顧曉霞 李潔 吳梅紅 彭小波 湛先保

·論著·

阿帕替尼對(duì)胰腺癌細(xì)胞株AsPC-1增殖、遷移和凋亡的影響

顧曉霞 李潔 吳梅紅 彭小波 湛先保

目的 探討阿帕替尼(Apatinib)對(duì)體外培養(yǎng)的胰腺癌AsPC-1細(xì)胞增殖、凋亡、遷移的影響。方法 以不同濃度Apatinib干預(yù)胰腺癌AsPC-1細(xì)胞，采用CCK-8法和流式細(xì)胞技術(shù)檢測(cè)細(xì)胞的增殖和凋亡，通過劃痕實(shí)驗(yàn)觀察Apatinib對(duì)胰腺癌細(xì)胞遷移能力的影響。結(jié)果 對(duì)照組及10、20、30、40、50μmol/L Apatinib處理組AsPC-1細(xì)胞的增殖抑制率分別為0、(1.45±0.68)%、(16.92±0.70)%、(23.84±0.84)%、(34.35±1.55)%、(37.33±0.81)%，細(xì)胞增殖隨Apatinib濃度的增加而顯著被抑制，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。對(duì)照組及20、40 μmol/L Apatinib處理組AsPC-1細(xì)胞的凋亡率分別為(9.44±0.18)%、(16.62±0.19)%、(25.42±0.41)%，細(xì)胞凋亡數(shù)量隨Apatinib濃度增加而顯著增多，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。對(duì)照組及20、40 μmol/L Apatinib處理組AsPC-1細(xì)胞的遷移率分別為(29.5±0.7)%、(17.4±0.9)%、(6.6±0.5)%，細(xì)胞遷移能力隨Apatinib濃度增加而顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論 Apatinib能有效抑制胰腺癌AsPC-1細(xì)胞的增殖、遷移，并誘導(dǎo)其凋亡。

阿帕替尼； 胰腺腫瘤； 細(xì)胞增殖； 細(xì)胞運(yùn)動(dòng)； 細(xì)胞凋亡

胰腺癌是高度惡性的腫瘤，5年生存率低于5%[1]，這主要與它早期局部或者遠(yuǎn)處轉(zhuǎn)移、臨床癥狀出現(xiàn)較晚、對(duì)傳統(tǒng)的化療和放射治療高度抵抗等因素有關(guān)[2-3]。吉西他濱是目前用于局部進(jìn)展和轉(zhuǎn)移性胰腺癌患者的標(biāo)準(zhǔn)化療藥物，但也只能延長(zhǎng)患者幾周的生存期[4]。Apatinib是一種新的、通過口服給予的血管上皮生長(zhǎng)因子受體-2(VEGFR-2)的選擇性抑制劑，可以有效抑制細(xì)胞內(nèi)VEGFR-2的磷酸化,也能抑制c-Kit和c-Src的激酶活性以及c-Kit和血小板源生長(zhǎng)因子受體(platelet derived growth factor receptor，PDGFR)的磷酸化[5]。研究顯示[6-9]，Apatinib在體外能抑制多種腫瘤細(xì)胞，如非小細(xì)胞肺癌、乳腺癌、膽管細(xì)胞癌等的增殖，也能明顯減緩裸鼠移植瘤的生長(zhǎng)，且毒性較小。但目前尚未見其對(duì)胰腺癌的研究報(bào)道。本研究應(yīng)用Apatinib干預(yù)胰腺癌AsPC-1細(xì)胞，觀察其對(duì)癌細(xì)胞增殖、凋亡、遷移的影響。

材料和方法

一、CCK-8法測(cè)定細(xì)胞增殖抑制率

人胰腺癌AsPC-1細(xì)胞由上海長(zhǎng)海醫(yī)院消化內(nèi)科實(shí)驗(yàn)室提供，常規(guī)復(fù)蘇、培養(yǎng)、傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞，調(diào)整細(xì)胞密度為5×104/ml，在96孔板每孔中加入100 μl細(xì)胞懸液，培養(yǎng)至細(xì)胞貼壁后更換含1% FBS的DMEM饑餓培養(yǎng)過夜，然后加入10、20、30、40、50 μmol/L的Apatinib，以不加Apatinib作為對(duì)照組，每組設(shè)6個(gè)復(fù)孔。培養(yǎng)24 h后每孔加入10 μl CCK-8液繼續(xù)培養(yǎng)2 h，上酶標(biāo)儀測(cè)定450 nm處的吸光度值(A450值)，繪制細(xì)胞生長(zhǎng)曲線。Apatinib由恒瑞醫(yī)藥有限公司提供，CCK-8試劑盒購(gòu)自上海威奧生物科技有限公司。

