腦星型細(xì)胞瘤中CD105、SKP2表達(dá)及臨床意義

郭欣茹 于新 王亞明 劉銳 尹豐 王淑為 張劍寧 劉爽 (海軍總醫(yī)院全軍神經(jīng)外科研究所, 北京 100048)

腦星型細(xì)胞瘤中CD105、SKP2表達(dá)及臨床意義

郭欣茹 于新 王亞明 劉銳 尹豐 王淑為 張劍寧*劉爽*
(海軍總醫(yī)院全軍神經(jīng)外科研究所, 北京 100048)

目的探討細(xì)胞膜糖蛋白 (CD105) 和S期激酶相關(guān)蛋白2 (SKP2)在腦星型細(xì)胞腫瘤組織中的表達(dá)及其臨床意義。方法應(yīng)用免疫組化方法檢測(cè)不同級(jí)別120例腦中樞神經(jīng)系統(tǒng)星型細(xì)胞瘤組織與25例正常人腦組織中CD105，SKP2蛋白的表達(dá)情況及相關(guān)性。結(jié)果CD105蛋白標(biāo)記的MVD在正常腦組織不表達(dá)，在各級(jí)別腫瘤細(xì)胞中表達(dá)陽性，并有逐級(jí)升高的趨勢(shì)，各組之間的差異與病理分級(jí)密切相關(guān)(P<0.01)。Skp2在正常腦組織基本不表達(dá)，在腫瘤細(xì)胞中表達(dá)陽性，隨病理分級(jí)逐漸升高，各組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。二者表達(dá)水平呈正相關(guān)。SKP2在腫瘤組織中的血管內(nèi)皮細(xì)胞及血管周圍也有陽性表達(dá)。結(jié)論CD105，SKP2表達(dá)陽性率都隨腫瘤惡性進(jìn)展增強(qiáng)，SKP2在血管內(nèi)皮細(xì)胞及周圍也有陽性表達(dá)；提示在腦中樞神經(jīng)系統(tǒng)星型細(xì)胞瘤中，二者的表達(dá)變化與腫瘤的惡性發(fā)展、血管生成方面有密切相關(guān)，有可能有協(xié)同作用。

中樞神經(jīng)系統(tǒng)星型細(xì)胞瘤; 細(xì)胞膜糖蛋白; S期激酶相關(guān)蛋白2

腦星型細(xì)胞瘤是發(fā)生于神經(jīng)外胚葉組織的原發(fā)腫瘤，在顱內(nèi)原發(fā)性腫瘤的發(fā)病率中占第 1 位，它生長(zhǎng)迅速且呈惡性侵襲性發(fā)展[1]。腫瘤的發(fā)生是正常細(xì)胞染色體多重?fù)p傷的復(fù)雜過程，伴隨著多種生理現(xiàn)象。其中廣泛的微小新血管形成是腫瘤生長(zhǎng)的生物學(xué)基礎(chǔ)，但其機(jī)制尚不清楚，研究發(fā)現(xiàn)這于多基因的多種調(diào)控機(jī)制有關(guān)。細(xì)胞膜糖蛋白(Endoglin, CD105)在新生血管形成中起重要作用[2]。細(xì)胞周期調(diào)控機(jī)制紊亂也是腫瘤產(chǎn)生的原因之一。S期激酶相關(guān)蛋白2 (S-phase kinase-associated protein 2, Skp2)是新近發(fā)現(xiàn)的致癌基因，在細(xì)胞周期調(diào)控中發(fā)揮重要作用[3]。目前沒有兩者聯(lián)系的相關(guān)報(bào)道。 本文應(yīng)用免疫組化方法，檢測(cè)CD105和Skp2在不同級(jí)別腦星型細(xì)胞瘤組織中的表達(dá)情況及相關(guān)性。探討二者在星型細(xì)胞瘤發(fā)生、發(fā)展中的相互關(guān)系和作用，以及臨床意義。

材料與方法

一、標(biāo)本來源

收集我科2010年5月至2012年5月期間，經(jīng)組織病理學(xué)診斷的資料完整的星型細(xì)胞腫瘤標(biāo)本120例。男性52例，女性48例，年齡12～77歲，平均年齡(42.6±3.24)歲。按照《WHO中樞神經(jīng)系統(tǒng)腫瘤分類》(2007年出版)標(biāo)準(zhǔn)診斷，其中Ⅰ級(jí)毛細(xì)胞型星形細(xì)胞瘤30例，Ⅱ級(jí)星形細(xì)胞瘤30例，Ⅲ級(jí)間變性星形細(xì)胞瘤30例，Ⅳ級(jí)多形性膠母細(xì)胞瘤30例，按病理分級(jí)分組。同期外傷手術(shù)切除的破碎腦組織25例做為正常對(duì)照組。所有患者術(shù)前均未接受放、化療。

