陳佳 王桂艷 張宇
(佳木斯大學(xué),佳木斯 154007)
北京鴨胚胎后腎間充質(zhì)干細(xì)胞分離培養(yǎng)及鑒定
陳佳 王桂艷 張宇
(佳木斯大學(xué),佳木斯 154007)
建立禽類后腎間充質(zhì)干細(xì)胞(Metanephric mesenchymal stem cells,MMSCs)體外培養(yǎng)體系,研究其生物學(xué)特性和多向分化潛能。采用酶消化法分離北京鴨胚胎MMSCs,繪制生長曲線,通過免疫熒光和RT-PCR對(duì)MMSCs進(jìn)行鑒定,誘導(dǎo)MMSCs向脂肪細(xì)胞和胰島細(xì)胞分化。結(jié)果表明,鴨胚胎MMSCs具有良好的增殖活力,表達(dá)間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs)特異性標(biāo)志物,并誘導(dǎo)分化為脂肪細(xì)胞和胰島細(xì)胞。北京鴨胚胎MMSCs在體外具有較強(qiáng)的自我更新能力及多向分化潛能,可作為組織工程的種子細(xì)胞進(jìn)行保存。
鴨;后腎間充質(zhì)干細(xì)胞;生物學(xué)特性;誘導(dǎo)分化
間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs)具有高度增殖、自我更新和多向分化潛能,廣泛存在于骨髓、脂肪、牙周膜、新生兒臍帶和胎盤組織中,目前大部分的研究集中于人和小鼠,關(guān)于家禽類胚胎后腎間充質(zhì)干細(xì)胞(Metanephric mesenchymal stem cells,MMSCs)的相關(guān)研究較少[1]。胚胎發(fā)育過程中,腎臟有3個(gè)相互連接、略為重疊的發(fā)育過程,即前腎、中腎和后腎,后腎發(fā)育為永久性的腎臟。后腎發(fā)育過程中,后腎間充質(zhì)干細(xì)胞有自我更新及多向分化能力,發(fā)育為所有腎單位及基質(zhì)細(xì)胞[2]。對(duì)胚胎后腎間充質(zhì)干細(xì)胞的研究有助于理解腎臟疾病發(fā)生、發(fā)展及恢復(fù)的病理生理機(jī)制,為腎臟組織工程研究提供充足的無免疫原性的腎臟種子細(xì)胞[3]。MMSCs在不同誘導(dǎo)條件下可分化為脂肪細(xì)胞、成骨細(xì)胞、肝樣細(xì)胞、上皮細(xì)胞和胰島細(xì)胞,作為前體細(xì)胞使其在組織工程創(chuàng)傷修復(fù)、細(xì)胞移植、支持造血、基因治療等方面具有廣闊的臨床應(yīng)用前景[4]。
北京鴨是家養(yǎng)鳥綱雁形目鴨科動(dòng)物,繁殖能力強(qiáng),有穩(wěn)定的遺傳特性,是北京地區(qū)特有的優(yōu)良品種,也是制作正宗北京烤鴨唯一的原材料。本研究選取北京鴨為實(shí)驗(yàn)材料,以胚胎后腎間充質(zhì)干細(xì)胞為研究對(duì)象,從形態(tài)學(xué)、細(xì)胞增殖、表面標(biāo)志物以及多向誘導(dǎo)分化潛能等方面進(jìn)行鑒定,為家禽干細(xì)胞研究及畜禽遺傳資源保存提供實(shí)驗(yàn)依據(jù)。
1.1 材料
北京鴨胚胎由中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所昌平實(shí)驗(yàn)基地種禽場提供。
DMEM/F12培養(yǎng)基、胰蛋白酶、EDTA二鈉鹽(Gibco),胎牛血清(FBS)(Hyclone),多聚甲醛(北京化工廠),兔抗雞Vimentin、CD44單克隆抗體(BIOSS),兔抗雞CD34單克隆抗體(Santa Cruz),EGF、bFGF、HGF、Active A(Peprotech),地塞米松、IBMX、胰島素、DAPI、lV型膠原酶、TritonX-100、β-毓基乙醇、尼克酞胺、L-谷氨酰胺、雙硫腙(Sigma),山羊血清、山羊抗兔FITC標(biāo)記二抗(北京中杉金橋),Trizol(Invitrogen),反轉(zhuǎn)錄試劑盒(TaKaRa)。
