EGFR及ADAM9在胰腺癌干細(xì)胞與分化細(xì)胞中的表達(dá)及意義

洪曉泉 林凡 王敏 王欣 秦仁義

·論著·

EGFR及ADAM9在胰腺癌干細(xì)胞與分化細(xì)胞中的表達(dá)及意義

洪曉泉 林凡 王敏 王欣 秦仁義

目的通過微球體培養(yǎng)富集胰腺癌干細(xì)胞，檢測其表皮生長因子受體(EGFR)和去整合素-金屬蛋白酶9(ADAM9)的表達(dá)，并探討其意義。方法運(yùn)用無血清條件培養(yǎng)基懸浮培養(yǎng)胰腺癌PANC1細(xì)胞，并連續(xù)傳代培養(yǎng)。將部分微球體接種于含血清及膠原底物的培養(yǎng)基中進(jìn)行分化誘導(dǎo)。收集微球體細(xì)胞及分化細(xì)胞，流式細(xì)胞儀檢測側(cè)群(SP)細(xì)胞比例；實(shí)時(shí)PCR及蛋白質(zhì)印跡法檢測細(xì)胞EGFR、ADAM9 mRNA和蛋白的表達(dá)。結(jié)果成功培養(yǎng)出胰腺癌PANC1細(xì)胞微球體，并能在體外連續(xù)傳代。微球體細(xì)胞分化誘導(dǎo)后能重新貼壁生長。微球體細(xì)胞和分化細(xì)胞中SP細(xì)胞比例分別為(5.40±0.38)%和(2.80±0.42)% ，兩者差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。微球體細(xì)胞與分化細(xì)胞相比，EGFR及ADAM9 mRNA表達(dá)分別上調(diào)約2.5和3.0倍(P<0.05)；微球體細(xì)胞EGFR及ADAM9蛋白相對(duì)表達(dá)量分別為0.90±0.09和0.64±0.07，顯著高于分化細(xì)胞的0.62±0.11和0.48±0.09(P<0.05)。結(jié)論微球體培養(yǎng)能富集胰腺癌干細(xì)胞，ADAM9可能通過EGFR信號(hào)通路在胰腺癌的發(fā)生、發(fā)展中起重要作用。

胰腺腫瘤； 干細(xì)胞； 受體，表皮生長因子； 去整合素-金屬蛋白酶9

研究[1]表明，去整合素-金屬蛋白酶9(a disintegrin and metalloprotease, ADAM9)在腫瘤細(xì)胞黏附、蛋白水解、信號(hào)轉(zhuǎn)導(dǎo)和血管生成等環(huán)節(jié)中起著重要作用。ADAM9通過參與表皮生長因子受體(epidermal growth factor receptor, EGFR)配體胞外功能區(qū)的脫落而發(fā)揮作用。EGFR和ADAM9的高表達(dá)與多種實(shí)體腫瘤的進(jìn)展和預(yù)后密切相關(guān)[2-4]，但在胰腺癌干細(xì)胞中的表達(dá)未見報(bào)道。本實(shí)驗(yàn)通過微球體培養(yǎng)富集胰腺癌干細(xì)胞，檢測ADAM9和EGFR在微球體細(xì)胞及其分化細(xì)胞中的表達(dá)，探索胰腺癌干細(xì)胞的調(diào)控機(jī)制。

材料與方法

一、微球體培養(yǎng)及分化誘導(dǎo)

人胰腺癌細(xì)胞系PANC1由本實(shí)驗(yàn)室保存。微球體培養(yǎng)參照文獻(xiàn)[5]并進(jìn)行適當(dāng)改進(jìn)。取105個(gè)PANC1細(xì)胞，重懸于含EGF(20 ng/ml)、bFGF(20 ng/ml)、胰島素(5 μg/ml)、B27(2%)的無血清條件培養(yǎng)基DMEM/F12(serum-free medium, SFM)常規(guī)培養(yǎng)，2 d更換1次培養(yǎng)液。微球體形成后，經(jīng)胰酶消化，40 μm篩網(wǎng)過濾，傳代培養(yǎng)。

將微球體細(xì)胞接種于膠原蛋白(按Bell等[6]方法提取)包被的6孔板，用含10%胎牛血清的DMEM/F12培養(yǎng)基常規(guī)培養(yǎng)2 d以分化誘導(dǎo)細(xì)胞，倒置顯微鏡下觀察細(xì)胞形態(tài)及生長情況。

