蛋白质组学技术差异蛋白单克隆抗体用于肺癌放射免疫显像的实验研究
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摘要
本课题将分子生物学、免疫学及核医学技术相结合,利用蛋白质组学(proteomics)技术比较研究肺腺癌细胞系(A549)与正常支气管上皮细胞系(HBE)之间的差异表达蛋白,筛选出部分在癌细胞中高表达的特征性膜蛋白,并利用免疫组化及western-blot技术对所选差异蛋白在肺腺癌组织中的表达情况进行检测。将筛选出的在肺腺癌细胞及癌组织中均高表达的蛋白质作为肺腺癌的特异性抗原,利用放射性核素99mTc标记其单克隆抗体(monoclonal antibody, McAb),进行肺腺癌的放射免疫显像(radioimmunoimag, RII)研究,可望建立一种分子水平早期诊断肺癌的影像检查方法。
近年国内外应用放射性核素标记肺癌相关抗原的McAb进行了广泛深入的动物实验及临床研究,但肺癌的RII研究进展较慢,主要原因之一就是抗原的因素,因肺癌缺乏特异性的抗原,抗原性较弱,抗原易突变等。因此寻找肺癌特异性、稳定高表达的抗原是肺癌RII显像的关键之一。
蛋白质是基因的体现者,是体内各种功能的真正执行者,其成分和功能的异常都会导致机体出现异常。体内各种紊乱或疾病(包括肿瘤)的发生、发展及转归都与蛋白质的异常有关。因此对机体(特定组织或细胞)的全部表达蛋白质进行研究(即蛋白质组学),有利于阐释肿瘤发生、发展的分子机制,寻找肿瘤特异性标志物及阐明药物治疗机理、确定新的药物治疗靶点等,在肿瘤的早期诊断、早期治疗方面具有广阔的应用前景。本课题采用蛋白质组学技术筛选出在肺腺癌细胞中特异性高表达的膜蛋白作为目标抗原,为肿瘤RII研究提供了一条新的思路,必将大大促进肺癌RII研究的进一步发展。
肿瘤细胞系由于样品制备相对容易,并在膜蛋白、信号传导通路及分泌蛋白等的研究中具有较大的优势,其已成为蛋白质组学良好的研究模型。从肺癌患者癌组织中分离出来的非小细胞肺癌细胞系(如A549细胞),与患者体内的癌细胞具有基本相似的生物学特性,因此对其蛋白质组水平的研究,有助于阐释肺癌发生、发展的分子机制,寻找到肺癌组织中特异性表达的蛋白质及诊断、治疗靶点等。
通过细胞比较蛋白质组学技术筛选、鉴定出来的差异表达蛋白质,是否在活体组织内也存在相同或类似的表达差异,需要进一步验证。免疫组化及western-blot是检测肿瘤组织中特异性表达蛋白较常用的方法。本研究采用此两种技术对肺腺癌组织及癌旁正常组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)和CD44蛋白(通过蛋白质组学技术鉴定出的在肺腺癌细胞系A549中特异性高表达的膜蛋白)的表达差异进行鉴定,一方面可验证两种蛋白在人肺腺癌组织及癌旁正常组织中是否也存在类似的表达差异,另一方面,确定差异表达蛋白(目标抗原)的存在,也是保证肺癌RII研究成功的必要条件。
McAb的标记及标记抗体的特性也是影响RII结果的主要因素。本研究选择物理特性理想的99mTc为标记核素,采用直接标记法标记McAb,并通过改变不同的标记要素筛选出最佳标记条件。对标记后的McAb,我们采用硅胶薄层层析法(TLC)测定其标记率、放射性比活度、放射化学纯度,对标记抗体的物理特性进行鉴定；通过室温下放置、加入不同竞争试剂及与血清混合放置等方法了解标记抗体的体外、体内稳定性；通过免疫荧光法对标记抗体的免疫活性进行判断。
由于人体肿瘤抗原的表达存在异质性,有些病人的某种肿瘤抗原不表达或表达量很少,而另一种肿瘤抗原可能表达量很多,如给予单一抗体进行RII则可能造成假阴性,将抗同一肿瘤不同抗原的几种单抗混合在一起同时给药后进行显像,即抗体的“鸡尾酒”法,可增加肿瘤部位的放射性聚集(提高靶/非靶比值),有利于减少假阴性的存在。本课题以针对肺癌组织两种特异性抗原(EGFR和CD44)的标记McAb分别进行单独及联合显像,并对显像效果进行对比分析。
方法
第一部分肺腺癌细胞系A549与正常支气管上皮细胞系HBE的比较蛋白质组学研究
取对数生长期的A549细胞和HBE细胞分别经细胞裂解后进行总蛋白的提取,并采用Bradford法进行蛋白定量。
将提取的蛋白质样品直接上样到IPG预制胶条(18cm pH3-10)上,先后放入IPGphor等电聚焦仪、垂直板电泳仪中进行双向凝胶电泳,将细胞总蛋白按等电点不同及分子量不同区分开。将二维电泳后的凝胶采用银染显色后,用ImageScanner光学扫描仪进行扫描,得到蛋白质点图像,并采用ImageMaster2D Elite5.01凝胶图象分析软件进行分析,了解A549细胞和HBE细胞总蛋白在2-DE图谱上的表达差异情况。
挖取部分仅在A549细胞中特异性表达或在A549细胞中表达量明显上调的蛋白质点,经酶解后,进行生物质谱分析,得到这些蛋白质点的肽质量指纹图谱。通过数据库搜寻,鉴定出这些在A549细胞中特异性或高表达的蛋白质。
对鉴定出的A549细胞特异性表达蛋白进行分析,了解它们在细胞中存在的部位及相关功能。并从中选择两种以上细胞膜蛋白作为本研究中RII研究的目标抗原。
第二部分免疫组化及western-blot对差异蛋白EGFR和CD44在肺腺癌组织中表达的检测
通过第一部分对A549细胞和HBE细胞的比较蛋白质组学研究,筛选出了一些在癌细胞中特异性表达或高表达的蛋白质,通过对这些蛋白质在细胞内的分布部位及功能等进行分析后,我们拟选择EGFR和CD44这两种膜蛋白进行后续的RII研究。因此采用了免疫组化及western-blot技术对两种蛋白在肺腺癌组织中是否也存在特异性、高表达进行检测。
1.取新鲜肺腺癌组织及癌旁正常肺组织(距肿瘤组织至少5cm以上),分别经石蜡包埋后行组织切片,并采用免疫酶组化SABC三步法进行免疫组化分析,酶选择辣根过氧化物酶,以DAB为显色剂。将显色后的癌组织及癌旁组织切片分别置于光学显微镜下观察,每组随机观察5个高倍视野,观察细胞染色情况,对于EGFR及CD44两种蛋白均以瘤细胞的胞膜及胞浆出现棕黄色,且着色强度高于背景非特异性染色者判断为阳性,并采用计数分析软件,计算阳性细胞的百分数。
2.western-blot分析：首先配置SDS-PAGE凝胶,并进行封胶、灌胶。然后从液氮中取已制备的肺腺癌组织及配对癌旁正常肺组织样品(共4组),分别进行蛋白质的裂解与定量后,直接上样进行PAGE。根据EGFR及CD44的相对分子量选择PAGE凝胶区域进行转膜,将蛋白质转移到NC膜上。然后分别加入两种蛋白的特异性一抗及HRP标记的二抗,最后加入HRP的底物DAB进行显色,并以β-actin为内对照,分析EGFR及CD44两种蛋白在癌组织及癌旁组织中的表达差异。
第三部分抗EGFR、CD44单克隆抗体的99mTc标记及标记抗体特性的鉴定
以2-巯基乙醇(2-ME)为还原剂,含SnCl2的亚甲基二膦酸盐(MDP)为弱结合配体,采用99mTc直接标记法分别标记EGFR-McAb、 CD44-McAb,通过改变不同的标记条件(如改变还原剂、弱结合配体的剂量,还原时间、温度,标记反应时间等),筛选出最佳标记条件。利用SephadexG50柱对标记抗体进行纯化,硅胶薄层层析法测定两种标记抗体的标记率、比活度及放射化学纯度。通过室温下放置不同时间及加入不同99mmTc竞争试剂后观察放射化学纯度的变化,了解标记抗体的体外稳定性,通过标记抗体与血清混合放置不同时间后放射化学纯度的变化情况,了解其体内稳定性。采用免疫荧光法对标记抗体免疫活性进行判断。
第四部分99mTc-EGFR-McAb及99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠的体内分布及SPECT显像研究取对数生长期的肺腺癌A549细胞,接种于裸鼠的前肢(左或右)外侧皮下(每只裸鼠接种A549细胞1.0×107/0.1m1),建立荷人肺腺癌裸鼠动物模型。将荷瘤裸鼠随机分为3组,分别经尾静脉注射99mTc-EGFR-McAb、99mTc-CD44-McAb及以上两种混合标记抗体,进行两种标记抗体单独及联合应用在荷人肺腺癌裸鼠的体内分布及单光子发射型计算机断层显像(single photon emission computed tomography,SPECT)研究。
结果
第一部分肺腺癌细胞系A549与正常支气管上皮细胞系HBE的比较蛋白质组学研究
对人肺腺癌细胞系A549和人正常支气管上皮细胞系HBE的总蛋白经2-DE分离及银染显色后的图谱分析,两种细胞的凝胶图上分别得到平均蛋白质点897±35个及882±29个。以HBE细胞的平均胶为参照胶,将其与A549细胞的平均胶进行匹配,匹配率为82.3%。通过ImageMaster2D Elite5.01凝胶图象分析软件分析,两种细胞的差异表达蛋白质点256个,表达量差异在三倍以上的点98个,其中仅在A549细胞中表达的蛋白质点15个,仅在正常组织中表达的蛋白质点21个。
