大骨節(jié)病軟骨中內(nèi)質(zhì)網(wǎng)應(yīng)激性蛋白分子表達(dá)變化研究*

陜西省人民醫(yī)院骨科病院(西安710068)

易　智　武世勛　凌　鳴　劉時(shí)璋　劉宗智　孫正明　靳占奎

?

大骨節(jié)病軟骨中內(nèi)質(zhì)網(wǎng)應(yīng)激性蛋白分子表達(dá)變化研究*

陜西省人民醫(yī)院骨科病院(西安710068)

易智武世勛凌鳴劉時(shí)璋劉宗智孫正明靳占奎

目的：探討活化轉(zhuǎn)錄因子6 (ATF6)和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)促凋亡蛋白C/EBP 同源蛋白(CHOP)在大骨節(jié)病軟骨組織中的表達(dá)變化及其與軟骨損傷的關(guān)系。方法：收集7例大骨節(jié)病患者術(shù)后關(guān)節(jié)軟骨標(biāo)本(KBD組)，7例骨關(guān)節(jié)炎病人關(guān)節(jié)軟骨(OA組)作為疾病對照，6例正常軟骨組織作為正常對照。采用SP免疫組化染色法檢測三組關(guān)節(jié)軟骨組織中AFT6和CHOP表達(dá)趨勢，分析其關(guān)節(jié)軟骨內(nèi)陽性表達(dá)率差異。 結(jié)果： KBD組和OA組中層ATF6陽性細(xì)胞表達(dá)率顯著高于正常軟骨組，而三組表層和深層軟骨ATF6陽性細(xì)胞表達(dá)率無統(tǒng)計(jì)學(xué)差異。OA組軟骨表層CHOP表達(dá)顯著高于KBD軟骨組和正常軟骨組，而KBD組和正常組間比較無統(tǒng)計(jì)學(xué)差異；KBD組軟骨和OA組中層CHOP陽性細(xì)胞表達(dá)率顯著高于正常軟骨組；OA組軟骨深層陽性率高于KBD組。KBD組與OA組ATF6主要表達(dá)于中層，正常軟骨組主要表達(dá)于中層和深層，KBD組與OA組CHOP主要表達(dá)于中層，正常軟骨組CHOP在三層表達(dá)無統(tǒng)計(jì)學(xué)差異。結(jié)論：KBD軟骨中層的ATF6和CHOP顯著上調(diào)，表明CHOP高表達(dá)可能與大骨節(jié)病軟骨損傷密切相關(guān)，而ATF6高表達(dá)與其軟骨損傷后反應(yīng)性細(xì)胞增殖有關(guān)。

主題詞骨關(guān)節(jié)病，原發(fā)肥大性/病理生理學(xué)軟骨，關(guān)節(jié)/病理生理學(xué)蛋白/代謝@活化轉(zhuǎn)錄因子6

大骨節(jié)病(Kashin-Beck disease, KBD)是一種特殊的地方性、以關(guān)節(jié)軟骨壞死為特征性疾病。前期研究結(jié)果表明:大骨節(jié)病和骨關(guān)節(jié)病(Osteoarthritis，OA)的軟骨損傷均與細(xì)胞過度凋亡有關(guān)[1]，人骨關(guān)節(jié)炎軟骨和動物模型軟骨中發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子免疫球蛋白重鏈結(jié)合蛋白呈現(xiàn)異常高表達(dá)[2]。已經(jīng)證實(shí)了ATF6(Activating transcription factor-6)過表達(dá)與軟骨細(xì)胞凋亡及內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)促凋亡蛋白C/EBP同源蛋白[3-4](CCAAT/enhancer-binding protein homologous protein，CHOP)介導(dǎo)的凋亡與關(guān)節(jié)軟骨退變有關(guān)。還有研究結(jié)果認(rèn)為OA軟骨內(nèi)ATF6可上調(diào)XBP1S[5]，進(jìn)而抑制內(nèi)質(zhì)網(wǎng)應(yīng)激性細(xì)胞凋亡。然而，目前尚未見到大骨節(jié)病軟骨組織AFT6和CHOP的表達(dá)變化的相關(guān)報(bào)道。本文以前人研究成果為基礎(chǔ)，探討了大骨節(jié)病軟骨組織損傷過程中內(nèi)ATF6和CHOP表達(dá)變化。

