秦本源，楊陽，張燕偉，劉敏，張萬鋒，王海珍，吳怡琦，張雪蓮，蔡春波，高鵬飛，郭曉紅，李步高，曹果清

豬骨骼肌衛(wèi)星細(xì)胞分離培養(yǎng)、鑒定及其生物學(xué)特性

秦本源，楊陽，張燕偉，劉敏，張萬鋒，王海珍，吳怡琦，張雪蓮，蔡春波，高鵬飛，郭曉紅，李步高，曹果清

（山西農(nóng)業(yè)大學(xué)動物科技學(xué)院，山西太谷 030801）

【目的】建立豬骨骼肌衛(wèi)星細(xì)胞體外分離、純化及鑒定的方法，并對其生物學(xué)特性進(jìn)行探討，為進(jìn)一步研究豬肌肉生長發(fā)育提供良好的細(xì)胞模型。【方法】選取1日齡仔豬背最長肌為材料，無菌狀態(tài)下將背最長肌剪碎為肉糜狀。此后采用濃度為0.2%的Ⅰ型膠原酶消化90 min，再加入濃度為0.25%的胰蛋白酶37 ℃聯(lián)合消化30 min。經(jīng)終止消化、過濾、重懸后將分離得到的細(xì)胞置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)。選用反復(fù)差速貼壁法對骨骼肌衛(wèi)星細(xì)胞進(jìn)行純化，第一次純化選擇在細(xì)胞接種2 h后，將未貼壁細(xì)胞轉(zhuǎn)移至新培養(yǎng)皿。上清液繼續(xù)培養(yǎng)18 h后，對衛(wèi)星細(xì)胞進(jìn)行第二次差速貼壁純化。當(dāng)細(xì)胞密度達(dá)70%—80%時可對細(xì)胞進(jìn)行傳代或凍存處理。利用細(xì)胞免疫熒光技術(shù)檢測P2代衛(wèi)星細(xì)胞標(biāo)志基因、的蛋白表達(dá)情況，并繪制衛(wèi)星細(xì)胞生長曲線。分別添加不同誘導(dǎo)分化液使衛(wèi)星細(xì)胞定向分化為肌細(xì)胞、脂肪細(xì)胞、成骨細(xì)胞，檢測成肌分化標(biāo)志基因的免疫熒光，鑒定衛(wèi)星細(xì)胞肌管形成情況；油紅O染色及油紅O定量鑒定衛(wèi)星細(xì)胞誘導(dǎo)成脂分化效果；茜素紅染色鑒定衛(wèi)星細(xì)胞成骨分化能力，qRT-PCR檢測成肌、成脂、成骨進(jìn)程中關(guān)鍵基因的表達(dá)?！窘Y(jié)果】通過兩步酶消化和反復(fù)差速貼壁法分離純化得到了純度較高的衛(wèi)星細(xì)胞，剛分離的細(xì)胞折光性強，貼壁后呈梭形或紡錘形，此后細(xì)胞延展并開始快速增殖。衛(wèi)星細(xì)胞特異標(biāo)志蛋白Pax7、MyoD細(xì)胞免疫熒光鑒定結(jié)果呈陽性，表明分離細(xì)胞為骨骼肌衛(wèi)星細(xì)胞。骨骼肌衛(wèi)星細(xì)胞增殖過程經(jīng)潛伏期、生長期最終達(dá)到平臺期，細(xì)胞生長曲線呈“S”型。當(dāng)細(xì)胞生長至90%密度時，衛(wèi)星細(xì)胞會出現(xiàn)自融合現(xiàn)象。對分離的骨骼肌衛(wèi)星細(xì)胞成肌誘導(dǎo)分化后，可見鄰近衛(wèi)星細(xì)胞融合形成大量粗長肌管，多核肌管呈方向性排列，成肌標(biāo)志蛋白MHC染色呈陽性。qRT-PCR結(jié)果顯示標(biāo)志基因在成肌誘導(dǎo)分化過程中二者均呈先上升后下降趨勢。經(jīng)成脂誘導(dǎo)后細(xì)胞形態(tài)變?yōu)槿切?，連續(xù)誘導(dǎo)發(fā)現(xiàn)脂滴出現(xiàn)并聚集成大脂滴，油紅O染色可見大量紅色葡萄樣脂滴。油紅O定量檢測結(jié)果表明，成脂誘導(dǎo)過程中甘油三酯含量呈穩(wěn)步上升趨勢，各時間點均存在極顯著差異（＜0.01）。qRT-PCR結(jié)果顯示，基因表達(dá)量在誘導(dǎo)中后期高表達(dá)；在誘導(dǎo)分化第6天達(dá)到最高，極顯著高于其余時間點（＜0.01）；和表達(dá)趨勢一致，均呈先升高后降低趨勢。誘導(dǎo)成骨分化后，發(fā)現(xiàn)細(xì)胞形態(tài)變?yōu)椴灰?guī)則狀，誘導(dǎo)后期細(xì)胞復(fù)層生長形成骨鈣結(jié)節(jié)，茜素紅染色可見圓形不透明鈣化結(jié)節(jié)，數(shù)量和密度較未誘導(dǎo)時期都明顯增加，結(jié)果表明細(xì)胞出現(xiàn)成骨向分化。成骨標(biāo)志基因、的表達(dá)量也隨誘導(dǎo)進(jìn)程呈穩(wěn)步上升趨勢，相比未誘導(dǎo)細(xì)胞差異極顯著（<0.01）?！窘Y(jié)論】建立了基于聯(lián)合酶消化和反復(fù)差速貼壁實現(xiàn)豬骨骼肌衛(wèi)星細(xì)胞分離和純化的方法，所得細(xì)胞增殖能力強且具有多向分化潛能，為豬骨骼肌衛(wèi)星細(xì)胞作為種子細(xì)胞用于未來組織工程研究提供了技術(shù)平臺。

