劉志剛，熊　敏※，唐　冰，李　鋒，余化龍，曾　云，陳　潔，何　寧，王志勇，韓　珩

(1.湖北醫(yī)藥學(xué)院附屬東風(fēng)醫(yī)院骨科研究所,湖北 十堰 442000； 2.華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院骨科,武漢 430030)

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人骨髓間充質(zhì)干細(xì)胞在體外向成骨細(xì)胞定向分化的研究分析

劉志剛1，熊敏1※，唐冰1，李鋒2，余化龍1，曾云1，陳潔1，何寧1，王志勇1，韓珩1

(1.湖北醫(yī)藥學(xué)院附屬東風(fēng)醫(yī)院骨科研究所,湖北 十堰 442000； 2.華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院骨科,武漢 430030)

摘要：目的探討人骨髓間充質(zhì)干細(xì)胞在體外向成骨細(xì)胞定向分化情況。方法取引產(chǎn)胎兒的人骨髓間充質(zhì)干細(xì)胞進(jìn)行原代細(xì)胞培養(yǎng)，誘導(dǎo)培養(yǎng)向成骨細(xì)胞分化，誘導(dǎo)后進(jìn)行形態(tài)學(xué)觀察，堿性磷酸酶(ALP)染色與生長(zhǎng)曲線測(cè)定。當(dāng)人骨髓間充質(zhì)干細(xì)胞培養(yǎng)生長(zhǎng)密度為80.0%左右時(shí)分為轉(zhuǎn)染組與對(duì)照組，對(duì)照組不進(jìn)行轉(zhuǎn)染，轉(zhuǎn)染組常規(guī)進(jìn)行轉(zhuǎn)染。結(jié)果原代細(xì)胞生長(zhǎng)2 h開(kāi)始貼壁，培養(yǎng)4 d左右胞體逐漸變得粗大。經(jīng)成骨誘導(dǎo)后細(xì)胞形態(tài)由長(zhǎng)梭形逐漸變?yōu)槎噙呅?，形成?xì)胞結(jié)節(jié)甚至團(tuán)塊狀。轉(zhuǎn)染組的胎兒骨髓間質(zhì)干細(xì)胞和未轉(zhuǎn)染對(duì)照組的生長(zhǎng)狀況對(duì)比差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，轉(zhuǎn)染細(xì)胞與未轉(zhuǎn)染細(xì)胞倍增時(shí)間分別為(23±4) h和(25±4) h。轉(zhuǎn)染組在轉(zhuǎn)染后第3、6、9、12日的ALP活性均明顯高于對(duì)照組[(0.12±0.02)%比( 0.10±0.02)%,(0.16±0.02)% 比(0.12±0.02)%,(0.20±0.03)% 比(0.13±0.02)%,(0.23±0.04)% 比(0.14±0.02)%]，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。結(jié)論胎兒骨髓間充質(zhì)干細(xì)胞在成骨細(xì)胞分化轉(zhuǎn)染的誘導(dǎo)下可促進(jìn)向成骨細(xì)胞進(jìn)行定向分化，為胎兒間質(zhì)干細(xì)胞分化調(diào)控和藥物作用機(jī)制研究奠定基礎(chǔ)。

關(guān)鍵詞：人骨髓間充質(zhì)干細(xì)胞；成骨細(xì)胞；定向分化；地塞米松；堿性磷酸酶

骨髓間充質(zhì)干細(xì)胞屬于成體干細(xì)胞，在骨髓中的含量很低，占骨髓內(nèi)單核細(xì)胞總數(shù)的1/104～1/105。但是其可在體外分離及大量擴(kuò)增培養(yǎng)，在不同的誘導(dǎo)劑條件可分化為成骨細(xì)胞、軟骨細(xì)胞和脂肪細(xì)胞，也充分證明了骨髓間充質(zhì)干細(xì)胞是多潛能間質(zhì)干細(xì)胞[1-3]。骨髓間充質(zhì)干細(xì)胞因具有獨(dú)特的優(yōu)越性而成為骨組織工程中理想的種子細(xì)胞，為骨缺損和骨壞死的修復(fù)提供了新方法[4-5]。同時(shí)骨髓成骨微環(huán)境是一個(gè)復(fù)雜的三維環(huán)境，骨髓微環(huán)境不僅為細(xì)胞間的相互溝通提供場(chǎng)所，也為成骨的增殖和分化提供必要條件，更重要的是它可以在骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞相互作用的基礎(chǔ)上產(chǎn)生調(diào)控功能，從而保證機(jī)體骨細(xì)胞的穩(wěn)定狀態(tài)[6-7]。骨髓間充質(zhì)干細(xì)胞的轉(zhuǎn)分化包含兩種形式:一種骨髓間充質(zhì)干細(xì)胞在不同環(huán)境下可分別向不同組織細(xì)胞分化[8]；另一種是骨髓間充質(zhì)干細(xì)胞向一種組織細(xì)胞分化后，在微環(huán)境改變的情況下又再向另外的組織細(xì)胞轉(zhuǎn)分化[9]。本研究主要探討人骨髓間充質(zhì)干細(xì)胞在體外向成骨細(xì)胞定向分化情況，現(xiàn)報(bào)道如下。