二、主要試劑與方法

CD105和 SKP2單克隆抗體、鏈霉親和素-生物素-過氧化物酶復(fù)合物(Strept Avidin-Biotin Complex, SABC)免疫組化試劑盒、3,3-二氨基聯(lián)苯胺顯色試劑盒均購(gòu)自中山金橋生物技術(shù)公司。采用蛋白免疫組織化學(xué)SABC方法及熒光染色。陰性對(duì)照用磷酸鹽緩沖液 (phosphate buffered solution, PBS)代替一抗抗體, 陽性對(duì)照公司提供。應(yīng)用雙盲法，光學(xué)顯微鏡觀察。

1.CD105：先于低倍鏡下選擇陽性染色最多的區(qū)域，然后高倍鏡下觀察計(jì)數(shù)：呈棕黃色的內(nèi)皮細(xì)胞或細(xì)胞簇，有或無管腔，只要與其它結(jié)締組織成分區(qū)別明顯，就為1個(gè)微血管。每例計(jì)數(shù)5個(gè)高倍視野下微血管數(shù)目，平均值作為該標(biāo)本的微血管密度(microvessel density, MVD)[4]。

2.SKP2：SKP2 染色陽性為胞核與胞質(zhì)內(nèi)出現(xiàn)棕黃色顆粒，采用陽性標(biāo)記指數(shù)(labeling index, LI)判定染色結(jié)果，即在每張切片陽性細(xì)胞最多的區(qū)域計(jì)數(shù)1000個(gè)細(xì)胞，按LI=陽性細(xì)胞數(shù)/總細(xì)胞數(shù)×100%，計(jì)算出每例的 LI。

三、統(tǒng)計(jì)學(xué)分析

實(shí)驗(yàn)數(shù)據(jù)采用SPSS 16.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理。組間采用χ2檢驗(yàn)。相關(guān)性分析應(yīng)用 Spearma等級(jí)相關(guān)分析，以P<0.01為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、CD105在各組中的表達(dá)

本研究發(fā)現(xiàn)，CD105在正常對(duì)照組中有表達(dá)。但在不同病理分組中，隨著腫瘤惡性程度的升高，既病理級(jí)別的升高，CD105標(biāo)記的微血管密度MVD顯著升高，并明顯高于于正常腦組織。(見圖1)Ⅰ-Ⅳ各組CD105標(biāo)染的微血管密度(CD105-MVD)分別為：8.12±2.98、14.87±3.36、18.87±3.89、23.12±3.45。各組與質(zhì)控組及各組間差異均有統(tǒng)計(jì)學(xué)意義(均P<0.01)(圖1, 表1)。

GroupnCD105?MVD(N/View)SKP2LI(%) Controlgroup2502．84±2．61 AstrocytomaⅠ308．12±2．98a10．21±2．35e AstrocytomaⅡ3014．87±3．34bi15．34±2．68fl AstrocytomaⅢ3018．87±3．8c，j19．91±1．8gm AstrocytomaⅣ3023．12±3．4dk25．69±3．64hn

aP<0.01,vscontrol group;eP<0.05,vscontrol group;bP<0.01,vscontrol group;fP<0.05,vscontrol group;cP<0.01,vscontrol group;gP<0.05,vscontrol group;dP<0.01,vscontrol group;hP<0.05,vscontrol group;iP>0.01,vsastrocytoma groupⅠ;lP<0.01,vsastrocytoma groupⅠ;jP>0.01,vsastrocytoma groupⅡ;mP<0.01,vsastrocytoma groupⅡ;kP<0.01,vsastrocytoma groupⅢ ;nP<0.01,vsastrocytoma groupⅢ.

二、SKP2 在各組中的表達(dá)

SKP2在正常對(duì)照組中基本不表達(dá)，僅有少數(shù)細(xì)胞內(nèi)有陽性染色。在各級(jí)腫瘤細(xì)胞中陽性表達(dá)且陽性表達(dá)指數(shù)逐級(jí)升高，見圖2。數(shù)據(jù)見表1。各組與正常組差異及各組間差異均有統(tǒng)計(jì)學(xué)意義(均P<0.05)。SKP2在血管內(nèi)皮細(xì)胞中及血管周圍也有陽性表達(dá)。

三、CD105和SKP2的表達(dá)以及與腦星型細(xì)胞瘤病理分級(jí)的關(guān)系

經(jīng)Spearma相關(guān)分析顯示，CD105和SKP2表達(dá)與腦星型細(xì)胞瘤的病理分級(jí)呈顯著正相關(guān)(均為P<0.01)，相關(guān)系數(shù)分別為0.786、0.823。且CD105和SKP2表達(dá)呈正相關(guān)(P<0.01)，相關(guān)系數(shù)為0.754。

四、CD105和SKP2 共表達(dá)

經(jīng)免疫熒光染色發(fā)現(xiàn)。CD105 (羅丹明染色顯示)和SKP2 (FITC染色顯示)兩者具有染色重合。如圖3顯示。則說明SKP2 可能參與了新血管生成。

圖1 CD105在星型細(xì)胞瘤組織中的表達(dá) (SABC, ×400)

Fig 1 The expression of CD105 in astrocytoma (SABC, ×400)

The positive expression of the CD105 MVD showed that the cytoplasm was dark brown (as red arrow showed).

