| Cell cycle is composed of interphase and mitotic phase,the duplication of genetic materials occurs in interphase,and the equal distribution of genetic materials and organelles occurs in mitotic period.Some cells remain in the resting stage without dividing after a series of mitotic,which is called GO phase.Cell cycle begins from this phase.The distribution of chromosome is completed by spindle apparatus which is composed of centrosome,microtublues and other motor proteins.Spindle apparatus pulls highly spiraled chromosomes to the opposite cell polarity.Centrosome is composed of two centrioles,each made up of microtublue triplet.Microtublue is a type of cytoskeleton with polarity,and is a long tubular structure composed by the dimer of?-tubulin and ?-tubulin.The ?-tubulin and ?-tubulin are highly homologous,with the?-tubulin being one of the basis in building microtublues.My project choose three different cells to discuss and research the distribution pattern in cell cycle of our interested gene and protein a-tubulin.The first kind of cells are from mouse early embryo,which undergo all the typical phases in cell cycle.Embryonic cells have strong dividing ability,and are generally considered to lack GO phase.We choose 1-cell,2-cell,4-cell and 8-cell mouse embryos as experimental materials to explore the cell function of ?-tubulin in cell cycle without GO phase.The second kind of cells are macrophages cell line Raw264.7,they have both differentiative capacity and mitotic capacities.We explore the dynamic changes of a-tubulin in the phase which cells keep the ability of dividing and in the stage which cells have the ability of differentiating,compared those with the dynamic changes of highly differentiated cells treated with nicotine.The third kind group of cells are fully differentiated macrophages from mouse's abdominal cavity,they only stay in GO phase.We studied the function of a-tubulin,and compared those primary macrophages with Raw264.7 to explore the similarities and differences in our protein of interest in extreme differentiation.Macrophages used in our project are deemed to be the first defense of immune system in mammals,which have highly athletic ability and phagocytic ability.Macrophages have the character of terminal differentiation,the relation of this character and a-tubulin is unclear.The relevant molecular mechanism of a-tubulin in this aspect is significant in medical biology.My project The current study also provides a new method in the preparation of anti animal blood serum,in order to get embryonic stem cells and use them to better observe the distribution pattern of a-tubulin in cell cycle.The results showed as follows.1.In mouse early embryos,the images taken by laser scanning confocal microscope shows:The 1-cell embryo was in dividing stage of cell cycle,with polymerized a-tubulin composed of spindle apparatus to take part in cell division.The 2-cell,4-cell,6-cell,7-cell and 8-cell embryos were in interphase,polymerized a-tubulin molecules compose centrosome linking complex to connect the two adjacent cells.As the division increased,the cross section of centrosome linking complex decreased progressively.RNA interference result shows the appearance of multiple centrosomes and multinuclear after a-tubulin knockdown,meaning a-tubulin plays an important role in cell division.2.In different macrophages,the pictures taken by inverted fluorescence microscope shows:In macrophage cell line Raw264.7 cultured 48 hours without nicotine treatment,the polymerized a-tubulin compose spindle apparatus to take part in mitotic period of cell cycle,or compose centrosome linking complex to connect two adjacent cells in interphase of cell cycle,or compose centrosome to participate in other cell progress.In mononuclear macrophages in mice leukemia cell Raw264.7 cultured 48 hours with nicotine treatment,cells were in GO phase,with polymerized ?-tubulin composed centrosomes.In primary mouse macrophages cultured 48 hours with or without nicotine treatment,all the cells were in GO phase,together with polymerized a-tubulin composed centrosomes.The datas show that the area of cells with and without nicotine treatment has significant difference,with the area of cells with nicotine treatment being bigger.3.The migration of primary mouse macrophages before and after nicotine treatment.Before primary mouse macrophages were treated with ncotine,there was not much changes in the length of the cell during migration.However,there was no body movement after nicotine treatment,the tail was fixed in one place but had elongated head,making the length of the cell longer.The treatment of nicotine would decrease the velocity of cell migration,but not perpetually,after six days,the velocity of cell would recover as before.The results show that the length of cells before and after nicotine treatment had significant differences,with those treated with nicotine being longer.4.The preparation of anti animal blood serum:We got rabbit anti pig serum successfully by injecting the rabbits with the solution made by pig spleen cells,in order to collapse the TE cells of pig blastocyst stage embryos in the future.Conclusion:My project use immunofluorescent staining,RNA interference,RT-PCR were used to study the dynamic distribution of ?-tubulin in cell cycle from the level of genes and proteins.MY project The current study shows that there are three polymerizing ways of ?-tubulin,one is spindle apparatus taking part in mitotic phase of cell cycle,the other is by building centrosome linking complex to connect two adjacent cells in interphase,and the last one is to build centrosome in GO phase or other polymer dispersing in cells.After a-tubulin knockdown,some embryos had multiple centrosomes and multinuclear,meaning the polymerization and reconciliation of a-tubulin play an important role in cell cycle.It could provide data for mitosis in early embryo development,and also could provide some molecular mechanisms for human early embryo development.Thus it may provide some new theoretical basis for human disease such as infertility and early embryo death. |
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