通过图像分析软件及肉眼观察,选择在A549细胞中表达明显上调(与在HBE细胞中的表达量差异在3倍以上)的蛋白质点9个进行质谱分析,获得蛋白质点的肽质量指纹图谱(peptide mass fingerprint, PMF)。利用Expasy中的Peptident查询软件应用上述的PMF数据搜索SWISS-PROT数据库,结果未搜索到匹配的蛋白质2个,另7个蛋白质点匹配的蛋白质分别为表皮生长因子受体(epidermal growth factor receptor, EGFR)，MDM2蛋白(murine double mimute2),CD44蛋白,细胞骨架蛋白CK8,不均一性核糖体蛋白H(heterogeneous nuclear ribonucleoprotein,hnRNP H),热休克蛋白60(heat shock proteins60,HSP60),磷酸甘油酸变位酶1(phosphoglycerate mutase1,PGAM1)。
对以上7种蛋白进行分析,可分为：信号传导分子,如EGFR、CD44蛋白；细胞骨架蛋白,如CK8；转录及翻译相关蛋白,如MDM2蛋白、hnRNP H;分子伴侣,如HSP60；与基本代谢相关的的酶类,如PGAM1。根据这些蛋白在细胞内的位置及分子特点,我们拟选择细胞膜蛋白EGFR、CD44蛋白进行后续RII研究。
第二部分免疫组化及western-blot对差异蛋白EGFR和CD44在肺腺癌组织中表达的检测
免疫组化结果显示,EGFR和CD44在肺腺癌组织的细胞膜及细胞浆内均有较强的表达,阳性表达细胞的胞膜及胞浆均呈棕黄色,而正常肺组织中细胞的胞膜及胞浆大多不着色。通过对阳性细胞计数分析,EGFR和CD44在肺腺癌组织中的表达水平明显高于癌旁正常肺组织。
以β-actin为内对照,采用western-blot法分别检测EGFR和CD44两种蛋白在肺癌组织及其配对的癌旁正常组织中的表达情况,结果表明,与配对癌旁正常肺组织比较,EGFR和CD44在肺腺癌组织中的表达有明显增强。
第三部分抗EGFR、CD44单克隆抗体的99mTc标记及标记抗体特性的鉴定
采用99mmTc直接标记法标记抗EGFR-McAb、CD44-McAb,经SephadexG50柱纯化后,用硅胶薄层层析法测定两种标记抗体的标记率分别为91.5%±3.8%、92.3%±4.1%,比活度分别为2.8±0.3MBq/μl、2.9±0.5MBq/μl及放射化学纯度分别为96.5%±2.8%、96.2%±3.1%。标记抗体的以上物理特性能满足放射免疫显像的要求。
通过改变不同的标记条件,观察抗体标记效果发现,还原剂2-ME的用量以其与抗体的摩尔比为2000：1最佳,抗体的还原时间不宜超过30min,标记时间以20min为宜,而标记反应的温度,弱结合配体的用量等对抗体标记率的影响不大。
标记抗体在室温下稳定性分析显示,标记抗体的放射化学纯度随放置时间延长略有下降,但24h均值仍大于85%。标记抗体与新鲜人血清37℃孵育后稳定性分析显示,4h后标记抗体的放射化学纯度略有降低,但仍超过88%。通过加入不同99mTc竞争试剂后观察标记抗体的体外稳定性发现,加入分子上含-SH的竞争试剂(如半胱氨酸)对标记抗体的稳定性影响较大。
通过免疫荧光法检测两种标记抗体的免疫活性,结果显示,99mTc-EGFR-McAb和99mTc-CD44-McAb免疫活性与标记前比较无明显降低。
第四部分99mTc-EGFR-McAb及99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠的体内分布及SPECT显像研究
99mTc-EGFR-McAb,99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠体内的分布实验显示,肿瘤部位的放射性摄取及瘤/血、瘤/肌肉的放射性比值均随注射后时间的延长逐渐增高,在16h达高峰,以后有所降低(16h各组的瘤/血、瘤/肌肉比值分别为3.55±0.58、16.45±4.28,3.37±0.42、12.85±3.62,5.15±0.68、23.85±5.86)。通过组间比较发现,混合抗体组肿瘤部位的放射性摄取及瘤/血、瘤/肌肉比值均明显高于两种标记抗体单独使用组(p<0.05),两种标记抗体单独使用组间则无显著性差异(p>0.05)。对裸鼠各主要脏器的放射性摄取分析显示,心、肝、脾、肺等血运丰富的脏器放射性摄取相对较多,而胃、肠、肌肉、骨骼、脑等放射性摄取相对较少,但均随时间的延长,放射性摄取均逐渐降低。
通过99mTc-EGFR-McAb和99mTc-CD44-McAb单独及联合应用对荷人肺腺癌裸鼠的SPECT显像研究发现,各组裸鼠的早期影像中(2-4h),肿瘤显示不佳,而血中放射性本底较高,肝、脾等显影明显。到注射标记抗体后16h,肿瘤部位有较多的放射性分布,显示最佳,而血中本底及肝、脾等放射性分布则明显降低。通过ROI技术测得16h各组的T/NT比值分别为2.74,2.29和5.53,组间比较显示,联合抗体组的T/NT比值明显高于一种抗体单独使用组(p<0.05)。以上显像结果与体内分布实验结果基本一致。
结论
本研究通过比较蛋白质组学技术,得到A549细胞系和HBE细胞系差异表达蛋白质点256个,初步鉴定了EGFR、MDM2蛋白、CD44蛋白、细胞骨架蛋白CK8、hnRNP H、HSP60、PGAM1共7种蛋白质在肺腺癌细胞系A549中存在特异性高表达,并用免疫组化及western-blot技术验证了EGFR、CD44两种膜蛋白在肺腺癌组织中也同样存在高表达。因此我们认为此两种膜蛋白可作为肺癌放射免疫显像研究的目标抗原。
采用99mTC直接标记法对EGFR、CD44两种膜蛋白的单克隆抗体进行标记,得到的标记抗体标记率、放射化学纯度高,比活度适当,能满足放射免疫显像的要求。
99mTc-EGFR-McAb和99mTc-CD44-McAb单独及联合应用于荷人肺腺癌裸鼠的体内分布及RII研究结果均显示,肿瘤部位有较多的放射性浓聚,能得到比较理想的靶/非靶比值。此结果肯定了通过蛋白质组学技术筛选、鉴定出的差异表达蛋白,作为肺癌特异性抗原在RII研究中的价值。另外,通过对比研究,证实标记抗体的联合使用明显优于单独使用,肿瘤的靶／非靶比值可以得到进一步提高。
本研究利用蛋白质组学、western-blot和免疫组化技术筛选、鉴定在肺癌细胞及组织中特异性高表达的蛋白质作为目标抗原,进行肺癌RII研究,为肺癌的RII研究提供了一条新的思路,也必将大大促进肺癌RII研究的发展。
多学科、跨学科的联合研究是未来科学发展的主流,本研究将分子生物学、免疫学技术及其研究成果应用于核医学领域,必将促进各学科的共同发展。
Molecular biology, immunology, and nuclear medicine technology were combined in the study. The comparative study on differential expression protein between human lung adenocarcinoma cell line A549and normal bronchial epithelial cell line HBE was comparatively researched by utilizing proteomics technology. And a fraction of overexpression characteristic membrane protein in cancer cell was screened, and its expression in lung adenocarcinoma tissue was verified by utilizing immunohistochemistry and western-blot technology. The screened overexpressed protein from adenocarcinoma of lung and cancer tissue were selected as a specific antigen of adenocarcinoma of lung, and radioactive nuclide99mTc was adopted to label its monoclonal antibody(McAb) for the study on Radioimmunoimaging(RⅡ). An image method for early diagnosis of lung cancer on the molecular level was expected to be established. It is of economic value and social significance for early diagnosis, early treatment, and raising survival rate of patient.