豬；衛(wèi)星細(xì)胞；分離培養(yǎng)；鑒定；分化

0 引言

【研究意義】骨骼肌衛(wèi)星細(xì)胞（satellite cells, SCs）位于肌纖維膜與基底膜之間，屬于肌源性干細(xì)胞[1]，具有較強的增殖、分化潛能。正常哺乳動物體內(nèi)，骨骼肌衛(wèi)星細(xì)胞通常處于靜息狀態(tài)，但當(dāng)肌肉組織受到高強度訓(xùn)練、損傷或其他外界刺激時，衛(wèi)星細(xì)胞便被激活[2]。此后衛(wèi)星細(xì)胞進(jìn)入細(xì)胞周期，通過增殖、遷移、分化、融合等一系列復(fù)雜的生物學(xué)過程，最后形成新的肌纖維，以恢復(fù)正常的組織結(jié)構(gòu)，維持骨骼肌的生長發(fā)育[3-5]。因此，衛(wèi)星細(xì)胞可作為組織工程的種子細(xì)胞對機體相關(guān)疾病的治療具有重大意義。豬骨骼肌衛(wèi)星細(xì)胞是探究豬肌肉形成過程的良好模型，建立體外肌衛(wèi)星細(xì)胞的分離培養(yǎng)體系對探索豬肌肉生長發(fā)育過程具有指導(dǎo)意義，也可為衛(wèi)星細(xì)胞移植在臨床中的應(yīng)用提供參考?！厩叭搜芯窟M(jìn)展】骨骼肌衛(wèi)星細(xì)胞最先由Mauro于1961年在青蛙的脛前肌中發(fā)現(xiàn)，電子顯微鏡下觀察到其位置分布緊貼于肌細(xì)胞的表面[6]。衛(wèi)星細(xì)胞的數(shù)量隨年齡的增加而逐漸減少，細(xì)胞核總數(shù)在出生后一段時期內(nèi)明顯下降，當(dāng)達(dá)到一定程度后可終身維持[7-8]。相較于動物幼齡期而言，成年后衛(wèi)星細(xì)胞含量較低，約占1%—5%[9]。先前研究已表明，各種分子標(biāo)記是不同階段細(xì)胞鑒定的基礎(chǔ)，骨骼肌衛(wèi)星細(xì)胞的特異性基因可作為骨骼肌衛(wèi)星細(xì)胞鑒定的常用方法。判定衛(wèi)星細(xì)胞最常用的標(biāo)志基因是、和[10]。配對盒基因7（pired box 7, Pax7）作為骨骼肌衛(wèi)星細(xì)胞的標(biāo)志基因，在靜息期和增殖期均有表達(dá)[11]，已有的研究結(jié)果證明對衛(wèi)星細(xì)胞的成肌分化是必須的，只有當(dāng)正常表達(dá)時才具備對肌肉損傷的修復(fù)功能[12]。生肌決定因子（myogenic determination gene, MyoD）可引導(dǎo)衛(wèi)星細(xì)胞向成肌細(xì)胞分化，缺陷型小鼠的衛(wèi)星細(xì)胞無法參與分化進(jìn)程，阻礙肌纖維的修復(fù)[13]。肌細(xì)胞生成素（myogenin, MyoG）主要在分化的中后期表達(dá)，可作為成肌分化的標(biāo)志，當(dāng)衛(wèi)星細(xì)胞融合形成肌管時，表達(dá)量的升高會促進(jìn)肌衛(wèi)星細(xì)胞終末分化。肌球蛋白重鏈（myosin heavy chain, MHC）基因最后表達(dá)，標(biāo)志著分化已進(jìn)入終末時期，肌管形成[14]。目前，關(guān)于衛(wèi)星細(xì)胞的分離主要有單根肌纖維法[15]和酶消化法[16]。其中酶消化法又可分為鏈霉蛋白酶消化法[17]和兩步酶消化法[18]，即采用膠原酶胰酶聯(lián)合消化，此法可將肌纖維中的衛(wèi)星細(xì)胞釋放，分離得到較多衛(wèi)星細(xì)胞。盡管酶消化法提高了衛(wèi)星細(xì)胞得率，但不可避免地引入了其它的非肌源性細(xì)胞，如成纖維細(xì)胞、紅細(xì)胞等[19]。因此，科學(xué)家們又對衛(wèi)星細(xì)胞的純化進(jìn)行了改良，當(dāng)前常用的純化方法主要有差速貼壁法[20]、Percoll梯度密度離心法[21]、流式細(xì)胞分選術(shù)[22]和免疫磁珠細(xì)胞分選術(shù)[23]等。骨骼肌衛(wèi)星細(xì)胞自被證實具有干細(xì)胞特性以來便引起了廣大研究者的關(guān)注，已有的研究結(jié)果發(fā)現(xiàn)，骨骼肌衛(wèi)星細(xì)胞除了可以向成肌方向分化外，在不同誘導(dǎo)環(huán)境下也可以成脂分化形成脂滴或成骨分化形成鈣沉淀[24-25]?！颈狙芯壳腥朦c】快速簡潔高效地分離高純度骨骼肌衛(wèi)星細(xì)胞成為了當(dāng)前研究的熱點，體外骨骼肌衛(wèi)星細(xì)胞的分離已在小鼠[26]、雞[27]、牛[28]、羊[29]等動物上成功獲得，但在豬上研究較少，對豬骨骼肌衛(wèi)星細(xì)胞生物學(xué)特性及多向分化潛能研究鮮有報道?！緮M解決的關(guān)鍵問題】故本研究選取1日齡仔豬背最長肌為材料，采用兩步酶消化法成功分離豬骨骼肌衛(wèi)星細(xì)胞并對其鑒定，此后分別誘導(dǎo)成肌、誘導(dǎo)成脂、誘導(dǎo)成骨檢測其多向分化潛能。分離培養(yǎng)高純度的骨骼肌衛(wèi)星細(xì)胞，有利于探明其細(xì)胞生物學(xué)特性，也為家畜遺傳資源改良和相關(guān)疾病治療奠定了良好的基礎(chǔ)。

