The Study Of CXCL12/CXCR4Biological Axis On Biological Behavior Of Tca8113Cell Lines And The Effects Of AMD3100on It

Background:Squamous cell carcinoma of the tongue was one of the most common malignant tumors in oral and maxillofacial tumors, which had the features of rapid growth, strong local invasion and higher lymphnode metastasis rate. Among the biological features, easier transfer to lymphnodes of neck area, especially, had an in important role on the prognosis of patients. Therefore, the study of proliferation, invasion and metastasis of Squamous cell carcinoma of the tongue in oral and maxillofacial tumors, could have a very important significance in improving rates of cure and survival of patients.Chemokines were kinds of small molecules secreted protein,which could stimulate chemotaxis of leukocytes, could attract neutrophils, monocytes/macrophages and other inflammatory cells moving to inflammation, could enhance the function of phagocytosis and killing of inflammatory cells, could promote inflammatory cells to releasing inflammatory proteins and inflammatory mediators.These inflammatory proteins and inflammatory mediators could involve in the process of immune regulation and immune pathology directly. Chemotactic factor CXCL12, which can be expressed in multiple organs, played an important role in development and immune system, and also had a strong chemotaxis on lymphocytes. CXCR4was the specific receptors of chemotactic factor CXCL12, and it can widely exist in many organs’surface, such as lymphocytes, endothelial cells.The newest researchs showed that the surface of a lot of malignant tumor cells can be detected with high expression of CXCR4receptors, such as breast cancer, colorectal cancer, gastric cancer etc. The surface of malignant tumor cells can highy express chemokine receptors, at the same time the certain organs of the body express its related ligands.The malignant tumor cells, which had the high expression of CXCR4receptors can appear organ-specific metastasis by the chemotaxis of chemotactic factor CXCL12.CXCL12/CXCR4biological axis had been shown to related to proliferation, invasion and metastasis of malignant tumor cells. Through strengthening the study of the relation of CXCL12/CXCR4biological axis and its biological behavior in malignant tumor cells, a new way of thought and method for tumor therapy can be provided.Objective:To study the influence of CXCL12/CXCR4biological axis to the biological behavior of squamous cell carcinoma of the tongue, this experiment aimed at through the application of certain concentration of chemokines CXCL12and AMD3100, as the inhibitor of CXCR4receptor, and study the squamous cell carcinoma of the tonge in vitro so as to detect the expression of CXCR4receptor of squamous cell carcinoma of the tonge. At the same time study the influence of CXCL12/CXCR4biological axis to proliferation, invasion and metastasis of squamous cell carcinoma of the tongue to provide some theoretical foundation for invasion and metastasis of squamous cell carcinoma of the tongue.Materials and methods:1. cell lines:Tongue squamous cell carcinoma cell line Tca8113purchased from the Chinese Academy of Sciences Shanghai Institute for Biological Sciences2. main materials:Medium RPMI-1640, fetal bovine serum (Hangzhou Sijiqing), CCK-8reagent (Sigma), DMSO (Dimethyl sulfoxide), CXCL12factor (Boaosen), AMD3100, CXCR4antibody (Boaosen), RNA extraction reagent TRIzol (American Invitrogen company), Taq DNA polymerase (Transgene), Matrigell (BD company, USA), Transwell cell and supporting plate (costar) etc.3. method:Tca8113cells of tongue squamous cell carcinoma were cultured in RPMI1640medium with10%fetal bovine serum, which contained100U/ml penicillin and100ug/ml streptomycin), Tca8113cells were cultured in saturated humidity incubator culture at37℃,with5%CO2.Every2-3days the solution was changed. It be digested by trypsin.of0.25%Making the cell slide, by the method of SP cell immunohistochemical to detect the expression of CXCR4receptors in Tca8113cell surfaceTo use the method of CCK-8to detect the proliferation of CXCL12/CXCR4biological axis on squamous cell carcinoma cell Tca8113of the tongue and to study the effect of AMD3100, which was the inhibitor of CRCR4receptor.The application of PCR