胰腺星状细胞对胰腺癌AsPC-1细胞侵袭转移的影响

高振军 吴恺 王兴鹏

·论著·

胰腺星状细胞对胰腺癌AsPC-1细胞侵袭转移的影响

高振军 吴恺 王兴鹏

目的探讨胰腺星状细胞(PSCs)对胰腺癌细胞侵袭转移的影响及基质细胞因子-1(SDF-1)在此过程中的作用。方法常规分离、培养PSCs，收集及浓缩PSCs条件培养液(PSC-CM)，应用不同浓度的PSC-CM、抗SDF-1抗体(anti-SDF-1)及两者联合应用处理AsPC-1细胞，采用MTT法检测AsPC-1细胞的增殖，Transwell小室检测细胞迁移，体外侵袭实验观察细胞侵袭能力。结果对照组及0.25、0.5、1 μg/μl PSC-CM组AsPC-1细胞增殖的吸光度值(A490)分别为0.437±0.041、0.472±0.048、0.553±0.057、0.690±0.051，PSC-CM呈剂量依赖性促进细胞增殖，其中0.5、1 μg/μl PSC-CM组与对照组间以及0.5 μg/μl与1 μg/μl PSC-CM组间的差异具有统计学意义(P值均<0.05)。对照组、anti-SDF-1组、PSC-CM组及PSC-CM+anti-SDF-1组细胞增殖的A490值分别为0.407±0.028、0.416±0.030、0.629±0.048、0.481±0.049；穿膜细胞数分别为(35.3±7.1)、(34.8±5.6)、(140.9±12.7)、(56.5±5.9)个；侵袭细胞数分别为(27.1±2.9)、(29.1±4.2)、(81.5±8.2)、(46.4±4.4)个。anti-SDF-1组与对照组间的差异无统计学意义，PSC-CM组细胞的增殖、迁移、侵袭能力较对照组显著增强(P<0.05或P<0.01)，PSC-CM+anti-SDF-1组细胞的增殖、迁移、侵袭能力较PSC-CM组显著降低，但仍显著高于对照组(P<0.05或P<0.01)。结论PSC-CM可促进AsPC-1的增殖、迁移及侵袭，其机制为部分通过SDF-1/CXCR4受体配体系统。

胰腺肿瘤； 星形细胞； 受体，CXCR4； 基质细胞衍生因子-1

胰腺癌预后很差，早期即可局部或远处转移，近年来发病率和病死率在国内外均有明显上升的趋势。肿瘤进展不仅取决于肿瘤细胞本身的生物学特性，而且与多种非肿瘤性基质细胞构成的微环境密切相关，基质与肿瘤细胞相互作用会进一步促进肿瘤细胞的生存、增殖和侵袭。在胰腺癌周围产生基质反应的细胞是胰腺星状细胞(pancreatic stellate cells，PSCs)[1-2]，其数量有时甚至超过癌细胞[3]。基质细胞衍生因子(stromal cell-derived factor 1，SDF-1)与它的特异性受体CXCR4作为最重要的受体配体复合体在胰腺癌的定向转移中起重要作用[4]。研究发现[5-6]，PSCs上清液可刺激肿瘤细胞增殖、侵袭及运动，但具体机制尚不清楚。本研究旨在探讨PSCs对胰腺癌细胞侵袭转移的影响及SDF-1/CXCR4系统在此过程中的作用。

材料与方法

一、PSCs的分离、培养

取胰腺癌根治术中的新鲜癌旁组织，在无菌环境、无血清培养液中剪成0.5 mm3大小的组织块。把组织块贴放在培养皿中，间隔0.5～1.0 cm，加入含20%胎牛血清(FCS)的DMEM/F12培养液，培养24 h后更换培养液，以后每2～3 d更换培养液一次。培养约3～5 d，组织块周围有星状细胞爬出，并形成克隆。小心去除组织块后用含乙二胺四乙酸(EDTA)的0.25%胰蛋白酶消化细胞，洗涤后置培养瓶中培养。待细胞贴壁生长至70%～80%融合时按1∶3比例传代。收集对数生长期PSCs，弃去培养液，用PBS冲洗2遍，每个培养瓶加无血清DMEM/F12培养液(serum-free medium, SFM)5 ml培养48 h，收集培养液，过滤去除细胞碎片，用YM3超滤离心管浓缩，混合。该浓缩的培养液取名为PSCs条件培养液(PSCs conditioned media, PSC-CM)，应用Bio-Rad法检测蛋白浓度后置-80°C保存备用。

