【作者】 明健；

【导师】 邱雪杉；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 前言白介素-7（Interleukin-7,IL-7）是一种能诱发造血细胞和恶性肿瘤发生和增殖的细胞因子,在乳腺癌细胞系IL-7通过磷脂酰肌醇-3-激酶（phosphatidylinositol3-kinase,PI3K）敏感径路诱发乳腺癌细胞的生长,这对研究乳腺癌的发生和发展有着重要意义。卵巢癌可刺激宿主免疫细胞分泌IL-7,血清和腹腔液IL-7升高是宿主免疫系统抗肿瘤作用的一种表现。IL-7作为一种具有潜在抗肿瘤作用的免疫调节活性因子,其在腹水中水平升高不及血清显著,推测腹腔局部可能存在某些免疫抑制因素。提高腹腔局部IL-7水平可对卵巢癌有治疗意义。目前IL-7在肿瘤中的作用不一,作用机制也不清楚。目的本研究探讨IL-7在肺癌中的表达,分析它们与各临床病理因素之间的关系,揭示IL-7在肺癌细胞侵袭和转移过程中的作用机制。实验材料和方法一、材料100例原发性非小细胞肺癌癌组织及癌旁正常肺组织标本均来自1980年到2005年在中国医科大学附属一院实行手术的病人,患者术前均未接受放化疗。标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,采用链霉素抗生物素蛋白-过氧化物酶免疫组化法（streptavidin-peroxidase,S-P法）检测组织中IL-7、IL-7R和血管内皮生长因子-D（vascular endothelial growth factor-D,VEGF-D）蛋白表达情况,微血管密度（microvessel density,MVD）和淋巴管密度（lymphaticvessel density,LVD）。以PBS代替一抗作为阴性对照。结果判定:IL-7、IL-7R和VEGF-D以细胞质中出现棕黄色颗粒为阳性显色。光镜下每张切片选取癌细胞较多的10个高倍视野计数100个癌细胞中的阳性细胞数,IL-7、IL-7R（Interleukin-7Receptor）和VEGF-D的评估为:无阳性细胞为“-”,阳性细胞数≤5%为“+”,阳性细胞数为5%-20%为“++”,＞20%为“+++”,其中“++”和“+++”为高表达,“-”和“+”为低表达。血管和淋巴管判定标准为:内皮细胞形成条状、裂隙状等孤立结构棕黄染色或有管腔者按一条血管或淋巴管计数。低倍光镜下确定3个微血管或淋巴管高密度区域（热点）,然后在高倍镜下分别计数每个热点中3个区域的CD34染色（用于标记MVD）和D2-40（用于标记LVD）染色阳性管腔数均值。MVD=（CD34阳性管腔数-D2-40阳性管腔数）均值;LVD=D2-40阳性管腔数均值。当计数相差10%以上时则重新计数。二、细胞培养分别用含10%新鲜胎牛血清的RPMI1640或DMEM培养基,在37℃、5%CO₂的条件下培养人肺巨细胞癌细胞系PG-LH7和NCI-H460、人肺腺癌细胞系A549和SPC-A1和人肺鳞癌细胞系SK-MES-1。三、逆转录-聚合酶链式反应（RT-PCR）提取细胞总RNA,按RT-PCR（TaKaRa）试剂盒方法进行逆转录反应,反应体系为20βL,取4βL的RT产物进行PCR反应,反应体系为20μL。PCR引物经在GeneBank上比对后在bio-spring公司合成。PCR产物琼脂糖凝胶电泳进行检测,采用凝胶成像分析系统进行半定量分析。四、Western Blot法在收集的细胞内加入裂解液充分裂解,低温高速离心（4℃,12000转/min,30min）,提取上清为总蛋白。上样蛋白量为60ug。电泳（12%SDS-PAGE凝胶）、转印（50V,120min）、5%正常小牛血清封闭,一抗c-Jun（1:200）、phosphorylated-c-Jun（p-c-Jun,1:200）、c-Fos（1:200）、VEGF（1:200）、VEGF-D（1:200）和β-actin（1:200）,抗体均购自Santa Cruz,USA,4℃孵育过夜,分别与各自对应的二抗（1:2500,chemicon,USA）室温孵育2h,3,3’-二氨基联苯胺（DAB）显色,结果经自动电泳凝胶成像分析仪（Chemi Imager 5500,Alpha Innotech,USA）采集,进行灰度值测定。五、染色质免疫沉淀法加入37%的甲醛使A549细胞内的蛋白和DNA充分交联,裂解细胞,超声剪切DNA片段（80w,30次）,免疫沉淀交联的蛋白和DNA,洗脱蛋白/DNA复合物,逆转蛋白/DNA复合物交联,纯化DNA,做PCR,重复三次,取平均值。六、免疫共沉淀法在收集的新鲜培养的细胞中加入裂解液,充分裂解后,每管分别加入100ul磁珠（Miltenyi Biotec,Germany）和20ul c-Jun抗体,使终体积为1ml,4℃孵育30min,用蛋白裂解液冲洗u柱（Miltenyi Biotec,Germany）,将蛋白样加入u柱,加入20ul的95℃预热上样缓冲液,孵育5min,用50ul的95℃预热上样缓冲液冲洗,收集液体,做Western blot,重复三次,取平均值。七、细胞迁移和侵袭能力检测在transwell小室（Corning公司）下室加入600ul含10%小牛血清DMEM培养基,上室中加入100ul无血清DMEM培养基,接种A549细胞数为2.5×10⁴个。37℃、5%CO₂孵箱中培养6hr后吸尽培养基,PBS清洗后,甲醇室温固定15min,用棉签轻擦掉微孔膜上表面的细胞,苏木素染色,室温干燥过夜。取下微孔膜,置载玻片上,镜下观察。细胞侵袭能力的测定用预冷的无血清培养基以1:7稀释Matrigel（BD Biosciences,USA）加入上室100ul,在室温下放置6h。使用前用培养基重新水化并吸净,其他与迁移实验相同,培养18hr后观察结果。八、MTT法检测细胞增殖将单个细胞悬液接种于96孔培养板中,每孔含10³个细胞,培养24h,每孔加入MTT（Methylthiazolyldiphenyl-tetrazolium bromide）溶液继续培养4h,加入DMSO,490nm波长下测定各孔光吸收值,以不含细胞的等体积培养基作对照。绘制细胞生长曲线。九、体内裸鼠移植实验将24只4周龄裸鼠背部皮下注射A549细胞（2×10⁷个/只）,1周后（有肿瘤形成）随机分成4组,每组6只。以后每周1次,每次50ul背部皮下注射。一组:注射PBS;二组注射IL-7（20mg/ml）;三组注射IL-7（20mg/ml）+IL-7R（50mg/ml）;四组注射IL-7R（50mg/ml）。10周后处死。观察瘤大小,并取瘤组织检测其他指标。十、数据分析使用SPSS13.0统计软件进行数据处理,以P＜0.05为有统计学意义。实验结果1、IL-7/IL-7R与各临床病理因素、VEGF-D表达、淋巴管密度、淋巴结转移和患者预后的关系免疫组化分析100例肺癌组织,其中IL-7蛋白高表达为61例（61%）,低表达者为39例（39%）;IL-7R蛋白高表达为62例（62%）,低表达者为38例（38%）;VEGF-D蛋白高表达为59例（59%）,低表达者为41例（41%）。IL-7和IL-7R的表达分别与各临床病理因素的关系:IL-7的表达水平与肺癌的分期呈正相关（P=0.001）,与淋巴结转移呈正相关（P＜0.001）;IL-7R的表达水平与肺癌的分期呈正相关（P=0.007）,与淋巴结转移呈正相关（P＜0.001）。