The Study Of Inhibition Of Bone Morphogenetic Protein 4 On Proliferating Glioma Cells

Glioma, as the name of gliocytoma, is the tumor from neuroe-pidermal layer, which is so called"neuroectoderm tumor"or"neuroepithelium tumor". These tumor stem from nerve cell, which has characteristics of rapid growth and easy recrudescence. The main gliomas therapies are operation, radiation, chemotherapy, X rays andγrays. But theorily, operation cann't remote the tumor completely, and cann't be used in the tumor of truncus encephalicus. The most of gliomas is resistant to irradition and chemotherapy. And the damage of the brain function cann't be ignored. The inducive differentiation therapy is a new method for tumor chemotherapy, which mainly relief the tumor development discrepancy , repress the tumor proliferation, reverse the malignant phenotype including proliferation, infil-tration and metastasis, and induce tumor differentiation into normal cells. The investigation is about the differentiation effect of medicine on gliomas. But when the body is used in medicine, the medicine concentration of the tumor is very low, and the secondary effect is obvious, which is useless in clinics. It has been reported that there were a lot of BMP4 proteins in dorsal and ventral telencephalon at the beginning of neuron and in choroid plexus in the late stage of neuron. It has also been suggested that BMPs is one of important extracellular signals in the development of cholinergic neuron cells. The neuron cell differentiation style is determined by BMPs, other extracellular signals and homology transcription factors which is determined by hereditary. BMP is multifunction factor, which is the super family of TGF-β, is a lot of parallel constitution and high conservation function protein , can induce the factors of bone and cartilage. And the factor has great effect on embryonic development, regeneration and repair about bone when the body grows, cartilage needs ossification, the early period of fracture and the repairment of cartilage. BMP is very important in the process of embryonic development, nerve development and repairment, hematopoietic tissue development and the induction of cell apoptosis.It has been reported that low consistency BMP4(1～5ng/m1) can induce neuron cell proliferation, however, high consistency BMP4 (10～100ng/ml)can suppress proliferation.Cell proliferation is one of the cell fundamental characteristics. Cell growth, DNA duplicate and cell division are included in proliferation by cell cycle. There are multiple regulation factors, and were many investigation about the apoptosis of cell cycle which is modulated by cAMP as the intracellular second signal. It has been found that the level of cAMP was lowest in M phase of cultured rabbit liver cells, and mitosis delay was induced by dibutyryl cAMP. Cell mitosis was suppressed by Dibutyryl cAMP in later G2 phase. The cell growth speed was reduced and cell proliferation was suppressed by relative factors of increasing intracellular cAMP. However, cAMP has cell division effect on thymus and lymphocyte of peripheral blood. The level of cAMP was increased when cancer cells undergo malignant degeneration.Objective: To investigate inhibitory effect of BMP4 with 20ng/ml on glioma proliferation and whether synergetic effect exist between BMP4 and cAMP.Methods:C6 cells are cultured in DMEM with 10% calf serum(CS) and take a photo in order to observe normal growth state of glioma cell;Vimentin immunocytochemistry was detected the glioma cells in order to identify the glioma cells;The suppressive effect of BMP4 with 20ng/ml on glioma proliferation was detected by immunocytochemistry, MTT, skin-prick test and flow cytometry;We compared BMP4, BMP4 with cAMP and control inorder to detect whether there was synergy effect between BMP4 and cAMP.Result:The normal growth state of glioma cells is like shuttle with long cytoplasmic projections on both side or multiple cytoplasmic projections in few cells. And the cells displayed monolayer growth;Vimentin immunocytochemistry showed us that Vimentin immunoreaction is positive in most of cells. This provides us that our glioma cells has the characteristics of infantilism star glioma cells;The result of immunocytochemistry is that the Brdu positive cells with 20ng/ml BMP4 and BMP4 with cAMP obviously diminished comparing the control (P<0.05). However,the ratio between the group of BMP4 with 20ng/ml and the group of BMP4 with cAMP is P>0.05.There is proliferation inhibition of BMP4 on glioma in vitro and there is no synergy between BMP4 and cAMP;The result of FCM is that the ratio of G1/G0 phase is rise to 73.42%, the ratio of S phase is descent to 18.91%, and the ratio of G2+M phase is 15.69%. These provide us that cell proliferation is suppressed by BMP4 with 20ng/ml;The result of MTT is that the absorbance of gliomas cells with 20ng/ml BMP4 is less than the control in the time of 24h,48h and 72h. The cell proliferation is inhibited. The amount of the gliomas cells with 20ng/ml BMP4 is less than the control;Skin-prick test showed that the recovery speed of BMP4 with 20ng/ml is slower than control in second day.The glioma cells proliferation is suppressed by BMP4 with 20 ng/ml.Conclusion:The gliomas cell proliferation was inhibied after treated by BMP4 with 20ng/ml;There was no synergy effect between BMP4 with 20ng/ml and cAMP to induce differentiation on glioma cancer cells.