張 薇， 楊金蓮， 胡中倩， 盧艷敏， 儲著朗， 余科科， 瞿成奎,3， 汪思應(yīng)△

(1安徽醫(yī)科大學(xué)病理生理學(xué)教研室，安徽 合肥 230032；2安徽醫(yī)學(xué)高等專科學(xué)校病理生理學(xué)教研室，安徽 合肥 230601；3美國凱斯西儲大學(xué)，克里夫蘭 44106)

SHP-2酪氨酸磷酸酶激活突變導(dǎo)致小鼠髓系異常增殖*

張 薇1，2▲， 楊金蓮1▲， 胡中倩1， 盧艷敏1， 儲著朗1， 余科科1， 瞿成奎1,3， 汪思應(yīng)1△

(1安徽醫(yī)科大學(xué)病理生理學(xué)教研室，安徽 合肥 230032；2安徽醫(yī)學(xué)高等?？茖W(xué)校病理生理學(xué)教研室，安徽 合肥 230601；3美國凱斯西儲大學(xué)，克里夫蘭 44106)

目的觀察激活突變SHP-2酪氨酸磷酸酶是否參與髓系異常增殖的發(fā)生。方法以野生型(WT)和SHP-2D61G/+突變型C57BL/6小鼠為研究對象，計數(shù)外周血白細(xì)胞，比較脾大小，流式細(xì)胞術(shù)檢測外周血及骨髓髓系來源細(xì)胞表面標(biāo)志分子(Mac-1、Gr-1)，并統(tǒng)計外周血Mac-1和Gr-1陽性細(xì)胞率及骨髓細(xì)胞中紅系 (Ter119)、髓系 (Mac-1、Gr-1)、T(CD3)、B(B220)淋巴細(xì)胞系的陽性細(xì)胞率，觀察骨髓造血干/祖細(xì)胞的集落形成(CFU)能力，Western blotting檢測外周血白細(xì)胞經(jīng)白細(xì)胞介素 3(IL-3)和5 μg/L，刺激后磷酸化的絲氨酸/蘇氨酸蛋白激酶B(p-Akt)和磷酸化的細(xì)胞外信號調(diào)節(jié)激酶(p-ERK)表達(dá)水平。結(jié)果SHP-2D61G/+突變16周齡組小鼠較WT組外周血白細(xì)胞數(shù)增多(Plt;0.05)，脾明顯增大，同時外周血白細(xì)胞的Mac-1和Gr-1陽性細(xì)胞率增加(Plt;0.05)，骨髓細(xì)胞中Mac-1和Gr-1的陽性細(xì)胞率也增多，但紅系、淋巴細(xì)胞系的變化不明顯。同時骨髓中粒-單核細(xì)胞集落形成單位(CFU-GM)較正常對照組明顯增加，白細(xì)胞經(jīng)IL-3刺激后Akt和ERK蛋白磷酸化水平升高。結(jié)論SHP-2D61G/+突變可能通過MAPK及PI3K的活化而導(dǎo)致小鼠髓系異常增殖。

SHP-2酪氨酸磷酸酶； 激活突變； 小鼠； 髓系增殖

SHP-2酪氨酸磷酸酶(SHP-2 tyrosine phosphatase)是蛋白酪氨酸磷酸酶家族中的一員，廣泛表達(dá)于各組織的細(xì)胞中。SHP-2在N末端含有2個SH2(即Src同源體2)結(jié)構(gòu)域，C末端上含有1個蛋白酪氨酸磷酸酶(protein tyrosine phosphatases,PTP)結(jié)構(gòu)域及2個酪氨酸殘基(Tyr542和Tyr580)[1]。正?；A(chǔ)狀態(tài)下，N-末端的SH2結(jié)構(gòu)域與PTP結(jié)構(gòu)域結(jié)合抑制了SHP-2的催化活性[2，3]。當(dāng)SHP-2經(jīng)特定的生長因子如血小板源性生長因子(platelet-derived growth factor,PDGF)、表皮生長因子(epidermal growth factor,EGF)、促生長因子(insulin-like growth factor, IGF)[4]或細(xì)胞因子如白細(xì)胞介素3(interleukin-3,IL-3)、粒細(xì)胞-巨噬細(xì)胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和促紅細(xì)胞生成素(erythropoietin,EPO)以及胰島素和干擾素刺激，以及N-SH2結(jié)構(gòu)域連接酪氨酸磷酸化的對接蛋白如Gab1、Gab2等，可解除自身抑制狀態(tài)，激活SHP-2的磷酸酶活性[5]；或者SHP-2的PTP結(jié)構(gòu)域與N-SH2結(jié)構(gòu)的突變導(dǎo)致PTP被部分或全部地解離出來，從而使SHP-2磷酸酶活性被不同程度地激活。而羧基端PTP結(jié)構(gòu)域是對SHP-2起調(diào)節(jié)功能的關(guān)鍵部位[6]，其功能主要是在受體或胞漿酪氨酸蛋白激酶(protein tyrosine kinases,PTKs)介導(dǎo)的信號途徑中起促進(jìn)作用，同時還發(fā)現(xiàn)SHP-2具有磷酸酶非依賴性接頭蛋白功能[7-11]。

