雙膦酸鹽對NFATc1及相關(guān)因子p-NF-κB、p-c-Jun在破骨細(xì)胞分化過程中的影響

董偉，彭宏峰，梁永強(qiáng)，馮曉潔，廖囡囡，戚孟春

論 著

雙膦酸鹽對NFATc1及相關(guān)因子p-NF-κB、p-c-Jun在破骨細(xì)胞分化過程中的影響

董偉，彭宏峰，梁永強(qiáng)，馮曉潔，廖囡囡，戚孟春

目的研究雙膦酸鹽(ALN)對破骨細(xì)胞(OC)分化過程中關(guān)鍵因子NFATc1及其信號分子p-NF-κB、p-c-Jun的影響。方法采用小鼠單核巨噬細(xì)胞RAW264.7誘導(dǎo)培養(yǎng)OC，細(xì)胞培養(yǎng)于培養(yǎng)皿，分A組(對照組)、B組(ALN組)。培養(yǎng)第2d采用Western blotting檢測NFATc1、p-NF-κB、p-c-Jun基因表達(dá)變化情況，培養(yǎng)第4d采用免疫熒光方法檢測NFATc1表達(dá)，培養(yǎng)第7d檢測各組OC生成及數(shù)目。采用牙本質(zhì)磨片接種RAW264.7細(xì)胞，分組及處理方法同上述培養(yǎng)皿培養(yǎng)過程，第9d檢測骨吸收功能。結(jié)果兩組細(xì)胞均有TRAP陽性多核OC生成，并在牙本質(zhì)磨片上形成吸收陷窩；但A組TRAP陽性多核細(xì)胞數(shù)目、吸收陷窩數(shù)目及陷窩面積均大于B組。免疫熒光檢測NFATc1表達(dá)A組高于B組；Western blotting檢測NFATc1、p-NF-κB、p-c-Jun表達(dá)B組較A組低，兩組有顯著性差異(P<0.01)。結(jié)論雙膦酸鹽通過下調(diào)p-NF-κB、p-c-Jun的表達(dá)，最終抑制NFATc1的表達(dá)，從而抑制OC生成和骨吸收功能。

阿侖膦酸鹽；破骨細(xì)胞；NFATc1；NF-κB；基因，jun

破骨細(xì)胞的功能分化過程是當(dāng)今研究的重點(diǎn)[1]，許多骨骼疾病如骨質(zhì)疏松癥[2]、類風(fēng)濕性關(guān)節(jié)炎、Paget's病、多發(fā)性骨髓瘤、惡性腫瘤的溶骨性骨轉(zhuǎn)移等，都與破骨細(xì)胞(osteoclasts，OC)增殖、骨吸收功能亢進(jìn)密切相關(guān)[3]。阿侖膦酸鹽(alendronate，ALN)作為第三代含氮雙膦酸鹽，是現(xiàn)階段治療上述疾病的首選藥物，除此之外還被用作預(yù)防和治療骨密度降低及成骨不全癥等疾病[4]。ALN主要通過抑制破骨細(xì)胞骨吸收功能、誘導(dǎo)破骨細(xì)胞凋亡來發(fā)揮作用，但確切機(jī)制及涉及的胞內(nèi)信號通路目前知之甚少，特別是對破骨細(xì)胞分化過程中關(guān)鍵信號分子NFATc1及其重要通路如NF-κB通路、MKK通路是否產(chǎn)生影響等。本研究在OC培養(yǎng)過程中加入ALN并檢測NFATc1的表達(dá)，分析影響其表達(dá)的p-c-Jun、p-NF-κB，以初步揭示雙膦酸鹽抑制OC的分子機(jī)制，為ALN的臨床應(yīng)用提供理論基礎(chǔ)。

1 材料與方法

1.1 實驗材料 DMEM培養(yǎng)基(Gibco，美國)，RANKL(receptor activator of nuclear factor κB ligand) (Biovision，美國)，阿侖膦酸鹽(Sigma，美國)，兔抗鼠NFATc1、p-c-Jun、p-NF-κB多克隆抗體(Santa Cruz，美國)，羊抗兔辣根過氧化物酶標(biāo)記IgG和RIPA(武漢博士德，中國)，抗酒石酸酸性磷酸酶(TRAP)染色試劑盒(Sigma，美國)，RAW264.7小鼠單核巨噬細(xì)胞系(中國科學(xué)院細(xì)胞庫)。

