崔英　沈曼茹　顏美珠　黃繼英　唐鄂　安敏　喻青　徐林芳　李曉翠　史先芳　高振軍

?

胰腺星狀細(xì)胞通過HGF/c-Met信號通路調(diào)控胰腺癌的侵襲轉(zhuǎn)移

崔英沈曼茹顏美珠黃繼英唐鄂安敏喻青徐林芳李曉翠史先芳高振軍

目的探討胰腺星狀細(xì)胞(PSCs)調(diào)控胰腺癌的遷移和侵襲轉(zhuǎn)移機(jī)制。方法檢測PSCs培養(yǎng)上清液中肝細(xì)胞生長因子(HGF)蛋白的含量。應(yīng)用PSCs上清液、PSCs上清液加HGF中和抗體或加c-Met特異性抑制劑PHA-665752分別處理人胰腺癌細(xì)胞株AsPC-1細(xì)胞，以不加PSCs上清液細(xì)胞作為對照組，采用MTT法檢測AsPC-1細(xì)胞的增殖，Transwell小室檢測細(xì)胞遷移能力，體外侵襲實(shí)驗(yàn)觀察細(xì)胞侵襲能力。 結(jié)果PSCs上清液中HGF蛋白含量為(4 213±543)ng/L。PSCs上清液組、PSCs上清液+HGF中和抗體組、PSCs上清液+c-Met抑制劑組及對照組細(xì)胞增殖的A490值分別為0.628±0.030、0.324±0.021、0.347±0.054及0.405±0.008，穿膜細(xì)胞數(shù)分別為(123.3±6.8)、(62.4±6.9)、(58.1±2.2)、(36.6±4.8)/400倍視野，侵襲細(xì)胞數(shù)分別為(70.0±2.3)、(42.5±4.6)、(42.7±2.8)、(36.4±3.5)/400倍視野。PSCs上清液組細(xì)胞增殖、穿膜及侵襲能力較對照組顯著升高，PSCs上清液+HGF中和抗體組及PSCs上清液+c-Met抑制劑組細(xì)胞增殖、穿膜及侵襲能力較PSCs上清液組顯著下降，差異均有統(tǒng)計學(xué)意義(P值均<0.01)，而PSCs上清液+HGF中和抗體組與PSCs上清液+c-Met抑制劑組之間的細(xì)胞增殖、穿膜及侵襲能力差異無統(tǒng)計學(xué)意義(P值均>0.05)。結(jié)論P(yáng)SCs可能是通過HGF/c-Met信號通路從而調(diào)控胰腺癌細(xì)胞的增殖、遷移和侵襲。

胰腺腫瘤；星形細(xì)胞；肝細(xì)胞生長因子；原癌基因蛋白質(zhì)c-Met

近年來胰腺癌的發(fā)病率有增高的趨勢[1-2]。由于發(fā)展迅速，早期即有局部及遠(yuǎn)處轉(zhuǎn)移，故胰腺癌預(yù)后極差。研究證實(shí)，胰腺星狀細(xì)胞(pancreatic stellate cells, PSCs)可促進(jìn)胰腺癌的侵襲轉(zhuǎn)移[3-4]，但其機(jī)制尚不清楚。本課題組既往的研究證實(shí)，SDF-1/CXCR4受體配體系統(tǒng)在PSCs促進(jìn)胰腺癌轉(zhuǎn)移侵襲中起重要作用，但抑制SDF-1/CXCR4途徑并不能完全抑制腫瘤的侵襲轉(zhuǎn)移，故可能存在其他調(diào)控機(jī)制。肝細(xì)胞生長因子(hepatocyte growth factor, HGF)/c-Met信號通路被認(rèn)為與許多惡性腫瘤的發(fā)生、發(fā)展密切相關(guān)，其中包括胃癌、肺癌、食道癌、乳腺癌、肝癌、結(jié)腸癌、前列腺癌、腎癌、頭頸部癌、卵巢癌等[5-6]。本研究旨在進(jìn)一步探討HGF/c-Met信號通路在胰腺癌侵襲轉(zhuǎn)移中的作用。