二、流式細(xì)胞儀檢測側(cè)群(side population，SP)細(xì)胞比例

取微球體細(xì)胞和分化細(xì)胞消化成單細(xì)胞懸液，調(diào)節(jié)細(xì)胞濃度為105/ml。各分為兩組，實(shí)驗(yàn)組加入Hoechst33342(Sigma公司)至終濃度5 μg/ml，對(duì)照組加入verapamil(Sigma公司)至終濃度150 μmol/L,孵育30 min后再加入同濃度的Hoechst33342，37℃水浴90 min，每15 min震蕩一次。孵育后1000 r/min離心5 min，棄上清，PBS洗2次，重懸于4℃的PBS液中，加入PI(Sigma公司)至終濃度2 μg/ml，流式細(xì)胞儀檢測SP細(xì)胞。實(shí)驗(yàn)重復(fù)3次。

三、實(shí)時(shí) PCR檢測細(xì)胞EGFR、ADAM9 mRNA表達(dá)

取微球體細(xì)胞和分化細(xì)胞，Trizol提取總RNA。采用cDNA第一鏈合成逆轉(zhuǎn)錄試劑盒(TOYOBO)轉(zhuǎn)錄cDNA。使用SYBR Green Master Mix在ABI Prism?7900HT型熒光定量PCR儀上進(jìn)行實(shí)時(shí) PCR檢測。EGFR上游序列5′-CACTTGCTACCCTGAGTTCATCCA-3′，下游序列5′-GGAAGCCTTGAAGCAGAACCAC-3′；ADAM9上游序列5′-TTGTGGGAACAGTGTGTTCAAGG-3′，下游序列5′-CCAATTCATGAGCAACAATGGAAG-3′；內(nèi)參GAPDH上游序列5′-GCACCGTCAAGGCTGAGAAC-3′，下游序列5′-TGGTGAAGACGCCAGTGGA-3′，引物均由上海生工生物工程技術(shù)服務(wù)有限公司合成。反應(yīng)條件：50℃ 2 min，95℃ 10 min；95℃ 15 s、60℃ 60 s，40個(gè)循環(huán)。每個(gè)樣本目的基因的循環(huán)閾值(Ct)與內(nèi)參Ct的差異為△Ct，實(shí)驗(yàn)樣本與對(duì)照樣本△Ct的差異為△△Ct。以2-△△Ct表示基因的相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次。

四、蛋白質(zhì)印跡法檢測EGFR、ADAM9蛋白表達(dá)

用細(xì)胞裂解液(50 μmol/L Tris-Cl、100 μmol/L Nacl、2 μmol/L EDTA、1% SDS、適量蛋白酶抑制劑)分別提取微球體細(xì)胞和分化細(xì)胞的總蛋白。常規(guī)行蛋白質(zhì)印跡法檢測。兔抗人ADAM9多抗工作濃度1∶2000，鼠抗人EGFR單抗工作濃度1∶1000，ECL顯色，LabworksTM掃描分析軟件系統(tǒng)檢測條帶灰度值，以目的蛋白與內(nèi)參比值作為蛋白的相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次。

五、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、微球體細(xì)胞及分化細(xì)胞的形態(tài)變化

大多數(shù)PANC1細(xì)胞在SFM培養(yǎng)中呈懸浮生長，少量細(xì)胞貼壁，4 ～5 d后產(chǎn)生少量體積較小的懸浮細(xì)胞球，6～7 d后形成致密且形態(tài)較一致的微球體(圖1a)，傳代同樣可形成微球體，且微球體的數(shù)量明顯增多，體積也增大(圖1b)。接種于膠原包被6孔板的微球體細(xì)胞在含血清的培養(yǎng)基中培養(yǎng)4 h后開始貼壁，2 d后大多數(shù)貼壁生長，細(xì)胞伸出偽足由圓形變成扁平形、長梭形或多角形(圖1c)。

圖1第一代PANC1微球體細(xì)胞(a)、第二代PANC1微球體細(xì)胞(b)及分化細(xì)胞(c)形態(tài)(×200)

二、SP細(xì)胞比例

微球體細(xì)胞的SP細(xì)胞比例為(5.40±0.38)%，顯著高于分化細(xì)胞的(2.80±0.42)%(P<0.05)。用verapamil預(yù)處理的對(duì)照組細(xì)胞的SP細(xì)胞比例分別為(0.52±0.11)%和(0.31±0.14)%，均較未處理細(xì)胞顯著減少(P值均<0.05，圖2)。

a: 第二代微球體細(xì)胞; b: 分化細(xì)胞; c: verapamil處理的第二代球體細(xì)胞; d: verapamil處理的分化細(xì)胞

