The Influence Exerted To The Biological Characteristics Of Human Bronchial Epithelium HBE And Lung Adenocarcinoma A549Cell Line By The Expression Alteration Of DP71

Dystrophin gene is located in the Xp21.1-21.3.Dystrophin Dp71is the whole protein which transcribed and translated by Dystrophin gene in initial gene promoter region of exon62and exon63.The translation of the protein Dp71retains a cysteine rich region and a carboxy terminal region of dystrophin protein. Dp71is the most widely distributed protein in non-muscle tissues of dystrophin protein family. Research on Dp71shows that the expression of Dp71can be detected in the embryonic pluripotent stem cells. Dp71is widely distributed in cell membrane while a certain amount of expression in the nuclei and cytoplasm can be detected too.At the end of3’,the shear mainly produced two kinds of shears:Dp71f and Dp71d. Dp71d shear can mainly be found in the nucleus and Dp71f shear mainly be found in cytoplasmic distribution. Related functional studies indicate that the two different shears plays the same important role in the PC12cell growth process.Binding with lamin B and nuclear protein emerin, Dp71may be involved in PC12cell division. The biological behavior of Dp71is also involved in PC12cell adhesion. Most of the current studies about Dp71focused on PC12cells and there are no biological function research in other cells could be found up to now.We build a Dp71expression cell model in the normal human bronchial epithelial cells (HBE) and made a study about its biological function. Expression and inhibiting expression of Dp71cell model are made by the selection of human lung adenocarcinoma A549cell line respectively. In order to provide the experimental basis for further study of the biological function of Dp71,analysis is made for the study of the biological function of Dp71in vivo xenograft model and in vitro cell model. Chapter I Establishment of Dp71over expression HBE cell lines and analysis of their biological charactersObjective:To establish HBE cell lines over expression Dp71f and Dp71d isoforms.To analyze the proliferation, invasion, migration capabilities of the five HBE cell lines. Also we try to find the molecules which can explain the biological characters’change of the five HBE cell lines.Methods:The HBE cell lines over expression Dp71f and Dp71d plasmids were obtained via G418selection. CCK8and clone formation were used to analyze its proliferation capabilities.Transwell and would scratch assays were performed to analyze their invasion capabilities.Flow cytometry assay were performed to analyze the cell cycles of the five HBE cell lines.Western blot was performed to find the target molecules which are responsible for the biological changes of the Dp71over expression cell lines.Results:HBE cell lines over expression Dp71d and Dp71f isoforms were settled, the5HBE cell lines used in chapter one were named HBE-Dp71d, Control-Dp71d, HBE-Dp71f, Control-Dp71f and parental HBE cells.Compared with the Control-Dp71d, Control-Dp71f and blank HBE cell lines, HBE-Dp71d and HBE-Dp71f cell lines proliferated faster; their invasion and migration capabilities were much stronger. FCS analysis revealed the G0/G1phase of HBE-Dp71d and HBE-Dp71f cell lines were much shorter. All the differences were statistically significant.Western blot analysis and q-RT-PCR revealed increased laminB1and Bcl2mRNA and protein expression in HBE-Dp71d and HBE-Dp71f cell lines.Conclusions:Compared with the Control-Dp71d, Control-Dp71f and blank HBE cell lines, HBE-Dp71d and HBE-Dp71f cell lines changed the following biological characters:they proliferate and migrate faster, they are more invasive, and the G0/G1phase of the HBE-Dp71d and HBE-Dp71f cell lines are much shorter. The increased lamin B1and Bcl2protein are highly possible for these altered phenotypes. Chapter Ⅱ Establishment of Dp71over expression A549cell lines and analysis of their biological charactersObjective:To establish A549cell lines over expression Dp71f and Dp71d isoforms. To analyze the proliferation, invasion, migration capabilities of the five A549cell lines. Also we try to find the molecules which can explain the biological characters’ change of the five A549cell lines.Methods:The A549cell lines over expression Dp71f and Dp71d plasmids were obtained via G418selection. CCK8and clone formation were used to analyze its proliferation capabilities.Transwell and would scratch assays were performed to analyze their invasion capabilities of the five A549cell lines.Results:A549cell lines over expression Dp71d and Dp71f isoforms were settled, the five A549cell lines used in chapter two were named A549-Dp71d, Control-Dp71d, A549-Dp71f, Control-Dp71f, and parental A549cells. Compared with the Control-Dp71d, Control-Dp71f and blank A549cell lines, A549-Dp71d and A549-Dp71f cell lines proliferated faster; their invasion and migration capabilities were much stronger. All the differences were statistically significant.Conclusions:Compared with the Control-Dp71d, Control-Dp71f and blank A549cell lines, A549-Dp71d and A549-Dp71f cell lines changed the following biological characters:they proliferate faster, they form more clones, they migrate faster and they are more invasive. Chapter Ⅲ Establishment of A549cell lines stably knocking down Dp71and analysis of its biological charactersObjective:To construct siRNA vectors, this can stably knock down Dp71expression in A549cells. To establish A549cell lines stably expression Dp71siRNA construct. To analyze the proliferation, invasion, migration capabilities of the three A549cell lines. Methods:Dp71siRNA interfering sequences were designed via siRNAonline software, Dp71siRNA plasmids were constructed.A549cell lines stably expression Dp71siRNA plasmids were obtained via G418selection. CCK8and clone formation were used to analyze its proliferation capabilities.Transwell and would scratch assays were performed to analyze their invasion capabilities of the three A549cell lines. Results:A549siRNA plasmids were successfully constructed. A549celllines stably expression Dp71siRNA construct were obtained, and the three A549cell lines were named A549-Dp71AS,A549-Dp71E and blank A549cells.Compared with the A549-Dp71E and blank A549cells, A549-Dp71AS cell lines proliferated slower; formed less clones, their invasion and migration capabilities were much weaker. All the differences were statistically significant.Conclusions:Compared with the A549-Dp71E and blank A549cells, A549-Dp71AS cell lines changed the following biological characters: they proliferate and migrate much slower, they are less invasive and all these differences were statistically significant. Chapter IV Stable RNA interference of Dp71gene inhibits human lung adenocarcinoma A549cells growth in vivoObjective:To investigate whether silencing the expression of Dp71can inhibit the growth of A549cells in nude mice model.Methods:The stably transfected A549-Dp71AS,A549, A549-Dp71E cell lines were injected subcutaneously into nude mice.During3week following period, the sizes and weights of tumors were measured.Results:The volume and weight of tumors in A549-Dp71AS group were significantly decreased while the latent period increased compared with those of other control groups (P<0.05). Conclusion:The in vivo experiments suggest knocking down Dp71expression in A549cells can significantly inhibit the tumor growth in nude mice model.