余慧鐳，殷曉雪，陳仲?gòu)?qiáng)，冷慧杰，宋純理，劉忠軍

(北京大學(xué)第三醫(yī)院骨科，北京 100191)

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基因沉默組織型轉(zhuǎn)谷氨酰胺酶抑制SaOS-2細(xì)胞成骨分化

余慧鐳，殷曉雪*，陳仲?gòu)?qiáng)，冷慧杰，宋純理，劉忠軍

(北京大學(xué)第三醫(yī)院骨科，北京 100191)

目的 研究組織型轉(zhuǎn)谷氨酰胺酶(tissue transglutaminase, TG2)是否參與人SaOS-2細(xì)胞系成骨分化過(guò)程。 方法 使用攜帶短發(fā)夾RNA(short hairpin RNA, shRNA)的慢病毒轉(zhuǎn)染SaOS-2細(xì)胞以敲減TG2表達(dá)，以SaOS-2細(xì)胞及轉(zhuǎn)染了含陰性對(duì)照shRNA病毒的SaOS-2作為對(duì)照組，分別進(jìn)行體外成骨誘導(dǎo)培養(yǎng)，并進(jìn)行以下檢測(cè)：誘導(dǎo)14 d后各組礦化情況(茜素紅染色)；誘導(dǎo)4、7 d后堿性磷酸酶活性及I型膠原、骨鈣素、骨形態(tài)發(fā)生蛋白-2(BMP-2)的mRNA表達(dá)，并與誘導(dǎo)前的表達(dá)水平相比較。 結(jié)果 SaOS-2細(xì)胞組及轉(zhuǎn)染陰性對(duì)照shRNA組在體外成骨誘導(dǎo)過(guò)程中I型膠原、骨鈣素、BMP-2的mRNA表達(dá)和ALP活性逐漸增加，14 d時(shí)形成明顯礦化結(jié)節(jié)，而TG2敲減后的SaOS-2細(xì)胞在誘導(dǎo)14 d時(shí)礦化水平顯著低于對(duì)照組，誘導(dǎo)7 d時(shí)ALP活性及I型膠原、骨鈣素、BMP-2的mRNA表達(dá)水平顯著低于對(duì)照組。結(jié)論 組織型轉(zhuǎn)谷氨酰胺酶參與SaOS-2細(xì)胞體外成骨分化及礦化。

組織型轉(zhuǎn)谷氨酰胺酶；SaOS-2細(xì)胞；成骨分化

轉(zhuǎn)谷氨酰胺酶是一組催化轉(zhuǎn)酰胺反應(yīng)的酶，由9個(gè)成員組成，其中組織型轉(zhuǎn)谷氨酰胺酶(tissue transglutaminase, TG2)分布廣泛，功能多樣，近年來(lái)研究表明它可能在礦化與骨化過(guò)程中發(fā)揮重要作用[1]。TG2在肥大軟骨細(xì)胞、成骨細(xì)胞、骨細(xì)胞和成牙質(zhì)細(xì)胞中均有表達(dá)[2-3]，與軟骨細(xì)胞分化和基質(zhì)礦化相關(guān)[4]，參與軟骨細(xì)胞向肥大前階段的轉(zhuǎn)化[5]。源于軟骨細(xì)胞的TGs能促進(jìn)前成骨細(xì)胞的礦化，而TG活性抑制劑能抑制體內(nèi)外的成骨細(xì)胞分化及礦化[6-7]。目前已經(jīng)在礦化組織基質(zhì)中發(fā)現(xiàn)多種TG2蛋白交聯(lián)活性的底物如I型膠原(collagen I, Col I)、纖連蛋白、骨橋蛋白等，它們可在TG2催化作用下進(jìn)行蛋白交聯(lián)反應(yīng)，形成穩(wěn)定的膠原網(wǎng)絡(luò)，促進(jìn)細(xì)胞外基質(zhì)的形成與礦化[8]。另外，TG2在一些成骨相關(guān)的病理過(guò)程中起重要作用，比如血管鈣化和胸椎黃韌帶骨化[9-10]。在血管鈣化發(fā)病過(guò)程中，TG2能誘導(dǎo)血管平滑肌細(xì)胞發(fā)生成骨細(xì)胞樣轉(zhuǎn)化[11]，提示TG2具有促成骨的作用。

