【作者】 黄礼年；

【导师】 肖伟；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 [研究背景]肺癌是当今世界上严重威胁人类生命与健康的最常见的恶性肿瘤之一,已成为各种癌症死亡的首要原因。其中约80%为非小细胞肺癌(non-small cell lung cancer, NSCLC)。80%的NSCLC患者在确诊时已属晚期,失去手术机会。NSCLC的治疗主要包括手术、化疗、放疗和分子靶向治疗,无论是何种治疗都需要一个指导和评判NSCLC治疗效果的有效指标。目前临床广泛采用的评价指标是TNM分期,这个分期固然为临床NSCLC的治疗和预后判断提供了一定的指导价值。但是在临床工作中经常发现在各个分期中有部分患者的预后与预期的不同。临床有相当一部分NSCLC患者(如：Ⅰ期NSCLC患者,术后5年生存率仅为60%-70%),尽管都得到了早期的诊断和治疗,但结果却短期内出现了复发和转移,无疑这些患者的肿瘤在生物学行为特点方面表现为更具有侵袭性。因此,单纯以TNM分期来判断肿癌预后和指导治疗显然是不够的。近年来研究发现,从肺癌的早期发生如支气管上皮增生到肺癌生长、侵袭、转移和预后复发都与血管生成有关。和其他实体瘤一样,新生血管为肺癌提供营养物质,促进肿瘤的生长,也是肿瘤侵袭转移的前提。肿瘤血管的生成涉及到多种基因的突变和不同细胞因子及受体的表达,受正负两方面因素的调控。在肿瘤组织,由于促血管生成因子和抗血管生成因子二者之间平衡失调,血管生成促进因子占优势。血管内皮生长因子(VEGF)是目前所知的作用最强的促血管生成因子,参与血管生成的多个环节。在促进肿瘤微血管生成中起着关键作用。NSCLC组织中VEGF的表达及肿瘤微血管密度(MVD)可能与NSCLC的生物学行为及预后相关,针对VEGF及其受体的靶向治疗有可能成为NSCLC的一种新的治疗途径。1983年Folkman首先提出肝素具有血管生成抑制作用。国外学者晚近研究发现,低分子肝素(LMWH)在改善肿瘤患者生活质量,提高患者生存期等方面起着积极作用,推测LMWH可能干扰肿瘤细胞向血管内膜的黏连与侵袭、肿瘤的血管生成等环节。目前有关肺癌组织中VEGF的表达和MVD的相关性及二者与肺癌临床分期、病理分型等的关系方面的研究及LMWH对人NSCLC A549细胞表达VEGF的影响的体外研究国内外报道尚少。本研究拟对此分别进行探讨。(1)运用免疫组化方法检测VEGF在肺癌中的表达并测定微血管密度(MVD),并结合肺癌临床病理学特征及预后进行分析,探讨VEGF表达与NSCLC肿瘤组织MVD的相关性；(2)通过分析VEGF表达及MVD与肺癌临床病理学特征及预后的关系,试图寻找对非小细胞肺癌的诊断和预后判断有价值的肿瘤标记物,为临床提供依据；(3)采用荧光定量-PCR(real-time PCR)等方法探讨LMWH对人非小细胞肺癌A549细胞血管内皮细胞生长因子表达水平的影响,为临床应用LMWH治疗NSCLC提供实验支持和理论依据。第一部分血管内皮生长因子在非小细胞肺癌中的表达及与血管生成的相关性研究目的：研究血管内皮生长因子(vascular endothelial growth factor, VEGF)在非小细胞肺癌组织中的表达,同时探讨VEGF对NSCLC血管生成的影响及两者之间可能具有的相关性,并探讨它们与非小细胞肺癌(non small cell lung cancer,NSCLC)生物学行为的关系。方法：选用1996年1月至1999年1月蚌埠医学院附属医院心胸外科手术切除并经病理学确诊的43例非小细胞肺癌癌组织,应用免疫组织化学S-P法检测组织中的VEGF的表达,并对组织中的血管进行染色、计数。应用SPSS13.0统计软件包进行统计处理。数值变量资料均以x±s表示,分别采用t检验,单因素方差分析对实验结果进行统计分析。分类变量资料采用χ2检验。结果：1、在43例NSCLC中VEGF表达阳性者23例,阳性表达率为53.50%(23／43)；43例NSCLC的MVD均值为31.98±14.93。2、NSCLC中10例腺癌组织VEGF的阳性表达率为50%(5／10),33例鳞癌组织VEGF的阳性表达率为59.4%(18／33)；经统计学分析二者之间的差异无显着性(P>0.05)。MVD的均值在磷癌中为30.91±15.25,腺癌中为35.50±15.28,二者之间的差异无统计学意义(P>0.05)。3、VEGF在Ⅰ期、Ⅱ期和Ⅲ期NSCLC中的阳性表达率分别为12.50%,66.67%和91.67%,临床中晚期(Ⅲ期)阳性表达率显着高于临床早期(Ⅰ期,Ⅱ期)(P<0.01)；MVD的均值在Ⅰ期、Ⅱ期和Ⅲ期NSCLC中分别为21.25±7.86,32.87±15.27和45.17±11.49,有统计学意义(P<0.01)。4、根据有无淋巴结转移,把43例NSCLC分为有淋巴结转移和无淋巴结转移两组。在无淋巴结转移组VEGF阳性表达率为11.11%(2／18),在淋巴结转移组VEGF阳性表达率为84.00%(21／25),有淋巴结转移组和无淋巴结转移组MVD值分别为39.56±14.73和21.44±7.44,在两组间VEGF阳性表达率、MVD值差异均有统计学意义(P<0.01)。5、在NSCLC组织中,VEGF表达阳性组的MVD为42.83±11.92,阴性组的MVD为19.50±5.73,二者相比差异有显着性(P<0.01)。结论：1、VEGF在NSCLC组织中高表达。VEGF的表达与NSCLC的瘤体大小、有无淋巴结转移及TNM分期相关,而与NSCLC的组织学分类无关。2、NSCLC组织中MVD值与淋巴结转移、肿瘤细胞分化程度及TNM分期有关,NSCLC组织中血管生成活跃,微血管密度明显增高。血管生成与NSCLC的发生、发展过程有着密切的联系。3、VEGF的阳性表达与NSCLC中的MVD值呈显着正相关,VEGF正向调节NSCLC的血管生成,以VEGF或其受体为靶点将有望成为非小细胞肺癌新的治疗途径。第二部分血管内皮生长因子、微血管密度与人非小细胞肺癌患者预后的相关性研究目的：研究血管内皮生长因子(vascular endothelial growth factor, VEGF)在非小细胞肺癌组织中的表达,同时探讨VEGF对NSCLC血管生成的影响及两者之间可能具有的相关性,并探讨它们与非小细胞肺癌(non small cell lung cancer,NSCLC)生物学行为及预后的关系,为肺癌的临床诊断、预测转移和治疗提供理论基础。