In recent years animal experiments and clinical researches in depth and in width were implemented through applying radioisotope to the labeling McAb of lung cancer-related antigen at home and abroad. But the study of RII advanced slowly, which partially due to lacking of specific antigen, weak antigenicity, and idiovariation of antigen. So a stable antigen with high expression was a key to lung cancer RII.
Protein was the embodiment of gene and real performer of various functions in vivo, and its abnormality of constituent and function could give rise to organic abnormality. Genesis, development, and turnover of various kinds of disorder or diseases including tumor were concerned with the abnormality of protein. So studying all the expressed protein in organism (or tissue or cell) was conductive to expounding molecule mechanisms for genesis and development of tumor, to searching specific marker of tumor, and to elucidating drug treatment mechanism and ascertaining new drug treatment target. It was a great prospect for early diagnosis and early treatment of tumor. Therefore the study promised a tremendous prospect for early diagnosis and treatment of tumor. Membrane protein with overexpression in lung adenocarcinoma cells was screened as target antigen with the application of proteomics, which created a new way for the RII study, and promoted the further development of RII research for lung cancer.
Due to the easy sample preparation for tumor cell line and its obvious advantages in study on membrane protein, signal transduction pathway and secretory protein, it has become an excellent study model of proteomics. Cell line of non-small cell lung cancer isolated from the cancer tissue of the patients with lung cancer (e.g. A549) had basically similar bionomics of cancer cell in the bodies of patients. Therefore, study on its proteome level was instrumental in elucidating mechanism of carcinogenesis and development of cancer, seeking the protein with specific expression in tissue of lung cancer as well as target of diagnosis and treatment.
Protein with differential expression, screened and identified through cell comparative proteomics technology, was needed to be examined as to whether they also possessed the same or similar expressive difference in living organism tissue. Immunohistochemistry and western-blot were two commonly used means to detect specifically expressive protein. Both of them were adopted in this research to detect the expressive difference of EGFR and CD44(specific overexpression membrane protein in A549detected by proteomics technology) in adenocarcinoma of lung and adjacent normal tissues. On the one hand, the possibility of similar expressive differences existing in adenocarcinoma of lung and adjacent normal tissues was verified. On the other, it was also a necessary condition for ensuring the success of RII research for lung cancer to ascertain the existence of differential expression protein (target antigen).
RII was also influenced by the labeling of monoclonal antibody and the property of the labeled antibody.99mTc, with its ideal physical characteristics, was chose as labeled nuclide in the study. The method of direct labeling was adopted to label monoclonal antibody. The best labeling condition was selected by changing different labeling elements. Silica gel thin-layer chromatography (TLC) was applied to measurement of labeling rate, radio specific activity, and radiochemical purity of the labeled monoclonal antibody and to identifying the physical characteristics of the labeled antigen; the stability in vivo and vitro of the labeled antibody were understood through adding different competitive agentia or mixing with blood serum at room temperature; the method of immunofluorescence was applied to judging the immunocompetence of the labeled antibody.
Due to heterogenicity of tumor antigen expression, some patients had certain tumor antigen with non expression or poor-expression, yet another tumor antigen with much expression, so false negative might be generated by single antigen RII. However, a number of mono-antibodies for different antigens of the same tumor could be mixed and visualized after simultaneous administration, which was the method of antibody "cocktail". Radio conerntration on tumor location could be increased, which was beneficial for reducing false-negative. This study was focused on visualizing the labeled monoclonal antibodies of the two specific antigens (EGFR and CD44) alone and in combination respectively, and results of visualization were compared and analyzed.
Method
Party1:Comparative proteomic research between human lung adeno-carcinoma cell line A549and normal bronchial epithelial cell line HBE
A549cells of exponential phase of growth and HBE cells underwent schizolysis respectively, and the total protein was extracted. Bradford method was applied to quantitating.
the extracted protein sample was directly put on the IPG prefabricated gelatin (18cm pH3-10), then in IPGphor and perpendicular plate electrophoresis consequently to undergo two-dimensional electrophoresis. Cell protein was discriminated in the light of different isoionic points and molecular weights. After two-dimensional electrophoresis and silver staining coloration the gelatin was scanned by ImageScanner to obtain protein spot map, and the image of the gel was analyzed through image analysis software, ImageMaster2D Elite5.01, thus the differences of cell protein expression of A549and HBE were known.
Some of the protein spots were excavated, which had specific over-expression in A549cells compared with that in HBE cells. Mass spectrometry analysis was conducted after enzymolysis, and peptid mass fingerprinting was obtained. Through database searching the overexpressed protein in A549cells was identified.
The overexpressed proteins in A549cells were analyzed to learn about their locations in cells and relevant functions. More than two kinds of cell membrane proteins were selected as target antigen of RII research.
Party2:Immunohistochemistry and Western-blot detection for the expressions of EGFR and CD44protein in the lung adenocarcinoma tissue
Through the research of comparative proteomics on A549and HBE cells in part one, some specific expression or overexpressed proteins were screened from cancer cells; through the analysis of the proteins' distribution and functions in cells, EGFR and CD44, the two membrane proteins, were chose for follow-up research of RII. So the immunohistochemistry and western-blot studies were employed to verifying whether the two proteins kept specificity and overexpression in adenocarcinoma of lung.
1. Fresh adenocarcinoma of lung and adjacent normal tissue (at least5cm from tumor tissue) were selected, imbedded with paraffin, and cut into tissue slices respectively. Immunoenzyme SABC was used to conduct immunohistochemistry analysis. Horseradish peroxidase was selected as biocatalyst, and DAB as developer. The cancer and adjacent cancer tissue slices of coloration were observed for cell staining respectively under light microscopes, and5high power fields in each group were observed. The Buffy tumor cell membrane and cell plasm with higher chromatosis intensity than background non-specific stain was taken as the positive expression of EGFR and CD44. Accounting analysis software was applied to computing percentage of positive ells.
2. western-blot analysis:first, SDS-PAGE gel was disposed, and then the sealing and automatic glues pouring were conducted. The prepared tissue samples of adenocarcinoma of lung and the corresponding adjacent normal tissue samples (4groups in all) were taken from liquid nitrogen, after proteinaceous schizolysis and quantitation; they were put to undergo PAGE. PAGE gel area was selected to conduct trarsmembrane according to relative molecular weight of EGFR and CD44, and then protein was transferred onto NC membrane. The specific first antibody and HRP-labeled second antibody were added, and then the substrate DAB of HRP for coloration was also added for analyzing the expression difference of EGFR and CD44in cancer tissue and adjacent tissue, with β-actin as internal control.
PART3:99mTc Labeling for EGFR-McAb and CD44-McAb and identification of characteristic of the labeled antibody
2-ME was served as reducing agent, and MDP containing SnCl2as weak bound ligand,99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. The best labeling condition was screened through changing various labeling conditions (for example, changing the dose of reducing agent or weak bound ligand, reduction time, temperature, and labeling of reaction time etc.) SephadexG50Column was utilized to purify labeled antibody. The labeling rate, specific activity, and radiochemical purity of two labeled antibodies were measured through silicon thin-layer chromatography. The stability in vitro of the labeled antibody was understood through observation of changes of radiochemical purity by placing the labeled antibody for different length of time at room temperature as well as by adding different99mTc competition reagent. The stability in vivo of the labeled antibody was understood according changes of radiochemical purity after mixing the labeled antibody and blood serum for different length of time. The immumofluorescence method was applied to evaluate immunocompetence of labeled antibody.
