馬　丹　張　麗　徐少勇

武漢大學(xué)中南醫(yī)院消化內(nèi)科1(430071)　湖北醫(yī)藥學(xué)院附屬人民醫(yī)院病理科2　消化內(nèi)科3

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Skp2在結(jié)直腸癌中的表達(dá)及其與VEGF、MVD的關(guān)系*

馬丹1張麗2徐少勇3#

武漢大學(xué)中南醫(yī)院消化內(nèi)科1(430071)湖北醫(yī)藥學(xué)院附屬人民醫(yī)院病理科2消化內(nèi)科3

背景：近年來(lái)結(jié)直腸癌的發(fā)病率和死亡率呈上升趨勢(shì)，從基因水平診斷和治療癌癥已成為一種新興的有效手段。目的：探討S期激酶相關(guān)蛋白2(Skp2)在結(jié)直腸癌中的表達(dá)及其與血管內(nèi)皮生長(zhǎng)因子(VEGF)、腫瘤微血管密度(MVD)的關(guān)系。方法：選取2014年3月—2015年3月湖北醫(yī)藥學(xué)院附屬人民醫(yī)院35例結(jié)直腸癌患者，以15例癌旁組織作為對(duì)照。采用免疫組化SP法檢測(cè)Skp2、VEGF表達(dá)和MVD值，分析其與臨床病理特征的關(guān)系。結(jié)果：結(jié)直腸癌組織中Skp2表達(dá)顯著高于癌旁組織(P=0.000)，且與腫瘤分化、淋巴結(jié)轉(zhuǎn)移呈正相關(guān)(P<0.05)，與性別、年齡、TNM分期無(wú)明顯相關(guān)性(P>0.05)。結(jié)直腸癌VEGF表達(dá)和MVD值顯著高于癌旁組織(P=0.019，P=0.002)，且與結(jié)直腸癌淋巴結(jié)轉(zhuǎn)移和TNM分期相關(guān)(P<0.05)。結(jié)直腸癌Skp2表達(dá)與VEGF表達(dá)、MVD值均呈正相關(guān)(r=0.569，P=0.000；r=0.481，P=0.017)。結(jié)論：結(jié)直腸癌組織Skp2異常高表達(dá)可能與腫瘤血管生成有關(guān)，Skp2、VEGF表達(dá)和MVD值可作為判斷結(jié)直腸癌惡性程度的重要指標(biāo)。

關(guān)鍵詞結(jié)直腸腫瘤；S期激酶相關(guān)蛋白質(zhì)類；血管內(nèi)皮生長(zhǎng)因子類；微血管密度

Expression of Skp2 and its Relationship with VEGF and MVD in Colorectal CarcinomaMADan1,ZHANGLi2,XUShaoyong3.1DepartmentofGastroenterology,ZhongnanHospitalofWuhanUniversity,Wuhan(430071);2DepartmentofPathology,3DepartmentofGastroenterology,People’sHospitalofHubeiUniversityofMedicine,Shiyan,HubeiProvince

Correspondence to: XU Shaoyong, Email: xsy@hbmu.edu.cn

Microvessel Density

結(jié)直腸癌是常見的消化道惡性腫瘤[1]，目前治療多采用手術(shù)切除輔以放化療的方法，但術(shù)后易轉(zhuǎn)移復(fù)發(fā)，病死率高。近年來(lái)，隨著分子生物學(xué)技術(shù)的迅猛發(fā)展，從基因分子水平診斷和治療癌癥已成為一種新興的有效治療手段。

腫瘤內(nèi)部血管的形成對(duì)腫瘤生長(zhǎng)、侵襲、轉(zhuǎn)移具有重要作用，是直徑超過2 mm的實(shí)體腫瘤生長(zhǎng)的必要條件[2]。S期激酶相關(guān)蛋白2(Skp2)因能特異性識(shí)別磷酸化的蛋白底物，介導(dǎo)其泛素化降解，調(diào)節(jié)細(xì)胞周期而廣受關(guān)注[3]。血管內(nèi)皮生長(zhǎng)因子(VEGF)在血管生成中起有關(guān)鍵作用，微血管密度(MVD)是腫瘤微血管形成的重要指標(biāo)。本研究采用免疫組化法檢測(cè)結(jié)直腸癌中Skp2、VEGF表達(dá)和MVD值，并分析Skp2與VEGF、MVD之間的關(guān)系，旨在為結(jié)直腸癌的分子生物治療提供新的靶點(diǎn)。