2001年以來陸續(xù)在Noonan綜合征(伴髓系增生癥)、幼年型粒單細(xì)胞白血病、兒童骨髓增生異常綜合癥和骨髓增生異常、B細(xì)胞急性淋巴細(xì)胞白血病、急性髓系白血病和一些實體瘤中發(fā)現(xiàn)有PTPN11(編碼SHP-2的基因)激活突變[12-17]。這些突變主要集中在SHP-2的N-SH2或PTP結(jié)構(gòu)域內(nèi)，其中N-SH2結(jié)構(gòu)域內(nèi)D61G/Y或E76K突變被認(rèn)為與白血病等腫瘤密切相關(guān)。但PTPN11突變引起疾病的機(jī)制尚不清楚。為此，Neel實驗室建立了SHP-2 D61G基因敲入小鼠模型，但在該模型小鼠中沒有發(fā)現(xiàn)白血病，僅出現(xiàn)Noonan綜合征表現(xiàn)[18]。本實驗中采用已建立的SHP-2D61G/+模型小鼠及正常對照組C57BL/6小鼠為研究對象，觀察SHP-2激活突變后對小鼠髓系增殖及相關(guān)信號通路的影響，以確定SHP-2激活突變后是否參與白血病早期的典型表型-髓系細(xì)胞異常增殖發(fā)生，為進(jìn)一步研究白血病提供新的線索。

材 料 和 方 法

1材料

1.1藥品與試劑 生理鹽水購于北京雙鶴藥業(yè)，胎牛血清、RPMI-1640購于Gibco，LTS1093小鼠白細(xì)胞分離液購于上海歐韋達(dá)公司， FITC(fluorescein isothiocyanate)標(biāo)記的Mac-1、Gr-1、CD3等抗體購于北京博奧森生物公司, Base methylcellulose medium(Methocult M 3134) 購于Stem Cell。Western 所用ERK、Akt、p-ERK,p-Akt等抗體購于Santa Cruz。

1.2動物 野生型(wild type,WT)小鼠采用C57BL/6小鼠，雌雄各半，8周齡及16周齡， SPF級，由安徽醫(yī)科大學(xué)動物中心提供，為WT組。C57BL/6品系SHP-2D61G/+小鼠由美國Case Western Reserve大學(xué)瞿成奎教授提供，為SHP-2D61G/+突變組。分別取8周齡(WT8，SHP-2D61G/+8)和16周齡小鼠(WT16，SHP-2D61G/+16)共分為4組進(jìn)行實驗，每組8只。

2方法

2.1外周血白細(xì)胞(white blood cell, WBC)計數(shù) 分別取WT組和SHP-2D61G/+突變組小鼠尾靜脈血約300 μL于2% EDTA-K2抗凝劑的采血管中，紅細(xì)胞破壞法(3%乙酸溶液∶血液=19∶1)獲得白細(xì)胞，應(yīng)用F2800微細(xì)胞儀計數(shù)小鼠白細(xì)胞。

2.2骨髓細(xì)胞的建立 將小鼠脫頸椎處死后．無菌分離雙側(cè)股骨和脛骨并剪去骨端,用3 mL IMDM (Gibco)培養(yǎng)液的注射器沖出骨髓，再以22號針頭抽吸吹打3次制成單細(xì)胞懸液,用白細(xì)胞稀釋液稀釋，作骨髓有核細(xì)胞計數(shù)后備用。

2.3脾指數(shù)測定 分別對2組小鼠稱重，麻醉小鼠摘眼球取血后脫頸處死，無菌條件下常規(guī)分離，取脾分別稱重。計算脾指數(shù)(spleen index,SI)= 脾重量(mg)/小鼠體重(g)值。