材料與方法

一、PSCs上清液的制備

胰腺癌根治術(shù)中獲取新鮮癌旁組織，在無菌環(huán)境、無血清培養(yǎng)液中剪成0.5 mm3大小的組織塊，貼放于培養(yǎng)皿中，間隔0.5～1.0 cm。加入含20%胎牛血清(FCS)的DMEM/F12培養(yǎng)液培養(yǎng)24 h后更換培養(yǎng)液培養(yǎng)3～5 d，每2～3 d更換培養(yǎng)液。待組織塊周圍有星狀細(xì)胞爬出并形成克隆后小心去除組織塊，用含乙二酰四乙酸(EDTA)的0.25%胰蛋白酶消化細(xì)胞，洗滌后置培養(yǎng)皿中培養(yǎng)。待細(xì)胞貼壁生長至70%～80%融合時按1∶3比例傳代。收集對數(shù)生長期PSCs，棄去培養(yǎng)液，用PBS沖洗2遍，加無血清DMEM/F12培養(yǎng)液5 ml培養(yǎng)48 h，收集上清液，過濾去除細(xì)胞碎片，置-80℃保存?zhèn)溆谩?/p>

二、PSCs培養(yǎng)上清液HGF蛋白含量的檢測

采用ELISA法定量測定PSCs上清液HGF蛋白含量，人HGF ELISA試劑盒購自江蘇晶美生物科技有限公司，按說明書操作。上Bio-rad比色儀測定各孔在490 nm處的吸光度值(A490值)。參照標(biāo)準(zhǔn)曲線計算樣本濃度。HGF最小檢測濃度為40 ng/L。

三、人胰腺癌AsPC-1細(xì)胞增殖的檢測

人胰腺癌細(xì)胞株AsPC-1購自中科院細(xì)胞庫，常規(guī)培養(yǎng)、傳代。取對數(shù)生長期AsPC-1細(xì)胞制成單細(xì)胞懸液，按每孔5×103個細(xì)胞(100 μl)接種于96孔板,貼壁生長后換無血清培養(yǎng)液(SFM)。實(shí)驗(yàn)分4組，分別為加入PSCs上清液、PSCs上清液+60μg/ml HGF中和抗體(Sigma-Aldrich公司)、PSCs上清液+100 nmol/L c-Met抑制劑PHA-665752(Selleck chemicals公司)的SFM，以單加SFM為對照組。每組設(shè)5個平行孔，培養(yǎng)24 h后每孔加入濃度為5 mg/ml的MTT 20 μl繼續(xù)培養(yǎng)4 h，吸棄上清液，每孔加入150 μl DMSO振蕩10 min，用酶標(biāo)儀測定各孔在490 nm波長處的吸光度值(A490值)。

四、細(xì)胞遷移實(shí)驗(yàn)

收集對數(shù)生長期AsPC-1細(xì)胞，應(yīng)用SFM制備2×105/ml的單細(xì)胞懸液。取Transwell小室，在隔膜的上室鋪100 μl/ml的纖連蛋白(Fn，Sigma-Aldrich公司)50 μl，各組上室加入200 μl的細(xì)胞懸液，下室分別加入500 μl PSCs上清液、PSCs上清液+60 μg/ml HGF中和抗體、PSCs上清液+100 nmol/L PHA-665752的SFM,以只加SFM為對照組。每組5個Transwell小室。常規(guī)培養(yǎng)12 h，取出隔膜，用棉簽輕輕擦去未穿膜細(xì)胞，用0.1%結(jié)晶紫染色20 min，高倍鏡下隨機(jī)取10個視野，計數(shù)穿膜細(xì)胞數(shù)，取均值。實(shí)驗(yàn)重復(fù)3次。

五、細(xì)胞侵襲實(shí)驗(yàn)

收集對數(shù)生長期AsPC-1細(xì)胞，加入SFM制備1×106/ml的單細(xì)胞懸液。應(yīng)用體外侵襲試劑盒ECM550(Chemicon公司)檢測細(xì)胞體外侵襲能力。各組上室加入300 μl細(xì)胞懸液，下室分別加入500 μl PSCs上清液、PSCs上清液+60μg/ml HGF中和抗體、PSCs上清液+100 nmol/L PHA-665752的含2% FCS培養(yǎng)液，以單加500 μl含2% FCS的培養(yǎng)液為對照組。每組5個小室。培養(yǎng)48 h后，用0.1%結(jié)晶紫染色20 min，高倍鏡下隨機(jī)計數(shù)10個視野的穿膜細(xì)胞數(shù)，取均數(shù)。實(shí)驗(yàn)重復(fù)3次。