圖2 SKP2在星型細(xì)胞瘤組織中的表達(dá) (SABC, ×400)

Fig 2 The expression of SKP2 in astrocytoma (SABC, ×400)

The positive expression of the SKP2 showed both cytoplasm and nucelus were dark brown (as arrow showed).

圖3 CD105和SKP2在腦膠質(zhì)母細(xì)胞瘤細(xì)胞中的共表達(dá)

Fig 3 The coexpression of CD105 and SKP2 in glioblastoma cells

A: TRITC(red, Zhongshan jinqiao)labeled the CD105 positive cells; B: SKP2 positive cells labeled by FITC (green, Zhongshan jinqiao); C: Nuclei of the cells labeled by DAPI (blue, Zhongshan jinqiao); D: It was merged A, B and C.

討 論

腦星型細(xì)胞瘤的發(fā)生、發(fā)展都依賴于微小新血管生成。新生血管為其生長(zhǎng)、擴(kuò)散和轉(zhuǎn)移提供營(yíng)養(yǎng)來源，是腫瘤快速增殖和轉(zhuǎn)移的推動(dòng)力[5]。血管生成是一個(gè)需要多基因調(diào)控的復(fù)雜過程。CD105在這個(gè)過程中起著重要的作用，是內(nèi)皮細(xì)胞增殖的主要標(biāo)記物之一。因?yàn)樗鼉H在增殖活躍的內(nèi)皮細(xì)胞表達(dá)，因此優(yōu)于其它的標(biāo)志物[6]。CD105在腫瘤新生血管內(nèi)皮細(xì)胞和腫瘤組織邊緣部分的血管內(nèi)皮細(xì)胞中高度表達(dá)，可成為抑制腫瘤血管生成以治療腫瘤的理想分子靶位[7]。本文研究顯示：CD105在正常組織中表達(dá)陰性。在腦星型細(xì)胞瘤中表達(dá)陽性，并隨病理級(jí)別的升高表達(dá)呈上升趨勢(shì)。各組與正常對(duì)照組差異有統(tǒng)計(jì)學(xué)意義。且表達(dá)和病理級(jí)別呈正相關(guān)。這表明：CD105的表達(dá)即腦星型細(xì)胞瘤新生血管的增生程度與腫瘤惡性進(jìn)展有關(guān)。

細(xì)胞 S 期激酶相關(guān)蛋白 2(Skp2)作為泛素蛋白酶體途徑的底物識(shí)別序列，能夠泛素化降解多種周期相關(guān)蛋白，參與細(xì)胞周期調(diào)控。并作為一種致癌基因與腫瘤的發(fā)生、發(fā)展、生物學(xué)行為及預(yù)后關(guān)系密切?？赏蔀閻盒阅[瘤基因治療的新靶點(diǎn)。Radhakrishnan等[8]在對(duì)胚胎成纖維細(xì)胞系NIH3T3細(xì)胞和人星形細(xì)胞瘤1321N1細(xì)胞的研究中發(fā)現(xiàn)，基因表達(dá)譜(gene expression profiling, GEP)分析致癌基因發(fā)現(xiàn)有絲分裂的信號(hào)包含SKP2的表達(dá)上調(diào)。Skp2在細(xì)胞周期調(diào)控機(jī)制中通過Galpha(12)和其同源受體傳輸致癌信號(hào)。Galeano等[9]新研究發(fā)現(xiàn)，通過調(diào)節(jié)SKP2上游基因RNA編輯酶 (adenosine deaminase that act on RNA, ADAR)2的活性，可以抑制膠質(zhì)母細(xì)胞瘤生長(zhǎng)。而且還發(fā)現(xiàn)ADAR2在星型細(xì)胞瘤中防止腫瘤在體內(nèi)生長(zhǎng)和調(diào)節(jié)重要的細(xì)胞周期通路中Skp2、P21、P27蛋白的作用在膠質(zhì)母細(xì)胞瘤中經(jīng)常改變。本研究發(fā)現(xiàn)，SKP在正常腦組織細(xì)胞中很少表達(dá)。僅有少數(shù)細(xì)胞內(nèi)有陽性染色。在各級(jí)腫瘤細(xì)胞中均有陽性表達(dá)且陽性表達(dá)指數(shù)隨病理級(jí)別升高。陽性表達(dá)與腫瘤病理分級(jí)呈正相關(guān)。說明SKP2 的表達(dá)與星型細(xì)胞瘤的惡性進(jìn)展、組織分化有密切關(guān)系。這于尹豐等[10]的研究結(jié)果相一致。由于還發(fā)現(xiàn)：SKP2在血管內(nèi)皮細(xì)胞中及血管周圍也有陽性表達(dá)。且SKP2表達(dá)和CD105的表達(dá)成正相關(guān)。說明CD105和SKP2的表達(dá)反應(yīng)了星型細(xì)胞瘤的惡性進(jìn)展、組織分化程度，可能與腫瘤的不良預(yù)后有關(guān)。并且說明二者在星型細(xì)胞瘤中的血管生成方面可能有協(xié)同作用。關(guān)于這方面的研究還沒有發(fā)現(xiàn)。這可以為腫瘤的發(fā)生、發(fā)展的調(diào)控網(wǎng)絡(luò)提供新的研究方向。也為星型細(xì)胞瘤的基因治療提供了新的線索。