PART4:Body distribution and SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung
A549cells at exponential phase of growth were inoculated subcutaneously into the forward limb (L or R) of the nude mice (1.0×107/0.1ml A549cells for each mouse). The animal model of nude mice bearing human adenocarcinoma of lung was established. The nude mice were divided into three groups at random.99mTc-EGFR-McAb,99mTc-CD44-McAb and the mixture of the two labeled antibodies were injected into the three groups of nude mice via vena caudalis respectively. And study on body distribution and SPECT imaging by applying the two labeled antibodies alone or in combination to nude mice bearing human adenocarcinoma of lung was conducted.
Result
PART1:Comparative proteomic research between human lung adenocarcinoma cell line A549and normal bronchial epithelial cell line HBE
According to the atlas analysis for total protein concerning A549and HBE of epithelial cell line of normal human bronchus through applications of2-DE isolation and silver staining,897±35protein spots and882±29protein spots were respectively obtained from the gels of the two cells. The average adhesive of HBE cells served as the reference adhesive, which was applied to the matching with the average adhesive of A549with the matching rate of82.3%.256protein spots of differential expression were found through the analysis of ImageMaster2D Elite5.01gel image analysis software, and98protein spots with above-treble differential expression were found. Among them,15spots were only expressed in A549and21spots were only expressed in normal tissue.
9protein spots, over-expressed in A549(three times more than the expression in HBE), were selected for mass spectrometry analysis. Protein spots peptide mass fingerprint (PMF) was obtained. Two protein spots were found unmatched through Searching SWISS-PROT data base by utilizing Peptident query software in Expasy and above-mentioned PMF data. The other7proteins with matched protein spots were epidermal growth factor receptor(EGFR), murine double mimute2, CD44protein, cytoskeletal protein CK8, heterogeneous nuclear ribonucleoprotein (hnRNP H),60(heat shock proteins (HSP60), and phosphoglycerate mutase1(PGAM1).
According to the analysis of the above7proteins, some were signal conducting molecules, such as EGFR and CD44protein; some were cytoskeletal protein, for example,CK8; some were transcription and translation-associated protein including MDM2protein and hnRNP H; some molecular chaperones, for instance, HSP60; And some were metabolism-associated enzymes such as PGAM1. Cell membrane protein EGFR and CD44protein ere selected for the follow-up RII study according to the intracellular position and molecule characteristic of these proteins.
PART2:Immunohistochemistry and Western-blot measurement for the expressions of EGFR and CD44protein in the lung adenocarcinoma tissue
According to the immunohistochemistry results, EGFR and CD44were found to have strong expressions in cell membrane and cytoplasm of lung adenocarcinoma tissue. The Buffy cell membrane and cell plasm were taken as the positive expression, while cell membrane and cell plasm of normal lung tissue were usually not stained. According to the counting analysis of positive cells, the expression levels of EGFR and CD44in lung adenocarcinoma tissue were remarkably higher than that of the adjacent normal lung tissue.
With β-actin as internal control, western-blot was applied to the detecting of expressions of EGFR and CD44in lung adenocarcinoma tissue and its matched adjacent normal lung tissue. Compared to the matched adjacent normal lung tissue, expressions of EGFR and CD44enhanced remarkably in lung adenocarcinoma tissue.
PART3:99mTc Labeling for EGFR-McAb and CD44-McAb and identification of characteristic of the labeled antibody
99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. Purified by SephadexG50Column and then measured through silicon thin-layer chromatography, the labeling rates of the two labeled antibodies were91.5%±3.8%and92.3%±4.1%, specific activities were2.8±0.3MBq/μl and2.9±0.5MBq/μl, and the radiochemical purities were.96.5%±2.8%and96.2%±3.1%.The above-mentioned physical characteristics of the labeled antibodies could meet the demand of radioimmunoimaging.
The labeling effect of the antibodies was observed after changing different labeling conditions. It was reveled that the dosage of reducing agent2-ME was optimal when the mole ratio of2-ME and antibodies amounted to2000:1. Reduction time of the antibodies was supposed to be controlled within30min.20min was the appropriate labeling time. While the labeling rates of the two labeled antibodies were not remarkably influenced by labeling reaction temperature and dosage of weak bound ligand.
According to the stability analysis of placement of the labeled antibody at room temperature, the radiochemical purity of the labeled antibody declined slightly with the extension of time. However,24h mean was still above85%. According to the stability analysis of the labeled antibody and fresh human blood serum after37℃incubation, the radiochemical purity of the labeled antibody declined slightly after4h, However, it was still above88%. The stability in vitro of the labeled antibody was observed after adding various99mTc competition reagents. The stability of the labeled antibody was remarkably influenced after adding competition reagent with molecule containing-SH (e.g. aminothiopropionic acid)
The immumofluorescence method was applied to evaluate immunocompetence of labeled antibody. Compared to the situation before the labeling, the immunocompetence of99mTc-EGFR-McAb and99mTc-CD44-McAb were shown no apparent declined.
PART4:Body distribution and SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung
According to experiment on the body distribution of99mTc-EGFR-McAb,99mTc-CD44-McAb, and their mix-antibody in the nude mice bearing human adenocarcinoma of lung, the radioactive uptake of the tumor location, tumor/blood activity ratio, tumor/muscle activity ratio increased gradually after the injection with the extension of time, reached the peak at16h, then dropped later.(16h tumor/blood activity ratio and tumor/muscle activity ratio of the three groups were3.55±0.58and16.45±4.28,3.37±0.42and12.85±3.62,5.15±0.68and23.85±5.86respectively) In the group of mix-antibody, the radioactive uptake of the tumor location, tumor/blood activity ratio, tumor/muscle activity ratio were found through groups comparison remarkably higher than those of the rest group applying the labeled antibodyalone(p<0.05).These values in the two groups applying the labeled antibody alone were shown no significant difference (p>0.05).According to the radioactive uptake analysis, among the main organs of the nude mice, the radioactive uptakes were relatively high in organs with affluent blood supply including heart, liver, spleen and lung, while the radioactive uptakes in stomach, intestine, muscles, skeleton, and brain were relatively low. However, the radioactive uptakes in all these organs declined gradually with the extension of time.
In accordance with SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung, the tumor stain was not good in the early imaging (2-4h) for each group of nude mice while radioactive background in the blood was high and apparent visualization was found in liver and spleen. Relatively high radioactive distribution was detected in tumor locations after16h of injection of labeled antibody, when the imaging was most apparent. While radioactive background in the blood as well as radioactive distribution in liver and spleen declined remarkably. T/NT ratios (16h) for each group measured through ROI technology were2.74,2.29and5.53respectively. Group comparison indicated that T/NT ratio for the group of mix-antibody was significantly higher than the groups applying single antibody (p<0.05). All above imaging results conformed to the body distribution experimental results.
Result
Comparative proteomics technology was applied to the study discovering151protein spots of differential expression in A549and HBE cell line. Overexpression of7proteins including EGFR, MDM2, CD44protein, cytoskeletal protein CK8, hnRNP H, HSP60, and PGAM1, was identified in A549.It was approved by immunohistochemistry and western-blot that two membrane proteins EGFR and CD44were overexpressed in the lung adenocarcinoma tissue. Therefore, the two membrane proteins could be served for lung cancer RⅡ study as target antigen.
99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. The high labeling rate, appropriate specific activity, and high radiochemical purity of two labeled antibodies were obtained, which could meet the demands of RII.
High radioactivity concentration in tumor location was approved by RII study results on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung, thus an ideal target/non-target ratio could be obtained. In addition, the combined application of the labeled antibody was obviously superior to its single application. Target/non-target ratio of the tumor could be further enhanced.