二、流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡

取對(duì)數(shù)生長(zhǎng)期細(xì)胞，以每孔5×105個(gè)AsPC-1細(xì)胞接種于6孔板，培養(yǎng)至細(xì)胞80%融合后饑餓培養(yǎng)過夜，然后加入20、40 μmol/L的Apatinib，以不加Apatinib作為對(duì)照組。培養(yǎng)24 h后用胰酶消化收集細(xì)胞，PBS清洗1次后重懸于300 μl PBS，加入300 μl的binding buffer、3μl AV-FITC和3 μl PI，混均后避光10 min，上流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡比例。

三、細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力

取對(duì)數(shù)生長(zhǎng)期細(xì)胞，以每孔5×105個(gè)AsPC-1細(xì)胞接種于6孔板，培養(yǎng)至細(xì)胞90%融合后饑餓培養(yǎng)過夜，用200 μl的移液槍頭在每孔中間劃一條橫線， PBS沖洗3次，然后加入20、40 μmol/L Apatinib，以不加Apatinib作為對(duì)照組。在培養(yǎng)即刻(0 h)及培養(yǎng)24 h時(shí)置顯微鏡下照相，每個(gè)樣本測(cè)量3個(gè)劃痕距離。細(xì)胞遷移率= [(0 h劃痕距離-24 h劃痕距離)/0 h劃痕距離]×100%。實(shí)驗(yàn)重復(fù)3次，取均值。

四、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、Apatinib對(duì)AsPC-1細(xì)胞增殖的影響

對(duì)照組及10、20、30、40、50 μmol/L Apatinib處理組AsPC-1細(xì)胞的增殖抑制率分別為0、(1.45±0.68)%、(16.92±0.70)%、(23.84±0.84)%、(34.35±1.55)%、(37.33±0.81)%，細(xì)胞增殖隨Apatinib濃度的增加而顯著被抑制，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

二、Apatinib對(duì)AsPC-1細(xì)胞凋亡的影響

對(duì)照組及20、40 μmol/L Apatinib處理組AsPC-1細(xì)胞培養(yǎng)24 h后的凋亡率分別為(9.44±0.18)%、(16.62±0.19)%、(25.42±0.41)%，細(xì)胞凋亡數(shù)量隨Apatinib濃度增加而顯著增多，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05，圖1)。

圖1 對(duì)照組(1A)及20、40 μmol/L Apatinib處理組(1B、1C)AsPC-1細(xì)胞的凋亡

三、Apatinib 對(duì)AsPC-1細(xì)胞遷移能力的影響

對(duì)照組及20、40 μmol/L Apatinib處理組AsPC-1細(xì)胞培養(yǎng)24 h后的遷移率分別為(29.5±0.7)%、(17.4±0.9)%、(6.6±0.5)%，細(xì)胞遷移能力隨Apatinib濃度增加而顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，圖2)。

討 論

血管形成是腫瘤的標(biāo)志之一，能夠促進(jìn)惡性腫瘤的生長(zhǎng)和轉(zhuǎn)移[10]，而腫瘤轉(zhuǎn)移是引起腫瘤相關(guān)死亡的主要原因。胰腺癌預(yù)后不良也和腫瘤轉(zhuǎn)移密切相關(guān)，大約50%的胰腺癌患者被確診時(shí)已有遠(yuǎn)處轉(zhuǎn)移。促進(jìn)腫瘤血管形成的通路主要有3條：VEGF/VEGFR、血管生成素(ANGPT)家族以及Notch-Notch配體通路，其中VEGF是血管形成的重要調(diào)節(jié)因子。VEGF通過和VEGFR-2結(jié)合發(fā)揮促血管生成的作用以及誘導(dǎo)VEGFR-2的下游分子的激活。VEGFR-2主要是在血管內(nèi)皮細(xì)胞上表達(dá)的，在很多腫瘤表達(dá)都是上調(diào)的，包括胰腺癌[11]。VEGF和VEGFR的過表達(dá)與腫瘤生長(zhǎng)速率加快、微血管密度增加、腫瘤增殖和轉(zhuǎn)移能力增強(qiáng)以及不良預(yù)后相關(guān)[12]。

