| [Background] Lung cancer has become the most common cause of cancer-related mortality in the world. Although the traditional therapeutic methods that utilizing chemotherapy, radiation therapy, and surgery are developing and improving continuously, the overall survival ratio of patients who suffered from lung cancer has changed little. Lung cancer patients are always in psychological stress reaction caused by blues, anxiety, dread, and their resistance is weakened. In addition, those patients are easier to complicate respiratory tract infection caused by the pressing and blocking of the tumor to the air tract. Human respiratory syncytial virus (hRSV) is not only a major cause of severe infections of lower respiratory tract in infants and young children worldwide, but also is considered as an important respiratory tract infection pathogen of adults in resent years, especially for the old and the immuno-suppressed. Professor Chen Hang-wei has been engaged in researches on hRSV for many years, and she has found that it can cause all kinds of severe pulmonary infections in adults. With RT-PCR technology, Professor Chen successfully amplified out hRSV N gene segment from primary lung cancer operation specimens. By comparing special hRSV IgM antibody of two patient groups, it is found that the positive ratio of lung cancer group is higher than that of infection group, and this result is related to K-ras oncogene mutation of lung cancer patients. We try to study the relationship between hRSV and lung cancer in this thesis. It is known that the biological factor is one of the carcinogens. But, hRSV is an enveloped negative strand RNA virus, a pneumovirus of the paramyxovirus family. The hRSV is not a traditional carcinogenic virus, so it will bring meaningful influence on clinical medicine to find out its effects and mechanism in cancer incidence and development. The viral genome RNA of hRSV is composed of 15,200 nucleotides, including 10 genes: NS1, NS2, N, P, M, SH, G, F, M2, and L, encoding 11 viral proteins. The M2 gene contains two open reading frames, encodes protein M2-1 and M2-2. Protein M2-1 is the virus'transcriptional processivity and antitermination factor and ensures efficient production of full-length mRNA, and it cannot be absent when reading-through the gene-conjunction. The protein also participates in the construction of nucleocapsid/polymerase protein and the assembly of the virus. It is obvious that M2-1 protein is very important for hRSV life course. So it is the focus of the study. The prokaryocyte expression plasmid of M2-1 gene has been constructed by our lab. It will be very helpful to this thesis. [Objective] To find out the possible affection of M2-1 gene on the human lung adenocarcinoma PAa and A549 cells, including proliferation ability, invasion and metastasis ability, tumorogenesis ability, angiogenesis ability, pathological morphology and ultrastructure, and research the molecular mechanism of the affection. Then, to discuss the relationship between hRSV infection and lung cancer. The study would provide a new idea for clinical work of lung cancer's diagnosis and therapy. [Methods] 1. Using gene recombination to acquire the eukaryote expression plasmid of M2-1 gene, and verify the correctness of pXJ/M2-1 via methods such as gene-sequencing. 2. To transfect the pXJ/M2-1 plasmid into the lung adenocarcinoma cells PAa and A549 mediated by liposome, screen out successfully transfected cells by G418, and use RT-PCR to validate that those cells can express M2-1 gene. 3. To detect the changes of those successfully transfected cells PAa and A549 in cell-cycle, abilities of proliferation, invasion, sticking to the bottom of the culturing-bottles and tumorogensis by FCM, MTT, Boyden chamber, pancreatin-digesting, sticking experiment, soft agar clones-forming experiment and nude mice tumorogenesis experiment. 4. Using optic microscope to observe the pathologic changes of PAa and A549 cells which are successfully transfected by pXJ/M2-1; to observe tumor tissue pathologic changes of PAa cells which are successfully transfected by pXJ/M2-1 and find out whether there are lung or liver's metastasis or not. 5. Using TEM to observe the nude mice heterologous grafts of the PAa cell and thesesuccessfully transfected by pXJ/M2-1, pXJ-41, compare and find out the changes of ultrastructure. 6. Using CD34-IHC, VEGF-ELISA, CD54-FCM and bFGF RT-PCR detection to observe changes in angiogenesis and metastasis ability of PAa and A549 cells which are successfully transfectied by pXJ/M2-1, and to analyze the reasons of those changes. 7. Using gene chip technique to analyze the different expression of PAa and A549 cells before and after M2-1 gene transfection in 15 classes, about 4,000 genes, such as oncogenes and tumor suppressor genes, cell cycle proteins, cell skeleton and movement, cell apoptosis, and use RT-PCR, ELISA, FCM methods to validate some of them. [Results] 1. The recombined M2-1 gene eukaryote expression plasmid was correctly conjuncted, and verified by gene-sequencing. The M2-1 gene is highly conservative. 2. The PAa and A549 cells that can steadily expressing M2-1 gene were acquired after liposome transfection, G418 screening and RT-PCR verification. 3. After M2-1 transfection, the ratios of G2/M-phase of PAa and A549 cell were multiplied, multiplication time was shortened, clone formation ability was enhanced, pancreatin-digesting time was shortened and the speed-sticking to the bottom of the culturing bottle was increased; the invitro invasion ability of the A549 cell was enhanced; and the PAa cell's proliferation rate in nude mice was expedited. 4.There were not evident changes in cell and tissue configuration observed under optic microscope, but there were local necroses in the PAa tumor tissue. There were 3 lung metastasis focuses in 2 of 10 nude mice grafted with PAa/M2-1 cells. But no metastasis occurred in the other two contrast groups. 5.Some changes of PAa/M2-1 group tumor cell were found under TEM, such as the nucleus/cytoplasm ratio increased, free ribosomes were abundant in the cytoplasm, but other organelles were infrequent, no osmiophilic multilamellar body was found, microvilli were rarefied and intercellular junctions were reduced, the cell in fission phase increased and apoptosis phenomenon could be seen. 6. The results of IHC, FCM, ELISA and RT-PCR detection suggested: microvessel density of PAa tumor tissue, PAa cell's CD54 expressing ratio, PAa and A549 cells'VEGF andbFGF-expressing quantity were increased after M2-1 gene transfection. 7. The results of gene chip inspect shows that there were more changes in gene-expressing in M2-1 gene transfected A549 cells than that in M2-1 gene transfected PAa cells; some genes are expressed higher, such as MAKP14; some are expressed lower, such as PIAS3; trends of some changes of those PAa and A549 cells were contrary, such as TFPI2. Among those changes, IL8, CD24, MAP4, OSF2 and EMP1 were validated by ELISA, FCM and RT-PCR. [Conclusion] 1. The hRSV M2-1 gene transfection promoted the proliferation, enhanced the invasion and metastasis of PAa and A549 cells, and can made tumor low differentiation and more malignant. 2. The molecular mechanism that hRSV M2-1 gene influences the human lung adenocarcinoma PAa and A549 cells is to express those genes that can promote the tumor proliferation, angiogenesis, metastasis etc highly; and to express those genes that can retard the tumor proliferation, angiogenesis, metastasis etc lowly. 3.The hRSV may promote lung cancer cells proliferating and invasion in vitro and in vivo by the effects of M2-1 gene. These findings break a new path for lung cancer patients'diagnosis and treatment in the future.
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