2.4小鼠白細(xì)胞分離培養(yǎng) 分離外周血單個核細(xì)胞的分層液比重是 1.077±0.001(骨髓中單個核細(xì)胞的分層液比重是1.120±0.001)的聚蔗糖(Ficoll)-泛影葡胺(Urografin)(F/H)分層液。無菌采取小鼠抗凝血1 mL，與等量Hanks液充分混勻；用滴管沿管壁緩慢疊加于2 mL分層液面上。2 000 r/min離心20 min；離心后管內(nèi)分為3層，上層為血漿和Hanks液，下層主要為紅細(xì)胞和粒細(xì)胞，中層為淋巴細(xì)胞分離液，在上、中層界面處有一以單個核細(xì)胞為主的白色云霧層狹窄帶，單個核細(xì)胞包括淋巴細(xì)胞和單核細(xì)胞；用毛細(xì)血管插到云霧層，吸取單個核細(xì)胞。置入另一短管中，加入5倍體積的Hanks液，1 500 r/min離心10 min，洗滌細(xì)胞2次，棄上清，加入含有10%胎牛血清的RPMI-1640，0.5 μg/L IL-3，重懸細(xì)胞，常規(guī)培養(yǎng)。

2.5流式細(xì)胞術(shù)檢測細(xì)胞表面標(biāo)記表達(dá) 外周血裂解紅細(xì)胞后或沖洗小鼠骨髓細(xì)胞，離心洗滌后，用含2%BSA的PBS制備成細(xì)胞懸液，加2 ng Fc受體阻斷抗體，室溫孵育15 min，離心洗滌1次，按1×106細(xì)胞/100μL，每管分別加FITC標(biāo)記的相關(guān)CD3等抗原抗體，冰浴30 min，加3 mL冰PBS后，離心洗滌，每管加500 μL PBS(1%BSA)重懸細(xì)胞，流式細(xì)胞儀檢測分析。

2.6骨髓粒-單核細(xì)胞集落生成單位的集落培養(yǎng)(colony-forming unit-granulocyte and macrophage ,CFU-GM) 用甲基纖維素半固體培養(yǎng)系統(tǒng)[19]，加入小鼠干細(xì)胞因子(mSCF) 50 μg/L、小鼠IL-3(mIL-3) 20 μg/L、人IL-6(hIL-6) 50 μg/L和人EPO(hEPO) 3×103U/L, 接種2×107細(xì)胞于35 mm培養(yǎng)皿中，常規(guī)條件培養(yǎng)14 d后倒置顯微鏡下觀察計數(shù)集落數(shù)目(細(xì)胞數(shù)gt;50為1個集落)，并根據(jù)細(xì)胞形態(tài)分類：來源于紅系造血祖細(xì)胞的為CFU-E，來源于粒單系的為CFU-GM，紅粒系混合的為CFU-GEM。

2.7外周血白細(xì)胞總蛋白的提取及Western blotting檢測磷酸化的細(xì)胞外信號調(diào)節(jié)激酶(phosphorylated- extracellular signal-regulated kinase,p-ERK)和磷酸化的絲氨酸/蘇氨酸蛋白激酶(phosphorylated -serine-threonine protein kinase B,p-Akt)的表達(dá)水平 正常生長狀態(tài)的白細(xì)胞饑餓8 h，IL-3 5 μg/L刺激0、5、15 min，細(xì)胞蛋白經(jīng)SDS-PAGE電泳，電轉(zhuǎn)移至NC膜上，用含2%BSA/TBST封閉液封閉2 h，加p-ERK和p-Akt抗體過夜(4 ℃)，TBST洗3次，每次10 min，再加經(jīng)辣根過氧化物酶標(biāo)記的相應(yīng)的Ⅱ抗(用封閉液1∶2 000稀釋)，搖床上緩慢搖動1 h，用TBST洗3次，每次10 min；用ECL系統(tǒng)進(jìn)行檢測，壓片，曝光。

3統(tǒng)計學(xué)處理

結(jié) 果

1SHP-2D61G/+突變小鼠外周血白細(xì)胞數(shù)增加

對SHP-2D61G/+突變小鼠及WT組小鼠的外周血白細(xì)胞計數(shù)觀察發(fā)現(xiàn)，16周齡SHP-2D61G/+突變的外周血白細(xì)胞數(shù)明顯比同齡WT組高(Plt;0.01),見圖1，提示SHP-2D61G/+突變導(dǎo)致了外周血白細(xì)胞的增殖。

Figure 1. Increasing peripheral white blood cells in the mice with gain-of -function mutant of SHP-2±s.**Plt;0.01 vs WT16.

2SHP-2D61G/+突變小鼠脾明顯增大

脾臟增大是髓系異常增殖的顯著特征之一。實驗中我們發(fā)現(xiàn)SHP-2D61G/+突變組小鼠的脾明顯較同齡的WT組增大、充血(圖2照片展示16周齡WT及SHP-2激活突變小鼠脾臟)，脾指數(shù)檢測也發(fā)現(xiàn)SHP-2D61G/+突變小鼠脾的相對增大程度較同齡WT組升高 (Plt;0.01),見圖2。以高齡鼠最為明顯。

Figure 2. Enlarged spleen in the mice with gain-of-function mutant of SHP-2±s.**Plt;0.01 vs WT16.