IL-7和IL-7R的表达水平与性别、年龄,组织分型,分化程度都不相关。IL-7和IL-7R的表达分别与VEGF-D表达水平的关系:IL-7的表达水平与VEGF-D表达水平呈正相关（P＜0.001）;IL-7R的表达水平与VEGF-D表达水平呈正相关（P=0.002）。IL-7、IL-7R和VEGF-D的表达与MVD和LVD的关系:IL-7高表达组的LVD（21.39±12.39）明显高于低表达组的LVD（11.10±9.48）,差异显著（P=0.003）;IL-7R高表达组的LVD（21.39±12.39）明显高于低表达组的LVD（11.10±9.48）,差异显著（P=0.041）;VEGF-D高表达组的LVD（21.39±12.39）明显高于低表达组的LVD（11.10±9.48）,差异显著（P＜0.001）。IL-7、IL-7R和VEGF-D的表达与MVD不相关。IL-7、IL-7R和VEGF-D的表达与患者预后的关系:IL-7高表达组的生存时间（21.39±12.39）明显低于低表达组的生存时间（11.10±9.48）,差异显著（P=0.008）;IL-7R高表达组的生存时间（21.39±12.39）明显低于低表达组的生存时间（11.10±9.48）,差异显著（P=0.031）;VEGF-D高表达组的生存时间（21.39±12.39）明显低于低表达组的生存时间（11.10±9.48）,差异显著（P=0.022）。2、IL-7/IL-7R促进肺癌细胞中VEGF-D的表达外源性人重组IL-7作用于肺癌IL-7R⁺细胞A549、SPC-A1、LH7和SK-MES-1后,VEGF-D的表达升高,VEGF-C的表达无明显变化;用抗IL-7R抗体作用于这些细胞后VEGF-D的表达降低。而无论是外源性人重组IL-7还是抗IL-7R抗体均对IL-7R^-细胞H460无影响。3、IL-7/IL-7R通过c-Fos/c-Jun通路促进VEGF-D的表达IL-7能促进c-Fos、c-Jun和p-c-Jun的表达;而阻断IL-7R能抑制c-Fos、c-Jun和p-c-Jun的表达。用AP-1特异性抑制剂SP600125作用后,VEGF-D的表达下降。说明IL-7/IL-7R通过c-Fos/c-Jun通路调控VEGF-D的表达。4、IL-7/IL-7R促进c-Fos/c-Jun异二聚体形成,并促进该二聚体与VEGF-D的DNA结合免疫共沉淀结果显示,IL-7能促进c-Fos/c-Jun异二聚体形成增多;而阻断IL-7R能抑制c-Fos/c-Jun形成异二聚体。染色质免疫沉淀结果显示,IL-7能促进c-Fos/c-Jun异二聚体与VEGF-D的DNA结合;而阻断IL-7R则抑制c-Fos/c-Jun异二聚体与VEGF-D的DNA结合。5、MTT结果显示IL-7能促进肿瘤细胞生长,而阻断IL-7R则抑制肿瘤细胞生长。6、在体内裸鼠抑制实验结果显示IL-7通过IL-7R调控AP-1复合物中c-fos,c-Jun表达及磷酸化,促进c-fos和c-Jun形成异二聚体,并与VEGF-D基因结合,调控VEGF-D基因转录,促进VEGF-D表达,从而促进肿瘤淋巴管形成及转移。结论1、IL-7/IL-7R高表达与非小细胞肺癌的分期、淋巴结转移和预后不良正相关,与VEGF-D的表达正相关,IL-7/IL-7R和VEGF-D高表达组肿瘤中的LVD明显高于低表达组。2、在肺癌IL-7R⁺细胞中IL-7通过IL-7R调控AP-1复合物中c-fos,c-Jun表达及磷酸化,促进c-fos和c-Jun形成异二聚体,并与VEGF-D基因结合,调控VEGF-D基因转录,促进VEGF-D表达。而IL-7对VEGF-C无调控作用。3、在体内实验中IL-7也通过该机制促进VEGF-D表达,从而促进肿瘤淋巴管形成及转移。更多还原

【Abstract】 ObjectiveInterleukin 7（IL-7） could induce the development and proliferation of haematopoietic cells and malignancies,including some forms of leukaemia and lymphoma.However,little was known about its involvement in solid tumours, including lung cancer.Some malignant cells,such as chronic lymphoblastic leukaemia cells,Burkitt’s lymphoma cells and colonic cancer cells were also capable of producing IL-7.Other solid tumours could express the IL-7 gene,including oesophageal,renal, head and neck squamous cell carcinoma,and Warthin’s tumour of the parotid gland.In addition,expression of IL-7 and IL-7R in breast cancer tissues has been shown recently, and IL-7R was positively correlated with tumours that had metastasised to the regional lymph nodes.Following the binding of IL-7R to its ligand,a series of intracellular phosphorylation events occurred,such as the activation of the Janus kinases（JAK-1 and JAK-3）,phosphoinositide 3 kinase（PI3K）,and the signal transducers and activators of transcription（STAT-5）.13 It was therefore tempting to speculate that certain unidentified downstream gene（s） of IL-7 may have a role in tumour metastasis.In this study,we studied the relationship between IL-7 and their impact on lung cancer patients’ outcomes.To explore the mechanism of IL-7/IL-7R inducing lymphangiogenesis and metastasis in lung cancer,we researched it in vivo and in vitro.MATERIALS AND METHODS1.Patients and Specimens.A total of 100 