摘要
肿瘤分子生物学研究在最近十余年来取得了巨大的进展,让我们能够更好地理解包括肺癌在内的人类恶性肿瘤的发病机理。人类基因序列测定已鉴定出许多原癌基因和肿瘤抑制基因,这对更好认清肺癌的形成、进展、转移及药物耐药等等分子事件。另外,在肺癌中还发现了许多重要的信号转导途径。新颖的抗癌药物不断更新,包括靶向治疗路径的鉴定与进展,一种或多种交叉的不同信号转导路径参与。联合传统的细胞毒类的化疗药物(非靶向)与生物治疗(靶向),提高肺癌患者,尤其是进展期的非小细胞肺癌的疗效及生存期,这一直是目前肺癌研究的热点。
肺癌一直是人类肿瘤相关死亡中首要原因。尽管过去几十年进行了大量的基础及临床研究,但是肺癌的5年生存率仍很低。进展期非小细胞肺癌的治疗在症状改善、生活质量的提高、生存期延长方面取得了一定的进步。以铂类为基础的联合化疗是当前标准治疗,反应率在30%至40%,平均生存期为8至9个月,尽管应用了更新的化疗药,如taxanes,gemcitabine,vinorelbine等,35%的进展期患者生存期为1年。
许多随机的Ⅲ期临床试验未能得出某一种铂类药治疗更有优势,因此肺癌治疗的药物发展在最近几年就转到了靶向生物治疗。这些研究中的药物作用于表皮生长因子受体、新生血管、信号转导途径、免疫治疗、疫苗及基因治疗等。靶向治疗为进展期非小细胞肺癌患者治疗带来了新的希望,研究高效、低毒的靶向治疗药物,提高生存期具有重要的现实意义。
不同恶性肿瘤中的微血管存在不同特征,不同肿瘤中新生的微血管的数量、形态、结构及构建也各不相同。由于缺少评估肿瘤微血管形态及分布的统一标准,肿瘤血管可能成为诊断肿瘤分化的标志,但这常常未引起病理专家的重视。新生肿瘤血管壁可能很厚或很薄,同一肿瘤进展过程中的不同时期,血管常常有形态学改变,这在肿瘤发生转移时更加明显。尽管不同肿瘤源性的内皮细胞(Tumor-derived endothelialcells,TEC)表达内皮细胞特异的标记,但是基因谱研究证实TEC与来源于肿瘤周围正常组织的内皮细胞存在很多差异。免疫治疗靶向针对血管内皮细胞抗原逐渐成为一种具有光明前景的抗新生血管生成治疗方法。长期以来缺乏TEC分离培养体系及此类细胞系,体外研究肿瘤血管生成时一直都使用源于正常血管内皮细胞甚至内皮细胞样细胞系。培养非小细胞肺癌血管内皮细胞(NSCLC-derived vascular endothelialcells,NSCLC-VECs)对研究肺癌的发生、浸润、转移以及开发新药均具有重要价值。
因此,本课题采用人非小细胞肺癌作为材料来源,摸索和建立NSCLC-VECs的分离培养方法,研究其体外生长特性。研究抗人血管生成素相关和肿瘤转移相关蛋白抗体(Human Angiostatin Interacting and Tumor Metastasis Invovling Protein,HAI-TMIP)对非小细胞肺癌来源的血管内皮细胞的增殖、迁移、凋亡能力的影响及新生血管的影响;初步探讨抗HAI-TMIP抗体对NSCLC-VECs作用机制,以期提供一种治疗非小细胞肺癌的备选抗体。
研究目的
摸索并建立人NSCLC-VECs的分离培养方法,研究其体外生长特性;研究人NSCLC-VECs中ATPaseF1α的表达;研究抗HAI-TMIP抗体对NSCLC-VECs的增殖、迁移、凋亡能力的影响及新生血管的影响;初步探讨抗HAI-TMIP抗体对NSCLC-VECs作用机制及相关的临床意义。
研究方法
1利用酶消化法分离出肺癌组织微血管片段,采用植块培养法培养原代内皮细胞,先后以局部消化法和差速黏附法进行纯化;应用光镜、免疫细胞化学及免疫荧光技术对所获得的人肺癌血管内皮细胞进行鉴定。
2采用细胞免疫荧光技术检测了NSCLC-VECs膜表面ATPaseF1α的表达情况;应用RT-PCR法、免疫印迹法(Western blotting)检测了ATPase基因在NSCLC-VECs中的表达情况。
3通过利用细胞增殖实验(CCK-8 assay)检测抗HAI-TMIP抗体抑制NSCLC-VECs的增殖作用;利用划痕愈合试验(Wound-healing assay)检测抗HAI-TMIP抗体抑制NSCLC-VECs迁移速度;利用FACS检测抗HAI-TMIP抗体对NSCLC-VECs凋亡的影响;利用鸡胚CAM实验(Chick embryo CAM assay)检测抗HAI-TMIP抗体抑制鸡胚新生血管生成。
实验结果
1所获得的人NSCLC-VECs呈FⅧ-RAg、CD34阳性;电镜下生长状态良好,原代生长缓慢,3～4周生长至融合,传代细胞第3～8d进入对数生长期,10d进入平台期。NSCLC-VECs传6～8代后细胞逐渐死亡。
2 NSCLC-VECs的RT-PCR扩增产物电泳主要显示2个清晰条带:β-actin条带和ATPaseF_1α条带。抽提出的NSCLC-VECs与正常支气管上皮细胞株的细胞膜蛋白经Dot-blot检验证实为细胞膜蛋白,排除了线粒体膜蛋白的干扰,免疫印迹法(Westernblottting)显示NSCLC-VECs胞膜有ATPascF1α的表达,而正常支气管上皮细胞株细胞膜上无ATPaseF1α表达。
3抗HAI-TMIP抗体、抗α亚单位抗体(阳性抗体对照)组中NSCLC-VECs的增殖抑制率均显著低于空白对照组(P＜0.05),两抗体组间无统计学差异(P＞0.05);与对照组比,抗HAI-TMIP抗体显著抑制NSCLC-VECs迁移速度(P＜0.05),随抗HAI-TMIP抗体浓度增加,抑制效果越明显(P＜0.05);加入抗HAI-TMIP抗体的NSCLC-VECs的凋亡率显著高于空白对照组(P＜0.05),与阳性对照组相比无统计学差异(P＞0.05)。
4与空白对照组比较,镜下抗HAI-TMIP抗体明显抑制鸡胚尿囊膜新生血管生成。
结论
首次成功建立人NSCLC-VECs的分离培养方法,获得了NSCLC-VECs部分生长特性,为研究非小细胞肺癌的侵袭、转移、进展等相关研究提供了直接实验材料。ATPaseF1α在NSCLC-VECs中有相对较高的表达水平。抗HAI-TMIP抗体可抑制NSCLC-VECs的增殖、迁移及新生血管的生成;抗HAI-TMIP抗体可增加NSCLC-VECs凋亡。因此抗HAI-TMIP抗体可能是靶向治疗非小细胞肺癌的抗体之一。