1材料與方法

1.1試劑與儀器DMEM培養(yǎng)基、青-鏈霉素溶液、十二烷基磺酸納(美國(guó)Gibco公司)、胎牛血清、胰蛋白酶、地塞米松(美國(guó)Promega公司)、乙二胺四乙酸、維生素C、β-甘油磷酸、二乙醇胺、D-磷酸硝基酚、二甲基亞砜(美國(guó)Sigma公司)。293T細(xì)胞株、質(zhì)粒T antigen和PCI1OA1由本實(shí)驗(yàn)室保存。倒置相差顯微鏡(日本Olympus公司)、CO2培養(yǎng)箱(美國(guó)Sheldon公司)、超凈工作臺(tái)(中國(guó)蘇凈集團(tuán))、低速冷凍離心機(jī)和高速冷凍離心機(jī)(美國(guó)Gigma公司)。

1.2人骨髓間充質(zhì)干細(xì)胞的分離和培養(yǎng)在產(chǎn)婦知情同意與醫(yī)院倫理批準(zhǔn)的前提下，取胎齡4個(gè)月的引產(chǎn)胎兒，乙醇消毒后取出四肢長(zhǎng)骨。無(wú)菌環(huán)境下剝離肌肉和骨膜，用微量一次性注射器沖洗骨髓腔。以離心半徑10 cm，1000 r/min 離心10 min，棄上清，加入15 mL DMEM培養(yǎng)液重懸細(xì)胞。將細(xì)胞的濃度調(diào)整至107個(gè)細(xì)胞/mL，重懸后加入裝有3 mL Ficoll淋巴分離液的離心管中，以離心半徑10 cm，2000 r/min 離心30 min。吸出培基和分離液之間的間質(zhì)干細(xì)胞層，再用DMEM培養(yǎng)基洗滌3次(離心半徑10 cm,1000 r/min，5 min)。重懸后將濃度調(diào)整至106個(gè)細(xì)胞/mL，種入25 cm2的塑料培養(yǎng)瓶中，常規(guī)在CO2培養(yǎng)箱進(jìn)行培養(yǎng)與換代。

1.3細(xì)胞轉(zhuǎn)染當(dāng)人骨髓間充質(zhì)干細(xì)胞培養(yǎng)生長(zhǎng)密度為80.0%左右時(shí)分為轉(zhuǎn)染組與對(duì)照組，對(duì)照組不進(jìn)行轉(zhuǎn)染。而轉(zhuǎn)染組在轉(zhuǎn)染前1日，按1×106個(gè)細(xì)胞/mL將293T細(xì)胞密度加入不含抗生素的DMEM 10 mL培養(yǎng)基種入10 cm的培養(yǎng)皿中，常規(guī)溶解SV40病毒質(zhì)粒和包裝質(zhì)粒PCI1OA1和Lipofectamin 2000，混勻后在室溫下孵育5～10 min，形成混合物。將前1日種板的293T細(xì)胞培基吸掉，加入3 mL上述混合物，孵育6 h后，加入新鮮培養(yǎng)基，培養(yǎng)48 h后將培養(yǎng)基用4.5 μm的濾膜過(guò)濾。然后加入50%合的原代間質(zhì)干細(xì)胞，同時(shí)加入800 mg/L的Polybene 5 μL。傳代后加入含0.5 mg/L的嘌呤霉素進(jìn)行篩選，繼續(xù)進(jìn)行傳代。

1.4成骨誘導(dǎo)分化將轉(zhuǎn)染細(xì)胞的轉(zhuǎn)染組和未轉(zhuǎn)染的P4代對(duì)照組細(xì)胞按1×104個(gè)細(xì)胞/mL密度接種12孔板，第2日換含誘導(dǎo)劑的培基(10%胎牛血清，5 mmol/L β-甘油磷酸、25 mg/L 維生素C、1 mmol/mL地塞米松)，傳代進(jìn)行培養(yǎng)。倒置顯微鏡下觀察細(xì)胞生長(zhǎng)的速度、形態(tài)，并進(jìn)行攝像保存。