cases of NSCLC were obtained from the 1^st January 1980 to the 31^st December 2005 at the First Affiliated Hospital of China Medical University,Shenyang, China.The turnout tissues in this study were from patients who had NSCLC proved by pathological diagnosis without distant metastasis.None of the 100 cases had received radiation therapy or chemotherapy before surgery.The TNM staging system of the UICC（1997） was used to classify the specimens.2.Immunohistochemistry and Quantitation of Blood and Lymphatic Vessel Densities.Four-micron thick sections were prepared from the paraffin-embedded tissues. Immunostaining was performed by the streptavidin-peroxidase（S-P） method （UltrasensitiveTM MaiXin,Fuzhou,China）.The primary antibodies were anti-IL-7, anti-IL-7R,anti-VEGF-D（1:100,1:100,1:150）,anti-D2-40 and anti-CD34 antibodies. The peroxidase reaction was developed with DAB.For negative control,the primary antibodies were replaced by non-immune serum.All the samples were evaluated by 2 independent pathologists.Intensity of immunohistochemical staining:-,negative;+,focal expression＜5%of cancer tissues; ++,focal expression in 5-20%of cancer tissues;and +++,diffuse expression＞20%of cancer tissues.The section with ++ and +++ staining of IL-7,IL-7R or VEGF-D was classified as high expression and section with - and + staining was assigned as low expression.The evaluation criteria used for determination of blood and lymphatic vessel staining as follows:yellow-brown stained endothelial cells with band or fissure-like isolated or clustered structures or with tubular lumen were counted as a single blood or lymphatic vessel.Within each section,we selected 3 tumour areas with the highest density of distinctly highlighted microvessels and lymphatic vessels（"hot spot"） when observed under low-power fields.Next,the average number of CD34- or D2-40-1abeled tubular lumens was counted under high-power fields[34].Microvessel density（MVD） = mean（CD34-1abeled tubular lumen number—D2-40-1abeled tubular lumen number）;LVD = mean D2-40-1abeled tubular lumen.If the difference between the numbers counted by the 2 pathologists was more than 10%,the lumens were recounted and a consensus between each observer was reached.3.Cell Culture.Human lung cancer cell lines A549 and SPC-A1（adenocarcinoma）,NCI-H460 and LH7（large carcinoma）,and SK-MES-1（squamous carcinoma） were maintained in Dulbcco’s Modifed Eagle Medium（DMEM） supplemented with 10%fetal bovine serum（Gibco,USA）4.RNA Isolation and Reverse Transcriptase-PCR.Total RNA was isolated using TRIZOL（Invitrogen,USA） according to the manufacturer’s instructions.The PCR products were amplified with 30 PCR cycles（1 min at 95℃;1 min at 53℃,1 min at 72℃）,and visualized by ethidium bromide staining after agarose gel electrophoresis.5.Western Blot Analysis.Total protein was extracted with lysis buffer（150mM NaCl,1%v/v NP-40,0.1% v/v SDS,2μg/ml aprotinin,1mM PMSF）,and 60μg of protein lysates were separated on a 12%v/v SDS-polyacrylamide electrophoresis gel,transferred to Polyvinylidene Fluoride（PVDF） membranes.Proteins were visualized with