材料與方法

1材料按照我國2010年《大骨節(jié)病診斷標(biāo)準(zhǔn)》納入病例，實(shí)驗(yàn)組包括來自陜西人民醫(yī)院骨科KBD病人的膝關(guān)節(jié)置換術(shù)后軟骨標(biāo)本7例(KBD組)，疾病對照組為性別、年齡與試驗(yàn)組相匹配的原發(fā)性骨關(guān)節(jié)炎患者膝關(guān)節(jié)置換術(shù)后軟骨標(biāo)本7例(OA組)，收集6例健康者截肢術(shù)后正常軟骨作為正常對照組(NC組)。所有標(biāo)本收集均征得患者及家屬知情同意，本研究通過陜西省人民醫(yī)院醫(yī)學(xué)倫理委員會批準(zhǔn)。

試劑有兔抗人ATF6抗體(北京博奧森,bs1634R)，兔抗人CHOP抗體(北京博奧森, bs8875R)，SP山羊抗兔二抗即用型試劑盒(北京中杉金橋，SP9012)。

2方法所有軟骨標(biāo)本收集后，即刻置入4%中性多聚甲醛溶液進(jìn)行固定，制備病理切片，然后采用常規(guī)SP免疫組織化學(xué)染色法進(jìn)行標(biāo)記陽性細(xì)胞[7]，將關(guān)節(jié)軟骨分為表、中、深層，然后分別統(tǒng)計(jì)每張染色切片中400倍鏡下一個視野內(nèi)不同軟骨細(xì)胞層的陽性細(xì)胞表達(dá)率。

結(jié)　果

1 KBD與OA和正常組關(guān)節(jié)軟骨細(xì)胞ATF6表達(dá)比較結(jié)果顯示：ATF6在三組軟骨細(xì)胞的細(xì)胞核及胞漿中均表達(dá)(圖1)。正常組軟骨表層ATF6表達(dá)陽性率低于OA組和KBD組，但統(tǒng)計(jì)學(xué)分析結(jié)果無差異(P=0.334)；三組中層之間ATF6表達(dá)陽性率有差異(F=15.108，P=0.001)，KBD組和OA組陽性表達(dá)率均明顯高于正常組(14.92%±2.27%)；三組軟骨組織深層ATF6陽性率比較無統(tǒng)計(jì)差異(F=1.911，P=0.155)。

圖1　三組軟骨細(xì)胞中層ATF6陽性表達(dá)率比較(A:正常，B:OA，C:KBD)

將三組軟骨組織表層、中層和深層陽性細(xì)胞表達(dá)率進(jìn)行統(tǒng)計(jì)分析，結(jié)果顯示：在正常組，三層細(xì)胞表達(dá)率有統(tǒng)計(jì)意義(F=4.059，P=0.021)，中層和深層陽性表達(dá)率均高于表層；在OA組，三層之間的表達(dá)率有統(tǒng)計(jì)學(xué)差異(F=29.408，P=0.001)，中層明顯高于表層和深層，而表層和深層的差異不明顯(P=0.575)；在KBD組，三層之間的表達(dá)率有統(tǒng)計(jì)學(xué)差異(F=50.162，P=0.001)，中層明顯高于表層和深層，而表層和深層的差異也不明顯(P=0.400)，說明KBD軟骨細(xì)胞內(nèi)均有ATF6表達(dá)，中層表達(dá)率最高,見表1。

表1　三組軟骨不同細(xì)胞層ATF6表達(dá)差異的比較±s，%)

2 KBD與OA和正常組關(guān)節(jié)軟骨細(xì)胞CHOP表達(dá)比較在光學(xué)顯微鏡下觀察免疫組化結(jié)果顯示：CHOP主要表達(dá)于三組軟骨細(xì)胞的細(xì)胞核及胞漿中(圖2)。計(jì)算三組軟骨不同分層CHOP表達(dá)陽性率：在表層，三組軟骨組織表達(dá)率有明顯差異(P=0.001)，OA組表達(dá)率明顯高于正常組和KBD組，但正常組和KBD組的統(tǒng)計(jì)學(xué)分析結(jié)果無差異(P=0.334)；在中層，三組之間的表達(dá)率有顯著差異(F=11.192，P=0.001)，KBD組和OA組陽性表達(dá)率均明顯高于正常組(P值分別為0.001和0.001)，而OA組和KBD組之間無統(tǒng)計(jì)學(xué)差異(P=0.700)；在深層，三組軟骨組織的CHOP陽性率存在差異(F=3.193，P=0.048)，表現(xiàn)在OA組高于KBD組(P=0.014)。