二、人胰腺癌AsPC-1细胞增殖的检测

人胰腺癌细胞株AsPC-1购自中科院细胞库，常规培养、传代。取对数生长期AsPC-1细胞制成单细胞悬液，按每孔5×103个细胞(每孔100 μl)接种于96孔板，贴壁生长后换SFM培养过夜。实验分5组，分别加入含0.25、0.5、1 μg/μl PSC-CM的SFM和含10%小牛血清(NCS)的培养液，以只加SFM作为对照组。另取上述细胞，分为4组，分别加入含1 μg/ml SDF-1中和抗体(SDF-1 neutralizing antibody, anti-SDF-1)、1 μg/μl 的PSC-CM、1 μg/μl的PSC-CM+1 μg/ml anti-SDF-1的SFM，以只加SFM为对照组。每组均设5个平行孔，继续培养24 h后每孔加入浓度为5 mg/ml的MTT20 μl培养4 h，吸弃上清液，每孔加入150 μl DMSO振荡10 min，用酶标仪测定各孔在490 nm波长处的吸光度(A490)值。

三、细胞迁移实验

收集对数生长期AsPC-1细胞，加入SFM制成2×105/ml的单细胞悬液。取Transwell小室，在膜的底面铺100 μg/ml的纤连蛋白(Fn,Sigma-Aldrich公司)50 μl，上室均加入200 μl的细胞悬液，下室分别加入500 μl含1 μg/ml anti-SDF-1、1 μg/μl PSC-CM、1 μg/μl PSC-CM+1 μg/ml的SFM，以单加500 μl SFM作为对照组。每组5个Transwell小室，常规培养12 h，加0.1%结晶紫染色20 min，置高倍显微镜下随机取10个视野，计数穿膜细胞数，取均值。实验重复3次。

四、AsPC-1细胞体外侵袭实验

收集对数生长期AsPC-1细胞，加入SFM制成1×106/ml的单细胞悬液。应用体外侵袭试剂盒ECM550(Chemicon公司)检测细胞体外侵袭能力。上室均加入300 μl的细胞悬液，下室分别加入500 μl含1 μg/ml anti-SDF-1、 1 μg/μl PSC-CM、 1 μg/μl PSC-CM+1 μg/ml anti-SDF-1的2% FCS培养液，以单加500 μl 2% FCS的培养液为对照组。每组5个小室，严格按试剂盒说明书操作，孵育48 h后随机计数10个高倍镜视野中浸润细胞的数量，取均值。实验重复3次。

五、统计学处理

结 果

一、AsPC-1细胞增殖的变化

对照组，0.25、0.5、1 μg/μl PSC-CM组及10% NCS组AsPC-1细胞增殖的A490值分别为0.437±0.041、0.472±0.048、0.553±0.057、0.690±0.051及0.591±0.056(F=27.116，P<0.05)，PSC-CM呈剂量依赖性促进细胞增殖，其中0.5、1 μg/μl PSC-CM组与对照组间以及0.5μg/μl与1 μg/μl PSC-CM组间的差异具有统计学意义(P值均<0.05)。10% NCS组的细胞增殖亦较对照组显著增加(P<0.05)。

对照组、anti-SDF-1组、PSC-CM组及PSC-CM+anti-SDF-1组细胞增殖的A490值分别为0.407±0.028、0.416±0.030、0.629±0.048、0.481±0.049，anti-SDF-1组与对照组的差异无统计学意义，PSC-CM组细胞增殖较对照组显著增加(P<0.05)，而PSC-CM+anti-SDF-1组细胞增殖较PSC-CM组显著下降，但仍高于对照组(P值均<0.05)。

二、AsPC-1细胞穿膜细胞数的变化

对照组、anti-SDF-1组、PSC-CM组及PSC-CM+anti-SDF-1组的穿膜细胞数分别为(35.3±7.1)、(34.8±5.6)、(140.9±12.7)、(56.5±5.9)个，anti-SDF-1组与对照组间的差异无统计学意义，PSC-CM组的穿膜细胞数较对照组显著增多(P<0.01)，而PSC-CM+anti-SDF-1组的穿膜细胞数较PSC-CM组显著减少，但仍高于对照组(P值均<0.01)。

三、AsPC-1细胞体外侵袭能力的变化

对照组、anti-SDF-1组、PSC-CM组及PSC-CM+anti-SDF-1组的侵袭细胞数分别为(27.1±2.9)、(29.1±4.2)、(81.5±8.2)、(46.4±4.4)个，anti-SDF-1组与对照组间的差异无统计学意义，PSC-CM组的侵袭细胞数较对照组均显著增多(P<0.01)，PSC-CM+anti-SDF-1组的侵袭细胞数较PSC-CM组显著减少，但仍高于对照组(P值均<0.01)。