方法：选用1996年1月至1999年1月蚌埠医学院附属医院心胸外科手术切除并经病理学确诊的43例非小细胞肺癌癌组织,应用免疫组织化学S-P法检测组织中的VEGF的表达,并对组织中的血管进行染色、计数。采用Kalpan-Meier方法,对病例随访资料进行生存分析并经Log-rank检验；COX模型(似然比检验)用于多因素预后分析。结果：1、采用Kaplan-Meier方法,对病例随访资料进行生存分析,NSCLC手术后生存期与TNM临床分期明显相关,分期越晚,生存率越低,差异有显着的统计学意义(P<0.01)；Ⅰ、Ⅱ与Ⅲ期两两比较亦具有显着的统计学差异(P<0.05),NSCLC与肿块大小亦明显相关,肿块直径大于3cm者术后生存期低于小于3cm者,具有显着的统计学差异(P<0.01)；结果亦显示术后生存期高MVD值组明显低于低MVD组,VEGF表达阳性组明显低于VEGF表达阴性组,差异有显着的统计学意义(P<0.01)。2、将MVD值、VEGF表达情况及患者年龄、性别、组织类型等临床生物学特征采用COX比例风险回归模型进行分析,显示MVD高表达组患者术后死亡的相对危险度是MVD低表达组患者的34.247倍(似然比检验,P<0.01),MVD值是影响NSCLC预后的一个独立危险指标。结论：VEGF的表达、MVD值与NSCLC的5年生存率密切相关。检测VEGF和MVD可以作为判断NSCLC的肿瘤血管生成、肿瘤侵袭、转移等生物学行为和预后的指标,MVD是NSCLC的独立预后预测因子。第三部分低分子肝素对人非小细胞肺癌A549细胞VEGF表达的影响目的：通过研究LMWH对A549细胞VEGF表达的影响,探讨LMWH对A549细胞侵袭、转移潜能的影响,为LMWH防治NSCLC提供实验依据。方法：1.观察LMWH对A549细胞增殖的影响。通过使用不同剂量LMWH处理A549细胞株,动态观察A549细胞生长情况,采用MTT法检测其增殖抑制情况,分析LMWH抗肿瘤效应。2.观察LMWH对A549细胞VEGF表达的影响。不同剂量LMWH处理A549细胞株,Western blotting技术检测VEGF蛋白的表达情况；荧光定量PCR法检测VEGF mRNA水平的表达情况,分析LMWH对A549细胞侵袭、转移相关的分子机制。结果：1.LMWH对人肺癌A549细胞增殖的抑制作用。MTT实验反映了细胞的生长状况,和各处理因素对A549肿瘤细胞增殖的影响。MTT染色结果显示,5IU／ml LMWH组24小时的抑制率为2.47%,与对照组比较无统计学意义(P>0.05),其余各组、各时间段对肺癌A549细胞增殖均表现出明显的抑制作用,随着LMWH剂量的增加和LMWH作用时间的延长,吸光度值(OD值)逐渐减少,A549细胞生长抑制率逐渐升高。LMWH作用24h及48h后,15IU／ml组的抑制率分别为8.01%和14.36%,500IU／ml组的抑制率分别为37.41%和45.93%,与对照组相比差异均有统计学意义(P<0.05)。经SNK两两比较,LMWH150IU／ml与500IU／ml剂量组间差异无统计学意义(P>0.05),其余各LMWH处理组之间差异均有统计学意义(P<0.05)；48hLMWH处理组对A549细胞的生长抑制率明显高于24h组。提示LMWH在一定的浓度、剂量范围内对A549细胞的生长抑制作用具有时间依赖效应和剂量依赖效应；当药物浓度达到150IU／ml以上时,即使增大剂量,对A549的抑制作用也无明显增强。2.LMWH对A549细胞VEGF表达的影响。荧光定量PCR检测显示,LMWH作用于A549细胞株后,随着LMWH浓度的增加,VEGF mRNA表达均下调,与对照组比较差异有显着性(P<0.05)。Western blot技术分析显示：VEGF蛋白表达水平明显受到抑制,与对照组比较有统计学意义(P<0.05)。实验结果表明LMWH能从基因和蛋白水平抑制VEGF的表达,并呈明显的量效关系。结论：1、LMWH能直接抑制人非小细胞肺癌A549细胞增殖,并呈剂量-效应和时间-效应依赖关系。2、LMWH在基因转录水平和蛋白翻译水平均可下调A549细胞VEGF的表达,提示LMWH具有抗血管生成作用。在抑制肺癌细胞增殖的同时可能抑制肿瘤血管的生成,因而可能对肺癌细胞的侵袭、转移潜能有抑制作用。通过本实验,进一步证实了LMWH具有较好的抗癌作用,并初步揭示了其作用机制,为临床应用LMWH治疗肺癌提供了理论依据。更多还原

【Abstract】 [Background]Lung cancer is one of the most common malignant tumor which seriously threaten human life and health in today’s world. Lung cancer has become the leading cause of death. Of about 80% patients is non-small-cell lung cancer (NSCLC) of which diagnosed at advanced stage, unresectable about 80%.The treatment of NSCLC include surgery, chemotherapy, radiotherapy and molecular targeted therapy, whatever the treatment requires a effective index of guide and judge for the treatment of NSCLC.TNM staging are widely used as the current indicators to evaluate the treatment and prognosis of NSCLC,which provide some guidance value. However, in clinical work, a part of NSCLC patients who although have been the early diagnosis and treatment appeared in the short term recurrence and metastasis, The biological behavior of tumor characteristics of these patients are no doubt more aggressive