3SHP-2D61G/+突變小鼠外周血及骨髓中髓系細(xì)胞比例升高

圖3結(jié)果顯示，SHP-2D61G/+突變組外周血增加的白細(xì)胞中，髓系來源(Mac-1和Gr-1陽性)比例高于對照正常小鼠，以高齡小鼠最為明顯。骨髓細(xì)胞表面標(biāo)記檢測也發(fā)現(xiàn)相同結(jié)果，SHP-2D61G/+突變組Mac-1和Gr-1陽性骨髓細(xì)胞比例明顯高于對照組(Plt;0.05),見圖4，提示小鼠髓系增殖異常。

4骨髓祖細(xì)胞集落形成實驗

取8周齡小鼠骨髓細(xì)胞，用甲基纖維素半固體培養(yǎng)系統(tǒng)檢測在多種細(xì)胞因子誘導(dǎo)下，不同造血祖細(xì)胞集落形成能力，結(jié)果發(fā)現(xiàn)SHP-2D61G/+突變組髓系來源祖細(xì)胞集落形成不僅數(shù)量明顯高于對照組(Plt;0.01),見圖5。從形態(tài)學(xué)上比較，突變組的集落亦大于對照組(結(jié)果未展示)。

Figure 3. Percentage of myeloid cells is increased in the mice with gain-of -function mutant of SHP-2±s.*Plt;0.05 vs WT16.

Figure 4. Percentage of myeloid cells is increased in the bone marrow of mice with gain-of-function mutant of SHP-2±s.n=5.*Plt;0.05 vs Mac-1.

5Westernblotting檢測p-ERK和p-Akt的表達(dá)水平

從圖6可以看出，經(jīng)IL-3 5 μg/L刺激后，SHP-2 D61G突變小鼠白細(xì)胞p-ERK和p-Akt的表達(dá)水平均有明顯上調(diào)，提示MAPK和PI3K途徑被明顯激活。

Figure 6. Phosphorylation of ERK and Akt was increased in the WBC of mice with gain-of-function mutant of SHP-2 induced by IL-3±s.n=5.**Plt;0.01 vs WT.

討 論

髓系異常增殖是白血病前期典型表現(xiàn)之一，研究髓系異常增殖應(yīng)該能為白血病發(fā)病的研究提供線索。臨床髓系異常增殖病可以單發(fā)，也可以伴發(fā)于其他疾病如Noonan綜合征之后或白血病前期。目前機(jī)制不清。研究發(fā)現(xiàn)10%的髓系增殖病患者體內(nèi)存在SHP-2激活突變，SHP-2是一個在細(xì)胞內(nèi)廣泛存在的酪氨酸磷酸酶，其在細(xì)胞生長、分化、遷移、凋亡的信號調(diào)控中起著重要的作用，尤其對胚胎干細(xì)胞的分化和造血細(xì)胞的分化也起著關(guān)鍵作用[1,4,20]。Neel等在研究SHP-2蛋白結(jié)構(gòu)時，利用計算機(jī)模擬技術(shù)認(rèn)為如果SHP-2 N-SH2結(jié)構(gòu)域內(nèi)第61位的天冬氨酸(D)突變成甘氨酸(G)，則N-SH2結(jié)構(gòu)域與其PTP結(jié)構(gòu)域的自身抑制作用將降低，SHP-2磷酸酶活性將升高。為進(jìn)一步研究SHP-2 D61G突變的功能，他們建立了SHP-2 D61G基因敲入小鼠模型，SHP-2 D61G純合子突變(SHP-2D61G/D61G)小鼠死于胚胎期，約50%雜合子突變(SHP-2D61G/+)小鼠生長發(fā)育成活，表現(xiàn)Noonan綜合征癥狀，但無白血病出現(xiàn)[18]。臨床Noonan綜合征患者常伴發(fā)髓系增殖，后期可能發(fā)生白血病。因此。我們在獲贈該小鼠后，觀察SHP-2D61G/+小鼠是否發(fā)生了髓系異常增殖及其機(jī)制。

