1誘導(dǎo)劑組則未發(fā)現(xiàn)TUNEL陽性染色細(xì)胞,認(rèn)為血清培養(yǎng)可誘導(dǎo)細(xì)胞凋亡而TGF-β1可阻止分化過程的細(xì)胞凋亡。Walenda等[14]2013年研究了不同濃度TGF-β1對(duì)hMSCs軟骨分化過程細(xì)胞增殖分化的影響,認(rèn)為1 ng/mL TGF-β1不影響細(xì)胞早衰,但因?yàn)門GF-β1能刺激細(xì)胞快速分裂而使細(xì)胞提早進(jìn)入復(fù)制抑制。本實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)誘導(dǎo)組細(xì)胞衰亡(第21天)早于空白組(第28天),這與Walenda的實(shí)驗(yàn)結(jié)果相似。但體外培養(yǎng)細(xì)胞早衰與培養(yǎng)時(shí)間有一定關(guān)系[15],因此,關(guān)于馬鹿茸MSCs的體外擴(kuò)增與分化生長(zhǎng)特性還需進(jìn)一步研究。
圖1 誘導(dǎo)組MSCs軟骨分化不同時(shí)間點(diǎn)細(xì)胞形態(tài)變化(100×)
圖2 誘導(dǎo)組MSCs軟骨分化阿利新藍(lán)染色結(jié)果(100×)
圖3 誘導(dǎo)組MSCs軟骨分化免疫組化檢測(cè)結(jié)果(100×)
鹿茸MSCs是鹿茸軟骨及骨細(xì)胞的細(xì)胞庫(kù),對(duì)比不同生長(zhǎng)期鹿茸間充質(zhì)細(xì)胞層發(fā)現(xiàn),從30-60 d,即生長(zhǎng)高峰,該組織層的厚度逐漸增加,而生長(zhǎng)高峰過后,其厚度逐漸變薄。這一形態(tài)的變化,可證實(shí)茸MSCs不斷向軟骨分化以維持鹿茸的快速生長(zhǎng)。C-myc基因上調(diào)表達(dá)時(shí)細(xì)胞處于增殖狀態(tài),但細(xì)胞處于分化狀態(tài)時(shí)c-myc基因則下調(diào)表達(dá)[16]。
圖4 qPCR檢測(cè)MSCs軟骨分化不同時(shí)間點(diǎn)c-myc表達(dá)量
圖5 半定量PCR檢測(cè)MSCs軟骨分化不同時(shí)間點(diǎn)c-myc表達(dá)變化
現(xiàn)已證實(shí),TGF-β1蛋白主要通過影響其信號(hào)通路下游基因SMAD4/SMAD3蛋白復(fù)合物來誘導(dǎo)干細(xì)胞軟骨分化。TGF-β1蛋白促使該復(fù)合物表達(dá)加強(qiáng),刺激細(xì)胞基質(zhì)分泌,誘導(dǎo)軟骨分化調(diào)控因子SRY-related gene 9(sox9)的轉(zhuǎn)錄,以加強(qiáng)軟骨的表面標(biāo)志collagen II蛋白的表達(dá)[17,18];另一方面TGF-β1蛋白能夠阻止該復(fù)合物與Wnt信號(hào)通路中LEF/TCF作用元件結(jié)合,抑制c-myc基因的激活[17,19],表現(xiàn)為分化過程c-myc基因的表達(dá)下調(diào)。本實(shí)驗(yàn)結(jié)果顯示,TGF-β1誘導(dǎo)P2鹿茸MSCs的軟骨分化過程中,軟骨分化的早期,空白組即非TGF-β1處理的間充質(zhì)細(xì)胞c-myc基因的表達(dá)量顯著高于誘導(dǎo)組基因的表達(dá)量。說明c-myc下調(diào)表達(dá)時(shí)鹿茸干細(xì)胞處于分化階段。
但c-myc基因下調(diào)表達(dá)也誘導(dǎo)細(xì)胞凋亡。該機(jī)制可通過抑癌基因P53完成。P53綁定在c-myc基因啟動(dòng)子區(qū),通過其組氨酸H4乙酰化阻止c-myc基因啟動(dòng),誘導(dǎo)細(xì)胞生長(zhǎng)停滯進(jìn)而凋亡。在一些細(xì)胞中c-myc介導(dǎo)的細(xì)胞凋亡不需要P53參與也能發(fā)生[20]。該理論解釋了鹿茸MSCs誘導(dǎo)組與空白組28 d后c-myc表達(dá)下調(diào)與細(xì)胞凋亡的因果關(guān)系。
C-myc 作為大多數(shù)惡性腫瘤的標(biāo)志性基因,在很多腫瘤組織中上調(diào)表達(dá)。本實(shí)驗(yàn)用HeLa細(xì)胞作為實(shí)驗(yàn)材料之一,目的是比較鹿茸MSCs與腫瘤細(xì)胞在c-myc基因表達(dá)量上的差異。當(dāng)cDNA以1∶10稀釋后,定量PCR結(jié)果未檢測(cè)到HeLa細(xì)胞c-myc基因的表達(dá)值,該結(jié)果表明鹿茸MSCs的基因表達(dá)量高于HeLa細(xì)胞。但原癌基因c-myc在HeLa細(xì)胞中是否像其它腫瘤細(xì)胞上調(diào)表達(dá)無相關(guān)文獻(xiàn)報(bào)道。
Liu等[21]在鼠MSCs軟骨分化過程,超表達(dá)Wnt11基因,結(jié)果發(fā)現(xiàn)MSCs G0/G1細(xì)胞周期停滯;聚集蛋白聚糖與Collagen II的表達(dá)量顯著高于對(duì)照組,同時(shí)sox9也大量表達(dá)。因此認(rèn)為除了TGF-β信號(hào)通路,非經(jīng)典的Wnt信號(hào)也參與了MSCs的軟骨分化進(jìn)程。
本實(shí)驗(yàn)以TGF-β1為誘導(dǎo)劑誘導(dǎo)鹿茸MSCs軟骨分化的過程,c-myc基因呈下調(diào)表達(dá)。這與體內(nèi)軟骨分化的結(jié)果是一致的[22-24]。但c-myc基因?qū)β谷咨L(zhǎng)再生的調(diào)控作用機(jī)制還需要對(duì)其信號(hào)通路做進(jìn)一步的研究。
在TGF-β1刺激下,塔里木馬鹿茸MSCs可以分化成軟骨;原癌基因c-myc下調(diào)表達(dá)誘導(dǎo)鹿茸MSCs分化;誘導(dǎo)分化軟骨細(xì)胞與MSCs進(jìn)入凋亡狀態(tài)。