1.2 方法
1.2.1 MMSCs的分離培養(yǎng)和傳代 取18日齡的北京鴨胚胎,解剖取出后腎,去除輸尿管芽,用磷酸鹽緩沖溶液(phosphate-buffered saline,PBS)反復(fù)沖洗,將組織塊剪碎成1 mm3大小,37℃ 用0.1% IV型膠原酶消化20 min,0.125%胰蛋白酶和0.02% EDTA消化10 min,用含10%FBS的DMEM/F12培養(yǎng)基終止消化[5],過200目篩,1200 r/min離心10 min,加入完全培養(yǎng)基(DMEM/F12,13%FBS,1% 200 mmol/L L-谷氨酰胺),將細(xì)胞密度調(diào)整為1 × 106個(gè)/ mL,接種到培養(yǎng)皿,置于37℃,5%CO2培養(yǎng)箱中[6]。細(xì)胞匯合至80%時(shí),吸棄培養(yǎng)基,PBS洗 2次,用0.125%胰蛋白酶和0.02%EDTA消化,觀察細(xì)胞形態(tài),有大量細(xì)胞收縮變圓發(fā)亮?xí)r,用完全培養(yǎng)基終止消化,進(jìn)行傳代[7]。
1.2.2 繪制生長曲線 取生長狀態(tài)良好的P5、P10和P15代MMSCs,胰酶消化收集細(xì)胞,以1.0 ×104個(gè)/ mL的細(xì)胞密度接種于24孔板,每日隨機(jī)選3個(gè)孔,計(jì)算細(xì)胞個(gè)數(shù),取平均值,連續(xù)計(jì)數(shù)8 d,繪制生長曲線,橫坐標(biāo)為培養(yǎng)時(shí)間,縱坐標(biāo)為細(xì)胞密度[8,9]。
1.2.3 MMSCs RT-PCR鑒定 選取P4、P8和P12 代MMSCs,Trizol法提取總RNA,測定總RNA濃度及純度[10],經(jīng)反轉(zhuǎn)錄體系合成cDNA[11],用Primer Premier 5.0軟件進(jìn)行特異性引物設(shè)計(jì)(表1),間充質(zhì)干細(xì)胞特異性標(biāo)記基因:Vimentin、CD73、CD44和CD29;造血干細(xì)胞特異性標(biāo)記基因:CD34和CD45;脂肪細(xì)胞特異性標(biāo)記基因:PPAR-γ和LPL;胰島細(xì)胞特異性標(biāo)記基因:PDX-1和Insulin;內(nèi)參基因:GAPDH。PCR產(chǎn)物進(jìn)行2.5%瓊脂糖凝膠電泳[12],紫外透射儀檢測目的基因是否表達(dá),凝膠成像系統(tǒng)拍照。
表1 RT-PCR反應(yīng)引物序列表
1.2.4 MMSCs免疫熒光鑒定 細(xì)胞匯合至60%時(shí),PBS洗3次,4%多聚甲醛固定20 min,PBS洗3次,0.25%Triton X-100通透15 min,PBS洗3次,山羊血清封閉1 h[13]。棄掉封閉液,加入一抗:兔抗雞Vimentin、CD44、CD34(1∶100),4℃孵育過夜。PBS洗3次,避光加入FITC標(biāo)記的山羊抗兔IgG(1∶100),室溫孵育1 h,PBS洗3次,1 μg/mL DAPI 避光染核20 min,PBS洗3次[14]。用激光掃描共聚焦顯微鏡觀察并拍照。
1.2.5 MMSCs向脂肪細(xì)胞誘導(dǎo)分化及鑒定 P6代MMSCs匯合至70%時(shí),隨機(jī)分為誘導(dǎo)組和對(duì)照組,對(duì)照組用完全培養(yǎng)基,誘導(dǎo)組用脂肪細(xì)胞誘導(dǎo)液(DMEM/F12 +10%FBS+10-6mol/L地塞米松+10 μg/mL胰島素+ 60 μmol/L吲哚美辛+ 0.5 mmol/L 3-1-甲基異丁基黃嘌呤(IBMX)+1%L-谷氨酰胺)[15],連續(xù)誘導(dǎo)12 d,觀察細(xì)胞形態(tài),油紅O染色[16]。RT-PCR檢測脂肪細(xì)胞特異性基因過氧化物酶體增殖因子活化受體-γ(Peroxisome proliferator activatedreceptor -γ,PPAR-γ)和脂蛋白脂酶(Lipoproteinlipase,LPL)的表達(dá)[17]。