實驗中我們發(fā)現(xiàn)SHP-2D61G/+突變組小鼠隨著年齡增大外周血白細(xì)胞數(shù)明顯較同周齡WT組增高，同時脾明顯增大， 16周齡時小鼠差異顯著和流式顯示外周血白細(xì)胞中髓系來源的細(xì)胞即Mac-1和Gr-1陽性細(xì)胞率在SHP-2D61G/+突變組也明顯高于WT組；同時骨髓細(xì)胞中髓系細(xì)胞(Mac-1、Gr-1)比例明顯增高，而紅細(xì)胞(Ter119)、T細(xì)胞(CD3)和B細(xì)胞(B220)淋巴細(xì)胞系變化不明顯；提示SHP-2 激活突變小鼠髓系發(fā)生異常增殖。機(jī)制研究時發(fā)現(xiàn)：SHP-2 激活突變小鼠骨髓造血祖細(xì)胞形成集落中CFU-GM比例明顯高于對照正常小鼠，并且集落明顯增大，提示髓系祖細(xì)胞對細(xì)胞因子反應(yīng)性增強(qiáng)。進(jìn)一步實驗結(jié)果發(fā)現(xiàn)SHP-2激活突變小鼠白細(xì)胞后經(jīng)IL-3刺激后磷酸化ERK及AKT水平均明顯高于對照組細(xì)胞，提示髓系異常增殖可能與其增強(qiáng)的MAPK及PI3K信號途徑有關(guān)。但SHP-2是酪氨酸磷酸酶，SHP-2激活突變往往磷酸酶活性升高，應(yīng)該導(dǎo)致下游蛋白磷酸化水平降低，為何經(jīng)細(xì)胞因子刺激后，突變細(xì)胞內(nèi)p-ERK及p-Akt水平升高呢，可能與SHP-2除了磷酸酶功能外，還具備接頭蛋白功能[21]。我們前期研究也發(fā)現(xiàn)SHP-2 D61G突變的肥大細(xì)胞對細(xì)胞因子IL-3反應(yīng)性增強(qiáng)，且可能是突變SHP-2與Gab2結(jié)合增多有關(guān)[22,23]。Gab的活化是MAPK途徑活化所必須的[21]。但詳細(xì)的機(jī)制以及與白血病發(fā)病的關(guān)系還有待進(jìn)一步研究闡明。

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AnactivatedmutantofSHP-2tyrosinephosphatasecausesmurinemyeloidabnormalproliferation

ZHANG Wei1,2, YANG Jin-lian1, HU Zhong-qian1, LU Yan-min1, CHU Zhu-lang1, YU Ke-ke1, QU Cheng-kui1, 3, WANG Si-ying1

(1DepartmentofPathophysiology,AnhuiMedicalUniversity,Hefei230032,China;2DepartmentofPathophysiology,AnhuiMedicalCollege,Hefei230601,China;3CaseWesternReserveUniversity,Cleveland44106,USA.E-mail:sywang@ahmu.edu.cn)

AIM: To investigate whether an activated mutant of SHP-2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODSWild-type (WT) and SHP-2D61G/+mutant mice aged 8 weeks and 16 weeks were used. The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively. The surface markers (Mac-1 and Gr-1 for myeloid, Ter119 for erythroid, CD3 for T-lymphocyte and B220 for B-lymphocyte) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry. The rate of Mac-1 or Gr-1 positive cells in the peripheral blood and the rate of Mac-1, Gr-1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed. The ability of colony formation unit (CFU) of the bone marrow was also observed by CFU assay. Finally, the expression of p-Akt and p-ERK in the peripheral blood leukocytes induced by interleukin-3 (IL-3, 5 μg/L) was detected by Western blotting.RESULTSThe number of leukocytes in peripheral blood of 16-week-old mice was more (Plt;0.01) and the spleens were bigger in mutant SHP-2D61G/+mice than those in WT mice. The rate of Mac-1 and Gr-1 positive cells in peripheral blood leukocytes of 16-week-old SHP-2D61G/+mice were dramatically increased (Plt;0.05). Mac-1 and Gr-1 positive cell rates in bone marrow of SHP-2D61G / +mice were much higher (Plt;0.05) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells. The number of CFU-GM (represents myeloid) was increased in mutant mice. The expression of p-Akt and p-ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL-3.CONCLUSIONThese results suggest that activated mutant SHP-2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.

SHP-2 tyrosine phosphatase; Activated mutant; Mice; Myeloid proliferation

1000-4718(2011)04-0682-06

R363

A

10.3969/j.issn.1000-4718.2011.04.012

2010-12-15

2011-02-25

國家自然科學(xué)基金資助項目(No. 30873046)；安徽省優(yōu)秀留學(xué)人才基金資助項目(No.2009-2010)

△通訊作者 Tel：0551-5167706；E-mail: sywang@ahmu.edu.cn

▲并列第1作者