Recent progress in molecular biology has enabled us to better understand the molecular mechanism underlying pathogenesis of human malignancy including lung cancer.Sequencing of human genome has identified many oncogenes and tumor suppressor genes,giving us a better understanding of the molecular events leading to the formation,progression,metastasis,and the development of drug resistance in human lung cancer.In addition,many signal transduction pathways have been discovered that play important roles in lung cancer.Novel strategy of anti-cancer drug development now involves the identification and development of targeted therapy that interrupts one or more than one pathways or cross-talk among different signal transduction pathways.In addition, efforts are underway that combine the traditional cytotoxic(non-targeted) agents with the biological(targeted)therapy to increase the response rate and survival in patients with lung cancer,especially advanced non-small cell lung cancer(NSCLC).
Lung cancer is the leading cause of cancer related death in both men and women.Despite decades of basic and clinical research,the 5-year survival of lung cancer is still low.The treatment of advanced NSCLC is largely palliative in order to achieve symptom control,better quality of life,and a small survival gain.Platinum based combination chemotherapy remains the current standard treatment,with a response rate of 30 to 40%,median survival of 8 to 9 months and 1-year survival of 35%in patients with advanced disease,despite the use of newer agents such as taxanes,gemcitabine and vinorelbine.
Manv randomized phaseⅢclinical trials have failed to demonstrate the superiority of any one platinum doublet over another.Therefore the focus of drug development in the treatment of lung cancer has shifted in recent years to the development of targeted biological therapy.This includes agents that affect the function of the epidermal growth factor receptor(EGFR),angiogenesis,signal transduction pathways,immunotherapy, vaccines and gene therapy.Targeted therapy has generated new hope that better treatment with higher efficacy and lower toxicities in specific patient population will ultimately lead to increased survival in patientscially advanced NSCLC.
Microvessels in malignant tumors exhibit different features.Newly formed microvessels in different tumors vary in number,morphology,structure and architecture. Due to the lack of defined criteria for assessing the morphology and distribution of tumor microvessels,pathologists often ignore tumor vessels that in contrast may serve as markers for differential diagnosis.The wall of newly formed microvessels can be either thick or thin.At different stages of the progression of a single tumor,vessels usually undergo a series of morphological changes which become even more evident when metastasis occurs.Endothelial cells derived from tumor vessels(TECs) exhibit differences from those from surrounding normal tissues as demonstrated by a global gene profiling study despite their expression of EC-specific markers.Immunotherapy targeting tumor vascular EC antigens is emerging as a promising antiangiogenesis therapy.The isolation and culture of TECs have been not established up to now.In the study of angiogenesis in vitro,normal vascular endothelial cells or endothelial cell-like cell lines were used.So,culturing NSCLC-derived vascular endothelial cells(NSCLC-VECs) has an important value in studying tumorigenesis,tumor infiltration,metastasis of NSCLC and developing new drugs.