1.2.6 MMSCs向胰島細(xì)胞誘導(dǎo)分化及鑒定 P6代MMSCs匯合至60%時(shí),隨機(jī)分為誘導(dǎo)組和對(duì)照組。對(duì)照組用完全培養(yǎng)基,誘導(dǎo)組用胰島細(xì)胞預(yù)誘導(dǎo)液(15 ng/mL bFGF+15 ng/mL EGF+2%B27+ DMEM/F12),3 d后換為誘導(dǎo)液(15 ng/mL HGF+30 ng/mL active A+1 mmol/L β-巰基乙醇+10 mmol/L 尼克酰胺+2% B27+ DMEM/F12)[18]。誘導(dǎo)15 d后誘導(dǎo)組和對(duì)照組進(jìn)行雙硫腙染色[19]。RT-PCR檢測胰島細(xì)胞特異性基因胰島素(Insulin)和胰島素促進(jìn)因子-1(Pancreatic duodenal homeobox-1,PDX-1)的表達(dá)情況[20]。
2.1 MMSCs的形態(tài)學(xué)觀察和傳代培養(yǎng)
原代細(xì)胞接種12 h后完全貼壁,40 h后開始增殖,呈長梭形或紡錘形漩渦狀生長。細(xì)胞形態(tài)良好,增殖迅速,傳代所需時(shí)間短,有利于傳代培養(yǎng)。MMSCs易被胰酶消化,傳代時(shí)可通過控制胰酶消化時(shí)間進(jìn)行純化,P3代后獲得較純的MMSCs,P18代后,細(xì)胞顯示衰老跡象(圖1),MMSCs共傳25代。
圖1 北京鴨胚胎MMSCs的形態(tài)學(xué)觀察
2.2 MMSCs生長曲線
P5、P10和P15代MMSCs增殖過程經(jīng)歷潛伏期,對(duì)數(shù)期,平臺(tái)期和衰退期,細(xì)胞增殖規(guī)律均呈典型的“S”形(圖2)。
圖2 北京鴨胚胎MMSCs生長曲線
2.3 MMSCs RT-PCR和免疫熒光鑒定
P4、P8和P12代MMSCs通 過RT-PCR鑒 定 Vimentin,CD29,CD44和CD73呈陽性,CD34和CD45為陰性,GAPDH為內(nèi)參基因(圖3)。免疫熒光鑒定MSCs特異性標(biāo)記物Vimentin和CD44陽性表達(dá),造血干細(xì)胞特異性標(biāo)記物CD34陰性表達(dá)(圖4)。
圖3 北京鴨胚胎MMSCs RT-PCR鑒定
2.4 MMSCs向脂肪細(xì)胞誘導(dǎo)分化及鑒定
MMSCs經(jīng)3 d誘導(dǎo)后,細(xì)胞增殖緩慢,形態(tài)呈扁平狀,胞質(zhì)內(nèi)出現(xiàn)少量脂滴,誘導(dǎo)8 d后,肥大細(xì)胞中脂滴更加明顯,脂滴數(shù)量隨誘導(dǎo)天數(shù)的增加而增加并由小的脂滴逐漸聚集成大的脂滴,油紅O染色呈陽性,脂滴被染成紅色。對(duì)照組形態(tài)無明顯變化,無脂滴出現(xiàn),油紅O染色呈陰性(圖5)。RT-PCR鑒定誘導(dǎo)組表達(dá)PPAR-γ和LPL,對(duì)照組不表達(dá)PPAR-γ和LPL(圖6)。
圖4 北京鴨胚胎MMSCs免疫熒光鑒定
圖5 MMSCs向脂肪細(xì)胞誘導(dǎo)分化的形態(tài)學(xué)觀察及油紅O染色
圖6 MMSCs向脂肪細(xì)胞誘導(dǎo)分化RT-PCR鑒定
2.5 MMSCs向胰島細(xì)胞誘導(dǎo)分化及鑒定