六、統(tǒng)計學(xué)處理

結(jié)　　果

一、PSCs上清液中HGF蛋白的含量

PSCs上清液中HGF蛋白的含量為(4 213±543)ng/L。

二、AsPC-1細(xì)胞增殖的變化

PSCs上清液組、PSCs上清液+HGF中和抗體組、PSCs上清液+c-Met抑制劑組及對照組培養(yǎng)24 h的細(xì)胞增殖A490值分別為0.628±0.030、0.324±0.021、0.347±0.054及0.405±0.008,PSCs上清液組較對照組顯著升高，PSCs上清液+HGF中和抗體組及PSCs上清液+c-Met抑制劑組較PSCs上清液組顯著下降，差異均有統(tǒng)計學(xué)意義(P值均<0.01)，PSCs上清液+HGF中和抗體組與PSCs上清液+c-Met抑制劑組間差異無統(tǒng)計學(xué)意義(P>0.05)。

三、AsPC-1細(xì)胞遷移能力變化

PSCs上清液組、PSCs上清液+HGF中和抗體組、PSCs上清液+c-Met抑制劑組及對照組的穿膜細(xì)胞數(shù)分別為(123.3±6.8)、(62.4±6.9)、(58.1±2.2)、(36.6±4.8)/400倍視野，PSCs上清液組較對照組顯著增加， PSCs上清液+HGF中和抗體組與PSCs上清液+c-Met抑制劑組較PSCs上清液組顯著減少，差異均有統(tǒng)計學(xué)意義(P值均<0.01)，而PSCs上清液+HGF中和抗體組與PSCs上清液+c-Met抑制劑組間的差異無統(tǒng)計學(xué)意義(P>0.05)。

四、AsPC-1細(xì)胞體外侵襲能力變化

PSCs上清液組、PSCs上清液+HGF中和抗體組、PSCs上清液+c-Met抑制劑組及對照組的侵襲細(xì)胞數(shù)分別為(70.0±2.3)、(42.5±4.6)、(42.7±2.8)、(36.4±3.5)/400倍視野，PSCs上清液組較對照組顯著增多，PSCs上清液+HGF中和抗體組及PSCs上清液+c-Met抑制劑組較PSCs上清液組顯著減少，差異均有統(tǒng)計學(xué)意義(P值均<0.01),而PSCs上清液+HGF中和抗體組與PSCs上清液+c-Met抑制劑組間的差異無統(tǒng)計學(xué)意義(P>0.05)。

討　　論

胰腺癌組織中有大量間質(zhì)組織，腫瘤-間質(zhì)之間的相互作用在腫瘤進(jìn)展中起著重要作用。1998年Bachem等[7]確定胰腺組織中胞質(zhì)含維生素A的細(xì)胞為PSCs，該細(xì)胞位于胰腺小葉間和腺泡周圍區(qū)，圍繞鄰近腺細(xì)胞基底部，在正常胰腺組織中處于靜止?fàn)顟B(tài)，活化時胞質(zhì)內(nèi)脂滴消失、呈α-SMA染色陽性的肌成纖維樣細(xì)胞。胰腺癌在病理組織學(xué)上大部分以腫瘤周圍纖維化增加為特點(diǎn)，且纖維化程度與腫瘤的侵襲轉(zhuǎn)移程度相關(guān)。大量實(shí)驗(yàn)證實(shí)促進(jìn)胰腺纖維化的細(xì)胞為PSCs[8-9]。但PSCs促進(jìn)胰腺癌侵襲轉(zhuǎn)移的機(jī)制尚未完全清楚。本課題組既往的實(shí)驗(yàn)證實(shí)，使用PSCs上清液處理AsPC-1細(xì)胞后可以促進(jìn)其增殖、遷移和侵襲，應(yīng)用SDF-1抗體中和后該促進(jìn)作用被抑制，提示PSCs分泌到培養(yǎng)上清中的SDF-1促進(jìn)胰腺癌細(xì)胞增殖、遷移和侵襲。但也發(fā)現(xiàn)可能存在其他一些因子影響癌細(xì)胞的增殖、轉(zhuǎn)移及侵襲[10]。