side. Therefore, it is obviously not enough only depending on TNM staging of lung cancer.Studies have found that tumor growth, invasion and metastasis are dependent on angiogenesis processes, which are closely related to the prognosis of cancer. The same as ohter solid tumors, turmor angiogenesis provide nutrients to promote tumor growth, tumor invasion and metastasis. Tumor angiogenesis involves a variety of different gene mutations and expression of cytokines and their receptors and is regulated both positive and negative factors. In tumor tissues, as pro-angiogenic factors are dominant. Vascular endothelial growth factor (VEGF) is widely recognized as the most important angiogenic factors, and plays an important role in the promotion of tumor microvessels and Lymhangiogenesis. Therefore, the understanding of lung VEGF expression and angiogenesis may help clarify the mechanism of growth and metastasis of lung cancer. Heparins may inhibit endothelial cell proliferation and inhibit angiogenesis, prevent cell adhesion, proliferation and migration, and blocking the violations of tumor from vessels and tissues.Up to now, there have been few reports on the expression of VEGF and microvessel density (MVD) in NSCLC, as well as their relations with the biological behavior and prognosis of NSCLC. The specific anti-tumor mechanism of heparin has not been entirely clarified. In this study, microvessel density (MVD) and VEGF expression were observed in non-small cell lung cancer (NSCLC) by immunohistochemical staining to explore the relationship of MVD/VEGF expression and NSCLC biological behavior and their effects on the prognosis of NSCLC. In this study, MTT and fluorescence quantitative PCR (real-time PCR) were performed to investigate the role of LMWH on VEGF expression in human NSCLC A549 cells. This study aimed to provide theoretical basis for the clinical application of LMWH in NSCLC. PartⅠExpression of VEGF and the Correlation with Angiogenesis in Non-small-cell Lung CancerObjective:To investigate the expression of vascular endothelial growth factor (VEGF) in NSCLC (non-small cell lung cancer) tissues, and study the correlations between the expression of VEGF, the angiogenesis and clinical path physiological characteristics of NSCLC.Methods:43 specimens of NSCLC were the surgical cases who were diagnosed pathologically from January 1996 to January 1999 in the cardio-chest surgical department of the first affiliated hospital of Bengbu Medical College. Immunohistochemical S-P method was used to detect the expression of VEGF and MVD in the organization, So as to provide theory basis for clinical diagnosis and treatment. All data presented were expressed as mean±SEM and analyzed by SPSS version 13.0 statistical software. Data were analyzed