1.5細(xì)胞生長(zhǎng)曲線的測(cè)定取生長(zhǎng)良好的第3代細(xì)胞和轉(zhuǎn)染后的細(xì)胞消化制備單細(xì)胞懸液，調(diào)整細(xì)胞濃度為1×104個(gè)細(xì)胞/mL接種于6 cm培養(yǎng)皿。常規(guī)進(jìn)行培養(yǎng)，每日取出3個(gè)培養(yǎng)皿，胰酶消化后計(jì)算每皿細(xì)胞數(shù)，多次測(cè)量取均值。以細(xì)胞數(shù)對(duì)數(shù)值為縱軸，培養(yǎng)時(shí)間為橫軸，繪制細(xì)胞生長(zhǎng)曲線。根據(jù)Patterson公式計(jì)算細(xì)胞群體倍增時(shí)間[10]。

1.6堿性磷酸酶 (alkaline phosphatase，ALP)活性測(cè)定取轉(zhuǎn)染的與未轉(zhuǎn)染細(xì)胞的細(xì)胞，用10%胎牛血清洗滌3次，0.04 g/L蛋白酶E消化并收集細(xì)胞。然后進(jìn)行細(xì)胞內(nèi)ALP活性測(cè)定，采用分光光度法進(jìn)行測(cè)定，400 nm處測(cè)定吸光度，測(cè)定3次，取平均值。

2結(jié)果

2.1顯微觀察原代細(xì)胞生長(zhǎng)2 h開(kāi)始貼壁，初期細(xì)胞呈長(zhǎng)梭形伸展貼壁，培養(yǎng)4 d左右后胞體逐漸變得粗大，有細(xì)長(zhǎng)的突起(圖1)。經(jīng)成骨誘導(dǎo)后，細(xì)胞形態(tài)由長(zhǎng)梭形逐漸變?yōu)槎噙呅?，胞核大而清晰，單核?個(gè)左右核仁，形成細(xì)胞結(jié)節(jié)甚至團(tuán)塊狀(圖2)。

圖1　原代人骨髓間充質(zhì)干細(xì)胞　(HE染色×20)

圖2　誘導(dǎo)劑誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞　(HE染色×20)

2.2細(xì)胞轉(zhuǎn)染效果將0.5 mg/L的嘌呤霉素加入轉(zhuǎn)染后的胎兒骨髓間質(zhì)干細(xì)胞，轉(zhuǎn)染組48 h后出現(xiàn)少量死亡細(xì)胞，72 h后細(xì)胞完全融合。而未轉(zhuǎn)染細(xì)胞的對(duì)照組全部死亡。而從繪制的生長(zhǎng)曲線可以看出，轉(zhuǎn)染組的胎兒骨髓間質(zhì)干細(xì)胞和未轉(zhuǎn)染對(duì)照組的生長(zhǎng)狀況對(duì)比差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)，轉(zhuǎn)染細(xì)胞與未轉(zhuǎn)染細(xì)胞倍增時(shí)間分別為(23±4) h和(25±4) h。見(jiàn)圖3。

圖3　未轉(zhuǎn)染人骨髓間充質(zhì)干細(xì)胞與轉(zhuǎn)染人骨髓間

2.3兩組ALP活性比較轉(zhuǎn)染組在轉(zhuǎn)染后第3、6、9、12日的ALP活性均明顯高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。見(jiàn)表1。

表1轉(zhuǎn)染組與對(duì)照組轉(zhuǎn)染后第3、6、9、12日ALP活性比較

組別3d6d9d12d對(duì)照組0.10±0.020.12±0.020.13±0.020.14±0.02轉(zhuǎn)染組0.12±0.020.16±0.020.20±0.030.23±0.04t3.2687.1529.55712.458P<0.01<0.01<0.01<0.01

ALP：堿性磷酸酶; 對(duì)照組:不進(jìn)行轉(zhuǎn)染

3討論

骨髓間充質(zhì)干細(xì)胞來(lái)源于中胚層，具有多向分化潛能，能向成骨細(xì)胞、內(nèi)皮細(xì)胞、脂肪細(xì)胞、成纖維細(xì)胞、網(wǎng)狀細(xì)胞等分化，是目前應(yīng)用較廣泛的重要種子細(xì)胞之一[10-11]。