[1]Li CY, Colin GM, Shirley K, et al. Clark. Identification of key tissue type for antler regeneration through pedicle periosteum deletion[J]. Cell Tissue Res, 2007, 328:65-75.
[2] Li CY, James MS, Dawn EC. Histological examination of antler regeneration in red deer(Cervus elaphus)[J]. The Anatomical Record Part A, 2005, 282A:163-174.
[3]Debra KB, Li CY, Geoff A, et al. Red deer cloned from antler stem cells and their differentiated progeny[J]. Biology of Reproduction,2007, 77:384-394.
[4]Li CY, James MS. Deer antlerogenic periosteum:a piece of postnatally retained embryonic tissue?[J]. Anat Embryol, 2001,204:375-388.
[5]Gao XH, Yang FH, Zhao HP, et al. Antler transformation is advancedby inversion of antlerogenic periosteum implants in sika deer(Cervus nippon)[J]. The Anatomical Record, 2010, 293:1787-1796.
[6]Andrea M, Istvan G, Ena K. Identification of differentially expressed genes in the developing antler of red deer Cereus alphas[J]. Mol Genet Genomics, 2007(277):237-248.
[7]Kierdorf U, Kierdorf H. Deer antlers-a model of mammalian appendage regeneration:An extensive review[J]. Gerontology,2011, 57:53-65.
[8]韓春梅, 高慶華, 李世軍, 等. 原癌基因c-myc在塔里木馬鹿茸不同生長(zhǎng)期的表達(dá)[J]. 中國(guó)獸醫(yī)學(xué)報(bào), 2012, 32(10):1536-1542.
[9]Goessler UR, Bugert P, Bieback K, et al. In vitro analysis of the expression of TGF-β- superfamily members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during differentiation in cell culture[J]. Cell Mol Biol Lett, 2005, 10(2):345-362.
[10]Martin F, Lehmann M, Schl?ger P, et al. Differentiation capacity of chondrocytes in microtissues depends on TGF-β subtype[J]. J Biochip Tissue Chip, 2012, S2:002. doi:10. 4172/2153-0777. S2-002.
[11]Erdogan K, Duygu G, Ayse C. Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells[J]. J Mol Hist, 2012, 43:497-508.
[12] Christopher GW, Tae KK, Anya T, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel[J]. Tissue Engineering, 2003, 9(4):679-688.
[13]Wang CY, Chen LL, Kuo PY, et al. Apoptosis in chondrogenesis of human mesenchymal stem cells:Effect of serum and medium supplements[J]. Apoptosis, 2010, 15:439-449.
[14]Walenda G, Abnaof K, Joussen S, et al. TGF-beta1 Does Not induce senescence of multipotent mesenchymal stromal cells and has similar effects in early and late passages[J]. PLoS One, 2013, 8(10):e77656. doi:10. 1371/journal. pone. 0077656.
[15]Fu RG, Wu JJ, Xue RL, et al. Premature senescence and cellular phenotype transformation of mesangial cells induced by TGFB1[J]. Renal Failure, 2013, 35(8):1142-1145.