然而研究表明另外一種TG家族成員FXIIIA在骨和軟骨細(xì)胞中也有表達(dá)并參與骨的礦化[12]，其功能可能與TG2有重疊或代償。為了進(jìn)一步明確TG2在成骨分化和礦化過(guò)程中的作用，本課題使用基因沉默技術(shù)，特異性敲減成骨細(xì)胞系SaOS-2中TG2 mRNA的表達(dá)，觀察是否會(huì)對(duì)細(xì)胞體外成骨分化產(chǎn)生影響。

1 材料與方法

1.1 細(xì)胞與培養(yǎng)

SaOS-2細(xì)胞由David R.Eyre教授(University of Washington Medical Center, Seattle, WA, USA)饋贈(zèng)，使用含10%胎牛血清(Gibco)的DMEM高糖培養(yǎng)液(Hyclone)培養(yǎng)于5%CO2，37℃培養(yǎng)箱中，每2 d更換培養(yǎng)液。進(jìn)行成骨誘導(dǎo)分化實(shí)驗(yàn)時(shí)，在細(xì)胞密度約為80%時(shí)將培養(yǎng)液更換為成骨誘導(dǎo)培養(yǎng)液，配方為DMEM高糖培養(yǎng)液中加入10%胎牛血清、10-8mol/L地塞米松、50 ng/mL抗壞血酸(Sigma)以及10 mmol/L β-甘油磷酸鈉(Sigma)。

1.2 慢病毒轉(zhuǎn)染

攜帶敲減TG2的shRNA的慢病毒(sc-37514-v)購(gòu)于Santa Cruz公司，用不影響基因表達(dá)的陰性對(duì)照慢病毒(sc-108080)作為對(duì)照。在12孔板上以1×104/孔接種SaOS-2細(xì)胞，24 h后細(xì)胞密度達(dá)到約50%時(shí)，在各孔中分別加入相應(yīng)病毒。6 d后，使用含嘌呤霉素的培養(yǎng)液篩選轉(zhuǎn)染成功的細(xì)胞。在篩選10 d后檢測(cè)TG2敲減效率。

1.3 半定量RT-PCR

細(xì)胞以5×104/皿接種培養(yǎng)于60 mm培養(yǎng)皿，到達(dá)干預(yù)時(shí)間時(shí)使用Trizol法提取RNA，取5 μg總RNA使用Goscript逆轉(zhuǎn)錄試劑盒(Promega)進(jìn)行逆轉(zhuǎn)錄。然后通過(guò)半定量RT-PCR檢測(cè)TG2、Col I、骨鈣素(osteocalcin, OCN)、骨形態(tài)發(fā)生蛋白-2(bone morphogenetic protein-2, BMP-2)的mRNA表達(dá)，以β-肌動(dòng)蛋白(β-actin)作為內(nèi)參。引物使用Primer Premier軟件設(shè)計(jì)并由奧科公司合成。引物序列及反應(yīng)條件見表1。

表1 半定量RT-PCR使用引物列表

1.4 蛋白質(zhì)印跡

細(xì)胞以5×104/皿接種培養(yǎng)于60 mm培養(yǎng)皿，到達(dá)干預(yù)時(shí)間時(shí)使用RIPA裂解液提取細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度，制備用于蛋白印跡檢測(cè)的蛋白樣品。取30 μg蛋白上樣，在10% SDS-PAGE凝膠中電泳，然后轉(zhuǎn)移至硝酸纖維素膜，于含5%牛血清白蛋白的封閉緩沖液中室溫封閉2 h后孵育TG2一抗(Santa Cruz)4℃過(guò)夜，然后孵育二抗常溫30 min，于紅外熒光掃描儀上進(jìn)行條帶顯像。以甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)作為內(nèi)參。對(duì)條件使用Image J軟件進(jìn)行灰度值分析。

1.5 堿性磷酸酶活性檢測(cè)

細(xì)胞以1×104/孔接種于12孔板，到達(dá)干預(yù)時(shí)間時(shí)使用RIPA裂解液提取細(xì)胞總蛋白，以BCA法測(cè)定蛋白濃度。使用LabAssayTM ALP試劑盒(Wako)測(cè)定蛋白樣品的堿性磷酸酶(alkaline phosphatase, ALP)活性。