by one-way analysis of variance (ANOVA) and student t test. And the data from categorical variables were analyzed by x2 test.Rusults:1. The VEGF-positive rate was 53.50%(23/43) in NSCLC; The MVDs were 31.98±14.93 in NSCLC.2. The positive expression rate of VEGF was 59.4%(18/33) in squamous cell carcinoma and 50%(5/10) in adenocarcinoma, the MVDs were 35.50±15.28 in adenocarcinoma and 30.91±15.25 in squamous cell carcinoma respectively, There was no significant difference among them (P＞0.05).3. The VEGF-positive rates were 12.50%,66.67%和91.67%, the MVDs were 21.25±7.86,32.87±15.27和45.17±11.49 in stageⅠ,ⅡandⅢof NSCLC, The differences of VEGF expression and the MVDs between stageⅠ,ⅡandⅢwere significant respectively (P＜0.01).4. the positive expression rates of VEGF were 84%(21/25) and11.11%(2/18), the MVDs were 39.56±14.73和21.44±7.44 in the group with lymph node metastasis and the group without lymph node metastasis. Between the two groups, the differences of VEGF expression and the MVDs were significant respectively (P＜0.01).5. The average MVD (42.83±11.915) in VEGF-positive expression group was significantly higher than the average MVD (19.50±5.726) in VEGF-negative expression group (p＜0.01)Conclusions:1. There are high expressions of VEGF in NSCLC; VEGF expression is significantly related to tumor size, TNM clinical stage and lymph node metastasis in NSCLC.2. MVD was closely related to the degree of histology, lymph node metastasis and TNM in NSCLC. MVD was closely related to malignancy and metastasis of NSCLC.3. The positive expression of VEGF and MVDs in NSCLC was significantly positively correlated, VEGF positively regulate angiogenesis in NSCLC, to target VEGF or its receptors will be expected to be a new treatment approach of NSCLC. PartⅡVEGF Expression、Microvessel Density Related to Prognosis of Human Non-small-cell Lung CancerObjective:To investigate the correlations between the expression of VEGF, the angiogenesis and prognosis of human NSCLC, in order to provide theory basis for clinical diagnosis, prognosis and treatment.Methods:43 specimens of NSCLC were the surgical cases who were diagnosed pathologically from January 1996 to January 1999 in the cardio-chest surgical department of the first affiliated hospital of Bengbu Medical College. Immunohistochemical S-P method was used to detect the expression of VEGF and MVD in the organization The survival time were analyzed by Kaplan-meier following Log-rank test. COX model (likelihood ratio test) was used for the multivariate prognostic analysis. A level of p＜0.05 was considered statistically significant.Rusults:1. Kaplan-Meier analysis showed that survival time of NSCLC patients was significantly correlated with TNM clinical stage, histological grade, lymph node metastasis