[16]Laura S, Gerard IE. The ups and down of myc biology[J]. Curr Opin Genet, 2010, 20(1):91-99.
[17]Michael BM, Maria F, Johannes Z, et al. Hypertrophy in mesenchymal stem cell chondrogenesis:Effect of TGF-β isoforms and chondrogenic conditioning[J]. Cells Tissues Organs, 2010,192:158-166.
[18]Furumatsu T, Tsuda M, Taniquchi N, et al. The Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding Protein/p300 recruitment[J]. Journal of Biological Chemistry,2010, 280:8343-8350.
[21]Liu S, Zhang E, Yang M, et al. Overexpression of Wnt11 promotes chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in synergism with TGF-β[J]. Mol Cell Biochem, 2014,390:123-131.
[19]Lim SK, Hoffmann FM. Smad4 cooperates with lymphoid enhancerbinding factor 1/T cell-specific factor to increase c-myc expression in the absence of TGF-β signaling[J]. PNAS, 2006, 103(49):18580-18585.
[20]Hoffman B, Liebermann DA. c-MYC in apoptosis and cancer[J]. Oncogene, 2008, 27:6462-6472.
[22]Chen WH, Lai MT, Wu AT, et al. In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes[J]. Arthritis Rheum, 2009, 60(2):450-459.
[23]Gurusinghe S, Strappe P. Gene modification of mesenchymal stem cells and articular chondrocytes to enhance chondrogenesis[J]. BioMed Research International, 2014, Article ID 369528. http://dx. doi. org/10. 1155/2014/369528.
[24]Paula AC, Martins TM, Zonari A, et al. Human adipose tissuederived stem cells cultured in xeno-free culture condition enhance C-MYC expression increasing proliferation but bypassing spontaneous cell transformation[J]. Stem Cell Res Ther, 2015, 6(1):76.
(責(zé)任編輯 馬鑫)
TGF-β1-induced Differentiation of Wapiti Antler Mesenchymal Stem Cells to Cartilage and Expression Profile of Gene c-myc
HAN Chun-mei1,3WANG Shan-shan1GAO Qing-hua1,2ZHENG Yong-fu1MA Meng-ting2ZHANG Qin3
(1. College of Animal Sciences,Tarim University,Alar 843300;2. Key Laboratory of Tarim Animal Husbandry Science & Technology,Xinjiang Production & Construction Group,Alar 843300;3. College of Animal Science and Technology,China Agricultural University,Beijing 100193)
Antler mesenchymal stem cells(MSCs)play a pivotal role on the antler regeneration and ossification. To investigate the chondrogenic differentiation of antler MSCs and the regulation role of the proto-oncogene c-myc in this process,the second passage cells(MSCs,P2)of 60 d antler from adult Tarim wapiti were induced to chondrogenesis by the stimulation of transforming growth factor TGF-β1(10 ng/mL)in vitro. The inducing effects were identified by Alcian blue staining and immunohistochemics,and the expressions of gene c-myc during this process were detected by qPCR. The results demonstrated that,on the day 9 after induction,some MSCs begun to change from spindle-shaped to rounded or polygon,and the chrysanthemums-shaped pattern of the original MSCs gradually changed to paving stone like;the cartilage capsules were observed on the day 14,and the cartilage extracellular matrix was obvious and cell apoptosis appeared on the day 21. While the cells in the control group were observed to have apoptosis on the day 28,and the cavitations were discovered in the cells. On the day 35,massive cell apoptosis of both groups were observed,and the cell refraction became weak and the gaps between cells became larger. Theidentification by Alcian blue staining revealed that heavy positive staining emerged in the cellular matrix from the day 14 after stimulating. The detection by immunohistochemistry demonstrated that positive brown Col II reactant was in the cellular matrix from the day 21 after stimulating,and the color became darker along with the culture time,mainly distributed in the cells and their surround matrix. In the cartilage differentiation process from the day 7 to 28,the expressions of gene c-myc in the cells of the induced group were significantly lower than that of control group(P<0.05),while there was no significant difference after the day 35(P>0.05). In conclusion,Tarim wapiti antler MSCs differentiated into cartilage under the stimulation of TGF-β1. The down-regulated expression of proto-oncogene c-myc induced the apoptosis of antler MSCs and then differentiation to chondrogenic cells.
wapiti antler;mesenchymal stem cells;differentiation;transforming growth factor β1;c-myc gene
10.13560/j.cnki.biotech.bull.1985.2016.03.018
2015-06-05
國(guó)家自然科學(xué)基金項(xiàng)目(30860188)
韓春梅,女,碩士,教授,研究方向:動(dòng)物遺傳育種與繁殖;E-mail:chunmeihan224@163.com
張勤,男,博士,教授,研究方向:動(dòng)物遺傳育種與繁殖;E-mail:qzhang@cau.edu.cn