horse-radish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG（Zhongshan,Beijing,China） followed by DAB.Subsequently,densitometric analyses of the bands were performed.6.Chromatin Immunoprecipitation（CHIP）.We performed the ChIP assay according to the instructions of the ChIP assay kit （Upstate,USA）.The procedure included DNA-protein cross-linking in chromatin, shearing DNA into smaller fragments,immunoprecipitation with anti-c-Jun antibody （negative control with normal rabbit IgG）,followed by PCR identification of associated DNA sequences.7.Co-Immunoprecipitation（CoIP）.The protein was extracted with cellular lysis buffer.Equal amounts of protein were incubated with c-Jun specific antibody immobilized onto protein G-bead for 1 h at 4℃with gentle rotation.Beads were washed extensively with lysis buffer,boiled,and microcentrifuged.Proteins were detected with c-Fos antibody by Western blot analysis.8.Cell Migration and Invasion Assays.For the migration assay,5×10⁴ cells were trypsinized,washed,resuspended in serum-free DMEM,and placed in the top portion of the chamber.The lower compartment of the chamber contained 10%v/v FBS as a chemo-attractant.The chambers were incubated for 6 h,then cells on the membrane were washed with PBS, and fixed in 100%methanol,stained with hematoxylin,photographed,and counted.For the invasion ability assay,pre-cooled serum-free DMEM was mixed with Matrigel（BD Biosciences,USA）（1:7 dilution）.The upper compartments were filled with 100μl of the mixture,and the Matrigel was allowed to solidify at room temperature for 3 h. Other procedures followed the migration protocol（BD Biosciences,USA）.The chambers were incubated for 24 h.9.In vitro cell growth assays.Cells were plated in a 96-well plate and MTT was added to each well.After incubation for 4h,the medium was replaced with dimethyl sulfoxide.The OD value of each well was measured with a test wavelength of 490nm.10.In vivo Nude mice model of lung cancer.24（4 weeks old） mice were inoculated with 2×10⁷ A549 human lung carcinoma cells under mouse dorsal skin.The mice were divided into four groups of six by random.After 1 weeks,each group were injected into the tail vein with 100ul complex once a week.First group:PBS;second group:IL-7（20mg/ml）;third group:anti-IL-7R antibody（20mg/ml）;fourth group:IL-7（20mg/ml）+ anti-IL-7R antibody（20mg/ml）. After 10 weeks,the mice were killed.11.Statistical Analysis.The statistical package SPSS13.0（SPSS incorporated,Chicago） was used for all analysis.Values of P＜0.05 were considered statistically significant.RESULTS1.Expression of IL-7/IL-7R Correlate with variable expression levels.Immunohistochemical analysis of 100 NSCLC specimens revealed that the overexpression rates of IL-7 and IL-7R were 61%and 62%,respectively;VEGF-D overexpression rates 59%.IL-7 and IL-7R expression level were correlated respectively with the stage of lung cancer（P = 0.001,P = 0.007）,lymph node metastases（P＜0.001,P＜0.001）.IL-7 and IL-TR expression level were not correlated with age,gender,the histological type, and differentiation of the cancer.IL-7 and IL-7R expression level were correlated with the expression