and tumor size. Furthermore, the mean survival time and median survival time of NSCLC in high MVD expression group and VEGF-positive expression group were significantly lower than that in low MVD expression and VEGF-negative expression group.2. COX model was used to analyze the multivariate prognosis of the patients’ age, sex, TNM clinical stage, and MVD/VEGF expression. Results showed that MVD was an independent factor affecting NSCLC prognosis. The relative death risk of patients with high MVD expression was 34.247-fold that of patients with low MVD expression (likelihood ratio test, p＜0.01)Conclusions:The expression of VEGF, MVDs and 5-year survival rate of NSCLC is closely related. Detection of VEGF and MVD may serve as a major parameter to judge the angiogenesis, invasion and metastasis of NSCLC and prognosis of NSCLC, MVD was independent prognostic predictors of NSCLC. Part III Effects of Low molecular weight heparin on VEGF Expression of Non-small Cell Lung Cancer A549 Cell LineObjective:To investigate the effects of the expression of VEGF in A549 cells treated by LMWH, to discuss the influence of the potency of invasion and metastasis of lung cancer cells, and to provide the experimental evidence of using LMWH, in the recess of prevention and therapy Non-small Cell Lung Cancer.Materials and Methods:1.The influence of cell proliferation on Non-small Cell Lung Cancer A549 cell line by LMWH. A549 cells were treated singly at the dosages of 0,5,15,50,150,500IU/ml, for 1～2day respectively. Thereafter, the proliferation of A549 cells was assessed by MTT method.,2. The effects of the expression of VEGF on A549 cells treated by LMWH.A549 cells were treated at different concentrations of LMWH. Then VEGF were detected by Western blotting, and levels of VEGF mRNA were analyzed by SYBR-PCR amplification.Results:1.Being treated with LMWH could obviously inhibit the growth of A549 cells, and the secession effects would be enhanced following increasing dosage or extending treated time. When the concentration of LMWH was between 5IU/ml and 150IU/ml, the MTT assay at 24 hours and 48 hours showed a significant inhibitory action of LMWH, which can inhibit the proliferation of A549 in dose-dependent and time-dependent manner in vitro.2. After being treated with different concentrations of LMWH, level of VEGF mRNA was be obviously decreased (vs. control group, P＜0.05). Level of VEGF protein deeply depressed down-regulated. (Vs. control group, P＜0.05)Conclusions:1.To treat A549 cells with LMWH would obtain regression of cells proliferation.2.LMWH could redress the expression of VEGF which could encouraging tumor vessel angiogenesis, thus to treat A549 cells with LMWH might sores the ability of lung cancer cells to invasion and metastasis.更多还原