1.6 礦化檢測(cè)

細(xì)胞以5×104/皿接種培養(yǎng)于60 mm培養(yǎng)皿，到達(dá)干預(yù)時(shí)間時(shí)棄去皿中培養(yǎng)液并用PBS清洗兩遍，使用4%中性甲醛4℃下固定20 min，隨后使用pH 4.2的1%茜素紅溶液室溫下染色30 min，洗去浮色后分別在大體及鏡下觀察并采圖。

1.7 轉(zhuǎn)谷氨酰胺酶活性檢測(cè)

細(xì)胞以1×104/孔接種于12孔板，到達(dá)干預(yù)時(shí)間時(shí)使用RIPA裂解液提取細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度。取蛋白樣品進(jìn)行轉(zhuǎn)谷氨酰胺酶活性檢測(cè)，具體檢測(cè)方法在前文中有詳細(xì)介紹[13]。

1.8 數(shù)據(jù)分析

數(shù)據(jù)分析使用SPSS 14.0進(jìn)行統(tǒng)計(jì)分析，各組數(shù)據(jù)均采用均值±標(biāo)準(zhǔn)差表示。組間資料分析采用單因素方差分析，以P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 TG2 shRNA抑制了SaOS-2細(xì)胞中TG2的表達(dá)和TG酶活性

為了解轉(zhuǎn)染TG2 shRNA的SaOS-2細(xì)胞(shTG2)中TG2表達(dá)敲減的效率，我們以轉(zhuǎn)染scrambled shRNA的SaOS-2細(xì)胞(shScr)和野生型SaOS-2細(xì)胞(wide-type, wt)作為對(duì)照，在mRNA及蛋白水平檢測(cè)TG2的表達(dá)水平以及TG酶活性，結(jié)果表明與wt組相比，shTG2組TG2的mRNA及蛋白表達(dá)水平均顯著降低(圖1a,1b)，蛋白表達(dá)定量分析顯示TG2蛋白表達(dá)降低約85%(圖1c)，TG活性相對(duì)于對(duì)照組降低60%(圖1d)，shScr組與wt組無(wú)明顯差異。

注：細(xì)胞接種于12孔板,分別用攜帶TG2 shRNA和scrambled shRNA的慢病毒轉(zhuǎn)染, A.半定量RT-PCR檢測(cè)shTG2, shScr和wt SaOS-2中TG2的mRNA表達(dá);B.蛋白印跡檢測(cè)shTG2, shScr和wt SaOS-2中TG2的蛋白表達(dá); C.三組細(xì)胞中TG2蛋白表達(dá)的半定量數(shù)據(jù)統(tǒng)計(jì)分析，數(shù)據(jù)均來(lái)源于3次重復(fù)實(shí)驗(yàn)，**P<0.01 vs wt; D. shTG2, shScr, wt, 0.25 μg TG標(biāo)準(zhǔn)品和0.5 μg TG標(biāo)準(zhǔn)品的TG活性檢測(cè)，數(shù)據(jù)均來(lái)源于6次重復(fù)實(shí)驗(yàn)，*P<0.05 vs wt。圖1 SaOS-2細(xì)胞TG2敲減效率Note. Cells were seeded in 12-well plate and infections of TG2 shRNA lentiviral particles and scrambled shRNA lentiviral particles were performed as described under “Methods and Materials”. A.Semiquantitative RT-PCR analysis on TG2 mRNA expression in shTG2,shScr and wild-type(wt) SaOS-2 cells. B.Western blotting analysis on TG2 expression in shTG2,shScr and wt SaOS-2 cells. C.Statistical analysis of TG2 protein expression. Data were combined from 3 duplicate experiments and reported as means ±SD. **P<0.01 vs wt(Dunnett test). D. TG activity analysis for shTG2, shScr, wt, 0.25 μg TG and 0.5 μg TG. Data were combined from 6 duplicate experiments and are reported as means ±SD. *P<0.05 vs wt(Dunnett test).Fig.1 Efficiency of TG2 knocking down in SaOS-2 cells by TG2 shRNA