圖2流式細(xì)胞儀分析側(cè)群細(xì)胞比例

三、EGFR、ADAM9 mRNA 和蛋白表達(dá)的變化

微球體細(xì)胞、分化細(xì)胞EGFR mRNA檢測值分別為25.45±0.11和26.87±0.21，2-△△Ct分別為1.0000和2.4967，微球體細(xì)胞較分化細(xì)胞上調(diào)約2.5倍；ADAM9 mRNA檢測值分別為25.87±0.09和27.55±0.35，2-△△Ct分別為1.0000和2.9897，微球體細(xì)胞較分化細(xì)胞上調(diào)約3.0倍(P<0.05)。

微球體細(xì)胞中EGFR及ADAM9蛋白相對(duì)表達(dá)量分別為0.90±0.09和0.64±0.07，分化細(xì)胞分別為0.62±0.11和0.48±0.09，微球體細(xì)胞的表達(dá)顯著高于分化細(xì)胞(P<0.05，圖3)。

a: 微球體細(xì)胞; b: 分化細(xì)胞

圖3第二代微球體細(xì)胞及分化細(xì)胞中EGFR和ADAM9蛋白的表達(dá)

討 論

胰腺癌侵襲轉(zhuǎn)移和多藥耐藥等惡性生物學(xué)行為是其難治性的根源所在。近年來研究發(fā)現(xiàn)，腫瘤惡性生物學(xué)行為來源于惡性腫瘤中的一個(gè)特殊亞群細(xì)胞——腫瘤干細(xì)胞。腫瘤干細(xì)胞具有自我更新、無限增殖、多向分化的能力，其惡性度遠(yuǎn)高于一般的腫瘤細(xì)胞。如何富集胰腺癌干細(xì)胞，進(jìn)一步闡明胰腺癌惡性生物學(xué)行為的分子生物學(xué)機(jī)制，尋找新的基因和蛋白治療靶點(diǎn)是目前研究的關(guān)鍵所在。

目前富集腫瘤干細(xì)胞的方法主要有微球體培養(yǎng)法[7-8]、流式儀分選SP細(xì)胞和含腫瘤干細(xì)胞特異表面標(biāo)志的亞群細(xì)胞。本實(shí)驗(yàn)應(yīng)用微球體培養(yǎng)法能使胰腺癌PANC1細(xì)胞在SFM中形成微球體，含有高比例的SP細(xì)胞，并可傳代培養(yǎng)，且在含血清的培養(yǎng)條件下發(fā)生貼壁分化，表明胰腺癌微球體細(xì)胞富集了部分胰腺癌干細(xì)胞。

EGFR是一種單鏈跨膜糖蛋白，是由原癌基因編碼的酪氨酸激酶ErbB家庭成員。EGFR具有酪氨酸蛋白激酶的活性，有七種主要的內(nèi)源性配體，這些配體以跨膜的蛋白質(zhì)前體形式存在，只有經(jīng)過蛋白酶水解，釋放出胞外段，才能作用于EGFR。EGFR被激活后可激活下游信號(hào)轉(zhuǎn)導(dǎo)通路RAS/MAPK、PI3K/AKt、STAT以及PLCγ等，這些通路的激活可能促進(jìn)細(xì)胞增殖，抑制細(xì)胞凋亡和分化，增加血管內(nèi)皮生長因子(VEGF)表達(dá)水平，促進(jìn)腫瘤侵襲和遠(yuǎn)處轉(zhuǎn)移[9-10]。ADAM家族是近年來發(fā)現(xiàn)的一類廣泛參與各種生理功能的I型跨膜蛋白家族，通過調(diào)節(jié)細(xì)胞間黏附和蛋白酶活性降解細(xì)胞外基質(zhì)，控制細(xì)胞黏附、移動(dòng)。ADAM9是ADAM家族中和腫瘤發(fā)生、發(fā)展最為密切相關(guān)的成員之一。已有研究[11]表明,ADAM9可以使ErbB的配體脫落，參與EGFR途徑信號(hào)轉(zhuǎn)導(dǎo)。O′Shea等[12]與Lendeckel等[13]相繼研究報(bào)道，人乳腺癌組織及乳腺癌MCF-7、MDA-MB453細(xì)胞株ADAM9 mRNA和蛋白表達(dá)增高，并與ErbB2表達(dá)呈正相關(guān)。本研究結(jié)果顯示，胰腺癌微球體細(xì)胞與分化細(xì)胞相比，ADAM9 mRNA及蛋白均高表達(dá)，同時(shí)伴有EGFR mRNA及蛋白的高表達(dá)，表明ADAM9在胰腺癌干細(xì)胞的惡性生物學(xué)行為中發(fā)揮著重要作用。因此，尋找以胰腺癌干細(xì)胞為靶點(diǎn)的特異性的ADAM9抑制劑有可能成為抗胰腺癌治療的新的策略。

[1] Edwards DR,Handsley MM,Pennington CJ.The ADAM metalloproteinases.Mol Aspects Med,2008,29:258-289.