In the study, proteomics, western-blot, and immunohistochemistry technology were applied to screening and identification the protein with overexpression in lung cancer cell and tissue as the target antigen for lung cancer RII study, which blazed a trail for and vigorously facilitated the development of the lung cancer RII study. Multi-disciplinary research and interdisciplinary research will be the mainstream of the future scientific development. Molecular biology, immunology and its research achievements were applied to the field of atomic medicine in the study, which are bound to expedite the development of related disciplines.
近年国内外应用放射性核素标记肺癌相关抗原的McAb进行了广泛深入的动物实验及临床研究,但肺癌的RII研究进展较慢,主要原因之一就是抗原的因素,因肺癌缺乏特异性的抗原,抗原性较弱,抗原易突变等。因此寻找肺癌特异性、稳定高表达的抗原是肺癌RII显像的关键之一。
蛋白质是基因的体现者,是体内各种功能的真正执行者,其成分和功能的异常都会导致机体出现异常。体内各种紊乱或疾病(包括肿瘤)的发生、发展及转归都与蛋白质的异常有关。因此对机体(特定组织或细胞)的全部表达蛋白质进行研究(即蛋白质组学),有利于阐释肿瘤发生、发展的分子机制,寻找肿瘤特异性标志物及阐明药物治疗机理、确定新的药物治疗靶点等,在肿瘤的早期诊断、早期治疗方面具有广阔的应用前景。本课题采用蛋白质组学技术筛选出在肺腺癌细胞中特异性高表达的膜蛋白作为目标抗原,为肿瘤RII研究提供了一条新的思路,必将大大促进肺癌RII研究的进一步发展。
肿瘤细胞系由于样品制备相对容易,并在膜蛋白、信号传导通路及分泌蛋白等的研究中具有较大的优势,其已成为蛋白质组学良好的研究模型。从肺癌患者癌组织中分离出来的非小细胞肺癌细胞系(如A549细胞),与患者体内的癌细胞具有基本相似的生物学特性,因此对其蛋白质组水平的研究,有助于阐释肺癌发生、发展的分子机制,寻找到肺癌组织中特异性表达的蛋白质及诊断、治疗靶点等。
通过细胞比较蛋白质组学技术筛选、鉴定出来的差异表达蛋白质,是否在活体组织内也存在相同或类似的表达差异,需要进一步验证。免疫组化及western-blot是检测肿瘤组织中特异性表达蛋白较常用的方法。本研究采用此两种技术对肺腺癌组织及癌旁正常组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)和CD44蛋白(通过蛋白质组学技术鉴定出的在肺腺癌细胞系A549中特异性高表达的膜蛋白)的表达差异进行鉴定,一方面可验证两种蛋白在人肺腺癌组织及癌旁正常组织中是否也存在类似的表达差异,另一方面,确定差异表达蛋白(目标抗原)的存在,也是保证肺癌RII研究成功的必要条件。
McAb的标记及标记抗体的特性也是影响RII结果的主要因素。本研究选择物理特性理想的99mTc为标记核素,采用直接标记法标记McAb,并通过改变不同的标记要素筛选出最佳标记条件。对标记后的McAb,我们采用硅胶薄层层析法(TLC)测定其标记率、放射性比活度、放射化学纯度,对标记抗体的物理特性进行鉴定；通过室温下放置、加入不同竞争试剂及与血清混合放置等方法了解标记抗体的体外、体内稳定性；通过免疫荧光法对标记抗体的免疫活性进行判断。
由于人体肿瘤抗原的表达存在异质性,有些病人的某种肿瘤抗原不表达或表达量很少,而另一种肿瘤抗原可能表达量很多,如给予单一抗体进行RII则可能造成假阴性,将抗同一肿瘤不同抗原的几种单抗混合在一起同时给药后进行显像,即抗体的“鸡尾酒”法,可增加肿瘤部位的放射性聚集(提高靶/非靶比值),有利于减少假阴性的存在。本课题以针对肺癌组织两种特异性抗原(EGFR和CD44)的标记McAb分别进行单独及联合显像,并对显像效果进行对比分析。
方法
第一部分肺腺癌细胞系A549与正常支气管上皮细胞系HBE的比较蛋白质组学研究
取对数生长期的A549细胞和HBE细胞分别经细胞裂解后进行总蛋白的提取,并采用Bradford法进行蛋白定量。
将提取的蛋白质样品直接上样到IPG预制胶条(18cm pH3-10)上,先后放入IPGphor等电聚焦仪、垂直板电泳仪中进行双向凝胶电泳,将细胞总蛋白按等电点不同及分子量不同区分开。将二维电泳后的凝胶采用银染显色后,用ImageScanner光学扫描仪进行扫描,得到蛋白质点图像,并采用ImageMaster2D Elite5.01凝胶图象分析软件进行分析,了解A549细胞和HBE细胞总蛋白在2-DE图谱上的表达差异情况。
挖取部分仅在A549细胞中特异性表达或在A549细胞中表达量明显上调的蛋白质点,经酶解后,进行生物质谱分析,得到这些蛋白质点的肽质量指纹图谱。通过数据库搜寻,鉴定出这些在A549细胞中特异性或高表达的蛋白质。
对鉴定出的A549细胞特异性表达蛋白进行分析,了解它们在细胞中存在的部位及相关功能。并从中选择两种以上细胞膜蛋白作为本研究中RII研究的目标抗原。
第二部分免疫组化及western-blot对差异蛋白EGFR和CD44在肺腺癌组织中表达的检测
通过第一部分对A549细胞和HBE细胞的比较蛋白质组学研究,筛选出了一些在癌细胞中特异性表达或高表达的蛋白质,通过对这些蛋白质在细胞内的分布部位及功能等进行分析后,我们拟选择EGFR和CD44这两种膜蛋白进行后续的RII研究。因此采用了免疫组化及western-blot技术对两种蛋白在肺腺癌组织中是否也存在特异性、高表达进行检测。
1.取新鲜肺腺癌组织及癌旁正常肺组织(距肿瘤组织至少5cm以上),分别经石蜡包埋后行组织切片,并采用免疫酶组化SABC三步法进行免疫组化分析,酶选择辣根过氧化物酶,以DAB为显色剂。将显色后的癌组织及癌旁组织切片分别置于光学显微镜下观察,每组随机观察5个高倍视野,观察细胞染色情况,对于EGFR及CD44两种蛋白均以瘤细胞的胞膜及胞浆出现棕黄色,且着色强度高于背景非特异性染色者判断为阳性,并采用计数分析软件,计算阳性细胞的百分数。
2.western-blot分析：首先配置SDS-PAGE凝胶,并进行封胶、灌胶。然后从液氮中取已制备的肺腺癌组织及配对癌旁正常肺组织样品(共4组),分别进行蛋白质的裂解与定量后,直接上样进行PAGE。根据EGFR及CD44的相对分子量选择PAGE凝胶区域进行转膜,将蛋白质转移到NC膜上。然后分别加入两种蛋白的特异性一抗及HRP标记的二抗,最后加入HRP的底物DAB进行显色,并以β-actin为内对照,分析EGFR及CD44两种蛋白在癌组织及癌旁组织中的表达差异。
第三部分抗EGFR、CD44单克隆抗体的99mTc标记及标记抗体特性的鉴定
以2-巯基乙醇(2-ME)为还原剂,含SnCl2的亚甲基二膦酸盐(MDP)为弱结合配体,采用99mTc直接标记法分别标记EGFR-McAb、 CD44-McAb,通过改变不同的标记条件(如改变还原剂、弱结合配体的剂量,还原时间、温度,标记反应时间等),筛选出最佳标记条件。利用SephadexG50柱对标记抗体进行纯化,硅胶薄层层析法测定两种标记抗体的标记率、比活度及放射化学纯度。通过室温下放置不同时间及加入不同99mmTc竞争试剂后观察放射化学纯度的变化,了解标记抗体的体外稳定性,通过标记抗体与血清混合放置不同时间后放射化学纯度的变化情况,了解其体内稳定性。采用免疫荧光法对标记抗体免疫活性进行判断。
第四部分99mTc-EGFR-McAb及99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠的体内分布及SPECT显像研究取对数生长期的肺腺癌A549细胞,接种于裸鼠的前肢(左或右)外侧皮下(每只裸鼠接种A549细胞1.0×107/0.1m1),建立荷人肺腺癌裸鼠动物模型。将荷瘤裸鼠随机分为3组,分别经尾静脉注射99mTc-EGFR-McAb、99mTc-CD44-McAb及以上两种混合标记抗体,进行两种标记抗体单独及联合应用在荷人肺腺癌裸鼠的体内分布及单光子发射型计算机断层显像(single photon emission computed tomography,SPECT)研究。
结果
第一部分肺腺癌细胞系A549与正常支气管上皮细胞系HBE的比较蛋白质组学研究
对人肺腺癌细胞系A549和人正常支气管上皮细胞系HBE的总蛋白经2-DE分离及银染显色后的图谱分析,两种细胞的凝胶图上分别得到平均蛋白质点897±35个及882±29个。以HBE细胞的平均胶为参照胶,将其与A549细胞的平均胶进行匹配,匹配率为82.3%。通过ImageMaster2D Elite5.01凝胶图象分析软件分析,两种细胞的差异表达蛋白质点256个,表达量差异在三倍以上的点98个,其中仅在A549细胞中表达的蛋白质点15个,仅在正常组织中表达的蛋白质点21个。