圖2 對(duì)照組(2A)及20、40 μmol/L Apatinib處理組(2B、2C)AsPC-1細(xì)胞的遷移

近年來(lái)拮抗血管形成成為腫瘤治療的研究熱點(diǎn)之一，抗血管藥物在很多實(shí)體腫瘤，如肺癌、乳腺癌、結(jié)腸癌、胃癌等的治療中效果顯著，目前很多抗血管藥物也已用于胰腺癌患者，如貝伐單抗(抗VEGF的單克隆抗體)。貝伐單抗聯(lián)合吉西他濱用于晚期胰腺癌患者的Ⅱ期臨床研究顯示，聯(lián)合治療的總生存期、無(wú)進(jìn)展生存期、客觀反應(yīng)率均較單用吉西他濱有較明顯改善，但隨后的Ⅲ期研究結(jié)果顯示在總生存期方面并沒有明顯改善[13]。多種VEGF信號(hào)通路抑制劑，如sunitinib、axitinib、sorafenib、vatalanib等也已在晚期胰腺癌患者中開展Ⅱ或Ⅲ期臨床研究。Apatinib在胃癌的Ⅲ期以及非小細(xì)胞肺癌的Ⅱ期試驗(yàn)中顯示有生存效益，且不良反應(yīng)較少，在肺癌、乳腺癌患者中能明顯改善患者預(yù)后。Peng等[14]研究結(jié)果顯示，Apatinib通過VEGF/VEGFR-2/PI3K/AKT信號(hào)通路抑制肝內(nèi)膽管癌細(xì)胞的增殖并誘導(dǎo)其凋亡。Doi等[15]也報(bào)道VEGF-A/VEGFR-2信號(hào)在多個(gè)胰腺癌細(xì)胞系的遷移和侵襲中起到了重要的作用。本研究結(jié)果顯示，Apatinib在體外可以抑制AsPC-1細(xì)胞的增殖、遷移并誘導(dǎo)其凋亡，且濃度越高，抑制作用越明顯。

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(本文編輯：屠振興)

Effect of apatinib on cell proliferation, migration and apoptosis in pancreatic cancer cell line AsPC-1

GuXiaoxia,LiJie,WuMeihong,PengXiaobo,ZhanXianbao.

DepartmentofOncology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

ZhanXianbao,Email：zhanxianbao@126.com

Objective To investigate the effect of apatinib on the proliferation, apoptosis and migration of pancreatic cancer cell line AsPC-1 in vitro. Methods Pancreatic cancer AsPC-1 cells were treated by apatinib in different concentrations. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, and the effect of apatinib on cell migration ability was observed by wound healing assay. Results In control and 10, 20, 30, 40 and 50umol/L apatinib treatment group, the inhibitory rates of AsPC-1 cells were 0,(1.45±0.68)%,(16.92±0.70)%,(23.84±0.84)%,(34.35±1.55)% and (37.33±0.81)%，respectively. Cell proliferation was obviously inhibited by apatinib as the concentration increased, and the differences were statistically significant (P<0.05). In control and 20, 40 umol/L apatinib treatment group, the apoptotic rates were (9.44±0.18)%,(16.62±0.19)% and (25.42±0.41)%, respectively. Number of apoptotic cells was obviously increased by apatinib as the concentration increased, and the differences were statistically significant (P<0.05). In control and 20, 40 umol/L apatinib treatment group, the migration ability was (29.5±0.7)% ,(17.4±0.9)% and (6.6±0.5)%, which was greatly decreased as the concentration increased, and the differences were statistically significant (P<0.05). Conclusions Apatinib can effectively inhibit the proliferation and migration of pancreatic cancer AsPC-1 cells and induce apoptosis.

Apatinib; Pancreatic neoplasms; Cell proliferation; Cell movement; Apoptosis

10.3760/cma.j.issn.1674-1935.2017.01.004

200433 上海，第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院腫瘤科

湛先保，Email: zhanxianbao@126.com

2016-11-13)