1 材料與方法

1.1 主要試劑

1.1.1 試驗動物 選取1日齡大白豬，體重1 kg，購自山西省清徐縣天祿豐種豬育種有限公司。試驗于2019年3—7月在山西農(nóng)業(yè)大學(xué)動物科技學(xué)院動物遺傳育種與繁殖實驗室完成。

1.1.2 主要試劑 DMEM高糖培養(yǎng)基、胎牛血清FBS、馬血清HS均購自Gibco公司；胰蛋白酶、青鏈霉素混合液、PBS、油紅O染液、茜素紅染液（pH= 4.2）、4%多聚甲醛、Triton X-100、DAPI均購自Solaibio公司；2%即用型山羊血清封閉液購自博士德生物工程有限公司；Ⅰ型膠原酶、地塞米松（DEX）、胰島素、3-異丁基-1-甲基黃嘌呤（IBMX）、羅格列酮、β-甘油磷酸鈉、抗壞血酸均購自美國Sigma公司；Anti-Pax7、Anti-MHC、Anti-MyoD購自Abcam公司；FITC標(biāo)記羊抗鼠IgG二抗購自武漢三鷹生物技術(shù)有限公司；反轉(zhuǎn)錄和實時熒光定量試劑盒購自日本TaKaRa公司；引物合成于上海生工生物工程有限公司。

1.2 骨骼肌衛(wèi)星細(xì)胞的分離及純化

無菌采集仔豬背最長肌組織，先后置于75%乙醇、含2%雙抗的PBS中清洗數(shù)次，在預(yù)冷的PBS中除去肉眼可見的血管、筋膜等結(jié)締組織，剪碎為1 mm3的組織塊。轉(zhuǎn)移組織塊至無菌離心管中，用含0.2%的Ⅰ型膠原酶置于搖床消化90 min，期間每隔15 min吹打消化液一次。再用0.25%胰蛋白酶37 ℃消化30 min，期間每隔10 min晃動混勻一次。消化結(jié)束后，用等體積完全培養(yǎng)基（10% FBS+1%雙抗+DMEM高糖）終止消化，依次過70目、200目細(xì)胞篩。濾液1 000 r/min離心5 min，重懸細(xì)胞并接種至60 mm細(xì)胞培養(yǎng)皿中。置于體積分?jǐn)?shù)為5% CO2、37 ℃培養(yǎng)箱中培養(yǎng)2 h后將上清吸至新皿中，記為P01。P01細(xì)胞繼續(xù)培養(yǎng)18—24 h后再次轉(zhuǎn)移上清，記為P02。其中，P01、P02即為純化后的骨骼肌衛(wèi)星細(xì)胞，培養(yǎng)48 h后首次換液，之后每2 d更換一次完全培養(yǎng)基。

1.3 骨骼肌衛(wèi)星細(xì)胞的傳代與凍存

待培養(yǎng)皿中細(xì)胞生長匯合至70%—80%時，按照1﹕2或1﹕3的比例傳代培養(yǎng)，隔天換液，倒置顯微鏡下觀察。衛(wèi)星細(xì)胞的凍存取消化后的細(xì)胞用細(xì)胞凍存液（20% FBS+10% DMSO+DMEM）重懸，吹打混勻后加入凍存管中，-80℃冰箱過夜，取出后投入液氮中保存。復(fù)蘇時從液氮中取出細(xì)胞，立刻放入37℃水浴鍋中快速融化，離心去上清后用新鮮的完全培養(yǎng)基重懸接入新培養(yǎng)皿。

1.4 骨骼肌衛(wèi)星細(xì)胞的免疫熒光染色鑒定

選P2代骨骼肌衛(wèi)星細(xì)胞接種于24孔細(xì)胞培養(yǎng)板，待細(xì)胞密度達(dá)到50%左右時開始細(xì)胞免疫熒光染色。PBS漂洗細(xì)胞3次后，用4%多聚甲醛固定30 min，蒸餾水沖洗3次，每次5 min。0.1% Triton X-100通透細(xì)胞30 min，蒸餾水洗滌3次，每次5 min。2%即用型山羊血清封閉1 h，直接滴加一抗（Anti-Pax7, 1﹕300；Anti-MyoD, 1﹕200）覆蓋孔底，4 ℃孵育過夜。移除一抗后用蒸餾水清洗，加入熒光二抗（羊抗鼠，1﹕100），室溫避光孵育1 h。吸去二抗后每孔滴加250 μL DAPI染液，熒光顯微鏡下觀察。

1.5 骨骼肌衛(wèi)星細(xì)胞生長曲線

取生長狀態(tài)良好的P3代衛(wèi)星細(xì)胞，0.25%胰酶消化制成單細(xì)胞懸液，以1×104個/孔接種于24孔細(xì)胞培養(yǎng)板。每天取3孔細(xì)胞消化，血球計數(shù)板計數(shù)，連續(xù)8 d，以培養(yǎng)時間為X軸，細(xì)胞密度為Y軸，繪制細(xì)胞生長曲線。

1.6 骨骼肌衛(wèi)星細(xì)胞成肌誘導(dǎo)及鑒定

選P3代骨骼肌衛(wèi)星細(xì)胞接種于6孔板，待細(xì)胞生長至80%—90%匯合時，改用成肌分化培養(yǎng)基（2% HS+1%雙抗+DMEM高糖培養(yǎng)基）繼續(xù)培養(yǎng)。隔2 d換一次液，觀察細(xì)胞生長狀況和肌管融合情況。