to detect the influence of a certainconcentration of CXCL12and its specific receptor AMD3100on expression of mRNA of CXCR4receptors.of squamous cell carcinoma cell Tca8113of the tongue.Through using Transwell Hands with Matrigel in vitro to study the influence of invasion and metastasis of CXCL12/CXCR4biology axis on the tongue squamous cell carcinoma and to study the effect of AMD3100,which is the inhibitor of CRCR4receptors.Results:1The squamous cell carcinoma cell line Tca8113of tongue was cultured in vitro with its good condion and regular growth.The growth of Tca8113curves to approximate the S shape. Generally the cultured cells of logarithmic grow phase could be used in experiment and subculture in the the2-3days of the cell growth, in which the cultured cells can be observed under the microscope closely arranged and with different shapes. About1week, the ability of cell proliferation and division begins to decine and shapes can be changed under the microscope.At the same time, the number of apoptotic cells increased.2The results of immunohistochemical showed:the receptor of CXCR4was expressed positively in the4group, among which the stimulate group of CXCL12factor was with the highest expression level, the group of AMD3100pre-incubated was with the lowest expression level. As well as the other two groups, the difference of expression level of CXCR4receptor was not significant. The receptor of CXCR4was highly expressed in cell membrane, as the expression in cytoplasmic and nucleolar was seldom.3.The methods of detection of CXCR4receptor mRNA in gene level through the application of RT-PCR showed that the expression of CXCR4receptor mRNA can be found in the4group,among which the stimulate group of CXCL12factor was with the highest expression level, the group of AMD3100pre-incubated was with the lowest expression level. As well as the other two groups, the difference of expression level of CXCR4receptor was not significant.The results are almost the same with immunohi stochemical.4. The effects of CXCL12/CXCR4biological axis on the proliferation behavior of squamous cell carcinoma of the tongue through the CCK-8kit show that the efferts are more obvious with the prolongation of time of100ng/mL concentration of CXCL12to the proliferation of Tca8113cell. Low concentrations of factor CXCL12had an unobvious effects on proliferation of Tca8113cells.At the same site of time, the group of stimulation of CXCL12factor was with the highest OD datas. It can have the different degrees of inhibition on the effects of factor CXCL12promoting Tca8113cells on proliferation by AMD3100of1μg/ml incubating each group of chemokines in advance. The most apparent effects of inhibiting the promotion on cell proliferation was the effects to group of CXCL12factor of100ng/ml concentration.5. After the pretreatment of48hours by each experimental group and through the application in vitro of Transwell cells with Matrigel showed us that the group of stimulation of CXCL12factor had the maximum numbers of cells of trans-membrane, as the group of AMD3100pre-incubated was with the minimum numbers of cells of trans-membrane.Between groups the differences were significant different and statistically significant.(P<0.05)Conclusion:1. The sueface of squamous cell carcinoma Tca8113cell of the tongue is highly expressed the CXCR4receptors.2.100ng/mL concentration of CXCL12factor can promote the effect of proliferation in squamous cell carcinoma Tca8113cell of the tongue.At the same time, AMD3100, as the inhibitor of CXCR4receptor can reduce this promotion of proliferation.3.100ng/mL concentration of CXCL12can promote the expression of CXCR4receptors in the surface of squamous cell carcinoma Tca8113cell of the tongue.4.100ng/mL concentration of CXCL12can promote the invasion and metastasis of squamous cell carcinoma Tca8113cell of the tongue, which is highly expressed the CXCR4receptor in the surface of the cells.AMD3100can inhibit the chemotaxis and invasion metastasis by blocking CXCR4receptors.5.CXCL12/CXCR4biological axis plays an important role in proliferation, invasion and metastasis of squamous cell carcinoma Tca8113cell of the tongue. Blocking the interactions between CXCL12and CXCR4may become a new research direction of.the treatment of malignant tumor...