對(duì)象與方法

一、研究對(duì)象

收集2014年3月—2015年3月湖北醫(yī)藥學(xué)院附屬人民醫(yī)院手術(shù)切除的結(jié)直腸癌石蠟標(biāo)本35例和癌旁組織15例(切緣距離癌組織至少10 cm)，診斷經(jīng)病理學(xué)檢查證實(shí)。所有病例術(shù)前均未行放療、化療以及其他腫瘤相關(guān)治療。其中男20例，女15例；年齡31～77歲，平均58歲；低分化腺癌7例，中高分化腺癌28例；根據(jù)TNM分期，Ⅰ+Ⅱ期8例，Ⅲ+Ⅳ期27例。入選患者均知情同意。

二、研究方法

Skp2兔抗人多克隆抗體、VEGF兔抗人多克隆抗體購(gòu)自Santa Cruz公司，CD34兔抗人單克隆抗體購(gòu)自Abcam公司，免疫組化試劑盒購(gòu)自欣博盛生物科技有限公司。

采用免疫組化SP法，全部標(biāo)本連續(xù)5 μm厚切片，微波抗原修復(fù)，3% H2O2消除內(nèi)源性過氧化氫酶，10%山羊血清封閉1 h后，分別加入一抗Skp2、VEGF(工作濃度1∶50)和CD34(工作濃度1∶100)，4 ℃過夜。DAB顯色，蘇木素復(fù)染1 min，脫水，透明，封片。以PBS代替一抗作為陰性對(duì)照。

結(jié)果判斷：①Skp2：細(xì)胞核呈棕黃色為Skp2表達(dá)陽(yáng)性，隨機(jī)選取5個(gè)高倍(×400)視野，每個(gè)視野計(jì)數(shù)100個(gè)細(xì)胞，計(jì)算陽(yáng)性細(xì)胞百分率作為Skp2表達(dá)[4]。②VEGF：細(xì)胞質(zhì)呈棕黃色為VEGF表達(dá)陽(yáng)性。高倍(×400)視野下，觀察陽(yáng)性細(xì)胞占視野細(xì)胞總數(shù)的百分比和染色強(qiáng)度。陽(yáng)性細(xì)胞數(shù)：0分：無(wú)細(xì)胞染色；1分：≤25%；2分：26%～50%；3分：≥51%；染色強(qiáng)度：0分：未著色；1分：細(xì)胞內(nèi)散在或少量淺黃色；2分：細(xì)胞內(nèi)片狀棕黃色；3分：細(xì)胞內(nèi)彌漫棕褐色。陽(yáng)性細(xì)胞數(shù)與染色強(qiáng)度之和為VEGF表達(dá)[5]。③MVD：先在低倍鏡(×40和×100)下尋找微血管密集區(qū)“熱點(diǎn)”，然后在高倍鏡(×200)下精確計(jì)數(shù)。凡被染成棕黃色的血管內(nèi)皮細(xì)胞或內(nèi)皮細(xì)胞團(tuán)，且與周圍微血管、腫瘤細(xì)胞或其他組織分界明確均可作為一個(gè)血管計(jì)數(shù)，但管腔面積大于8個(gè)紅細(xì)胞或管壁帶有較厚肌層除外。選取3個(gè)視野，取其均值作為該病例的MVD值[6]。

三、統(tǒng)計(jì)學(xué)分析

結(jié)果

一、Skp2、VEGF表達(dá)和MVD值

結(jié)直腸癌和癌旁組織中Skp2表達(dá)分別為35.5%±12.9%、2.2%±0.7%，VEGF表達(dá)分別為4.3±0.8、0.8±0.4，MVD值分別為57.6±13.0、22.5±0.4。結(jié)直腸癌中Skp2、VEGF表達(dá)和MVD值均明顯高于癌旁組織(P=0.000，P=0.019，P=0.002)(圖1)。