1.2 OC的培養(yǎng)及實驗分組 RAW264.7細(xì)胞以1×105/孔的密度接種于48孔培養(yǎng)板中，DMEM培養(yǎng)基(含15%胎牛血清、100U/ml青霉素、100μg/ ml鏈霉素)培養(yǎng)，細(xì)胞貼壁后分為兩組，對照組加入100ng/ml RANKL；處理組加入100ng/ml RANKL+5×10-7mol/L ALN，定期于倒置相差顯微鏡下觀察。

1.3 抗酒石酸酸性磷酸酶(TRAP)染色 接種于48孔板的細(xì)胞于處理后第7d收獲，按TRAP染色試劑盒操作步驟進(jìn)行染色，顯微鏡100倍放大倍數(shù)下隨機(jī)選取5個視野計數(shù)TRAP染色陽性細(xì)胞(細(xì)胞核≥3 個)數(shù)目，5個視野計數(shù)取平均值為該細(xì)胞爬片的破骨細(xì)胞數(shù)目。

1.4 牙本質(zhì)磨片制備及吸收陷窩檢測 將人離體牙制備成直徑約0.5cm，厚度約0.2mm的牙本質(zhì)磨片，打磨拋光表面，消毒滅菌后將RAW264.7細(xì)胞接種其上，細(xì)胞貼壁后分為兩組(同1.2細(xì)胞處理方法)。取處理后第9天的牙本質(zhì)磨片，2.5%戊二醛4℃固定7min，1mol/L氫氧化胺中以50Hz超聲清洗5min，再經(jīng)蒸餾水超聲清洗5min×3次；隨后2.5%戊二醛固定2h，1%鋨酸固定2h，乙醇逐級脫水，醋酸異戊酯置換，CO2臨界點(diǎn)干燥，鍍金，掃描電鏡(HITACHIS-4800)觀察吸收陷窩。在500倍放大倍數(shù)下每孔牙本質(zhì)磨片上隨機(jī)選取5個視野，用醫(yī)學(xué)數(shù)碼圖像分析系統(tǒng)Med6.0測量五個視野吸收陷窩總數(shù)目及總面積。

1.5 免疫熒光法檢測NFATc1表達(dá) 取培養(yǎng)至第4d的RAW264.7細(xì)胞，PBS沖洗，4%多聚甲醛固定10min，0.5%Triton穿孔15min，1%BSA封閉30min，PBS沖洗5min×2次。加入5%BSA稀釋的兔抗鼠NFATc1多克隆一抗(1:25)過夜。PBS沖洗，加入5%BSA稀釋的FITC標(biāo)記的羊抗兔IgG，37℃孵育2h，5μg/ml DAPI染色2min，于共聚焦顯微鏡下觀察。

1.6 Western blotting檢測NFATc1、p-NF-κB、p-c-Jun蛋白表達(dá) 兩組細(xì)胞培養(yǎng)第2天，提取細(xì)胞總蛋白BCA法定量。上樣量35μg，采用12%SDS-PAGE凝膠電泳分離，采用濕法轉(zhuǎn)膜至PVDF膜，5%BSA室溫封閉1h，分別加入1μg/ml兔抗鼠NFATc1、p-NF-κB、p-c-Jun多克隆一抗孵育過夜，羊抗兔辣根過氧化物酶標(biāo)記IgG二抗結(jié)合室溫孵育1h。TBST洗膜后按化學(xué)發(fā)光試劑盒說明書制備反應(yīng)液，室溫孵育5min。用Image J分析軟件對條帶吸光度值(A)進(jìn)行半定量分析，以目的蛋白條帶(A)值/內(nèi)參GAPDH蛋白條帶(A)值的比值表示目的蛋白的相對量。

1.7 統(tǒng)計學(xué)處理 采用SPSS 11.0軟件進(jìn)行統(tǒng)計分析。計量數(shù)據(jù)以表示，組間比較采用SNK-q檢驗，P<0.05為差異有統(tǒng)計學(xué)意義。

圖1 倒置相差顯微鏡下觀察RAW264.7細(xì)胞生長情況(×400)Fig.1 RAW264.7 cell growth observed under inverted phase contrast microscope (×400) A. Incubated for 1 day; B. Incubated for 3 days; C. Incubated for 7 days

2 結(jié) 果

2.1 倒置相差顯微鏡觀察OC生成情況 培養(yǎng)開始時，貼壁的RAW264.7呈圓形鋪于培養(yǎng)板底壁(圖1A)。培養(yǎng)至第3天，在誘導(dǎo)因子RANKL的作用下，RAW264.7開始相互融合(圖1B)。培養(yǎng)至第7d，細(xì)胞融合成多核OC(圖1C)。