肝細(xì)胞生長因子主要來源于基質(zhì)成纖維細(xì)胞[11]，是一種多功能的細(xì)胞因子，具有強(qiáng)大的促分裂、組織成形、誘導(dǎo)上皮細(xì)胞遷移、侵襲以及誘發(fā)血管生成的作用[12]，其生物學(xué)活性由c-Met受體蛋白介導(dǎo)。當(dāng)HGF與其受體c-Met結(jié)合后，胞質(zhì)中的酪氨酸殘基可發(fā)生磷酸化，進(jìn)而激活c-Met蛋白激酶結(jié)構(gòu)域中的酪氨酸激酶(PTK),活化的PTK可引起c-Met羧基末端酪氨酸的磷酸化[13]。HGF/c-Met介導(dǎo)的信號轉(zhuǎn)導(dǎo)通路可使細(xì)胞-細(xì)胞間的黏附作用減弱，增強(qiáng)整合素的功能活性，促進(jìn)細(xì)胞與細(xì)胞外基質(zhì)成分的黏附，從而刺激多種類型癌細(xì)胞的遷移、侵襲[14]。

本研究結(jié)果顯示，應(yīng)用含有HGF的PSCs上清液處理AsPC-1細(xì)胞后可促進(jìn)細(xì)胞增殖、遷移及侵襲；應(yīng)用HGF中和抗體后PSCs上清液對腫瘤細(xì)胞增殖、遷移和侵襲作用被抑制；應(yīng)用c-Met抑制劑處理PSCs上清液后AsPC-1細(xì)胞增殖、遷移和侵襲作用也被明顯抑制，表明HGF可能通過HGF/c-Met信號通路調(diào)控胰腺癌的增殖、侵襲及轉(zhuǎn)移，該通路是除了SDF-1/CXCR4受體配體系統(tǒng)外另一個與胰腺癌的惡性生物學(xué)行為密切相關(guān)的信號通路。

[1]Maisonneuve P，Lowenfels AB. Epidemiology of pancreatic cancer:an update[J].Dig Dis, 2010,28(4-5):645-656. DOI: 10.1159/000320068. Epub 2010 Nov 18.

[2]Yadav D, Lowenfels AB. The epidemiology of pancreatitis and pancreatic cancer[J].Gastroenterology,2013,144(6):1252-1261. DOI: 10.1053/j.gastro.2013.01.068.

[3]Hwang RF,Moore T,Arumugam T,et al.Cancer-associated stromal fibroblasts promote pancreatic tumor progression[J].Cancer Res,2008,68(3):918-926. DOI 10.1158/0008-5472.CAN-07-5714.

[4]Bachem MG,Zhou S,Buck K,et al.Pancreatic stellate cells-role in pancreas cancer[J].Langenbecks Arch Surg,2008,393(6):891-900. DOI: 10.1007/s00423-008-0279-5.

[5]Eder JP,Vande Woude GF,Borner SA,et al.Noval therapeutic inhibitors of the c-Met signaling pathway in cancer[J].Clin Cancer Res,2009,15(7):2207-2214. DOI: 10.1158/1078-0432.CCR-08-1306. Epub 2009 Mar 24.

[6]Gayyed MT, Abd El-Maqsoud NM, El-Hameed El-Heeny AA, et al. c-MET expression in colorectal adenomas and primary carcinomas with its corresponding metastases[J].J Gastrointest Oncol,2015,6(6):618-627. DOI: 10.3978/j.issn.2078-6891.2015.072.

[7]Bachem MG, Schneider E, Gross H, et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans[J]. Gastroenterology,1998, 115(2): 421-432.

[8]Bachem MG, Schunemann M, Ramadani M, et al. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells[J]. Gastroenterology,2005, 128(4): 907-921.

[9]Apte M,Pirola RC,Wilson JS. Pancreatic stellate cell: physiologic role, role in fibrosis and cancer[J].Curr Opin Gastroenterol,2015,31(5):416-423. DOIi: 10.1097/MOG.0000000000000196.