通过图像分析软件及肉眼观察,选择在A549细胞中表达明显上调(与在HBE细胞中的表达量差异在3倍以上)的蛋白质点9个进行质谱分析,获得蛋白质点的肽质量指纹图谱(peptide mass fingerprint, PMF)。利用Expasy中的Peptident查询软件应用上述的PMF数据搜索SWISS-PROT数据库,结果未搜索到匹配的蛋白质2个,另7个蛋白质点匹配的蛋白质分别为表皮生长因子受体(epidermal growth factor receptor, EGFR)，MDM2蛋白(murine double mimute2),CD44蛋白,细胞骨架蛋白CK8,不均一性核糖体蛋白H(heterogeneous nuclear ribonucleoprotein,hnRNP H),热休克蛋白60(heat shock proteins60,HSP60),磷酸甘油酸变位酶1(phosphoglycerate mutase1,PGAM1)。
对以上7种蛋白进行分析,可分为：信号传导分子,如EGFR、CD44蛋白；细胞骨架蛋白,如CK8；转录及翻译相关蛋白,如MDM2蛋白、hnRNP H;分子伴侣,如HSP60；与基本代谢相关的的酶类,如PGAM1。根据这些蛋白在细胞内的位置及分子特点,我们拟选择细胞膜蛋白EGFR、CD44蛋白进行后续RII研究。
第二部分免疫组化及western-blot对差异蛋白EGFR和CD44在肺腺癌组织中表达的检测
免疫组化结果显示,EGFR和CD44在肺腺癌组织的细胞膜及细胞浆内均有较强的表达,阳性表达细胞的胞膜及胞浆均呈棕黄色,而正常肺组织中细胞的胞膜及胞浆大多不着色。通过对阳性细胞计数分析,EGFR和CD44在肺腺癌组织中的表达水平明显高于癌旁正常肺组织。
以β-actin为内对照,采用western-blot法分别检测EGFR和CD44两种蛋白在肺癌组织及其配对的癌旁正常组织中的表达情况,结果表明,与配对癌旁正常肺组织比较,EGFR和CD44在肺腺癌组织中的表达有明显增强。
第三部分抗EGFR、CD44单克隆抗体的99mTc标记及标记抗体特性的鉴定
采用99mmTc直接标记法标记抗EGFR-McAb、CD44-McAb,经SephadexG50柱纯化后,用硅胶薄层层析法测定两种标记抗体的标记率分别为91.5%±3.8%、92.3%±4.1%,比活度分别为2.8±0.3MBq/μl、2.9±0.5MBq/μl及放射化学纯度分别为96.5%±2.8%、96.2%±3.1%。标记抗体的以上物理特性能满足放射免疫显像的要求。
通过改变不同的标记条件,观察抗体标记效果发现,还原剂2-ME的用量以其与抗体的摩尔比为2000：1最佳,抗体的还原时间不宜超过30min,标记时间以20min为宜,而标记反应的温度,弱结合配体的用量等对抗体标记率的影响不大。
标记抗体在室温下稳定性分析显示,标记抗体的放射化学纯度随放置时间延长略有下降,但24h均值仍大于85%。标记抗体与新鲜人血清37℃孵育后稳定性分析显示,4h后标记抗体的放射化学纯度略有降低,但仍超过88%。通过加入不同99mTc竞争试剂后观察标记抗体的体外稳定性发现,加入分子上含-SH的竞争试剂(如半胱氨酸)对标记抗体的稳定性影响较大。
通过免疫荧光法检测两种标记抗体的免疫活性,结果显示,99mTc-EGFR-McAb和99mTc-CD44-McAb免疫活性与标记前比较无明显降低。
第四部分99mTc-EGFR-McAb及99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠的体内分布及SPECT显像研究
99mTc-EGFR-McAb,99mTc-CD44-McAb单独及联合应用在荷人肺腺癌裸鼠体内的分布实验显示,肿瘤部位的放射性摄取及瘤/血、瘤/肌肉的放射性比值均随注射后时间的延长逐渐增高,在16h达高峰,以后有所降低(16h各组的瘤/血、瘤/肌肉比值分别为3.55±0.58、16.45±4.28,3.37±0.42、12.85±3.62,5.15±0.68、23.85±5.86)。通过组间比较发现,混合抗体组肿瘤部位的放射性摄取及瘤/血、瘤/肌肉比值均明显高于两种标记抗体单独使用组(p<0.05),两种标记抗体单独使用组间则无显著性差异(p>0.05)。对裸鼠各主要脏器的放射性摄取分析显示,心、肝、脾、肺等血运丰富的脏器放射性摄取相对较多,而胃、肠、肌肉、骨骼、脑等放射性摄取相对较少,但均随时间的延长,放射性摄取均逐渐降低。
通过99mTc-EGFR-McAb和99mTc-CD44-McAb单独及联合应用对荷人肺腺癌裸鼠的SPECT显像研究发现,各组裸鼠的早期影像中(2-4h),肿瘤显示不佳,而血中放射性本底较高,肝、脾等显影明显。到注射标记抗体后16h,肿瘤部位有较多的放射性分布,显示最佳,而血中本底及肝、脾等放射性分布则明显降低。通过ROI技术测得16h各组的T/NT比值分别为2.74,2.29和5.53,组间比较显示,联合抗体组的T/NT比值明显高于一种抗体单独使用组(p<0.05)。以上显像结果与体内分布实验结果基本一致。
结论
本研究通过比较蛋白质组学技术,得到A549细胞系和HBE细胞系差异表达蛋白质点256个,初步鉴定了EGFR、MDM2蛋白、CD44蛋白、细胞骨架蛋白CK8、hnRNP H、HSP60、PGAM1共7种蛋白质在肺腺癌细胞系A549中存在特异性高表达,并用免疫组化及western-blot技术验证了EGFR、CD44两种膜蛋白在肺腺癌组织中也同样存在高表达。因此我们认为此两种膜蛋白可作为肺癌放射免疫显像研究的目标抗原。
采用99mTC直接标记法对EGFR、CD44两种膜蛋白的单克隆抗体进行标记,得到的标记抗体标记率、放射化学纯度高,比活度适当,能满足放射免疫显像的要求。
99mTc-EGFR-McAb和99mTc-CD44-McAb单独及联合应用于荷人肺腺癌裸鼠的体内分布及RII研究结果均显示,肿瘤部位有较多的放射性浓聚,能得到比较理想的靶/非靶比值。此结果肯定了通过蛋白质组学技术筛选、鉴定出的差异表达蛋白,作为肺癌特异性抗原在RII研究中的价值。另外,通过对比研究,证实标记抗体的联合使用明显优于单独使用,肿瘤的靶／非靶比值可以得到进一步提高。
本研究利用蛋白质组学、western-blot和免疫组化技术筛选、鉴定在肺癌细胞及组织中特异性高表达的蛋白质作为目标抗原,进行肺癌RII研究,为肺癌的RII研究提供了一条新的思路,也必将大大促进肺癌RII研究的发展。
多学科、跨学科的联合研究是未来科学发展的主流,本研究将分子生物学、免疫学技术及其研究成果应用于核医学领域,必将促进各学科的共同发展。
Molecular biology, immunology, and nuclear medicine technology were combined in the study. The comparative study on differential expression protein between human lung adenocarcinoma cell line A549and normal bronchial epithelial cell line HBE was comparatively researched by utilizing proteomics technology. And a fraction of overexpression characteristic membrane protein in cancer cell was screened, and its expression in lung adenocarcinoma tissue was verified by utilizing immunohistochemistry and western-blot technology. The screened overexpressed protein from adenocarcinoma of lung and cancer tissue were selected as a specific antigen of adenocarcinoma of lung, and radioactive nuclide99mTc was adopted to label its monoclonal antibody(McAb) for the study on Radioimmunoimaging(RⅡ). An image method for early diagnosis of lung cancer on the molecular level was expected to be established. It is of economic value and social significance for early diagnosis, early treatment, and raising survival rate of patient.