2.2 TRAP染色及定量分析 對照組OC生成數(shù)量顯著高于處理組(P<0.01)，提示ALN可顯著抑制OC的生成(圖2)。

2.3 牙本質(zhì)吸收陷窩觀察及定量分析 兩組比較，對照組OC吸收陷窩數(shù)目較多，陷窩總面積較大，兩組間吸收陷窩數(shù)目及總面積均有顯著性差異(P<0.01，圖3，表1)。上述結(jié)果表明，ALN可有效抑制OC的骨吸收功能。

圖2 細(xì)胞培養(yǎng)第7天的TRAP染色比較(×200)Fig. 2 TRAP staining of osteoclasts on the 7th day (×200)A. Control group; B. ALN-treated group

圖3 細(xì)胞培養(yǎng)第9天骨吸收陷窩形成情況(SEM×500)Fig. 3 Resorption lacunaes formed on dentin slices after culture for 9 days (SEM×500) A. Control group; B. ALN-treated group

2.4 免疫熒光檢測NFATc1表達(dá) 兩組細(xì)胞培養(yǎng)至4d，免疫熒光檢測見NFATc1表達(dá)(圖4)，對照組NFATc1熒光強(qiáng)度(5.42±0.63)明顯大于處理組(1.26±0.15)(P<0.01)。

2.5 Western blotting檢測NFATc1，p-NF-κB，p-c-Jun蛋白的表達(dá) Western blotting檢測兩組細(xì)胞NFATc1、p-NF-κB、p-c-Jun蛋白含量變化，處理組NFATc1、p-NF-κB、p-c-Jun蛋白表達(dá)較對照組明顯減弱，條帶灰度值分別下降47.1%、39.3%、18.4%；結(jié)果顯示，ALN可抑制OC細(xì)胞NFATc1、 p-NF-κB、p-c-Jun的表達(dá)。

表1 TRAP陽性破骨細(xì)胞計數(shù)及牙本質(zhì)磨片吸收陷窩計數(shù)和陷窩面積Tab.1 The number of TRAP(+) osteoclasts, number and size of resorption lacunaes formed on dentin slices in each group

圖4 免疫熒光化學(xué)法檢測兩組NFATc1的表達(dá)(×400)Fig. 4 Detection of NFATc1 expression by immunofluorescent chemistry (×400) A, E. NFATc1 expression labelled by FITC; B, F. Cell nucleus labelled by DAPI; C, G. Integrated image; D, H. Normal vision without fluorescence

圖5 Western blotting檢測兩組NFATc1、p-NF-κB、p-c-Jun蛋白表達(dá)Fig.5 Detection of NFATc1, p-NF-κB, p-c-Jun protein expression in two groups of cells by Western blotting

3 討 論

OC是骨吸收時唯一的效應(yīng)細(xì)胞，由多功能造血干細(xì)胞在誘導(dǎo)因子(RANKL)的誘導(dǎo)下相互融合形成的多核巨細(xì)胞。在OC形成過程中，眾多因子及信號通路發(fā)揮重要作用[5-7]，其中活化T細(xì)胞核因子NFATc1為破骨細(xì)胞分化的重要信號分子[8]，而NF-κB及MKK是影響NFATc1較為關(guān)鍵的信號通路分子[9-10]。

在OC分化中，當(dāng)RANKL與其受體即OC前體上的RANK相互作用后，引起了下游一系列信號因子的級聯(lián)作用[11]。首先，引發(fā)鈣/鈣調(diào)磷酸酶/活化T細(xì)胞核因子(calcium/calcineurin/NFAT)活化，導(dǎo)致胞質(zhì)內(nèi)Ca2+波動，Ca2+/鈣調(diào)蛋白依賴的鈣調(diào)磷酸酶被激活，使胞質(zhì)內(nèi)NFATc1蛋白異位進(jìn)入細(xì)胞核[12]；同時，NF-κB參與作用的經(jīng)典途徑(classical pathway)和替代途徑(alternative pathway)被激活。在經(jīng)典途徑中，由IKKβ和NEMO/IKKγ組成的IKK復(fù)合物是關(guān)鍵信號因子，它的作用是誘導(dǎo)在細(xì)胞質(zhì)中與無活性NF-κB形成復(fù)合物的抑制蛋白IκB磷酸化，隨后NF-κB被釋放，形成二聚體p65/cRel[13]；而替代途徑的關(guān)鍵蛋白是NF-κB誘導(dǎo)激酶NIK(NF-κB-inducing kinase)，其激活I(lǐng)κB激酶IKKα，促使NF-κB釋放并形成二聚體RelB/p52[13]。上述兩途徑形成的NF-κB二聚體轉(zhuǎn)移至細(xì)胞核中與NFATc2共同作用于NFATc1基因啟動子，誘發(fā)NFATc1基因初始表達(dá)，隨后NFATc1基因表達(dá)進(jìn)入自發(fā)擴(kuò)增階段；與此同時，在RANKL刺激下，另一通路MKK信號通路啟動，JUN被激活，NF-κB磷酸化，最終c-Jun磷酸化，c-Jun與c-Fos形成催化蛋白-1(AP-1)，c-Fos被激活，選擇性地募集到NFATc1基因啟動子上，與NF-κB、NFATc2共同促進(jìn)NFATc1基因的表達(dá)，進(jìn)而促使多核OC前體的形成[14-15]。隨后，OC前體活化形成成熟的破骨細(xì)胞，發(fā)揮其骨吸收功能。

