The Expression of CyclinA、CyclinD1 and P27 in Different Situations of Astrocytes
|School||Huazhong University of Science and Technology|
|Course||Human Anatomy and Embryology|
|Keywords||cell cycle astrocytes contact inhibition CyclinA CyclinD1 P27|
The phenomenon of contact-inhibition plays an important physiological meaning on controlling the shapes and sizes of organs and tissues in the organism and the numbers of all kinds of cells.when the tissues and organs grows up into some degree,they will stops to grow but not to unrestrictedly grow . In vitro assay ,we also find that the cells will stop to divide when they divide and grow into some degree,arriving to col-contact.But the mechanism of action of contact inhibition is still unknown. In general,owing to the increasing number of cell dissipate divide factors, or the release or sequestration of soluble factors secreted bu cells at contact-inhibition. In 2001,Nakatsuji’s research shows that: contact dependent inhibition of cell proliferation is mediated through modulation of cell cycle related protein expression,cell density regulates the expression levels of a number of critical cell cycle proteins that likely control progression through the cell cycle. In order to examine the mechanisms of contact inhibition,we study the contact inhibition and cell cycle regulation of mutual relations making the astrocyte as the model.The regulation of cell cycle is basic physiological functions of cells,cell cycle regulation is not only control cell division and proliferation,but also determine the cell differentiation and death; the same time,cell cycle regulation has many important physiological functions and a crucial role. Therefore,we have reasons to believe that there may be control of the gradual progress of the cell cycle through the entire period of cell growth and development . The cell cycle regulatory proteins is nuclear protein which interrelated constraints of different functions,according to the role of cell cycle control proteins ,it can be divided into cell cycle control proteins (including Cyclin, CDK,and pRb) and cell cycle negative control proteins (CKI family). As a group of protein with cyclic changes in the appearance and disappearance,cell cycle proteins (Cyclin) are wide range of molecules which controlled the cell cycle eukaryoteing from yeast to humans. The regulation of Cyclin plays an important role in the normal cell cycle. Currently,there are 20 kinds of Cyclin found in mammalian cells. A clear regulation of the cell cycle is mainly CyclinA, B, C, D and E,Cyclin molecules are highly conserved evolutionarily,which molecular structure has a "Cyclin box" sequence,Cyclin and CDK combined into complex regulating of cell cycle or playing the function of transcription; The content of CDK is relatively constant in various stages cell cycle,the activity of CDK is dependent on the regulation of Cyclin. CDK must be combined with the corresponding of Cyclin and had activity or inactivity depent on phosphorylation or dephosphorylation in the certain parts, Cyclin and related molecules of cell cycle regulation is hot spots in the research. Negative cell cycle control protein (CKIfamily), including P21, P27, P57, etc., can inhibit activity of most known Cyclin-CDK complex kinase,and play important role on the early and late G1 phase, S phase and other phase,the CKI,which distribution and function vary in different cells,is the main study in nowadays.To examine the mechanisms of contact inhibition,we chose three types of cell cycle associating protein with CyclinA,CyclinD1 and P27 as a marker to observe them in the process of proliferation and in expression of contact inhibition,and discuss the interaction between the contact inhibition and cell cycle regulation. In this study,we detect expression of CyclinA,CyclinD1 and P27 protein under the semi-convergence state and confluence state of astrocytes in vitro conditions. The purified astrocytes were subcultured at the confluence of state and semi-convergence state then do the research under the following step. First,we put semi-convergence state and confluence state of astrocytes react with its antibody respectively by using immunohistochemical staining,the results were statistically analyzed. Second,we collected the semi-convergence and confluence of cells by using RT-PCR technique,lysising centrifugation into RNA samples,reversing transcription into cDNA,amplifying products and running on the gel,developing an image,and the results were statistically analyzed. Results : Immunohistochemical staining show that compared with the semi-confluent cells, CyclinA was significantly decreased (p<0.05) and CyclinD1 was significantly decreased (p <0.05),while P27 was significantly higher (p <0.01) in the confluence state; RT-PCR show that compared with the semi-confluent cells, CyclinA was significantly decreased (p<0.01) and CyclinD1 was significantly decreased (p<0.05),while P27 was significantly higher (p <0.01) in the confluence state.Conclusion: When the astrocytes in semi-confluence state of vitro case,the cell cycle positive regulator CyclinA,CyclinD1 are high express,while the cell cycle negative regulator P27 is low express,indicating that cell are in the period of proliferation;When the astrocytes in the confluence state,the CyclinA, CyclinD1 are low express and P27 is high express,indicating that cell are in the period of proliferation inhibition.