In recent years animal experiments and clinical researches in depth and in width were implemented through applying radioisotope to the labeling McAb of lung cancer-related antigen at home and abroad. But the study of RII advanced slowly, which partially due to lacking of specific antigen, weak antigenicity, and idiovariation of antigen. So a stable antigen with high expression was a key to lung cancer RII.
Protein was the embodiment of gene and real performer of various functions in vivo, and its abnormality of constituent and function could give rise to organic abnormality. Genesis, development, and turnover of various kinds of disorder or diseases including tumor were concerned with the abnormality of protein. So studying all the expressed protein in organism (or tissue or cell) was conductive to expounding molecule mechanisms for genesis and development of tumor, to searching specific marker of tumor, and to elucidating drug treatment mechanism and ascertaining new drug treatment target. It was a great prospect for early diagnosis and early treatment of tumor. Therefore the study promised a tremendous prospect for early diagnosis and treatment of tumor. Membrane protein with overexpression in lung adenocarcinoma cells was screened as target antigen with the application of proteomics, which created a new way for the RII study, and promoted the further development of RII research for lung cancer.
Due to the easy sample preparation for tumor cell line and its obvious advantages in study on membrane protein, signal transduction pathway and secretory protein, it has become an excellent study model of proteomics. Cell line of non-small cell lung cancer isolated from the cancer tissue of the patients with lung cancer (e.g. A549) had basically similar bionomics of cancer cell in the bodies of patients. Therefore, study on its proteome level was instrumental in elucidating mechanism of carcinogenesis and development of cancer, seeking the protein with specific expression in tissue of lung cancer as well as target of diagnosis and treatment.
Protein with differential expression, screened and identified through cell comparative proteomics technology, was needed to be examined as to whether they also possessed the same or similar expressive difference in living organism tissue. Immunohistochemistry and western-blot were two commonly used means to detect specifically expressive protein. Both of them were adopted in this research to detect the expressive difference of EGFR and CD44(specific overexpression membrane protein in A549detected by proteomics technology) in adenocarcinoma of lung and adjacent normal tissues. On the one hand, the possibility of similar expressive differences existing in adenocarcinoma of lung and adjacent normal tissues was verified. On the other, it was also a necessary condition for ensuring the success of RII research for lung cancer to ascertain the existence of differential expression protein (target antigen).
RII was also influenced by the labeling of monoclonal antibody and the property of the labeled antibody.99mTc, with its ideal physical characteristics, was chose as labeled nuclide in the study. The method of direct labeling was adopted to label monoclonal antibody. The best labeling condition was selected by changing different labeling elements. Silica gel thin-layer chromatography (TLC) was applied to measurement of labeling rate, radio specific activity, and radiochemical purity of the labeled monoclonal antibody and to identifying the physical characteristics of the labeled antigen; the stability in vivo and vitro of the labeled antibody were understood through adding different competitive agentia or mixing with blood serum at room temperature; the method of immunofluorescence was applied to judging the immunocompetence of the labeled antibody.
Due to heterogenicity of tumor antigen expression, some patients had certain tumor antigen with non expression or poor-expression, yet another tumor antigen with much expression, so false negative might be generated by single antigen RII. However, a number of mono-antibodies for different antigens of the same tumor could be mixed and visualized after simultaneous administration, which was the method of antibody "cocktail". Radio conerntration on tumor location could be increased, which was beneficial for reducing false-negative. This study was focused on visualizing the labeled monoclonal antibodies of the two specific antigens (EGFR and CD44) alone and in combination respectively, and results of visualization were compared and analyzed.
Method
Party1:Comparative proteomic research between human lung adeno-carcinoma cell line A549and normal bronchial epithelial cell line HBE
A549cells of exponential phase of growth and HBE cells underwent schizolysis respectively, and the total protein was extracted. Bradford method was applied to quantitating.
the extracted protein sample was directly put on the IPG prefabricated gelatin (18cm pH3-10), then in IPGphor and perpendicular plate electrophoresis consequently to undergo two-dimensional electrophoresis. Cell protein was discriminated in the light of different isoionic points and molecular weights. After two-dimensional electrophoresis and silver staining coloration the gelatin was scanned by ImageScanner to obtain protein spot map, and the image of the gel was analyzed through image analysis software, ImageMaster2D Elite5.01, thus the differences of cell protein expression of A549and HBE were known.
Some of the protein spots were excavated, which had specific over-expression in A549cells compared with that in HBE cells. Mass spectrometry analysis was conducted after enzymolysis, and peptid mass fingerprinting was obtained. Through database searching the overexpressed protein in A549cells was identified.
The overexpressed proteins in A549cells were analyzed to learn about their locations in cells and relevant functions. More than two kinds of cell membrane proteins were selected as target antigen of RII research.
Party2:Immunohistochemistry and Western-blot detection for the expressions of EGFR and CD44protein in the lung adenocarcinoma tissue
Through the research of comparative proteomics on A549and HBE cells in part one, some specific expression or overexpressed proteins were screened from cancer cells; through the analysis of the proteins' distribution and functions in cells, EGFR and CD44, the two membrane proteins, were chose for follow-up research of RII. So the immunohistochemistry and western-blot studies were employed to verifying whether the two proteins kept specificity and overexpression in adenocarcinoma of lung.
1. Fresh adenocarcinoma of lung and adjacent normal tissue (at least5cm from tumor tissue) were selected, imbedded with paraffin, and cut into tissue slices respectively. Immunoenzyme SABC was used to conduct immunohistochemistry analysis. Horseradish peroxidase was selected as biocatalyst, and DAB as developer. The cancer and adjacent cancer tissue slices of coloration were observed for cell staining respectively under light microscopes, and5high power fields in each group were observed. The Buffy tumor cell membrane and cell plasm with higher chromatosis intensity than background non-specific stain was taken as the positive expression of EGFR and CD44. Accounting analysis software was applied to computing percentage of positive ells.
2. western-blot analysis:first, SDS-PAGE gel was disposed, and then the sealing and automatic glues pouring were conducted. The prepared tissue samples of adenocarcinoma of lung and the corresponding adjacent normal tissue samples (4groups in all) were taken from liquid nitrogen, after proteinaceous schizolysis and quantitation; they were put to undergo PAGE. PAGE gel area was selected to conduct trarsmembrane according to relative molecular weight of EGFR and CD44, and then protein was transferred onto NC membrane. The specific first antibody and HRP-labeled second antibody were added, and then the substrate DAB of HRP for coloration was also added for analyzing the expression difference of EGFR and CD44in cancer tissue and adjacent tissue, with β-actin as internal control.
PART3:99mTc Labeling for EGFR-McAb and CD44-McAb and identification of characteristic of the labeled antibody
2-ME was served as reducing agent, and MDP containing SnCl2as weak bound ligand,99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. The best labeling condition was screened through changing various labeling conditions (for example, changing the dose of reducing agent or weak bound ligand, reduction time, temperature, and labeling of reaction time etc.) SephadexG50Column was utilized to purify labeled antibody. The labeling rate, specific activity, and radiochemical purity of two labeled antibodies were measured through silicon thin-layer chromatography. The stability in vitro of the labeled antibody was understood through observation of changes of radiochemical purity by placing the labeled antibody for different length of time at room temperature as well as by adding different99mTc competition reagent. The stability in vivo of the labeled antibody was understood according changes of radiochemical purity after mixing the labeled antibody and blood serum for different length of time. The immumofluorescence method was applied to evaluate immunocompetence of labeled antibody.