讨 论

肿瘤局部微环境在肿瘤的进展中起重要作用[7-8]。肿瘤微环境中存在着复杂的趋化因子及其受体系统，其中，起首要作用的是SDF-1。SDF-1多以自分泌或旁分泌形式刺激肿瘤细胞及肿瘤间质细胞，使肿瘤细胞存活和生长。近来研究提示，间质细胞是SDF-1的重要来源，肿瘤相关性成纤维细胞(carcinoma associated fibroblasts, CAFs)能分泌SDF-1，通过与CXCR4作用来直接刺激肿瘤生长，并能介导内皮前体细胞进入肿瘤而促进肿瘤血管生成，进一步促进肿瘤的增殖及侵袭、转移[9]。SDF-1是分泌型蛋白质，间质成纤维细胞产生后释放到细胞间隙，在局部微环境中通过旁分泌方式直接刺激造血或非造血性正常或恶性细胞的迁移[10-11]。胰腺癌中有大量间质组织，胰腺癌-间质之间的相互作用在肿瘤进展中也起重要作用[2]。

本研究结果显示，应用PSCs培养上清处理AsPC-1细胞后可促进其增殖，与文献报道[6,12]相似。本研究结果还显示，应用PSCs培养上清处理AsPC-1细胞后可促进AsPC-1细胞迁移、侵袭，而应用抗SDF-1抗体后，PSCs培养上清对AsPC-1细胞增殖、迁移、侵袭的促进作用被抑制，提示PSC-CM可能通过PSCs分泌的SDF-1促进胰腺癌细胞增殖、迁移及侵袭，表明SDF-1/CXCR4轴通过旁分泌调控机制在PSCs与胰腺癌细胞的相互作用中扮演重要角色。本研究结果还显示，尽管抗SDF-1抗体可明显抑制PSC-CM所引起的促AsPC-1细胞增殖、迁移及侵袭作用，但处理后的AsPC-1细胞的增殖、迁移、侵袭能力仍显著高于对照组，提示在PSC-CM中尚存在其他一些可溶性因子具有促进胰腺癌细胞增殖、迁移及侵袭的作用，有待于进一步深入研究。

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EffectofpancreaticstellatecellsoninvasionandmetastasisofhumanpancreaticcancercelllineAsPC-1

GAOZhen-jun,WUKai,WANGXing-peng.

DepartmentofGastroenterology,ZhongshanHospitalQingpuBranch,FudanUniversity,Shanghai201700,China

Correspondingauthor:WANGXing-peng,Email:xpwcn@public7.sta.net.cn

ObjectiveTo investigate the effect of pancreatic stellate cells on invasion and metastasis of human pancreatic cancer cell AsPC-1, and to determine the role of SDF-1 in this process.MethodsPSCs were routinely isolated and cultured, and PSCs conditioned media(PSC-CM) was collected and concentrated. Different concentrations of PSC-CM, anti SDF-1 and their combination were used to treat AsPC-1 cells, and MTT assay was applied to detect the proliferation of pancreatic cancer cells. Transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. In vitro invasion assay was used to determine the invasion of AsPC-1 cells.ResultsA490values of AsPC-1 cell in control group and 0.25, 0.5, 1 μg/μl PSC-CM group were 0.437±0.041, 0.472±0.048, 0.553±0.057, 0.690±0.051, and PSC-CM promoted cell proliferation in a dose dependant manner. The difference between 0.5, 1 μg/μl PSC-CM group and control group, and between 1 μg/μl and 0.5 μg/μl PSC-CM group was statistically significant (P<0.05).A490values of control group, anti SDF-1 group, PSC-CM group and PSC-CM+anti SDF-1 group were 0.407±0.028, 0.416±0.030, 0.629±0.048, 0.481±0.049. The numbers of penetrating cells were 35.3±7.1, 34.8±5.6, 140.9±12.7, 56.5±5.9, and the numbers of invasive cells were 27.1±2.9, 29.1±4.2,81.5±8.2, 46.4±4.4. The difference between anti SDF-1 group and control group was not statistically significant. The proliferation, migration and invasion of pancreatic cancer cells in PSC-CM group was significantly higher than those of control group (P<0.05 orP<0.01). The proliferation, migration and invasion of pancreatic cancer cells in PSC-CM+anti SDF-1 group was significantly lower than those of PSC-CM group, but they were significantly higher than those of control group (P<0.01).ConclusionsPSCs can promote proliferation, migration and invasion of pancreatic cancer cells AsPC-1, and the mechanism may be partly due to SDF-1/CXCR4 receptor ligand axis.

Pancreatic neoplasm; Astrocytes; Receptors, CXCR4; Stromal cell-derived factor 1

2013-04-24)

(本文编辑：屠振兴)

10.3760/cma.j.issn.1674-1935.2013.04.009

201700 上海，复旦大学附属中山医院青浦分院消化科(高振军)；上海交通大学附属第一人民医院消化科(吴恺、王兴鹏)

王兴鹏，Email: xpwcn@public7.sta. net. cn