將三組軟骨組織表層、中層和深層陽性細(xì)胞表達(dá)率進(jìn)行統(tǒng)計(jì)分析，結(jié)果顯示：在正常組，三層細(xì)胞表達(dá)率差異有統(tǒng)計(jì)意義(P=0.021)，中層陽性表達(dá)率均高于表層(P=0.021)，表層和深層無統(tǒng)計(jì)學(xué)差異；在OA組，三層之間的表達(dá)率有統(tǒng)計(jì)學(xué)差異(P=0.001)，中層明顯高于表層和深層，而表層和深層的差異不明顯(P=0.376)；在KBD組，三層之間的表達(dá)率有統(tǒng)計(jì)學(xué)差異(P=0.001)，中層明顯高于表層和深層，而表層和深層的差異也不明顯(P=0.198)，說明KBD軟骨組織內(nèi)CHOP主要表達(dá)于中層，見表2。

表2　三組軟骨不同細(xì)胞層CHOP表達(dá)差異的比較±s，%)

討　論

既往研究結(jié)果表明：KBD患者關(guān)節(jié)內(nèi)發(fā)生了顯著的軟骨細(xì)胞死亡，且早期主要與軟骨細(xì)胞過度凋亡有關(guān)，凋亡包括線粒體途徑、死亡受體途徑、JNK/P38途徑、內(nèi)質(zhì)網(wǎng)應(yīng)激四種途徑，發(fā)表的研究結(jié)果表明線粒體途徑、死亡受體途徑、JNK/P38途徑[7,8]均可能與KBD的病理變化有關(guān)，目前尚不明確內(nèi)質(zhì)網(wǎng)應(yīng)激途徑是否與KBD有關(guān)。

目前認(rèn)為，ATF6主要與軟骨細(xì)胞的增生分化有關(guān)，還可以抑制內(nèi)質(zhì)網(wǎng)介導(dǎo)的細(xì)胞凋亡[3,5]。在我們的研究中，ATF6表達(dá)上調(diào)提示，KBD軟骨組織中軟骨細(xì)胞過度凋亡后，ATF6代償性表達(dá)增加，試圖減少軟骨細(xì)胞過度丟失，可能是一種對傷害性刺激的保護(hù)性反應(yīng)。還需要我們進(jìn)一步通過細(xì)胞分子生物學(xué)實(shí)驗(yàn)加以驗(yàn)證。

Gadd153/CHOP復(fù)合物可以通過降低Bcl-2含量和促進(jìn)谷胱甘肽，促使內(nèi)質(zhì)網(wǎng)內(nèi)鈣離子代謝異常，從而誘發(fā)細(xì)胞凋亡[9]。內(nèi)質(zhì)網(wǎng)長期處于這種應(yīng)激狀態(tài)，最終可以促發(fā)凋亡性細(xì)胞死亡[10]。有研究報(bào)道:CHOP還可以同過增強(qiáng)死亡受體5表達(dá)誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激性細(xì)胞凋亡[11]。內(nèi)質(zhì)網(wǎng)應(yīng)激可通過線粒體途徑誘導(dǎo)細(xì)胞凋亡[12]。軟骨組織退變與細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激性過度凋亡及細(xì)胞內(nèi)保護(hù)性反應(yīng)抑制[13]有關(guān)。軟骨細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡機(jī)制與晚期糖化終產(chǎn)物聚集有關(guān)[14]。寡霉素A可通過CHOP介導(dǎo)的DR5過表達(dá)進(jìn)而增強(qiáng)TRAIL誘導(dǎo)的細(xì)胞凋亡[15]。本研究中發(fā)現(xiàn)KBD軟骨組織細(xì)胞中CHOP表達(dá)異常增高，結(jié)合已發(fā)表的研究結(jié)果，我們認(rèn)為KBD軟骨組織細(xì)胞中CHOP高表達(dá)與其軟骨組織中細(xì)胞過度凋亡有關(guān)。

進(jìn)一步明確內(nèi)質(zhì)網(wǎng)應(yīng)激是否導(dǎo)致KBD軟骨細(xì)胞容易損傷或阻止了損傷后修復(fù)，及其內(nèi)在分子學(xué)機(jī)制，是我們今后的主要研究方向之一。

[1]Wang SJ, Guo X, Zuo H,etal. Chondrocyte apoptosis and expression of Bcl-2, Bax, Fas, and iNOS in articular cartilage in patients with Kashin-Beck disease[J]. J Rheumatol， 2006, 33(3): 615-619.

[2]謝靖, 楊冠.骨關(guān)節(jié)炎軟骨細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激[J].生物技術(shù)通訊, 2013，(6):787-791.

[3]Han XF, Zhang P, Jiang R,etal. Explore on the effect of ATF6 on cell growth and apoptosis in cartilage development[J]. Histochem Cell Biol， 2014, 142:497-509.

