The Function And Mechanism Study Of CyclinG2 In Vitro Cell And It's Expression In Gastric Carcinoma Tissue

Cell cycle regulation has a close relationship with cell proliferation and tu-morigenesis. In recent years, the study of cell cycle regulation is the foreland and hotspot field in cell biology and genetics. Cyclins are important proteins in regulating cell cycle, which form the holo - enzyme complex with CDK as a triggering factor in the cell cycle, the assembly and activation, degradation of cyclin - Cdk holo - enzyme complex is the main molecular basis of cell cycle regulation. They are positive regulating proteins and play a very important role in the development of neoplasma. CKI are negative regulators taking part in cell cycle, which can directly or indirectly combine with the corresponding Cdks to inhibit their activity. Cyclin - CDK - CKI constitutes a complex network regulating cell cycle. If the dynamic balance were destroyed, cell would proliferate abnormally, apoptosis, necrosis, carcinogenesis.All different cyclins found in mammalian cells contain a 100 amino - acid homologous region named cyclin box, which is the molecular structure marker of cyclins. The expression level of cyclins shows cell cycle phase specialty. It can be regulated by transcription and the degree of protein hydrolysis. CyclinG ( Cyc G) , a recent addition to the cyclin family, has homology to fisson yeast cigl, B - type cyclins, and human cyclin A and I. The molecular structure of G - type cyclins differs from other cyclins. NH2 terminus lacks a "destruction box " that controls the ubiquitin - dependent degration but contains an epithdermal growth factor receptor - like autophosphorylation sequence in addition to cyclin box. The structure hints that it is not only a cyclin in cell cycle regulation but also an intracelluar signal transducer molecule. CycG has not yet been matched with a Cdk binding partner, and it's biologic function is still elusive. CycG is the onlyknown cyclin that is transcriptionally activated by the p53 tumor suppressor gene. It forms a complex with B' regulatory subunits of protein phosphatase2A following it's induction by p53. CycG does not exert a tumor - suppressive role but plays a growth promoting role. Although the molecular structure of the two kinds of cycG has much similarity and identity, their biological function differs obviously. The physiological function of cyclinG2 and which kind of CDK is combined with it during the whole process of cell cycle is still unknown. But a series of research hint that it may be a negative regulator in cell cycle regulation.In order to study the function of cyclinG2 in vitro culturing cell line, we used pIRES - G2 eukaryotic expression vector transfecting human gastric cell line SGC - 7901 and human embryo kidney HEK - 293 cells by lipofectamine Plus reagent, and studied the function of cyclinG2 expression on the cell proliferation in vitro, further investigated the regulation mechanism of cyclinG2. At the same time, we made a study on the expression level change of cyclinG2 in normal gastric tissue and different type and different stage of gastric carcinoma tissue.Material and method1 Material: pIRESneo vector was purchased from Clonetech, plasmid extraction and purification kit was purchased from Qiagen company ;RPMI 1640,, DMEM fetal calf serum were obtained from Gibco/BRL; Lipofectamine Plus and G418 were purchased from Life Technologies;Ultrasensitive?S -P kit, mouse monoclonal antibody p21WAFl (in use) , DAB staining kit were purchased from MaiXin company. Mouse monoclonal antibody p21WAF1 (concentrated) ,horseradish - peroxidase - conjugated goat anti mouse secondary antibody were purchased from Beijing ZhongShan company, Enhanced chemilluminescence (ECL) kit from Santa Cruze,DC Protein assay kit from Bio -Rad company.2 Cell line and tissue sample: The human gastric cancer cell line SGC -790land human embryo kidney cell line HEK -293 cell were kept in cell biolo-gy laboratory. Forty case of different type and different stage of gastric carcinoma sample and ten case of normal gastric tissue were from the gastri...