連續(xù)誘導(dǎo)8 d，視野中看到大量細(xì)胞融合時進(jìn)行細(xì)胞免疫熒光染色。經(jīng)固定、通透、封閉后，加入一抗（Anti-MHC, 1﹕200）4 ℃過夜，第二天加入熒光二抗避光孵育1 h。滴加DAPI染液后，熒光顯微鏡觀察。

1.7 骨骼肌衛(wèi)星細(xì)胞成脂誘導(dǎo)及鑒定

取P3代骨骼肌衛(wèi)星細(xì)胞接種于6孔板，當(dāng)細(xì)胞培養(yǎng)至80%融合時，更換完全培養(yǎng)基為成脂誘導(dǎo)分化液（10% FBS+5 μg·mL-1胰島素+1 μmol·L-1DEX +0.5 mmol·L-1IBMX +1μmol·L-1羅格列酮+1%雙抗+DMEM高糖培養(yǎng)基）誘導(dǎo)3 d，之后改用成脂誘導(dǎo)維持液（10% FBS+5 μg·mL-1胰島素+1%雙抗+DMEM高糖培養(yǎng)基）維持9 d。每隔3 d更換新鮮誘導(dǎo)維持培養(yǎng)液。

油紅O染色鑒定細(xì)胞成脂分化能力。PBS緩沖液漂洗細(xì)胞3次，用4%多聚甲醛室溫固定10 min，棄去并加入新多聚甲醛再次固定1 h。蒸餾水沖洗3次，加入60%異丙醇靜置5 min，開蓋完全干燥。加入1 mL油紅O染液（油紅O儲液﹕蒸餾水=3﹕2），37 ℃染色30—40 min，棄去油紅O染液，蒸餾水洗滌4次，顯微鏡下觀察染色結(jié)果。

油紅O定量檢測，每孔加2 mL異丙醇萃取脂滴，37 ℃恒溫?fù)u床孵育15 min，酶標(biāo)儀檢測510 nm處OD值。

1.8 骨骼肌衛(wèi)星細(xì)胞成骨誘導(dǎo)及鑒定

取P3代骨骼肌衛(wèi)星細(xì)胞接種于6孔板，細(xì)胞密度達(dá)到90%后誘導(dǎo)成骨。成骨誘導(dǎo)培養(yǎng)液（10% FBS+10 mmol·L-1β-甘油磷酸鈉+0.1 μmol·L-1DEX+50 mg·L-1抗壞血酸+1%雙抗+DMEM高糖培養(yǎng)基）培養(yǎng)3周，期間每3 d換一次液，觀察細(xì)胞形態(tài)變化。

對誘導(dǎo)細(xì)胞茜素紅染色。多聚甲醛固定15 min，棄去固定液后蒸餾水洗3次，待液體完全吸干凈后加入茜素紅染色液，室溫染色30 min。棄去染液加蒸餾水保持濕潤，顯微鏡下觀察。

1.9 RNA提取及相關(guān)基因qRT-PCR檢測

成肌誘導(dǎo)組（收集0 d、誘導(dǎo)2、4、6、8 d細(xì)胞）、成脂誘導(dǎo)組（收集0 d、誘導(dǎo)3、6、9、12d細(xì)胞）、成骨誘導(dǎo)組（收集0 d、誘導(dǎo)7、14、21 d細(xì)胞）分別提取細(xì)胞RNA，并按照說明書要求反轉(zhuǎn)錄。采用qRT-PCR的方法分別檢測成肌誘導(dǎo)、成脂誘導(dǎo)、成骨誘導(dǎo)后相關(guān)基因的相對表達(dá)量，以18S為內(nèi)參基因，引物序列見表1。

表1 qRT-PCR引物

1.10 數(shù)據(jù)分析

所有試驗均設(shè)置3個生物學(xué)重復(fù)，數(shù)據(jù)結(jié)果使用SPSS Statistics 21.0軟件單因素方差分析進(jìn)行顯著性檢驗，不同時間點標(biāo)志基因表達(dá)量的差異采用Duncan’s法進(jìn)行多重比較，＜0.05表示差異顯著，＜0.01表示差異極顯著。

2 結(jié)果

2.1 骨骼肌衛(wèi)星細(xì)胞形態(tài)學(xué)觀察

分離的原代骨骼肌衛(wèi)星細(xì)胞接種至培養(yǎng)皿2 h后，其中的成纖維細(xì)胞、血細(xì)胞等雜細(xì)胞已貼壁。倒置顯微鏡下觀察，剛分離出來的衛(wèi)星細(xì)胞呈球形，折光性強（圖1-A）。此時，轉(zhuǎn)移細(xì)胞懸液到新培養(yǎng)皿中，對骨骼肌衛(wèi)星細(xì)胞進(jìn)行第一次純化。18 h后再次轉(zhuǎn)移上清液，衛(wèi)星細(xì)胞的純度再次提升。多次純化后，觀察發(fā)現(xiàn)部分衛(wèi)星細(xì)胞貼壁并向四周延展，部分細(xì)胞周圍有小的突起（圖1-B）。繼續(xù)培養(yǎng)24 h，大多數(shù)衛(wèi)星細(xì)胞已貼壁，細(xì)胞呈梭形或紡錘形，細(xì)胞飽滿且折光性較強（圖1-C）。首次更換新鮮培養(yǎng)基后，可見衛(wèi)星細(xì)胞數(shù)量明顯增多，且細(xì)胞形態(tài)也變得多樣，相鄰衛(wèi)星細(xì)胞開始相互接觸（圖1-D）。

當(dāng)衛(wèi)星細(xì)胞生長達(dá)70%時，可按照1﹕1的比例傳代培養(yǎng)或凍存（圖1-E）。由于細(xì)胞增殖能力不斷增強，細(xì)胞逐漸拉長變細(xì)形成有規(guī)律的平行排列（圖1-F）。繼續(xù)培養(yǎng)可見細(xì)胞方向性排列更明顯，并能自發(fā)融合形成多核肌管（圖1-G）。復(fù)蘇后的骨骼肌衛(wèi)星細(xì)胞與凍存前形態(tài)相似，細(xì)胞生長狀態(tài)良好且增殖能力較強，表明試驗所用的凍存體系可用來保存骨骼肌衛(wèi)星細(xì)胞（圖1-H）。