誘導(dǎo)組在無血清的預(yù)誘導(dǎo)液中,細(xì)胞增殖減慢,形態(tài)無明顯變化,換誘導(dǎo)液后,形態(tài)發(fā)生變化,少量細(xì)胞聚團(tuán)生長,隨誘導(dǎo)時(shí)間的延長,細(xì)胞團(tuán)簇?cái)?shù)量逐漸增加、變大且折光性較強(qiáng)。對(duì)照組細(xì)胞數(shù)量增加,形態(tài)無明顯變化,無聚團(tuán)現(xiàn)象。誘導(dǎo)處理15 d后,誘導(dǎo)組雙硫腙染色呈陽性,細(xì)胞團(tuán)被染成棕紅色,表明誘導(dǎo)細(xì)胞的胞漿中富含鋅離子,符合胰島細(xì)胞的特征。對(duì)照組雙硫腙染色呈陰性(圖7)。RT-PCR鑒定誘導(dǎo)組表達(dá)Insulin和PDX-1,對(duì)照組呈陰性表達(dá)(圖8)。
圖7 MMSCs向胰島細(xì)胞誘導(dǎo)分化的形態(tài)學(xué)觀察及雙硫腙染色
圖8 MMSCs向胰島細(xì)胞誘導(dǎo)分化RT-PCR鑒定
有研究表明低密度接種有利于細(xì)胞增殖[21]。本研究發(fā)現(xiàn),接種密度過低,細(xì)胞增殖緩慢,難以形成集落樣生長,細(xì)胞容易老化;接種密度過高,細(xì)胞增殖較快,短時(shí)間內(nèi)傳代次數(shù)增多,細(xì)胞受機(jī)械和化學(xué)損傷嚴(yán)重,容易出現(xiàn)空泡、核收縮現(xiàn)象。所以本試驗(yàn)低代次以1∶2傳代,高代次以2∶3傳代。胰酶對(duì)細(xì)胞的傷害性很大,要嚴(yán)格控制消化時(shí)間,細(xì)胞與胰酶充分接觸后,將胰酶吸出來,當(dāng)細(xì)胞變圓回縮時(shí),立刻加入終止液,避免了培養(yǎng)液中殘留的胰酶對(duì)細(xì)胞的傷害。
MMSCs生長曲線呈典型的“S”形,經(jīng)歷潛伏期、對(duì)數(shù)期、平臺(tái)期和衰退期,符合細(xì)胞體外生長的一般規(guī)律[22]。傳代培養(yǎng)過程中存在機(jī)械和化學(xué)損傷,細(xì)胞需要有適應(yīng)和恢復(fù)時(shí)間,即潛伏期,在這之后,細(xì)胞已完全適應(yīng)新的環(huán)境并進(jìn)入對(duì)數(shù)期。由于細(xì)胞密度增大,細(xì)胞與細(xì)胞之間存在接觸抑制,細(xì)胞生長緩慢進(jìn)入平臺(tái)期,隨著接觸抑制逐漸明顯,細(xì)胞數(shù)量呈下降趨勢進(jìn)入衰退期。細(xì)胞代次越高,受外部環(huán)境的影響越大,增殖能力逐漸減弱,細(xì)胞逐漸老化。
迄今為止,還沒有發(fā)現(xiàn)MMSCs特異性標(biāo)記基因,只能根據(jù)MMSCs的形態(tài)學(xué)特征和報(bào)道過的MSCs表面標(biāo)記基因進(jìn)行鑒定,本實(shí)驗(yàn)選擇免疫熒光和RTPCR檢測MMSCs與MSCs是否具有相似的生物學(xué)特性,進(jìn)而證實(shí)分離培養(yǎng)的MMSCs即為后腎來源的間充質(zhì)干細(xì)胞。
本研究用地塞米松、胰島素、IBMX和吲哚美辛共同作用誘導(dǎo)MMSCs分化為脂肪細(xì)胞。誘導(dǎo)后胞質(zhì)內(nèi)形成大量脂滴,油紅O染色是鑒定脂滴的常用方法。LPL是脂肪分化前期的標(biāo)記基因,PPAR-γ是脂肪分化中期的標(biāo)記基因,RT-PCR檢測LPL和PPAR-γ陽性表達(dá),表明MMSCs在適當(dāng)條件下能夠誘導(dǎo)成脂肪細(xì)胞,為脂肪組織工程研究提供種子細(xì)胞。