二、Skp2、VEGF和MVD與結(jié)直腸癌臨床病理特征的關(guān)系

Skp2蛋白高表達(dá)與結(jié)直腸癌分化程度和淋巴結(jié)轉(zhuǎn)移相關(guān)(P<0.05)，與性別、年齡、TNM分期無(wú)關(guān)(P>0.05)；VEGF蛋白高表達(dá)和MVD值與淋巴結(jié)轉(zhuǎn)移和TNM分期相關(guān)(P<0.05)，與性別、年齡、腫瘤分化程度無(wú)關(guān)(P>0.05)(表1)。

三、Skp2表達(dá)與VEGF、MVD的相關(guān)性

Spearman等級(jí)相關(guān)分析顯示，結(jié)直腸癌中Skp2表達(dá)與VEGF表達(dá)、MVD值均呈正相關(guān)(r=0.569，P=0.000；r=0.481，P=0.017)。

討論

泛素-蛋白酶體途徑介導(dǎo)的蛋白質(zhì)降解是真核生物調(diào)節(jié)細(xì)胞內(nèi)蛋白水平的重要生理機(jī)制之一，SCF復(fù)合體(Skp1-Cullin-F-box)是泛素連接酶的重要組成部分，Skp2是F-box蛋白家族成員之一，在泛素-蛋白酶體降解通路中起特異性識(shí)別降解底物的作用[7]。研究表明，Skp2在多種惡性腫瘤中的表達(dá)明顯升高[8-10]。結(jié)直腸癌中Skp2過度表達(dá)，可識(shí)別磷酸化p27并引導(dǎo)其降解，從而使細(xì)胞從G1期進(jìn)入S期，調(diào)控腫瘤的生長(zhǎng)。此外，Skp2還與腫瘤侵襲、轉(zhuǎn)移密切相關(guān)[7]。

Skp2(×400) A：結(jié)直腸癌；B：癌旁組織；　VEGF(×400) C：結(jié)直腸癌；D：癌旁組織；　CD34(×200) E：結(jié)直腸癌；F：癌旁組織

特征例數(shù)Skp2表達(dá)(%)P值VEGF表達(dá)P值MVDP值性別 男2034.7±13.70.2094.0±0.70.88252.6±10.00.290 女1542.7±11.04.9±0.867.5±13.3年齡(歲) ≤50840.3±17.20.3574.6±0.80.24757.8±13.50.929 >502734.0±11.54.1±0.957.3±13.2分化程度 中-高2833.4±12.60.0134.2±0.90.19156.9±10.10.665 低749.5±7.04.8±0.759.9±22.6淋巴結(jié)轉(zhuǎn)移 無(wú)825.3±6.10.0453.6±0.50.00053.2±7.80.007 有2742.2±6.95.0±0.666.9±13.7TNM分期 Ⅰ+Ⅱ期837.5±12.50.0903.2±0.40.00046.5±9.60.015 Ⅲ+Ⅳ期2734.2±8.25.5±0.862.3±10.8