2.2 骨骼肌衛(wèi)星細(xì)胞免疫熒光鑒定

應(yīng)用細(xì)胞免疫熒光技術(shù)檢測P2代骨骼肌衛(wèi)星細(xì)胞蛋白Pax7、MyoD的表達(dá)情況，并用DAPI染核。結(jié)果發(fā)現(xiàn)，Pax7和MyoD均呈陽性表達(dá)（圖2-A、2-D），經(jīng)DAPI染色后細(xì)胞核呈藍(lán)色（圖2-B、2-E）。其中，Pax7熒光強度較強，表明其在細(xì)胞內(nèi)的表達(dá)量可能較高（圖2-C），MyoD熒光強度較弱，說明其在細(xì)胞中表達(dá)量可能較低（圖2-F），綜上確證本次試驗成功分離了骨骼肌衛(wèi)星細(xì)胞，且細(xì)胞純度較高。

2.3 骨骼肌衛(wèi)星細(xì)胞生長曲線

骨骼肌衛(wèi)星細(xì)胞生長曲線呈“S”型。體外培養(yǎng)骨骼肌衛(wèi)星細(xì)胞前2 d生長較緩慢，為潛伏期。3—5 d細(xì)胞開始迅速增殖，進(jìn)入指數(shù)生長期。此后由于細(xì)胞密度增大，細(xì)胞相互接觸造成生長減慢，達(dá)到平臺期。

A：剛分離的骨骼肌衛(wèi)星細(xì)胞呈球形，折光性強；B：部分衛(wèi)星細(xì)胞開始細(xì)胞貼壁，向四周延展；C：分離培養(yǎng)48 h衛(wèi)星細(xì)胞呈梭形或紡錘形；D：分離培養(yǎng)72 h衛(wèi)星細(xì)胞數(shù)量增加出現(xiàn)匯合；E：細(xì)胞生長至70%時的形態(tài)；F：培養(yǎng)5 d細(xì)胞拉長變細(xì)呈有規(guī)律排列；G：培養(yǎng)7 d衛(wèi)星細(xì)胞自發(fā)融合，形成多核肌管；H：復(fù)蘇第3次傳代后凍存的骨骼肌衛(wèi)星細(xì)胞

A：Pax7呈陽性表達(dá)；B：DAPI染色細(xì)胞核；C：A與B疊加圖片；D：MyoD呈陽性表達(dá)；E：DAPI染色細(xì)胞核；F：D與E疊加圖片

2.4 骨骼肌衛(wèi)星細(xì)胞成肌誘導(dǎo)

利用2% HS對骨骼肌衛(wèi)星細(xì)胞誘導(dǎo)成肌分化，加入誘導(dǎo)成肌分化液48 h后可見細(xì)胞融合并出現(xiàn)少量短粗肌管（圖4-A）。隨后，細(xì)胞融合更加廣泛，形成大量多核肌管（圖4-B）。繼續(xù)誘導(dǎo)至4 d，顯微鏡下觀察可見肌管相互融合，且肌管之間平行排列（圖4-C）。MHC細(xì)胞免疫熒光結(jié)果為陽性（圖4-D），DAPI染色可見肌管內(nèi)存在多個細(xì)胞核（圖4-E），表明本試驗衛(wèi)星細(xì)胞成肌誘導(dǎo)成功（圖4-F）。

對誘導(dǎo)0、2、4、6、8 d細(xì)胞進(jìn)行qRT-PCR檢測，結(jié)果顯示標(biāo)志基因和在誘導(dǎo)的各階段均有表達(dá)（圖5）。在成肌誘導(dǎo)分化過程中二者均呈先升高后下降趨勢，都在第4天達(dá)到最大值，且極顯著高于誘導(dǎo)0 d（＜0.01）。

圖3 骨骼肌衛(wèi)星細(xì)胞生長曲線

A：誘導(dǎo)48 h，衛(wèi)星細(xì)胞融合出現(xiàn)少量肌管；B：誘導(dǎo)72 h，肌管數(shù)量增多；C：誘導(dǎo)4 d后，肌管之間相互融合；D：分化后MHC免疫熒光；E：DAPI染核；F：D和E疊加

不同大寫字母表示差異極顯著（P＜0.01）。下同

2.5 骨骼肌衛(wèi)星細(xì)胞成脂誘導(dǎo)

選取P2代細(xì)胞誘導(dǎo)成脂分化72 h后發(fā)現(xiàn)，細(xì)胞形態(tài)由梭形變?yōu)槿切位蚨噙呅危▓D6-A）。誘導(dǎo)6 d，顯微鏡下可見細(xì)胞質(zhì)逐漸透亮，有少量脂滴出現(xiàn)（圖6-B）。繼續(xù)誘導(dǎo)至9 d，可見細(xì)胞逐漸變圓，體積增大，脂滴數(shù)量明顯增多（圖6-C）。隨著時間的延長，誘導(dǎo)12 d后，脂滴聚集融合形成大脂滴（圖6-D）。用油紅O分別對誘導(dǎo)3、6、9和12 d的衛(wèi)星細(xì)胞染色，結(jié)果可見大小不等的紅色葡萄樣脂滴（圖6-E—H），肉眼觀察培養(yǎng)板底部也出現(xiàn)紅色沉淀（圖6-I—L）。

A-D：分別為成脂誘導(dǎo)3、6、9、12 d衛(wèi)星細(xì)胞形態(tài)學(xué)觀察（100×）；E-H：誘導(dǎo)細(xì)胞油紅O染色，脂滴呈紅色（100×）；I-L：肉眼觀察誘導(dǎo)后培養(yǎng)皿底可見紅色沉淀