level of VEGF-D（P＜0.001,P = 0.002） respectively.IL-7,IL-7R,and VEGF-D expression level were correlated respectively with LVD（P = 0.003,P= 0.041,and P＜0.001）,but they were not correlated respectively with MVD.Clinically, compared to the NSCLC with low expression of IL-7,tumours with high expressions of IL-7,IL-7R,and VEGF-D were more p shorter survival（P＜0.05）.2.IL-7/IL-7R Increase the Expression of VEGF-D in Lung Cancer Cell Lines.Treatment of IL-7R⁺ lung cancer A549,SPC-A1,LH7,and SK-MES-1 cells with recombinant human IL-7 increased the expression of VEGF-D,while the expression of VEGF-C were not affected.Interestingly,the expression of VEGF-D were not affected in IL-7R- lung large carcinoma H460 cells after IL-7 stimulation.We then detected the expression of VEGF-D of the five cells after blocking IL-7R.Blockage of IL-7R in A549,SPC-A1,LH7,and SK-MES-1 cells decreased the expression of VEGF-D, which were not affected by the IL-7.However,blocking IL-7R in H460 cells did not affect the expression of VEGF-D.3.IL-7/IL-7R Induce VEGF-D via c-Fos/c-Jun Pathway.Incubation of A549 cells with IL-7 increased the expression of c-Fos and c-Jun protein,while blocking IL-7R with sc-662 decreased the expressions of c-Fos,c-Jun, and p-c-Jun.We then inhibited the activity of c-Fos/c-Jun with a specific AP-1 inhibitor SP600125.The analyses showed that the expressions of VEGF-D were decreased significantly after treatment with SP600125.These results imply that IL-7/IL-7R up-regulate VEGF-D via c-Fos/c-Jun.4.IL-7/IL-7R Lead to the Formation of c-Fos/c-Jun Heterodimer.Using CoIP approach,we found that c-Fos/c-Jun heterodimer was increased after incubation with the IL-7,and blocking IL-7R with sc-662 decreased the dimer formation.5.IL-7/IL-7R Enhance the DNA Binding Activity of AP-1 to the Promoter of VEGF-D.CHIP analysis demonstrated that AP-1 could bind to VEGF-D promoter.Then we detected AP-1 DNA binding activity in A549 cell after incubation with either the IL-7 with or without sc-662.IL-7 enhanced AP-1 binding to VEGF-D promoter,while blocking IL-7R reduced AP-1 binding to VEGF-D promoter.6.IL-7 promote the proliferation of the cells.The results of the MTT assay showed that the proliferation of the A549 cells was significantly higher with IL-7 than without IL-7.7.In vivo Nude mice model of lung cancer assays.It demonstrated that,in vivo,IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1（c-Fos/c-Jun）-dependent pathway to upregulate lymphangiogenesis and lymphatic node metastasis.Conclusions1.IL-7/IL-7R high expression level were positive correlated with the stage, lymphatic node metastasis,and lower survival of lung cancer.In IL-7/IL-7R and VEGF-D high expression groups LVD were higher than in low expression groups.2.In human lung cancer cells,this study demonstrated that IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1（c-Fos/c-Jun）-dependent pathway.3.In vivo Nude mice model of lung cancer assays demonstrated that,in vivo,IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1 （c-Fos/c-Jun）-dependent pathway to upregulate lymphangiogenesis and lymphatic node metastasis.更多还原