PART4:Body distribution and SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung
A549cells at exponential phase of growth were inoculated subcutaneously into the forward limb (L or R) of the nude mice (1.0×107/0.1ml A549cells for each mouse). The animal model of nude mice bearing human adenocarcinoma of lung was established. The nude mice were divided into three groups at random.99mTc-EGFR-McAb,99mTc-CD44-McAb and the mixture of the two labeled antibodies were injected into the three groups of nude mice via vena caudalis respectively. And study on body distribution and SPECT imaging by applying the two labeled antibodies alone or in combination to nude mice bearing human adenocarcinoma of lung was conducted.
Result
PART1:Comparative proteomic research between human lung adenocarcinoma cell line A549and normal bronchial epithelial cell line HBE
According to the atlas analysis for total protein concerning A549and HBE of epithelial cell line of normal human bronchus through applications of2-DE isolation and silver staining,897±35protein spots and882±29protein spots were respectively obtained from the gels of the two cells. The average adhesive of HBE cells served as the reference adhesive, which was applied to the matching with the average adhesive of A549with the matching rate of82.3%.256protein spots of differential expression were found through the analysis of ImageMaster2D Elite5.01gel image analysis software, and98protein spots with above-treble differential expression were found. Among them,15spots were only expressed in A549and21spots were only expressed in normal tissue.
9protein spots, over-expressed in A549(three times more than the expression in HBE), were selected for mass spectrometry analysis. Protein spots peptide mass fingerprint (PMF) was obtained. Two protein spots were found unmatched through Searching SWISS-PROT data base by utilizing Peptident query software in Expasy and above-mentioned PMF data. The other7proteins with matched protein spots were epidermal growth factor receptor(EGFR), murine double mimute2, CD44protein, cytoskeletal protein CK8, heterogeneous nuclear ribonucleoprotein (hnRNP H),60(heat shock proteins (HSP60), and phosphoglycerate mutase1(PGAM1).
According to the analysis of the above7proteins, some were signal conducting molecules, such as EGFR and CD44protein; some were cytoskeletal protein, for example,CK8; some were transcription and translation-associated protein including MDM2protein and hnRNP H; some molecular chaperones, for instance, HSP60; And some were metabolism-associated enzymes such as PGAM1. Cell membrane protein EGFR and CD44protein ere selected for the follow-up RII study according to the intracellular position and molecule characteristic of these proteins.
PART2:Immunohistochemistry and Western-blot measurement for the expressions of EGFR and CD44protein in the lung adenocarcinoma tissue
According to the immunohistochemistry results, EGFR and CD44were found to have strong expressions in cell membrane and cytoplasm of lung adenocarcinoma tissue. The Buffy cell membrane and cell plasm were taken as the positive expression, while cell membrane and cell plasm of normal lung tissue were usually not stained. According to the counting analysis of positive cells, the expression levels of EGFR and CD44in lung adenocarcinoma tissue were remarkably higher than that of the adjacent normal lung tissue.
With β-actin as internal control, western-blot was applied to the detecting of expressions of EGFR and CD44in lung adenocarcinoma tissue and its matched adjacent normal lung tissue. Compared to the matched adjacent normal lung tissue, expressions of EGFR and CD44enhanced remarkably in lung adenocarcinoma tissue.
PART3:99mTc Labeling for EGFR-McAb and CD44-McAb and identification of characteristic of the labeled antibody
99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. Purified by SephadexG50Column and then measured through silicon thin-layer chromatography, the labeling rates of the two labeled antibodies were91.5%±3.8%and92.3%±4.1%, specific activities were2.8±0.3MBq/μl and2.9±0.5MBq/μl, and the radiochemical purities were.96.5%±2.8%and96.2%±3.1%.The above-mentioned physical characteristics of the labeled antibodies could meet the demand of radioimmunoimaging.
The labeling effect of the antibodies was observed after changing different labeling conditions. It was reveled that the dosage of reducing agent2-ME was optimal when the mole ratio of2-ME and antibodies amounted to2000:1. Reduction time of the antibodies was supposed to be controlled within30min.20min was the appropriate labeling time. While the labeling rates of the two labeled antibodies were not remarkably influenced by labeling reaction temperature and dosage of weak bound ligand.
According to the stability analysis of placement of the labeled antibody at room temperature, the radiochemical purity of the labeled antibody declined slightly with the extension of time. However,24h mean was still above85%. According to the stability analysis of the labeled antibody and fresh human blood serum after37℃incubation, the radiochemical purity of the labeled antibody declined slightly after4h, However, it was still above88%. The stability in vitro of the labeled antibody was observed after adding various99mTc competition reagents. The stability of the labeled antibody was remarkably influenced after adding competition reagent with molecule containing-SH (e.g. aminothiopropionic acid)
The immumofluorescence method was applied to evaluate immunocompetence of labeled antibody. Compared to the situation before the labeling, the immunocompetence of99mTc-EGFR-McAb and99mTc-CD44-McAb were shown no apparent declined.
PART4:Body distribution and SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung
According to experiment on the body distribution of99mTc-EGFR-McAb,99mTc-CD44-McAb, and their mix-antibody in the nude mice bearing human adenocarcinoma of lung, the radioactive uptake of the tumor location, tumor/blood activity ratio, tumor/muscle activity ratio increased gradually after the injection with the extension of time, reached the peak at16h, then dropped later.(16h tumor/blood activity ratio and tumor/muscle activity ratio of the three groups were3.55±0.58and16.45±4.28,3.37±0.42and12.85±3.62,5.15±0.68and23.85±5.86respectively) In the group of mix-antibody, the radioactive uptake of the tumor location, tumor/blood activity ratio, tumor/muscle activity ratio were found through groups comparison remarkably higher than those of the rest group applying the labeled antibodyalone(p<0.05).These values in the two groups applying the labeled antibody alone were shown no significant difference (p>0.05).According to the radioactive uptake analysis, among the main organs of the nude mice, the radioactive uptakes were relatively high in organs with affluent blood supply including heart, liver, spleen and lung, while the radioactive uptakes in stomach, intestine, muscles, skeleton, and brain were relatively low. However, the radioactive uptakes in all these organs declined gradually with the extension of time.
In accordance with SPECT imaging study on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung, the tumor stain was not good in the early imaging (2-4h) for each group of nude mice while radioactive background in the blood was high and apparent visualization was found in liver and spleen. Relatively high radioactive distribution was detected in tumor locations after16h of injection of labeled antibody, when the imaging was most apparent. While radioactive background in the blood as well as radioactive distribution in liver and spleen declined remarkably. T/NT ratios (16h) for each group measured through ROI technology were2.74,2.29and5.53respectively. Group comparison indicated that T/NT ratio for the group of mix-antibody was significantly higher than the groups applying single antibody (p<0.05). All above imaging results conformed to the body distribution experimental results.
Result
Comparative proteomics technology was applied to the study discovering151protein spots of differential expression in A549and HBE cell line. Overexpression of7proteins including EGFR, MDM2, CD44protein, cytoskeletal protein CK8, hnRNP H, HSP60, and PGAM1, was identified in A549.It was approved by immunohistochemistry and western-blot that two membrane proteins EGFR and CD44were overexpressed in the lung adenocarcinoma tissue. Therefore, the two membrane proteins could be served for lung cancer RⅡ study as target antigen.
99mTc direct labeling method was applied to the labeling of EGFR-McAb and CD44-McAb respectively. The high labeling rate, appropriate specific activity, and high radiochemical purity of two labeled antibodies were obtained, which could meet the demands of RII.
High radioactivity concentration in tumor location was approved by RII study results on single and combined application of99mTc-EGFR-McAb and99mTc-CD44-McAb to nude mice bearing human adenocarcinoma of lung, thus an ideal target/non-target ratio could be obtained. In addition, the combined application of the labeled antibody was obviously superior to its single application. Target/non-target ratio of the tumor could be further enhanced.
In the study, proteomics, western-blot, and immunohistochemistry technology were applied to screening and identification the protein with overexpression in lung cancer cell and tissue as the target antigen for lung cancer RII study, which blazed a trail for and vigorously facilitated the development of the lung cancer RII study. Multi-disciplinary research and interdisciplinary research will be the mainstream of the future scientific development. Molecular biology, immunology and its research achievements were applied to the field of atomic medicine in the study, which are bound to expedite the development of related disciplines.
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