油紅O定量檢測結(jié)果表明，隨著成脂誘導(dǎo)的推進(jìn)，甘油三酯含量呈穩(wěn)步上升趨勢，誘導(dǎo)3 d表達(dá)量最低，12 d后含量最高，且各時間點均存在極顯著差異（＜0.01）。結(jié)合油紅O染色及定量結(jié)果，表明衛(wèi)星細(xì)胞成脂誘導(dǎo)分化成功（圖7）。

檢測、、和的表達(dá)情況，結(jié)果如圖8所示，分化各時間點與0 d相比差異極顯著（＜0.01）。其中表達(dá)量隨著誘導(dǎo)分化時間的推進(jìn)，該基因表達(dá)量前期逐步上升并保持穩(wěn)定。主要在分化中期表達(dá)，表達(dá)量呈先升高后下降趨勢。和表達(dá)情況相似，第3天達(dá)到最高，隨后逐漸下降。

2.6 骨骼肌衛(wèi)星細(xì)胞成骨誘導(dǎo)

在成骨誘導(dǎo)培養(yǎng)基中培養(yǎng)7 d后，衛(wèi)星細(xì)胞逐漸收縮變?yōu)椴灰?guī)則形態(tài)（圖9-A）。誘導(dǎo)14 d后，細(xì)胞部分復(fù)層生長，細(xì)胞間可見鈣化沉淀（圖9-B）。繼續(xù)誘導(dǎo)至21 d，細(xì)胞立體感增強，出現(xiàn)大量礦化結(jié)節(jié)（圖9-C）。茜素紅染色可見隨著分化時間的延長，呈圓形不透明的鈣化結(jié)節(jié)數(shù)量和密度都明顯增加（圖9-D—E）。染色后培養(yǎng)皿底部也出現(xiàn)了顆粒狀沉淀，進(jìn)一步表明成骨誘導(dǎo)成功（圖9-F—G）。

圖7 油紅O定量結(jié)果

圖8 成脂誘導(dǎo)分化后PPARγ、CEBP/β、FABP4和HSL表達(dá)情況

A-C：成骨誘導(dǎo)7、14、21 d衛(wèi)星細(xì)胞顯微鏡下觀察（100×）；D-F：茜素紅可將誘導(dǎo)后細(xì)胞形成的鈣化結(jié)節(jié)染成紅色（100×）；G-I：肉眼觀察誘導(dǎo)后皿底出現(xiàn)紅色沉淀結(jié)節(jié)

Fig .9 Osteogenic differentiation of skeletal muscle satellite cells and Alizarin Red staining

誘導(dǎo)骨骼肌衛(wèi)星細(xì)胞成骨分化，檢測成骨基因骨鈣素（）和Runt相關(guān)轉(zhuǎn)錄因子2（）的表達(dá)情況，如圖10所示。誘導(dǎo)成骨分化過程中，成骨標(biāo)志基因的表達(dá)水平均上調(diào)，都在第3周達(dá)到最高，與0 d相比差異極顯著（＜0.01）。

圖10 成骨誘導(dǎo)后BGLAP和RUNX2表達(dá)情況

3 討論

骨骼肌衛(wèi)星細(xì)胞作為靜息狀態(tài)的肌源性干細(xì)胞，參與骨骼肌的生長與修復(fù)過程，對維持機體穩(wěn)態(tài)具有重要作用[30]。骨骼肌衛(wèi)星細(xì)胞分離培養(yǎng)的關(guān)鍵在于使數(shù)量較多且活力較好的衛(wèi)星細(xì)胞釋放，因此有學(xué)者對衛(wèi)星細(xì)胞的純化方式進(jìn)行了改進(jìn)。單根肌纖維法是將完整的肌纖維從肌束中剝離，從而使衛(wèi)星細(xì)胞游離出來的一種方法[31]。但該方法分離衛(wèi)星細(xì)胞所需時間較長且獲得細(xì)胞數(shù)量有限，因而應(yīng)用受限。鑒于骨骼肌衛(wèi)星細(xì)胞存在于基膜與肌纖維之間，只有基膜分解才有助于衛(wèi)星細(xì)胞的獲取[32]，因此如今體外分離衛(wèi)星細(xì)胞常用酶消化法。傳統(tǒng)的單一酶消化法不能完全解離衛(wèi)星細(xì)胞，選用膠原酶胰酶的兩步酶消化法可彌補先前缺點，保證衛(wèi)星細(xì)胞的產(chǎn)出率[33]。運用酶消化法很難避免成纖維細(xì)胞的出現(xiàn)，可通過純化的方式提高衛(wèi)星細(xì)胞的純度。其中流式分選術(shù)用于人和小鼠等模式動物細(xì)胞分選較多，但基于設(shè)備的限制及細(xì)胞表面特異分子標(biāo)志價格昂貴，并未在家養(yǎng)動物中推廣。Percoll梯度密度離心法步驟繁瑣、易污染，多次離心后會導(dǎo)致細(xì)胞流失。差速貼壁法是根據(jù)衛(wèi)星細(xì)胞與雜細(xì)胞粘附時間上的差異，成纖維細(xì)胞貼壁速度快，通過多次差速得到純度較高的衛(wèi)星細(xì)胞，此法簡單易行且對細(xì)胞無損傷[34]。