[4]Uehara Y, Hirose J, Yamabe S,etal. Endoplasmic reticulum stress-induced apoptosis contributes to articular cartilage degeneration via C/EBP homologous protein[J]. OsteoarthritisCartilage, 2014, 22(7):1007-1017.

[5]Guo FJ,Xiong ZY,Lu XJ,etal. ATF6 upregulates XBP1S and inhibits ER stress-mediated apoptosis in osteoarthritis cartilage[J]. Cellular Signalling, 2014, 26 :332-342.

[6]武世勛，郭雄，張峰，等.大骨節(jié)病與骨關(guān)節(jié)病軟骨組織死亡相關(guān)因子表達(dá)的比較[J].J South Med Univ, 2014, 34(12): 1785-1789.

[7]Liu JT, Guo X, Ma WJ,etal. Mitochondrial function is altered in articular chondrocytes of an endemic osteoarthritis, KashineBeck disease[J]. Osteoarthritis Cartilage, 2010, 18:1218-1226.

[8]Han J, Guo X, Tan WH,etal. The expression of p-ATF2 involved in the chondeocytes apoptosis of an endemic osteoarthritis, Kashin-Beck disease[J]. BMC Musculoskeletal Disorders, 2013, 14:209.

[9]Iqbal J, Meyer PN, Smith L,etal. BCL2 predicts survival in germinal center B-cell-like diffuse large B-cell lymphoma treated with CHOP-like therapy and rituximab[J]. Clin Cancer Res, 2011,17(24):7785-7795.

[10]Jwa M, Chang P. PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1alpha-mediated unfolded protein response[J]. Nat Cell Biol, 2012,14(11):1223-1230.

[11]Yamaguchi H, Wang HG. CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells[J]. J Biol Chem, 2004,279(44):45495-45502.

[12]Shiraishi H, Okamoto H, Yoshimura A,etal. ER stress-induced apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1[J]. J Cell Sci, 2006,119(19):3958-3966.

[13]Koji T，Jun H，Kei S,etal. Enhanced apoptotic and reduced protective response in chondrocytes following endoplasmic reticulum stress in osteoarthritic cartilage[J]. Int J Exp Pathol, 2011, 22(5):434-442.

[14]Yamabe S，Hirose J，Uehara Y,etal. Intracellular accumulation of advanced glycation end products induces apoptosis via endoplasmic reticulum stress in chondrocytes[J]. Febs Journal, 2013, 280(7):1617-1629.

[15]Long H, Jang JH, Choi HG,etal. Oligomycin A enhances apoptotic effect of TRAIL through CHOP-mediated death receptor 5 expression[J]. Molecular Carcinogenesis, 2013, 52(2):85-93.

(收稿：2016-04-20)

Changed expression of protein molecule related to ER stress in KBD cartilage

Department of Orthopaedics of the Shaan’xi Province People’s Hospital

(Xi’an 710068)Yi ZhiWu ShixunLing Minget al

Objective: To investigate the expression of ATF6 and CHOP in KBD cartilage changes and its relationship with KBD cartilage injury.Methods: 7 specimens of articular cartilage were collected from KBD patients under operation (KBD group), 7 cases from patients suffered from osteoarthritis (OA group) as disease control, 6 normal cartilage as normal controls. Expression of AFT6 and CHOP as key regulators in ER stress of three groups articular cartilage was detected using immunohistochemical staining,significant differences expression among the articular cartilage was analyzed. Results:In middle layer, ATF6-positive expression rate in KBD group and OA group was significantly higher than this of normal cartilage group, while in the surface and deep, ATF6 positive cells among three groups was no statistical difference。In surface layer,CHOP-positive expression rate in OA cartilage was significantly higher than this of KBD and normal, while no statistical difference was found between KBD and the normal group; while in middle layer, CHOP-positive expression rate in KBD cartilage group and OA groupwas significantly higher than normal group, positive rate of deep in OA cartilage was higher than KBD group。 ATF6 of KBD and OA group was mainly expressed in the middle layer, whilemiddle and deep in normal cartilage, CHOP of KBD and OA group was mainly expressed in the middle layer.Conclusion: Both ATF6 and CHOP in middle layer of KBD cartilage was increased significantly, indicating that CHOP may be closely related to the KBD cartilage damage; while ATF6 was related with react Aive cell proliferation after cartilage injury.

Osteoarthropathy,primary hypertrophic/physiopathologyCartilage,articular/physiopathlogyEggwhite/metabolism@AFT6

R684.1

A

10.3969/j.issn.1000-7377.2016.09.004

*陜西省社會發(fā)展科技攻關(guān)項(xiàng)目(2015SF138)