[10]高振軍，吳愷，王興鵬.胰腺星狀細(xì)胞對胰腺癌AsPC-1細(xì)胞侵襲轉(zhuǎn)移的影響[J].中華胰腺病雜志,2013,13(4):244-247. DOI:10.3760/cma.j.issn.1674-1935.2013.04.009.

[11]Yen BL,Yen ML,Hsu PJ,et al. Multipotent human mesenchymal stromal cells mediate expansion of myeloid-derived suppressor cells via hepatocyte growth factor/c-Met and STAT3[J].Stem Cell Reports,2013,1(2):139-151. DOI: 10.1016/j.stemcr.2013.06.006. eCollection 2013.

[12]Nakamura T,Mizuno S. The discovery of hepatocyte growth factor(HGF) and its significance for cell biology,life sciences and clinical medicine[J].Proc Jpn Acad Ser B Phys Biol Sci,2010,86(6):588-610.

[13]Gelsomino F,Rossi G,Tiseo M.MET and small-cell lung cancer[J].Cancers(Basel),2014,6(4):2100-2115. DOI: 10.3390/cancers6042100.

[14]Matsumura A,Kubota T,Taiyoh H,et al. HGF regulates VEGF expression via the c-Met receptor downstream pathways,PI3K/Akt,MAPK and STAT3, in CT26 murine cells[J]. Int J Oncol,2013,42(2):535-542. DOI: 10.3892/ijo.2012.1728.

(本文編輯：呂芳萍)

The regulation of invasion and metastasis of human pancreatic cancer by pancreatic stellate cells via the HGF/c-Met pathway

CuiYing,ShenManru,YanMeizhu,HuangJiying,TangE,AnMin,YuQing,XuLinfang,LiXiaocui,ShiXianfang,GaoZhenjun.

DepartmentofGastroenterology,ZhongshanHospitalQingpuBrach,FudanUniversity,Shanghai201700,China

Correspondingauthor:GaoZhenjun,Email：cui_yin@126.com

ObjectiveTo investigate the regulation mechanism of pancreatic stellate cells (PSCs) on invasion and metastasis of human pancreatic cancer through the pathway of HGF/c-Met. MethodsHepatocyte growth factor (HGF) level in PSCs supernatant was detected. PSCs supernatant, PSCs supernatant plus anti-HGF antibody, PSCs supernatant plus c-Met specific inhibitor PHA 665752 were used to treat human pancreatic cancer AsPC-1 cells, and untreated cells served as controls. MTT assay was applied to detect cell proliferation. Transwell chamber migration assay was employed to detect cell migration. In vitro invasion assay was used to determine cell invasion. ResultsThe level of HGF in PSCs supernatant was (4 213±543)ng/L.A490value for cell proliferation in PSCs supernatant, PSCs supernatant+anti-HGF, PSCs supernatant+c-Met inhibitor and control group were 0.628±0.030, 0.324±0.021, 0.347±0.054 and 0.405±0.008. The number of penetrating cells per 400 high power field was 123.3±6.8, 62.4±6.9, 58.1±2.2 and 36.6±4.8, respectively. The number of invasive cells per 400 high power field was 70.0±2.3, 42.5±4.6, 42.7±2.8 and 36.4±3.5. The proliferation, migration and invasion of pancreatic cancer AsPC-1 cells in PSCs supernatant group were significantly higher than those in the control group(allP<0.01). The proliferation, migration and invasion of pancreatic cancer AsPC-1 cells in PSCs supernatant+anti-HGF and PSCs supernatant+c-Met inhibitor group were significantly lower than those in PSCs supernatant group(P<0.01), but there was no difference between the PSCs supernatant+anti-HGF group and PSCs supernatant+c-Met inhibitor group(allP>0.05). ConclusionsPSCs can promote cell proliferation, migration and invasion of pancreatic cancer AsPC-1 cells via regulating HGF/c-Met pathway.

Pancreatic neoplasms;Astrocytes;Hepatocyte growth factor;Proto-oncogene proteins c-Met

10.3760/cma.j.issn.1674-1935.2016.04.005

201700上海，復(fù)旦大學(xué)附屬中山醫(yī)院青浦分院消化科

高振軍，Email：cui_yin@126.com

2016-01-06)