So,we established the method for isolating and culturing NSCLC-VECs and to observe their characteristic in vitro studied the effects of HAI-TMIP antibody on human NSCLC-VECs and its related mechanism.Maybe,it will turn out a new potential antibody to target for the treatment of non-small cell lung cancer.
Objective
To establish the method for isolating and culturing NSCLC-VECs and to observe their characteristic in vitro.To detect the expression of ATPaseF1αin NSCLC-VECs.To investigate the suppressing effect of Human Angiostatin Interacting and Tumor Metastasis Invovling Protein(HAI-TMIP) antibody on proliferation activity,metastasis activity of NSCLC-VECs and vascular tube formation in the chick embryo chorioallantoic membrane (CAM) assay,and to study the enhancing effect of HAI-TMIP antibody on apoptosis of NSCLC-VECs.To discuss the related mechanism of the effects of HAI-TMIP antibody on NSCLC-VECs.
Methods
1.Enzyme method was used to isolate mierovascular fragments from human non-small cell lung cancer tissue,and tissue nubbles cultivation was used to isolate mierovascular endothelial cells,then purified by local digestion and differential conglutination.NSCLCDECs were identified by optical microscope, immunocytochemistry and immunofluorescence.
2.The membrane expression level of ATPaseF1αwas examined by immunofluorescence in NSCLCDECs.The expression of ATPaseF1αmRNA of NSCLCDECs was exhibited by real time PCR.The expression of ATPaseFla peptide on the pericellular membrane of NSCLCDECs was observed by immunocytochemistry technique.We utilized sucrose density gradient centrifugalization in order to dissociate plasma membrane fraction and extracted pericellular membrane protein by the way of Pierce—Kit reforming method.The extracted protein was validated by COX-1 MoAb so as to deplete the interference of mitochondrial membrane proteinum.Then the distinction of the pericellular membrane expression of ATPaseF1αbetween NSCLCDECs and normal tracheal epithelium cell line was detected by Western blotting assay.
3.The suppressing effect of HAI-TMIP antibody on proliferation of NSCLCDECs is evaluated by CCK-8 assay,and the similar effect on metastasis activity is surveyed by Wound-healing assay.FACS was used to detect the enhancing effect of HAI-TMIP antibody on apoptosis of NSCLC-VECs.The suppressing effect of HAI-TMIP antibody on vascular tube formation in the chick embryo chorioallantoic membrane (CAM) assay were identified by optical microscope.
Results
1.The NSCLCDECs expressed FⅧ-RAg,CD34.They grew well found by optical microscope and could be passaged in vitro.Its growth curve show S-shape.
2.Immunofluorescence analysis showed midrange and higher expression of ATPaseF1αin the membrane of NSCLCDECs.Positive ATPaseF1αmRNA expression was confirmed by RT-PCR in NSCLCDECs.The amplification production electrophoresis of RT-PCR displayed two distinct strap:β-actin and ATPaseF1α.The conspicuous positive expression of ATPaseF1αpeptide on the pericellular membrane of NSCLCDECs was irrefutably observed by immunocytoehemistry technique.The extracted protein was validated by COX-1 MoAb so that we confirm the authenticity of the above extraction of pericellular membrane proteinum.Western blot assay showed that there is positive expression of ATPaseF1αin the pericellular membrane protein of NSCLCDECs while negative expression in the normal tracheal epithelium cell.
3.Compared with blank control group,the result of CCK-8 assay demonstrate that HAI-TMIP antibody showed predominance depressant effect on cell proliferation.The result of Wound-healing assay revealed that HAI-TMIP antibody compared with blank control group obviously restrain the migration of NSCLCDECs(P＜0.05).The proliferation activity and metastasis activity would lysis along with the augmentation of HAI-TMIP antibody concentration(P＜0.05).It presented a dose-dependent effect(P＜0.05).The apoptosis rate of NSCLCDECs in HAI-TMIP antibody group was higher than blank control group(P＜0.05).There were no significant differences between HAI-TMIP antibody group and positive control group(P＞0.05).
4.Compared with blank control group,HAI-TMIP antibody obviously blocked vascular tube formation in the chick embryo chorioallantoic membrane(CAM) assay. Conclusions
A practical method was first established for isolating NSCLCDECs and is useful in the studies of tumor angiogenesis.The pericellular membrane expression of ATPaseF1αin NSCLCDECs is relatively higher while negative expression in the normal tracheal epithelium cell.HAI-TMIP antibody may repress the proliferation and migration of NSCLCDECs and make them undergo apoptosis.HAI-TMIP antibody also can inhibit vascular tube formation.HAI-TMIP antibody can be studied for further antiangiogenic characteristics in vitro and in vivo and could be used to prevent tumor angiogenesis.Maybe, it will turn out a new potential antibody to target for the treatment of non-small cell lung cancer.
引文
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