郭曉鐘 任麗楠 劉旭 劉峰 鄒德莉
·論著·
內(nèi)分泌腺來源的血管內(nèi)皮生長因子對胰腺癌MiaPaCaⅡ細胞惡性表型的調(diào)控作用
郭曉鐘 任麗楠 劉旭 劉峰 鄒德莉
目的探討內(nèi)分泌腺來源的血管內(nèi)皮生長因子(EG-VEGF)對胰腺癌細胞MiaPaCaⅡ增殖、遷移和凋亡等的影響。方法應(yīng)用100、200 ng/ml EG-VEGF處理MiaPaCaⅡ細胞24、48、72、96 h,采用MTT方法檢測細胞增殖,細胞劃痕實驗法檢測細胞爬行距離百分比,流式細胞儀檢測細胞凋亡。結(jié)果經(jīng)0、100、200 ng/ml EG-VEGF處理72 h后,MiaPaCaⅡ細胞的增殖分別為0.253±0.012、0.374±0.013、0.383±0.015,EG-VEGF顯著促進MiaPaCaⅡ細胞的增殖(P<0.05)。0、100 ng/ml EG-VEGF處理24 h后,MiaPaCaⅡ細胞爬行距離百分比分別為(39.0±2.7)%和(79.0±5.4)%(P<0.05);細胞凋亡率分別為(27.40±3.45)%和(13.21±4.65)%(P<0.05)。結(jié)論EG-VEGF可明顯促進MiaPaCaⅡ細胞增殖和遷移,抑制其凋亡。
胰腺腫瘤; 血管內(nèi)皮生長因子; 表型
胰腺癌是消化系統(tǒng)常見的惡性腫瘤之一,早期診斷十分困難,預(yù)后極差,一旦確診其手術(shù)切除機會僅10%~15%,總體術(shù)后5年生存率1%~4%左右,因此尋找切實可行的診治方法對胰腺癌診治尤為重要。
內(nèi)分泌腺來源的血管內(nèi)皮生長因子(endocrine glands-derived vascular endothelial growth factor,EG-VEGF,又稱PK1)是近年來發(fā)現(xiàn)的具有組織特異性的血管內(nèi)皮生長因子,已經(jīng)發(fā)現(xiàn)其在結(jié)直腸癌和前列腺癌等多種類固醇相關(guān)的腫瘤組織中有表達,且可調(diào)控腫瘤細胞的生物學(xué)功能。EG-VEGF對胰腺癌細胞是否存在調(diào)控作用目前尚無文獻報道。因此,本實驗旨在探討EG-VEGF對人胰腺癌細胞惡性表型的調(diào)控作用。
一、MTT法檢測胰腺癌細胞的增殖
人胰腺癌高轉(zhuǎn)移細胞系MiaPaCaⅡ由本實驗室保存。取對數(shù)生長期細胞,以1.5×103個/孔接種于96孔板中,分為對照組、EG-VEGF(Peprotech 公司) 100 ng/ml和200 ng/ml組,每組3個復(fù)孔。常規(guī)培養(yǎng)細胞4 h后,更換培養(yǎng)基,分別加入0、100、200 ng/ml的EG-VEGF繼續(xù)培養(yǎng)24、48、72、96 h,每孔加入5 g/L MTT(Sigma公司)20 μl,繼續(xù)培養(yǎng)4 h,棄上清,加150 μl DMSO,震蕩10 min,酶標(biāo)儀上測各孔490 nm波長吸光度值(A490),以單純培養(yǎng)液調(diào)零。
二、劃痕實驗檢測胰腺癌細胞的遷移
取對數(shù)生長期細胞接種于6孔板中,分為對照組和100 ng/ml EG-VEGF組,每組設(shè)3個復(fù)孔。待細胞長滿瓶底時,取黃色槍尖垂直瓶底制造人工劃痕,拍照并記錄初始劃痕距離,更換細胞培養(yǎng)液去除脫落細胞,分別加入0、100 ng/ml的EG-VEGF,24 h后觀察細胞爬行距離。細胞爬行距離百分比=細胞爬行的距離/初始劃痕距離×100%
三、流式細胞儀檢測細胞凋亡
細胞以無血清DMEM培養(yǎng)過夜,再在培養(yǎng)上清中分別加入0、100 ng/ml的EG-VEGF,24 h后收集培養(yǎng)上清及貼壁細胞,4℃ PBS重懸細胞,按照細胞凋亡檢測試劑盒說明書進行Annexin Ⅴ染色,流式細胞儀收集10 000個細胞,測細胞凋亡率。凋亡率=(早期凋亡細胞+ 晚期凋亡細胞)/總細胞數(shù)×100%。
四、統(tǒng)計學(xué)處理
一、EG-VEGF對MiaPaCaⅡ細胞增殖的影響
經(jīng)100、200 ng/ml EG-VEGF處理72 h后,A490值分別為0.374±0.013和0.383±0.015,較未經(jīng)處理組的0.253±0.012有顯著增高(P值均<0.05),但兩種濃度間對細胞增殖的影響無顯著差異(圖 1)。
二、EG-VEGF對MiaPaCaⅡ細胞遷移的影響
0、100 ng/ml EG-VEGF處理細胞24 h后,細胞爬行距離百分比分別為(39.0±2.7)%和(79.0±5.4)%,兩組差異具有統(tǒng)計學(xué)意義(P<0.05,圖2)。
圖1 EG-VEGF對MiaPaCaⅡ細胞增殖的影響
圖2EG-VEGF對MiaPaCaⅡ細胞遷移能力的影響(實線為初始劃痕距離,虛線為24 h后細胞遷移距離)
三、EG-VEGF對MiaPaCaⅡ細胞凋亡的影響
0、100 ng/ml EG-VEGF處理24 h后,細胞凋亡率分別為(27.40±3.45)%和(13.21±4.65)%,兩組差異具有統(tǒng)計學(xué)意義(P<0.05,圖 3)。