[2] Sung SY,Kubo H,Shigemura K,et al.Oxidative stress induces ADAM9 protein expression in human prostate cancer cells.Cancer Research,2006,66:9519-9526.

[3] Mazzocca A,Coppari R,De Franco R,et al.A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions.Cancer Res,2005,65:4728-4738.

[4] Lendeckel U,Kohl J,Arndt M,et al.Increased expression of ADAM family members in human breast cancer and breast cancer cell lines.J Cancer Res Clin Oncol,2005,131:41-48.

[5] Dontu G,Abdallah WM,Foley JM,et al.In vitro propagation and transcriptional profiling of human mammary stem/progenitor cell properties.Genes Dev,2003,17:1253-1270.

[6] Bell E,Ivarsson B,Merrill C.Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different prolixferative potential in vitro.Proc Natl Acad Sci USA,1979,76:1274-1278.

[7] Al-Hajj M,Wicha MS,Benito-Hernandez A,et al.Prospective identification of tumorigenic breast cancer cells.Proc Natl Acad Sci USA,2003,100:3983-3988.

[8] Ponti D,Costa A,Zaffaroni N,et al.Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.Cancer Res,2005,65:5506-5511.

[9] Zhou BB,Peyton M,He B,et al.Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer.Cancer Cell,2006,10:39-50.

[10] Ciardiello F,Tortora G.EGFR antagonists in cancer treatment.N Engl J Med,2008,358:1160-1174.

[11] Hynes NE, Lane HA. ERBB receptors and cancer:the complexity of targeted inhibitors. Nat Rev Cancer,2005,5:341-354.

[12] O′Shea,MeKie N,Buggy Y,et al.Expression of ADAM9 mRNA and protein in human breast cancer.Int J Cancer,2003,105:754-761.

[13] Lendeckel U,Kohl J,Arndt M,et al.Increased expression of ADAM family members in human breast cancer and breast cancer cell lines.Cancer Res Clin Oncol,2005,131:41-48.

2010-05-12)

(本文編輯：呂芳萍)

ExpressionandsignificanceofEGFRandADAM9inpancreaticcancerstemcellsanddifferentiatedcells

HONGXiao-quan,LINFan,WANGMin,WANGXin,QINRen-yi.

DepartmentofBiliopancreaticSurgery,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan430030,China

WANGXin,Email:lfan1904@126.com

ObjectiveTo enrich pancreatic cancer stem cells through culturing mammospheres, and to detect the expressions of epidermal growth factor receptor(EGFR) and a disintegrin and metalloprotease 9(ADAM9) and investigate their significance.MethodsPANC1 cells were cultured in serum-free conditioned medium to continuously generate mammospheres, and parts of mammospheres were cultured on a collagen substratum to induce differentiation. Mammospheres cells and differentiated cells were collected, flow cytometry was used to detect the proportion of side population (SP) cells, and the expressions of EGFR, ADAM9 mRNA and protein were detected by real-time PCR and Western blotting.ResultsPANC1 cells mammospheres were successfully generated and could be passed continuously. After differentiation, mammospheres cells could regain the ability of adherent growth. The proportion SP cells in mammospheres cells and differentiated cells were (5.40±0.38)% and(2.80±0.42)%, and the difference was statistically significant (P<0.05). Compared with differentiated cells, the expression of EGFR and ADAM9 mRNA of mammospheres cells up-regulated 2.5 and 3.0 folds (P<0.05). The expressions of EGFR and ADAM9 protein of mammospheres cells were 0.90±0.09 and 0.64±0.07, which were significantly higher than those in differentiated cells (0.62±0.11 and 0.48±0.09,P<0.05).ConclusionsMammosphere cells contained higher proportion of pancreatic cancer stem cells. ADAM9 may play an important role in the occurrence and development of

pancreatic cancer through the EGFR signaling pathway.

Pancreatic neoplasms; Stem cells; Receptor,epidermal grouth factor; A disintegrin and metalloprotease

10.3760/cma.j.issn.1674-1935.2011.02.002

國家自然科學(xué)基金(30772172)

430030 武漢，華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院膽胰外科

王欣，Email: lfan1904@126.com