在體外，建立切實(shí)可行的誘導(dǎo)處理分化條件，提高骨髓間充質(zhì)干細(xì)胞向神經(jīng)元細(xì)胞誘導(dǎo)處理的效率，是骨髓間充質(zhì)干細(xì)胞最終應(yīng)用于臨床治療的基礎(chǔ)工作。但骨髓中骨髓間充質(zhì)干細(xì)胞的含量很低，并隨年齡增加而減少[12]。要利用骨髓間充質(zhì)干細(xì)胞就必須實(shí)現(xiàn)其體外分離培養(yǎng)和克隆。骨髓間充質(zhì)干細(xì)胞具有在塑料培養(yǎng)瓶中貼壁生長(zhǎng)的特性，同時(shí)操作方法簡(jiǎn)單、易掌握，是一個(gè)較好的分離方法。同時(shí)認(rèn)為在分離骨髓間充質(zhì)干細(xì)胞時(shí)選擇Percoll分離液對(duì)細(xì)胞進(jìn)行分離較好，用Percoll分離液對(duì)大鼠骨髓作梯度離心，除去大部分血細(xì)胞，收集單個(gè)核細(xì)胞；用貼壁法培養(yǎng)除去剩余的非骨髓間充質(zhì)干細(xì)胞，該方法所得細(xì)胞純度較高。

通過(guò)顯微鏡下觀察發(fā)現(xiàn)，胎兒骨髓間質(zhì)干細(xì)胞的形態(tài)與文獻(xiàn)報(bào)道[13]的成人的骨髓間充質(zhì)干細(xì)胞C的形態(tài)一致，初期細(xì)胞呈長(zhǎng)梭形伸展貼壁，培養(yǎng)4 d左右胞體逐漸變得粗大，有細(xì)長(zhǎng)的突起。傳代后生長(zhǎng)明顯加快，2～7 d為對(duì)數(shù)生長(zhǎng)期，細(xì)胞倍增時(shí)間為(23±4) h，主要在于胎兒處于發(fā)育早期，細(xì)胞增殖能力更強(qiáng)。

骨髓間質(zhì)干細(xì)胞的可以自發(fā)的向成骨細(xì)胞分化，在體外含10%胎牛血清普通培養(yǎng)條件下骨髓間質(zhì)干細(xì)胞還可部分自發(fā)地分化成脂肪細(xì)胞，不過(guò)傳代至5～6代細(xì)胞即開(kāi)始出現(xiàn)老化，增殖緩慢，不能形成骨結(jié)節(jié)[13]。為此本研究用SV40病毒大、小T片段轉(zhuǎn)染原代培養(yǎng)的胎兒骨髓間質(zhì)干細(xì)胞，用嘌呤霉素進(jìn)行篩選，得到的細(xì)胞具有更好的活力。骨髓間質(zhì)干細(xì)胞的體外成骨分化很大程度上依賴于誘導(dǎo)條件，如地塞米松、β-甘油磷酸鈉和維生素C作用于骨髓間質(zhì)干細(xì)胞，在特定的濃度下則是誘導(dǎo)其向成骨細(xì)胞分化的基本輔劑。其中地塞米松可以誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化表達(dá)骨鈣素，誘導(dǎo)其ALP活性，刺激細(xì)胞外膠原間充質(zhì)的生物合成、鈣的沉積和鈣化結(jié)節(jié)的形成[14]。而β-甘油磷酸鈉提供磷離子作為ALP作用的底物，誘導(dǎo)和激活A(yù)LP。而在維生素C存在時(shí)，β-甘油磷酸鈉對(duì)ALP的活性增加無(wú)明顯作用。只有在β-甘油磷酸鈉時(shí)，骨髓間充質(zhì)干細(xì)胞才發(fā)生礦化結(jié)節(jié)的沉積。而地塞米松的存在不僅促進(jìn)骨髓間充質(zhì)細(xì)胞生長(zhǎng)和分化，而且調(diào)節(jié)成骨細(xì)胞分泌胰島素樣生長(zhǎng)因子，促進(jìn)細(xì)胞外間充質(zhì)膠原合成。本研究骨髓間質(zhì)干細(xì)胞經(jīng)成骨誘導(dǎo)后，細(xì)胞形態(tài)由長(zhǎng)梭形逐漸變?yōu)槎噙呅?，胞核大而清晰，單核?個(gè)左右核仁，形成細(xì)胞結(jié)節(jié)甚至團(tuán)塊狀。轉(zhuǎn)染組48 h后出現(xiàn)少量死亡細(xì)胞，72 h后細(xì)胞完全融合。轉(zhuǎn)染組的胎兒骨髓間質(zhì)干細(xì)胞和未轉(zhuǎn)染對(duì)照組的生長(zhǎng)狀況對(duì)比差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.01)，轉(zhuǎn)染細(xì)胞的倍增時(shí)間為(25±4) h。