本試驗選用Ⅰ型膠原酶和胰蛋白酶聯(lián)合消化法分離骨骼肌衛(wèi)星細(xì)胞，并對消化時間進(jìn)行嚴(yán)格控制。剛分離出的骨骼肌衛(wèi)星細(xì)胞呈圓形，通過反復(fù)差速貼壁兩次以逐步去除成纖維細(xì)胞、血細(xì)胞等雜細(xì)胞，實現(xiàn)對衛(wèi)星細(xì)胞的純化。重新接種6 h后，部分衛(wèi)星細(xì)胞開始貼壁延展，繼續(xù)培養(yǎng)24 h，細(xì)胞呈現(xiàn)長梭形或紡錘形，接種3 d可見貼壁細(xì)胞數(shù)量明顯增多，部分細(xì)胞出現(xiàn)匯合。接種5 d后，細(xì)胞密度達(dá)到90%，此時細(xì)胞呈有規(guī)律的方向性生長，繼續(xù)培養(yǎng)則衛(wèi)星細(xì)胞出現(xiàn)自融合現(xiàn)象，臨近細(xì)胞會融合成肌管。分離純化結(jié)果與Baquero等研究結(jié)果基本一致[35-36]。

目前對骨骼肌衛(wèi)星細(xì)胞的鑒定有多種方法，其中公認(rèn)度最高的是免疫細(xì)胞化學(xué)染色法，該法原理就是抗原抗體反應(yīng)，因靈敏度高、特異性強等優(yōu)點被廣泛使用[37]。已有報道證實95%以上的衛(wèi)星細(xì)胞都表達(dá)，此外還能調(diào)控衛(wèi)星細(xì)胞處于靜息或激活狀態(tài)，因此可作為鑒定骨骼肌衛(wèi)星細(xì)胞的標(biāo)志物[38]。本試驗參考FENG等[39]的方法，通過細(xì)胞免疫熒光法對衛(wèi)星細(xì)胞特異性基因、的蛋白表達(dá)情況進(jìn)行檢測，結(jié)果可見標(biāo)志物染色呈陽性，且陽性細(xì)胞多，表明分離培養(yǎng)的衛(wèi)星細(xì)胞純度高、活性好。

骨骼肌衛(wèi)星細(xì)胞對培養(yǎng)要求較高，大量的研究結(jié)果表明，衛(wèi)星細(xì)胞的體外培養(yǎng)需要添加合適濃度的血清，高濃度的血清有助于細(xì)胞增殖，血清濃度的降低會促進(jìn)衛(wèi)星細(xì)胞的分化。STARKEY的研究發(fā)現(xiàn)，低血清濃度可使衛(wèi)星細(xì)胞自發(fā)向成肌細(xì)胞方向分化，但并不能自行分化為脂肪細(xì)胞、骨細(xì)胞等非成肌細(xì)胞[40]。已有的研究結(jié)果證實，在添加堿性成纖維細(xì)胞生長因子的培養(yǎng)基中，有利于衛(wèi)星細(xì)胞的增殖[41]。本試驗最終選擇在高糖培養(yǎng)基中添加10%濃度胎牛血清來培養(yǎng)骨骼肌衛(wèi)星細(xì)胞，當(dāng)細(xì)胞增殖匯合到一定密度后，即使高濃度血清培養(yǎng)細(xì)胞也會出現(xiàn)不可逆分化，最終融合成肌管的現(xiàn)象，因此衛(wèi)星細(xì)胞的傳代應(yīng)選擇細(xì)胞密度70%左右時進(jìn)行。本試驗結(jié)合之前方法用2%馬血清誘導(dǎo)衛(wèi)星細(xì)胞分化，結(jié)果顯示誘導(dǎo)4 d可見大量較長較粗肌管形成，MHC染色呈陽性，進(jìn)一步確證骨骼肌衛(wèi)星細(xì)胞的成肌分化。

在模式動物中的研究中，已證實體外培養(yǎng)的小鼠衛(wèi)星細(xì)胞可直接分化成脂肪細(xì)胞、骨細(xì)胞和軟骨細(xì)胞等[42]。目前，對豬骨骼肌衛(wèi)星細(xì)胞多向分化潛能的研究較少。本試驗參考“激素雞尾酒”法對衛(wèi)星細(xì)胞誘導(dǎo)成脂，在添加胰島素、地塞米松、IBMX和羅格列酮等誘導(dǎo)因子條件下培養(yǎng)衛(wèi)星細(xì)胞[43]。對細(xì)胞成脂能力的鑒定主要選用油紅O染色，油紅O染色呈陽性可作為脂滴沉積的標(biāo)志[44]。結(jié)果發(fā)現(xiàn)誘導(dǎo)3 d衛(wèi)星細(xì)胞形態(tài)發(fā)生變化，此后更換為維持培養(yǎng)基繼續(xù)誘導(dǎo)可見大量脂滴出現(xiàn)，油紅O染色及定量結(jié)果都表明成脂誘導(dǎo)成功。對于骨骼肌衛(wèi)星細(xì)胞成骨誘導(dǎo)，多在培養(yǎng)基中補充β-甘油磷酸鈉、抗壞血酸和地塞米松來促進(jìn)鈣質(zhì)形成，誘導(dǎo)結(jié)節(jié)鈣化[45]。利用茜素紅可與鈣發(fā)生顯色反應(yīng)的特性，可結(jié)合染色結(jié)果判定細(xì)胞成骨礦化結(jié)節(jié)積累情況，進(jìn)而鑒定細(xì)胞成骨分化效果[46]。本試驗結(jié)果表明誘導(dǎo)衛(wèi)星細(xì)胞成骨分化后，茜素紅染色可見大量骨鈣結(jié)節(jié)形成，同時熒光定量結(jié)果顯示誘導(dǎo)成骨成功，這與REN等[47]在山羊上的研究結(jié)果相似。

眾所周知，骨骼肌衛(wèi)星細(xì)胞作為機體干細(xì)胞的一種，具有可塑性強、多潛能等優(yōu)點[48]。衛(wèi)星細(xì)胞體外分離培養(yǎng)體系的建立，可為豬骨骼肌生長發(fā)育、肉質(zhì)改良提供研究基礎(chǔ)。同時為畜禽遺傳資源保存提供了新方法，也為今后基因組學(xué)研究提供相應(yīng)的實驗材料。此外本試驗還證明了豬骨骼肌衛(wèi)星細(xì)胞的多向分化潛能，拓寬了衛(wèi)星細(xì)胞在組織工程和再生醫(yī)學(xué)上的研究價值和廣泛的應(yīng)用前景。

