The Human Brain Astrocytic Glioma Cells In Primary Culture And The Experimental Study Of Its Invasion

Background and objectiveAstrocytic glioma is the most common primary brain tumor and accounts for more than 60% of all central nervous system (CNS) neoplasmas. One of the main biological features of astrocytic glioma is the local invasion of its constituent neoplastic cells into the surrounding brain tissue. Complete surgical resection is usually curative. For incompletely resected or recurrent tumors,radiotherapy or chemotherapy is administered. Astrocytic gliomas have a high recurrence rate. So the researches of astrocytic glioma invasion and the prevention of recurrence has become the important aspects in glioma treatments.The original culture is the culture in vitro for the first time after it's obtaining from the donor cells. Just because of tissues and cells in vitro,primary cultured cells' biological traits not seen tremendous changes,they still have twice the genetic characteristics and closest to the organization and response in vivo growth characteristics. The experiment use tissues after human astrocytic glioma surgery and the tissues to be sheared,digested,cultivated ,then to be gone down to posterity and accredited. At the same time, the cell's growth activity will be observed and a kind of convenience,easy,cheap and high performance method will be erected. So this will provide a theoretical basis foundation on the astrocytic glioma study of genesis, development,the biological behavior of tumor and therapy.Aggressive is the features of astrocytic gliomas and it is also the key to difficult healing in clinical conventional therapy. Boyden chamber is an invasion of tumor cells in vitro invasion of determination. We put the certain number cells on polycarbonate microporous membrane and record the number of transmembrane cells in plastic substrate existing or not. That the tendency and invasion of astrocytic glioma primary culture cells can be observed.This study aims to evaluate the human astrocytic glioma cells in primary culture methods. To establish the astrocytic glioma original cell culture method in short-term in vitro through the different levels of astrocytic glioma cells cultured and observe the relationship between the growth and invasion of the cultured cells with the level of tumors. So as to provide a theoretical basis to astrocytic glioma treatment or prevention of astrocytic glioma recurrence.Materials and methods1. Take the fresh tissue and make pathology exameWe use a part of tissue for pathology exame,another about 1cm×1cm×1cm is to be wrapped up asepsisly. Than we put the tissue into cultivate room,and the tissue which was proved astrocytic glioma will be cultured continuely.2. Handing of astrocytic gliomasThe tissue was divided and use for cell culture and routine pathological study. Separating astrocytic glioma tissue into cells with mechanical methods.3. Cell cultureThe astrocytic glioma cells were cultured with PRMI 1640 as the culture medium,substituted with 10% vol fatal bovine serium(FBS),100U/ml penicillin,and 100ug/ml streptomycin. The cells were grown in a nonhumidified in cubator at 37℃with 5% CO₂ for several days,with periodic medium changes. Once the cells reached confluence,the were removed by trypsin(0.25%) to culture and to count cell and to do down to the future genetation.4. Cell morphology We can observe the cells of different phases and all groups with invert microscope.5. Immunocytochemistry studiesThe primary culture cells were send for immunocytochemistry studies(GFAP> CollagenIV,nestin).6. Experiment of Boyden chamberIn the same times and conditions,to determine the tendency and invasion of astrocytic glioma primary culture III and IV grades cells with Boyden chamber and observe the tendency and invasion of astrocytic glioma primary culture cells between the 2 kinds of cells.7. All data were analysised by SPSS10.0 stastistical package, we used t-test. The level of significant difference was a=0.05.Results1. Routine pathological study revealed that the 22 examples are all astrocytic glioma patients. They are 3 astrocytic gliomas of grade I (pilocytic astrocytoma,subependymal giant cell astrocytoma),4 astrocytic gliomas of grade II (diffuse astrocytic glioma),7 astrocytic gliomas of grade III(anaplasia astrocytic glioma) and 8 astrocytic gliomas of grade IV(glioblastoma).The immunohistochemistry examination in astrocytic glioma section is that GFAP is positive.2. We successfully applied to both physical and chemical methods to cultivate primary astrocytic glioma cells. There are different cell growth activity in different levels of tumor. Astrocytic glioma cells of grade I can not survive, grade II can survive but not to go down to posterity,grade III and IV can survive and to go down to posterity for 4 or 6 generations(about 28 days).3. Observatining in morphology:in innverted microscope,cell morphologys is diverse and in different sizes. The cells' appearance is star,triangle,diamond and fusiform. The higher the level of tumor, the shorter time of cell adherence and the strong growth.4. Immunocytochemistry examination: GFAP and nestin are positive, Collagen IV is negative. These test and cell appearance demonstration that the cells we cultured are astrocytic glioma cells.5. The tendency and invasion of experimental results with Boyden chamber is: astrocytic glioma cells of grade III and IV in primary culture has the ability of tendency and invasion. Compared with glioma cells of grade III,the number of grade IV cells traversed Matrigel was increased obviously in experiment group (P<0.05) .Conclusions1. Human astrocytic glioma cells were cultured successfully and we establish a convenient and utility method in astrocytic gliomas primary culture.2. The cells can be proved human astrocytic glioma cells by morphology and immunocytochemistry examination3. The primary culture cells' biological activity,reproductive activity increased with high grade.4. The primary culture cells can be used for Boyden chamber experiment.5. Astrocytic glioma cells of grade III and IV in primary culture has the ability of tendency and invasion.6. The tendency and invasion in astrocytic glioma cells of grade IV is higher obviously than astrocytic glioma cells of grade III.