| 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. MethodParty1:Comparative proteomic research between human lung adeno-carcinoma cell line A549and normal bronchial epithelial cell line HBEA549cells 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 tissueThrough 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 antibody2-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 lungA549cells 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.ResultPART1:Comparative proteomic research between human lung adenocarcinoma cell line A549and normal bronchial epithelial cell line HBEAccording 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 tissueAccording 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 antibody99mTc 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 lungAccording 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.ResultComparative 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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