4 結(jié)論

本試驗成功分離豬骨骼肌衛(wèi)星細(xì)胞，通過細(xì)胞免疫熒光檢測骨骼肌衛(wèi)星細(xì)胞標(biāo)志蛋白對其進(jìn)行了鑒定。建立了豬骨骼肌衛(wèi)星細(xì)胞體外分離、純化以及鑒定的方法，同時利用不同誘導(dǎo)劑分別對其誘導(dǎo)成肌、成脂、成骨，探索了骨骼肌衛(wèi)星細(xì)胞的生物學(xué)特性。對研究豬肌肉生長發(fā)育過程具有一定科學(xué)意義，也為后續(xù)探究豬肌肉衛(wèi)星細(xì)胞功能和分子機制提供了試驗材料。

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Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells

QIN BenYuan, YANG Yang, ZHANG YanWei, LIU Min, ZHANG WanFeng, WANG HaiZhen, WU YiQi, ZHANG XueLian, CAI ChunBo, GAO PengFei, GUO XiaoHong, LI BuGao, CAO GuoQing

(College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi)

【Objective】 The aim of this study was to establish a method for isolation, purification and identification of porcine skeletal muscle satellite cells in vitro, and to explore its biological characteristics, in order to provide a reliable cell model for further research of muscle growth and development in pigs. 【Method】In this study, themuscle of 1 day old pig was selected and cut into meat emulsion in aseptic state. After that, it was digested with 0.2% type I collagenase for 90 min, and then digested with 0.25% trypsin for 30 min at 37 ℃. After termination of digestion, filtration and resuspension, the isolated cells were cultured in a 37 ℃ and 5% CO2cell incubator. The skeletal muscle satellite cells were purified by repeated differential adherence technique. The first purification selection was performed after cell culturing for 2 h, and non-adherent cells were transferred to a new culture dish. After the supernatant was further cultured for 18 h, the satellite cells were purified again. The cells were subcultured or frozen when the cell density reached 70% - 80%. Cell immunofluorescence technique was used to detect the protein expression of marker genes ofandof P2 satellite cell, and the growth curve was determined. The satellite cells were differentiated into myocytes, adipocytes and osteoblasts by adding different inducing differentiation fluids. The protein expression of myoblast differentiation marker genewas detected by using cell immunofluorescence to identify myotube formation in satellite cells. Oil red O staining and triglyceride content were quantified to identify the adipogenic differentiation effect in satellite cells. Alizarin red staining was used to identify the osteogenic differentiation ability in satellite cells, which were detected the expression of key genes during myogenesis, adipogenesis and osteogenesis by qRT-PCR. 【Result】 The results showed that the satellite cells with higher purity were isolated and purified by two-step enzymatic digestion and repeated differential adherence technique. The cells that were originally isolated with highly refractive, and were fusiform or spindle-shaped after adherence, after which the cells extended and began to proliferate. The results of cell immunofluorescence identification of satellite cells specific marker proteins Pax7 and MyoD were positive, indicating that the isolated cells were skeletal muscle satellite cells. Skeletal muscle satellite cell proliferation underwent the incubation period, and the growth period and finally reached the plateau phase. The satellite cells were self-fusion when the cells grew to 90% density. After myogenic induction and differentiation of the satellite cells, a large number of myotubes were formed by the adjacent satellite cells. Multinucleated myotubes were regularly arranged and the myoblast marker protein MHC staining was positive. The qRT-PCR results showed that the marker genes ofandboth increased first and then decreased during the process of myoblast differentiation. After adipogenic induction, the cell morphology changed into triangle, and lipid droplets appeared and aggregated into large lipid droplets with continuous induction. Oil red O staining observed a large number of red grape-like lipid droplets. Oil red O staining quantitative results showed that the triglyceride content steadily increasing during the adipogensis. There were extremely significant differences among each time point (＜0.01). The qRT-PCR results showed that the expression level ofgene was high in the middle and the late stage of induction. Thegene reached the highest at the 6th day of induction and was significantly higher than that at other time points (＜0.01). A similar dynamic was observed with the relative expression level ofandin the differentiating cells. Their expression tended to increase first and then decrease. After inducing osteogenic differentiation, it was found that the cell morphology became irregular. Cells formed bone nodules after inducing, compared with the period without induction, the alizarin red staining showed that the number and density of round opaque calcified nodules were significantly increased. The results showed that the cells appeared osteogenic differentiation. The expression levels of osteogenic marker genesandalso showed a steady upward trend during the inducing procession, which was significantly different from that without inducing cells (＜0.01). 【Conclusion】 This study established a method for isolation and purification of pig skeletal muscle satellite cells based on combined enzyme digestion and differential adherent technique. The obtained cells had strong proliferation ability and multi-directional differentiation potential. The results provided a technical platform for pig skeletal muscle satellite cells as seed cells for future tissue engineering research.

pig; satellite cell; isolation culture; identification; differentiation

2019-09-29；

2020-01-13

國家自然科學(xué)基金（31872336）、三晉學(xué)者支持計劃專項經(jīng)費資助（2016；2017）、山西省“1331”工程資助、山西省農(nóng)業(yè)重點研發(fā)項目（201803D221022-1）、山西農(nóng)業(yè)大學(xué)畜牧學(xué)學(xué)科建設(shè)專項課題資助

秦本源，Tel：18503442342；E-mail：923726358@qq.com。楊陽，Tel：13007050956；E-mail：yangyangh@163.com。秦本源與楊陽為同等貢獻(xiàn)作者。通信作者曹果清，Tel：13403665105；E-mail：anniecao710502@ aliyun.com

（責(zé)任編輯 林鑒非）