Effect Of Transfected And Expression Of Respiratory Syncytial Virus M_2-1 Gene On The Growth Of Human Lung Adenocarcinoma PAa Cell In Vivo And Vitro

Human respiratory syncytial virus(RSV)is a major pathogen of the lower respiratory tracts of infants world wide.RSV is an enveloped RNA virus and is a member of the family paramyxoviridae, classified within the genus pneumovirus. In recent years, the infectious adults with RSV have increasingly reported, especially the elderly and the immunocomprimised.It was reported that the RSV N gene was able to be amplified by RT-PCR in resected carcinomas of the lung and also has the signifcant concordance with the finding in detecting the mutation of K-ras gene in resected lung carcinoma at the same time. These findings suggested that there was some relationship between RSV and carcinoma of the lung.The RSV M2 protein, found only in the pneumoviruses, is a nonglycosylated structural protein. M2-1 full length genome has 595 nucleotides, encodes the 22-kD structural protein which is highly conserved in the M2 proteins of the members of the pneumovirus genus. Recently, it has been demonstrated the M2-1 protein is a transcriptional processivity factor that is involved in RNA transcription elongation.The M2-1 protein also decreases RNA transcription termination and facilitates read-through of RNA transcription at each gene junction, thereby acting as a transcription antiterminator, and play an important role to control and regulate the transcription and replication of RSV . We canconclude that M2-1 gene has some influences on the proliferation of host cell. On the basis of having constructed the M2-1 eukaryotic plasmid (PXJ-41/M2-1), we trsnsfect the recombinant PXJ-41 plasmid which carried the M2-1 cDNA fragments to the human lung adenocarcinoma PAa cell lines and then observe the effect on the growth of human adenocarcinoma in vivo and vitro.Objectives To observe the effect on the growth behavior of human adenocarcinoma cell lines in vivo and vitro after transfected with RSV m2-1 gene and establish a basement on further explore the molecular mechanism of RSV in the lung carcinogenesis. Methods 1.Identification the recombinant eukaryotic plasmid (PXJ- 41/M2-1); 2.To transfect PAa cell;3.The identification the RSV M2-1 gene expression was performed by RT-PCR, immunofluorescence and western blot; 4. The growth of human lung adenocarcinoma in vivo and vitro were observed by the means of MTT curve flow cytometry (FCM) enzyme digestion and injected nude mouse. Results 1. The recombinant PXJ-41/M2-1 were digested by EcorI and XhoI, the 620bp bands of M2-1 gene were shown; 2. By RT-PCR amplication , the 620bp bands of M2-1 gene were shown; 3. Expression of M2-1 protein in positive transfected PAa cell were detected by Western blot, the specific bands 22KD were shown; 4. The positive transfected PAa cell grow faster than the control one which only transfected by the plasmid PXJ-41 vector; 5. Colony-forming efficiency and Colony- forming unit of the transfected PAa with PXJ-41 /M2-1 were increased; 6. An ability of adherence to the wall was decreased; 7. The rations of PAa cells inthe S phase of cell cycle were decreased, but it was increased in G2/M phase; 8. The tumorogensising time of the transfected PAa cell in the nude mouse was delayed, but its growing speed was increased.There is no significant difference between the transfected PAa cell and non-transfected PAa cell in the metastasis and tumorogensis, when it was injected to the nude mouse, respectively. Conclusion 1. M2-1 gene were expressed in PAa cell successfully; 2. `The M2-1 protein promoted the PAa cell growth to some extent;3. We found that the lung adenocarcinoma has a tendency of transformation to the squamous carcinoma; 4. To establish a fundation to further study the biology behavior of the RSV M2-1 protein and the relationship with lung cancer.