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2015-05-20；

2015-08-31)

AbstractPatients who have sustained brain injury or had developmental brain lesions present a non-negligible risk for developing delayed epilepsy. Finding therapeutic strategies to prevent development of epilepsy in at-risk patients represents a crucial medical challenge. Noncoding microRNA molecules (miRNAs) are promising candidates in this area. Indeed, deregulation of diverse brain-specific miRNAs has been observed in animal models of epilepsy as well as in patients with epilepsy, mostly in temporal lobe epilepsy (TLE). Herein we review deregulated miRNAs reported in epilepsy with potential roles in key molecular and cellular processes underlying epileptogenesis, namely neuroinflammation, cell proliferation and differentiation, migration, apoptosis, and synaptic remodeling. We provide an up-to-date listing of miRNAs altered in epileptogenesis and assess recent functional studies that have interrogated their role in epilepsy. Last, we discuss potential applications of these findings for the future development of disease-modifying therapeutic strategies for antiepileptogenesis.

TheexpressionofCD105andSKP2inastrocytomaofbrainsanditsclinicalsignificance

GUOXinru,YUXin,WANGYaming,LIURui,YINFeng,WANGShuwei,ZHANGJianning,LIUShuang

TheNavyGeneralHospitalofPLA,NeurosurgicalInstituteofPLA,Beijing100048, China

ObjectiveThis paper aims to find the expression and clinical significance of Endoglin (CD105) and S-phase kinase-associated protein 2 (Skp2) in astrocytoma of brains.MethodsImmunohistochemistry was used to detect CD105 and SKP2 protein expression and the correlation in different levels of 120 human astrocytoma tissues and 25 normal brain tissues.ResultsMicrovessel density (MVD) marked by CD105 protein did not express in normal brain tissue but it was expressed positively at all levels of astrocytoma. The positive percentage was gradually increased with the pathological grade and they were also closely correlated (P<0.01). Skp2 expression in normal brain tissue was not obvious. The expression in astrocytoma tissues was also gradually increased with pathological grade and they were also correlated (P<0.05) with significant statistical differences (P<0.01). Skp2 also expressed positive in tumor tissue vascular endothelial cells and perivascular tissnes.ConclusionCD105 and SKP2 expression rates are enhanced with the malignant progression of tumors, and vascular endothelial cells are positive expressed. It prompts that the expression changes of CD105 and SKP2 in the human brain astrocytoma are closely related with the development of malignant tumors and angiogenesis. There may be a synergistic effect.

Astrocytomas; CD105; SKP2

Involvement of microRNAs in epileptogenesis

CattaniAA1,AlleneC2,SeifertV1,RosenowF3,HenshallDC4,FreimanTM1

1DepartmentofNeurosurgery,GoetheUniversity,Frankfurt,Germany;2ICM-Brain&SpineInstitute,Paris,France;3DepartmentofEpileptology,Goethe-University,Frankfurt,Germany;4Physiology&MedicalPhysicsDepartment,RoyalCollegeofSurgeonsinIreland,Dublin,Ireland.

1671-2897(2016)15-217-04

·論著·

R 739.41

A

郭欣茹，主管技師，碩士，E-mail: 郭欣茹@189.cn

*通訊作者： 張劍寧，主任醫(yī)師，博士生導(dǎo)師，E-mail: jnzhang2005@163.com；劉爽，副研究員，E-mail: shuangff@sina.com

10.1111/epi.13404. [Epub ahead of print]