糖尿病的發(fā)病率逐年升高,將MSCs誘導(dǎo)分化為胰島細(xì)胞,移植到患者體內(nèi)己經(jīng)成為治療糖尿病的新療法[23]。HGF能促進(jìn)胰島細(xì)胞形成,Activin A能誘導(dǎo)胰島素陽性細(xì)胞的分化,尼克酰胺促進(jìn)細(xì)胞分化成熟和聚集,增加胰島細(xì)胞數(shù)量,加快胰島細(xì)胞團(tuán)的形成。雙硫腙染色呈陽性,表明細(xì)胞團(tuán)含有豐富的鋅離子,RT-PCR檢測胰島細(xì)胞標(biāo)記基因Insulin和PDX-1呈陽性表達(dá),說明誘導(dǎo)的細(xì)胞符合胰島細(xì)胞特征,可為胰島細(xì)胞移植治療糖尿病提供種子資源。
本研究成功建立了北京鴨胚胎后腎間充質(zhì)干細(xì)胞體外分離培養(yǎng)體系,通過RT-PCR和免疫熒光鑒定MSCs的特異性標(biāo)記基因,對(duì)MMSCs自我增殖和多向分化潛能進(jìn)行鑒定,證實(shí)分離培養(yǎng)的MMSCs為后腎來源的間充質(zhì)干細(xì)胞。
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(責(zé)任編輯 李楠)
Isolation,Culture and Identification of Beijing Duck Embryonic Metanephric Mesenchymal Stem Cells
Chen Jia Wang Guiyan Zhang Yu
(University of Jaimusi,Jiamusi 154007)
This study aimed to establish in vitro culture system for the poultry metanephric mesenchymal stem cells(MMSCs), and study their biological characteristics and multi-directional differentiation potential. Enzyme digestion method was applied to separate Beijing duck embryo MMSCs and to draw the growth curve. MMSCs were identified by immunofluorescence and RT-PCR. MMSCs were induced to differentiate into adipocytes and islet cells. The results showed that MMSCs had solid proliferative activity and could express MSCs specific markers, and be induced to differentiate into adipocytes and islet cells. In summary, Beijing duck embryo MMSCs have a strong self-renewing ability and multi-directional differentiation potential in vitro, and can be saved as seed cells for tissue engineering.
duck;metanephric mesenchymal stem cell;biological characteristics;induced differentiation
10.13560/j.cnki.biotech.bull.1985.2015.06.028
2014-10-06
國家自然科學(xué)基金項(xiàng)目(31472099 ),國家家養(yǎng)動(dòng)物種質(zhì)資源平臺(tái)項(xiàng)目(2014年)
陳佳,女,碩士研究生,研究方向:藥物化學(xué);E-mail:chenjia321@126.com
王桂艷,女,副教授,研究生導(dǎo)師,研究方向:藥物化學(xué);E-mail:guiyan713@126.com