腫瘤的生長(zhǎng)、侵襲、轉(zhuǎn)移均與血管有關(guān)，VEGF為血管生成過程中最關(guān)鍵的刺激因子。研究發(fā)現(xiàn)，結(jié)直腸癌中VEGF表達(dá)與MVD值呈高度正相關(guān)，兩者的過度增高與惡性腫瘤復(fù)發(fā)率高和總生存率低密切相關(guān)[11]。目前Skp2是否能促進(jìn)腫瘤血管新生及其機(jī)制均未見相關(guān)報(bào)道。Skp2可激活Sp1激動(dòng)子，增加Sp1基因轉(zhuǎn)錄[12]，在非小細(xì)胞肺癌、胃癌、前列腺癌等惡性腫瘤中Sp1轉(zhuǎn)錄因子表達(dá)較正常組織增高，并增強(qiáng)VEGF表達(dá)[13]。此外，Skp2還可激活c-Myc的轉(zhuǎn)錄[14]，結(jié)直腸癌中c-Myc癌基因通過增強(qiáng)缺氧誘導(dǎo)因子(HIF)-1α、VEGF表達(dá)來(lái)促進(jìn)腫瘤血管新生[2,15]。有文獻(xiàn)證實(shí)以Skp2過表達(dá)轉(zhuǎn)染子使Skp2過表達(dá)后，MMP-2、MMP-9表達(dá)上調(diào)，肺癌細(xì)胞A549侵襲能力增加[16]；胃癌細(xì)胞MGC830沉默Skp2后，MMP-2和MMP-9表達(dá)下調(diào)[10]。而MMP-2和MMP-9在腫瘤血管新生中發(fā)揮重要作用[17]。上述研究結(jié)果說明Skp2可通過上調(diào)VEGF表達(dá)促進(jìn)結(jié)直腸癌血管新生。

本研究發(fā)現(xiàn)，Skp2在結(jié)直腸癌中高表達(dá)，其表達(dá)與結(jié)直腸癌淋巴結(jié)轉(zhuǎn)移和分化程度密切相關(guān)，與Li等[4]、Uddin等[18]的研究結(jié)果基本一致；VEGF在結(jié)直腸癌中高表達(dá)，MVD值升高，兩者與淋巴結(jié)轉(zhuǎn)移和TNM分期相關(guān)。此外，本研究還發(fā)現(xiàn)結(jié)直腸癌Skp2表達(dá)與VEGF、MVD呈正相關(guān)。推測(cè)在結(jié)直腸癌的發(fā)生、發(fā)展過程中，Skp2可能參與腫瘤血管生成的調(diào)控，有望成為結(jié)直腸癌抗腫瘤血管生成新的治療靶點(diǎn)。后續(xù)研究將利用RNAi基因干擾技術(shù)沉默結(jié)直腸癌細(xì)胞中Skp2基因的表達(dá)，并觀察腫瘤血管生成的情況，綜合多方面實(shí)驗(yàn)數(shù)據(jù)，進(jìn)一步探討Skp2在結(jié)直腸癌中的作用。

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(2015-10-19收稿；2015-11-27修回)

DOI:10.3969/j.issn.1008-7125.2016.06.005

Background: In recent years, the incidence and mortality of colorectal carcinoma have been increasing rapidly. Diagnosing and treating cancer at gene level have become a new effective approach. Aims: To investigate the expression of S-phase kinase-associated protein 2 (Skp2) and its relationship with vascular endothelial growth factor (VEGF) and tumor microvessel density (MVD) in colorectal carcinoma. Methods: Thirty-five colorectal carcinoma patients from March 2014 to March 2015 at People’s Hospital of Hubei University of Medicine were enrolled. Fifteen para-cancerous tissue samples were served as controls. The expressions of Skp2, VEGF and MVD value were determined by immunohistochemical SP, and their relationships with clinicopathological characteristics were analyzed. Results: The expression of Skp2 in colorectal carcinoma was significantly higher than that in para-cancerous tissue (P=0.000), and was correlated with tumor differentiation and lymph node metastasis (P<0.05), but not with gender, age and TNM stage (P>0.05). Expression of VEGF and MVD value were significantly higher than those in para-cancerous tissue (P=0.019,P=0.002), and were correlated with lymph node metastasis and TNM stage (P<0.05). Expression of Skp2 was positively correlated with the expression of VEGF and MVD value in colorectal carcinoma (r=0.569,P=0.000;r=0.481,P=0.017). Conclusions: The abnormal high expression of Skp2 is involved in the angiogenesis of colorectal carcinoma. Skp2, VEGF expressions and MVD value may be served as important markers for malignancy degree of colorectal carcinoma.

Key wordsColorectal Neoplasms;S-Phase Kinase-Associated Proteins;Vascular Endothelial Growth Factors;

*本課題由湖北省科技廳自然基金資助項(xiàng)目資助(2011CDB127)

#本文通信作者，Email: xsy@hbmu.edu.cn