2.2 TG2敲減抑制了SaOS-2細(xì)胞體外礦化

基質(zhì)礦化是成骨分化的重要特征之一，為了探究TG2在SaOS-2細(xì)胞體外礦化中的作用，我們將shTG2、shScr和wt三組細(xì)胞分別培養(yǎng)于成骨誘導(dǎo)液及普通培養(yǎng)液，14 d后進(jìn)行茜素紅染色，在大體像及鏡下觀察，三組細(xì)胞在普通培養(yǎng)14 d后均為陰性染色，成骨誘導(dǎo)培養(yǎng)14 d后wt及shScr出現(xiàn)陽(yáng)性染色，而shTG2染色明顯較弱(圖2)。

注：茜素紅染色檢測(cè)shTG2, shScr和wt SaOS-2在普通培養(yǎng)液或成骨誘導(dǎo)培養(yǎng)液中培養(yǎng)14 d后基質(zhì)礦化，觀察大體圖及鏡下圖(×100)，紅色代表陽(yáng)性染色，從三次重復(fù)實(shí)驗(yàn)中選取代表圖像。(標(biāo)尺=200 μm)圖2 TG2敲減抑制SaOS-2細(xì)胞礦化Note. ShTG2,shScr and wt SaOS-2 cells were cultured in osteo-inductive medium or general medium for 14 days and the mineralization of the cultured cells was assessed using alizarin red staining. The bright red region represents positively stained region. All of the cultures were taken photos in both general view and microscopic view(×100), representative images from three separate experiments. Scale bar=200 μm.Fig.2 TG2 knockdown suppressed the mineral deposition in SaOS-2 cells

2.3 TG2敲減抑制SaOS-2細(xì)胞體外成骨分化

Col I、OCN及ALP參與成骨分化過(guò)程，并對(duì)胞外基質(zhì)礦化有重要作用。在之前的研究中我們發(fā)現(xiàn)SaOS-2細(xì)胞以較低的水平表達(dá)Col I，OCN的mRNA并具有一定的ALP活性，而在誘導(dǎo)7 d后這些成骨指標(biāo)水平顯著提高[13]。本研究中結(jié)果顯示shTG2組細(xì)胞在誘導(dǎo)培養(yǎng)7 d后ALP活性雖較誘導(dǎo)前升高，但顯著低于相同處理的shScr和wt組(圖3a)，Col I和OCN的mRNA表達(dá)也在誘導(dǎo)培養(yǎng)7 d后顯著低于shScr和wt組(圖3b，3c)，這表明TG2敲減抑制了成骨性分化。BMP-2是一種成骨分化因子并且參與細(xì)胞成骨分化的過(guò)程，與對(duì)照組相比，shTG2組細(xì)胞在誘導(dǎo)前及誘導(dǎo)4 d時(shí)BMP-2輕度降低，而在7 d時(shí)顯著降低(圖3b，3c)，表明BMP-2可能參與TG2對(duì)成骨分化的調(diào)節(jié)。

3 討論

SaOS-2細(xì)胞是一種成熟的用于研究人成骨分化的體外實(shí)驗(yàn)?zāi)Ｐ?，它在培養(yǎng)于成骨誘導(dǎo)培養(yǎng)液時(shí)會(huì)進(jìn)行成骨分化及礦化，表現(xiàn)為Col I、OCN、ALP活性等成骨指標(biāo)水平增高及形成礦化基質(zhì)[14]。在之前的研究中我們發(fā)現(xiàn)，SaOS-2細(xì)胞在誘導(dǎo)培養(yǎng)7 d后，TG2的表達(dá)和TG活性都顯著增加，而且SaOS-2細(xì)胞的成骨分化過(guò)程會(huì)被TG活性抑制劑所阻滯[13]，以小鼠前成骨細(xì)胞系MC3T3-E1為模型的實(shí)驗(yàn)也得到了類似結(jié)果[6]，但是TG2是否在成骨分化中起作用仍不明確，由于TG家族的其他成員也能發(fā)揮出TG活性，所以在SaOS-2細(xì)胞的成骨分化過(guò)程中起作用的TG活性有可能是由其他成員提供。另外，TG2(-/-)小鼠并沒(méi)有表現(xiàn)出異常的骨骼表型，提示TG2在骨骼系統(tǒng)的發(fā)育中可能并不重要[15]。然而，近期研究表明基因敲除實(shí)驗(yàn)的結(jié)果可能會(huì)受代償網(wǎng)絡(luò)的影響[16]，所以TG2敲除小鼠的正常骨骼表型可能是由于FXIIIA、TGF-β或其他因子的代償作用。由于轉(zhuǎn)錄水平的敲減不會(huì)引起這種代償作用[16]，那么本研究使用shRNA敲減TG2可能會(huì)為認(rèn)識(shí)TG2在成骨中的作用提供進(jìn)一步的證據(jù)，以闡明TG2是否參與SaOS-2細(xì)胞成骨分化及礦化。SaOS-2細(xì)胞在使用shRNA敲減TG2后，TG2的蛋白表達(dá)降低了約85%，TG活性降低60%，這表明TG2敲減模型成功建立并且TG2是SaOS-2細(xì)胞中TG酶活性的重要來(lái)源。