ALP活性增強(qiáng)是成骨細(xì)胞分化成熟的重要標(biāo)志。ALP是成熟成骨細(xì)胞的標(biāo)志酶之一，其主要作用為水解有機(jī)磷酸酶，啟動(dòng)鈣化。其活性的增加反映了成骨細(xì)胞成熟的程度增高[15]。本研究結(jié)果顯示，轉(zhuǎn)染組不同時(shí)間點(diǎn)的ALP活性均明顯高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，表明分離的細(xì)胞是骨髓間質(zhì)干細(xì)胞，用SV40病毒基因穩(wěn)定轉(zhuǎn)染的細(xì)胞形態(tài)和成骨特性與未轉(zhuǎn)染的細(xì)胞無(wú)明顯差異，多次傳代后仍保持良好的增殖及分化狀態(tài)。而未轉(zhuǎn)染的細(xì)胞傳至第5～6代后就逐漸老化，失去增殖和分化的能力。

總之，胎兒間質(zhì)干細(xì)胞系是一種很好的研究細(xì)胞增殖、分化調(diào)控和藥物作用機(jī)制的細(xì)胞模型。胎兒骨髓間充質(zhì)干細(xì)胞在地塞米松、β-甘油磷酸鈉和維生素C的誘導(dǎo)下在體外可向成骨細(xì)胞進(jìn)行定向分化，為以后進(jìn)一步進(jìn)行胎兒間質(zhì)干細(xì)胞分化調(diào)控和藥物作用機(jī)制研究打下了基礎(chǔ)。

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歡 迎 閱 讀《 醫(yī) 學(xué) 綜 述 》半月刊6-106

The Basic Research Analysis of Direct Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Osteoblasts in VitroLIUZhi-gang1,XIONGMin1,TANGBing1,LIFeng2,YUHua-long1,ZENGYun1,CHENJie,HENing1,WANGZhi-yong1,HANHeng1.(1.DepartmentofOrthopedicsInstitute,AffiliatedDongfengHospitalofHubeiMedicalCollege,Shiyan442000,China; 2.DepartmentofOrthopedics,TongjiHospitalAffiliatedtoTongjiMedicalCollegeofHuazhongUniversityofScienceandTechnology,Wuhan430030,China)

Abstract:ObjectiveTo discuss human bone marrow mesenchymal stem cells differentiation into osteoblasts in vitro.MethodsHuman bone marrow mesenchymal stem cells of induced labor fetus were taken for primary culture to induce osteoblast differentiation,the morphology was observed,the alkaline phosphatase(ALP) staining and the growth curve were determined.When human bone marrow mesenchymal stem cell culture growth density was about 80.0%,they were divided into two groups:transfection group and control group,the control group was not transfected,the transfection group was givenconventional transfection.ResultsThe primary cell were adherent growth after 2 h and showed stretch fusiform adherent cells after cultured for 4 days.The cell body gradually became thick and showed elongated protrusions.After inducing,the cells showed morphology.The cells growth conditions between the transfected fetal bone marrow mesenchymal stem cells and the untransfected cells showed no significant difference(P>0.05).The doubling time of the transfected cells and non-transfected cells were (23±4) h and (25±4) h respectively.The ALP activity of the transfection group was significantly higher at different times [(0.12±0.02)% vs ( 0.10±0.02)%,(0.16±0.02)% vs (0.12±0.02)%,(0.20±0.03)% vs (0.13±0.02)%,(0.23±0.04)% vs (0.14±0.02)%] (P<0.01).ConclusionHuman bone marrow mesenchymal stem cells directly differentiated into osteoblasts in vitro under induction,which can be the basis for further fetal interstitial stem cell differentiation regulation and study on the drug action mechanisms.

Key words:Human mesenchymal stem cells; Osteoblasts; Directed differentiation; Dexamethasone; Alkaline phosphatase

收稿日期：2015-02-11修回日期：2015-06-11編輯：伊姍

基金項(xiàng)目：十堰市科學(xué)技術(shù)研究與開(kāi)發(fā)項(xiàng)目計(jì)劃(14Y50)

doi：10.3969/j.issn.1006-2084.2015.18.056

中圖分類號(hào)：R468-2

文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：1006-2084(2015)18-3412-03