圖3 MiaPaCaⅡ細胞的凋亡檢測圖
腫瘤是在各種致瘤因素作用下因基因調(diào)控失調(diào)導(dǎo)致細胞異常增生而形成的。腫瘤的發(fā)生和發(fā)展以及演變是受到多種因素調(diào)控的復(fù)雜過程。細胞因子能在細胞間傳遞信息,通過促進細胞黏附、血管生成和細胞外基質(zhì)的降解等多種途徑參與腫瘤的形成,并促進細胞增殖和浸潤轉(zhuǎn)移。但這些細胞因子缺乏組織特異性,故在治療過程中受到一定的限制。
EG-VEGF是2001年被確認的組織特異性的血管內(nèi)皮生長因子[1],它特異地作用于生成類固醇的內(nèi)分泌腺的內(nèi)皮組織,如卵巢、胎盤、腎上腺等,可誘導(dǎo)血管內(nèi)皮細胞增殖和遷移。目前研究表明,EG-VEGF對多種腫瘤的生長、凋亡和轉(zhuǎn)移發(fā)揮重要的調(diào)控作用[2-7]。胰腺細胞也具有合成類固醇的能力。有文獻初步顯示,EG-VEGF主要分布在男性的胰島部位和女性的外分泌部位,其表達水平由高到低依次為女性胰腺癌患者、男性胰腺癌患者、正常女性、正常男性[8-9]。Goi等[4]報道,EG-VEGF可促進結(jié)腸癌細胞的浸潤和轉(zhuǎn)移以及血管生成。本結(jié)果顯示,重組人EG-VEGF可在一定范圍內(nèi)促進胰腺癌細胞的增殖,促進癌細胞的遷移,抑制癌細胞的凋亡,一定程度上調(diào)控胰腺癌細胞的惡性表型,提示EG-VEGF在胰腺癌的生長過程中具有重要的調(diào)控作用。
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2010-07-28)
(本文編輯:屠振興)
MalignantphenotyperegulatedbyendocrineglandsderivedVEGFinpancreaticcancercellsMiaPaCaⅡ
GUOXiao-zhong,RENLi-nan,LIUXu,LIUFeng,ZOUDe-li.
DepartmentofGastreonterology,GeneralHospitalofShenyangMilitaryCommend,Shenyang110840,China
GuoXiao-zhong,Email:guoxiaozhong1962@163.com
ObjectiveTo evaluate the endocrine glands derived VEGF(EG-VEGF) influence on growth, migration and apoptosis of pancreatic cancer cells MiaPaCaⅡ.MethodsMiaPaCaⅡ were treated by 100, 200 ng/ml EG-VEGF for 24, 48, 72, 96 h, and MTT assay was used to determine the proliferation; and cell scratch experiment was used to investigate the percentage of cell migration distance, flow cytometry was used to measure the apoptosis of the cancer cells.ResultsAfter MiaPaCaⅡ cells were treated by 0, 100, 200 ng/ml EG-VEGF for 72 h, the proliferation of MiaPaCaⅡ was 0.253±0.012,0.374±0.013,0.383±0.015, respectively EG-VEGF could significantly promote the proliferation of MiaPaCaⅡ(P<0.05). After MiaPaCaⅡ cells were treated by 0, 100 ng/ml EG-VEGF for 24 h, the percentage of cell migration distance was (27.40±3.45)% and (13.21±4.65)%,respectively with statistical difference (P<0.05),EG-VEGF could significantly promote the migration ability of MiaPaCaⅡ cells and inhibite the apoptosis.ConclusionsAfter EG-VEGF treatment, the growth and migration ability of MiaPaCaⅡ cells increases, apoptosis decreases.
Pancreatic neoplasms; Vascular endothelial growth factor; Phenotype
10.3760/cma.j.issn.1674-1935.2011.02.007
110840 沈陽,沈陽軍區(qū)總醫(yī)院消化內(nèi)科
郭曉鐘,Email:guoxiaozhong1962@163.com