TG2敲減后，SaOS-2細(xì)胞礦化能力顯著降低，說(shuō)明TG2在其礦化中起重要作用。成骨誘導(dǎo)后SaOS-2細(xì)胞中Col I、OCN的表達(dá)以及ALP活性的升高也被TG2所抑制，說(shuō)明TG2表達(dá)的減少抑制了SaOS-2細(xì)胞的成骨分化。

注：shTG2, shScr和wt SaOS-2分別在成骨誘導(dǎo)液中培養(yǎng)0、4、7 d。 A.堿性磷酸酶活性檢測(cè)，數(shù)據(jù)均來(lái)源于6次重復(fù)實(shí)驗(yàn)，*P<0.05 vs wt; B.半定量RT-PCR檢測(cè)Col I、OCN和BMP-2的mRNA表達(dá); C. Col I、OCN和BMP-2的mRNA表達(dá)的灰度值統(tǒng)計(jì)分析,數(shù)據(jù)均來(lái)源于3次重復(fù)實(shí)驗(yàn)，*P<0.05 vs wt , **P<0.01 vs wt。圖3 TG2敲減抑制SaOS-2細(xì)胞成骨分化Note. Cells were cultured in osteo-inductive medium for 0, 4 and 7 days. A. Alkaline phosphatase activity was measured as described in “Methods and Materials”. Data were combined from 6 duplicate experiments and reported as means±SD. **P<0.01 vs wt (Dunnett test). B. Semiquantitative RT-PCR analysis of BMP-2, Col I and OCN mRNA expression in the shTG2, shScr and wt cells. C. Statistical analysis of BMP-2, Col I and OCN mRNA expression. Data were collected from 3 duplicate experiments and reported as means±SD. *P<0.05 vs wt cells (Dunnett test), **P<0.01 vs wt cells (Dunnett test).Fig.3 TG2 knockdown suppressed the osteoblastic differentiation of SaOS-2 cells

TG活性參與礦化組織發(fā)育過(guò)程已經(jīng)得到共識(shí)[4]，并且其機(jī)制已經(jīng)進(jìn)行了一些研究。I型膠原-纖連蛋白交聯(lián)產(chǎn)物是成骨分化過(guò)程中胞外基質(zhì)形成所不可缺少的[17]，而TG活性在I型膠原-纖連蛋白交聯(lián)產(chǎn)物的組裝及成熟過(guò)程中起重要作用[6]。本實(shí)驗(yàn)中發(fā)現(xiàn)TG2敲減后TG活性顯著降低，Col I表達(dá)減少，細(xì)胞外基質(zhì)礦化明顯受抑，所以TG2可能通過(guò)TG活性參與調(diào)節(jié)成骨性分化。另外有研究表明，TG2對(duì)膠原的修飾能促進(jìn)人成骨細(xì)胞的黏附、增殖、分化及礦化[18]，這也為TG2通過(guò)蛋白交聯(lián)活性參與人成骨分化過(guò)程提供了更多的證據(jù)。