ALN是現(xiàn)階段治療骨質(zhì)疏松等疾病的常用臨床藥物。眾多學(xué)者通過動物實驗及細(xì)胞實驗兩水平均證實了其對OC分化及骨吸收功能的抑制作用，但其是否對NFATc1表達(dá)產(chǎn)生抑制，并影響相關(guān)通路的作用，國內(nèi)外研究并未給出確切結(jié)論。本研究結(jié)果表明，ALN通過抑制p-NF-κB、p-c-Jun的表達(dá)，影響NF-κB參與通路及MKK信號通路，最終下調(diào)NFATc1基因表達(dá)，從而起到抑制OC生成及骨吸收功能的作用。本實驗部分揭示了雙膦酸鹽對OC生成抑制作用的分子學(xué)機(jī)制，將更好地為雙膦酸鹽在臨床中的應(yīng)用提供實驗依據(jù)。然而，ALN是否會通過影響鈣/鈣調(diào)磷酸酶/活化T細(xì)胞核因子(calcium/ calcineurin/NFAT)通路，導(dǎo)致胞質(zhì)內(nèi)Ca2+波動受阻，從而阻礙NFATc1發(fā)生核異位還需要進(jìn)一步實驗驗證。

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Effect of bisphosphonates on NFATc1 and correlators p-NF-κB and p-c-Jun in osteoclast differentiation

DONG Wei, PENG Hong-feng, LIANG Yong-qiang, FENG Xiao-jie, LIAO Nan-nan, QI Meng-chun*
School of Stomatology, North China University of Science and Technology, Tangshan 063000, China
*< class="emphasis_italic">Corresponding author, E-mail: qimengchun@163.com

, E-mail: qimengchun@163.com
This work was supported by the National Natural Science Foundation of China (81270965) and the Natural Science Foundation of Hebei Province (C2011401044)

ObjectiveTo study the effect of alendronate (ALN) on NFATc1 and correlated signaling factors p-NF-κB and p-c-Jun in osteoclast differentiation.MethodsOsteoclasts were inductively cultivated with mouse mononuclear macrophage RAW264.7, and they were divided into 2 groups: group A (control group) and group B (ALN-treated group). The protein expressions of NFATc1, p-NF-κB and p-c-Jun were determined with Western blotting at the 2nd day of cultivation; the expression of NFATc1 was assessed by immunofluorescent assay on the 4th day; and the osteoclast formation was examined on the 7th day of cultivation. RAW264.7 cells were inoculated on abrasive dentine disk, and divided into 2 groups and treated as mentioned above. The resorption function of osteoclast was observed on the 9th day of inoculation.ResultsTRAP positive multinuclear osteoclasts were observed, and resorption lacunaes formed in the abrasive dentine disks of the 2 groups. More TRAP positive multinuclear cells and resorption lacunaes in large size were found in group A than those in group B. Immunofluorescence assay showed the expression of NFATc1 was higher in group A than in group B. The gene expressions of NFATc1, p-NF-κB and p-c-Jun were lower in group B than in group A (P<0.01) as determined with Western blotting.ConclusionBy down-regulating the expressions of p-NF-κB and p-c-Jun, ALN may strongly inhibit the osteoclast formation and its resorption function, thus inhibiting NFATc1 expression.

alendronate; osteoclast; NFATc1; NF-κB; genes, jun

R329.24

A

0577-7402(2015)10-0778-04

10.11855/j.issn.0577-7402.2015.10.02

2014-11-25；

2015-08-20)

(責(zé)任編輯：張小利，沈?qū)?

國家自然科學(xué)基金(81270965)；河北省自然科學(xué)基金(C2011401044)

董偉，醫(yī)學(xué)碩士，講師。主要從事骨組織工程方面的研究

063000 河北唐山 華北理工大學(xué)口腔醫(yī)學(xué)院(董偉、彭宏峰、梁永強(qiáng)、馮曉潔、廖囡囡、戚孟春)

戚孟春，E-mail: qimengchun@163.com