BMP-2是TGF-β家庭的成員并且通過(guò)多種信號(hào)通路包括MAPK、Wnt等參與骨骼發(fā)育和骨折修復(fù)[19]，本實(shí)驗(yàn)中我們發(fā)現(xiàn)SaOS-2細(xì)胞分化過(guò)程中BMP-2表達(dá)的增加被TG2敲減所抑制，那么BMP-2的表達(dá)降低可能參與了TG2敲減對(duì)成骨分化的抑制?？傊?，我們的結(jié)果直接證明了TG2在SaOS-2細(xì)胞的礦化和成骨性分化中起重要作用，并且其機(jī)制可能包括調(diào)節(jié)細(xì)胞外基質(zhì)組裝和BMP-2的表達(dá)。

TG2和FXIIIA是TG家族中表達(dá)于骨、軟骨和牙齒等礦化組織的成員[1; 20]，近期有研究表明在小鼠MC3T3-E1細(xì)胞成骨分化中起主要作用的是FXIIIA而不是TG2。然而我們?cè)谥暗膶?shí)驗(yàn)中發(fā)現(xiàn)TG2的表達(dá)水平在SaOS-2細(xì)胞成骨分化過(guò)程中逐漸增加[13]，本實(shí)驗(yàn)中我們發(fā)現(xiàn)特異性沉默TG2能抑制SaOS-2細(xì)胞的成骨性分化和礦化，所以TG2在SaOS-2細(xì)胞成骨分化中起重要作用。我們的結(jié)果與上述研究的差別可能是由于兩種細(xì)胞的不同或者細(xì)胞種屬的不同。本研究確定了TG2與SaOS-2細(xì)胞體外成骨分化和礦化之間的關(guān)系，在TG2基因沉默后，SaOS-2細(xì)胞的成骨分化和礦化部分受抑，說(shuō)明TG2是人成骨分化過(guò)程中所必需的因子。另外，TG2對(duì)人成骨細(xì)胞的調(diào)節(jié)作用可能與胞外基質(zhì)的裝配和BMP-2的表達(dá)有關(guān)。我們的研究為認(rèn)識(shí)TG2在人成骨分化和礦化中的作用提供了進(jìn)一步的證據(jù)。

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Knockdown of tissue transglutaminase in SaOS-2 cell line inhibits its osteoblastic differentiation and mineralization

YU Hui-lei, YIN Xiao-xue*, CHEN Zhong-qiang, LENG Hui-jie,SONG Chun-li, LIU Zhong-jun

(Department of Orthopedics, Peking University Third Hospital, Beijing 100191, China)

Objective To investigate whether TG2 plays an important role in the osteoblast differentiation and mineralization. Methods TG2 mRNA of SaOS-2 cells was knocked down using a lentivirus stably expressing short-hairpin(sh) RNA targeting TG2.Then the cells were cultured in osteo-inductive medium for 14 d to measure mineralization and for 7 d to measure the levels of osteoblastic differentiation markers including ALP activity and mRNA of collagen I, osteocalcin(OCN) and BMP-2.The wild-type SaOS-2 cells and scrambled shRNA-transducted SaOS-2 cells served as the controls. Results The controls displayed an increasing trend of the level of ALP activity and mRNA of collagen I, osteocalcin and BMP-2,and notable mineralization at 14 d.When TG2 was knocked down, ALP activity, mRNA of collagen I, osteocalcin and BMP-2 at 7d,and mineralization at 14 d were all significantly lower in comparison with the corresponding values in the controls.Conclusion TG2 is involved in the differentiation and mineralization of osteoblasts in vitro.

Tissue transglutaminase;SaOS-2 cell line; Osteoblastic differerntiation

YIN Xiao-xue.E-mail: luckyemail2008@sina.com

國(guó)家自然科學(xué)基金(編號(hào)：81101334)。

余慧鐳(1992 -)，男，碩士，主要從事成骨細(xì)胞方面的研究，E-mail: yuhuileixs@126.com

殷曉雪(1972-)，女，博士，副研究員，主要從事成骨細(xì)胞及細(xì)胞外基質(zhì)研究。E-mail: luckyemail2008@sina.com

研究報(bào)告

Q95-33

A

1005-4847(2016)05-0448-06

10.3969/j.issn.1005-4847.2016.05.002

2016-03-08