| Background and objectiveThe glioma of human is the most common primary tumor and accounts for more than 40%of all central nervous system(CNS) neoplasmas.It is easy to recurrence, poor prognosis,mainly because invasive growth of gliomas are the biological characteristics which lead to various treatment measures difficult to achieve the purpose of healing.Calcium ion is a key ionic signal of the tumor cell's invasion and metastasis.Magnesium as a natural calcium antagonist,and its impact on tumor cell infiltration is also worthy of attention.Tumor cells and matrix elements participate in the completion of invasion and metastasis of the tumors,in which the basic steps of extra cell matrix(ECM) and vascular basement membrane degradation and destruction.The experiments show that the invasion and me- tastasis of tumor cells is closely related to the capacity of producing proteaseinduced degradation of ECM and the basement membrane.In tumor tissues, matrix metalloproteinases(MMPs) take part in the invasion of tumor growth and metastasis by regulating tumor cells adhesion with ECM,degradation of extracellular matrix in tumor cells and increasing tumor angi- ogenesis.Adding calcium and magnesium ions to the culture medium of tumor cells in vitro can change the the ability of secreting MMPs of tumor cells,for which could have an impact on the invasiveness of tumor cells.Primary culture also known as first culture which is obtained from the donor cells in vitro.Primary culture of cells from tissues and cells in vitro has biological traits did not change significantly,the closest and response of the growth characteristics in vivo.At present,there are parts of human glioma cells were successfully cultured,which are reported in most of astrocytomas,and ependymoma cells,especially human ependymoma cells in primary culture has not been reported in China.The traditional separation of the glioma cells is the enzymatic digestion and mechanical dispersion method, this experiment used "mechanical dispersion method + trypsin digestion" integrated separation method for the first time to cultivate the cells of astrocytoma and ependymoma. Contrast the characteristics of three methods of their own,with a view to finding a more low-priced and effective method of primary culture.This experiment is designed to regulate the calcium and magnesium ions' concentration changes in vitro to explore the impact on human malignant glioma cells' invasion ability,in order to further explore the molecular mechanism of glioma's invasiveness.Materials and methods1.Take the fresh tissue and make pathology exameWe use a part of the tissue for pathology exame,the rest about 1cm×1cm×1cm is to be wrapped up asepsisly.Then we put the tissue into cultivate room,and the tissue which was proved astrocytic glioma or ependymoma will be cultured continuely.2.Handing of gliomasSeparating astrocytic glioma tissue into cells with mechanical dispersion & trypsin digestion method.The cell suspension was filtrated by 200 mesh filter and centrifugalized with 800～1000 r/min×5min,then removed the supernatant liquid and the rest cells were used for cell culture.3.Cell cultureThe glioma cells were cultured with DMEM as the culture medium,substituted with 10%vol fatal bovine serium(FBS).The cells were grown in a nonhumidified in cubator at 37℃with 5%CO2 for several days,with periodic medium changes every 3 to 4 days.Once the cells reached confluence,they were removed by trypsin(0.25%) to count cell and to do down to the future genetation.To continue to foster the cells,after the morphology and immunocytochemical identification, choose the cells of third generation which grow well to disposal of DMEM with FBS culture medium,adding fresh DMEM without FBS in the culture medium, and add in the calcium or magnesium ions,regulate the calcium concentration were 0.8mmol/L,1.2mmol/L 1.6 mmol/L,2.0 mmol/L,adjusting the concentration of magnesium ions were 0.5 mmol/L,1.0 mmol/L,1.5 mmol/L, 2.0 mmol/L,and which does not contain calcium and magnesium ions as the blank control group.After various treatments for 24 h,cells and the medium samples were harvested,then we can use them respectively to do the Boyden chamber experiments and gelatin zymography.4.Cell morphologyWe can observe the cells of different phases and all groups with invert microscope.5.Immunocytochemistry identificationThe primary culture cells were send for immunocytochemistry studies(astrocytic glioma:GFAP,CollagenⅣ,nestin;ependymoma:GFAP,CD99,EMA).6.Experiment of Boyden chamberTo observe the invasiveness of glioma cells under the influence of calcium and magnesium ions of different levels,we use theⅢandⅣgrades of astrocytic glioma cells,ⅡandⅢgrades of ependymoma cells to do Boyden chamber experiment.Then we can examine the changes of the invasiveness of the cells which cultured during the same time and under different culture conditions. 7.Gelatin zymography detected the expression of MMP-2 enzyme and plasminogen. Gelatin zymography assay at the different concentrations of calcium and magnesium ions under the influence of the different levels of glioma cells with MMP-2 enzyme and the expression of plasminogen,which determine the different concentration of calcium and magnesium ions in vitro could affect the invasiveness of glioma cells8.Statistical analysis:All datas were analyzed by SPSS10.0 stastistical package,we used analysis of variance.The level of significant difference was a=0.05.Results1.Routine pathological study revealed that the 20 examples are all astrocytic glioma patients.(16 cases survived).There are7 astrocytic gliomas of gradeⅢ(anaplasia astrocytic glioma) and 9 astrocytic gliomas of grade(glioblastoma). 11examples are all ependymoma patients(9 cases survived).There are7 ependymomas(gradeⅡ) and 2 anaplastic ependymomas(gradeⅢ).2.Both physical and chemical methods were successfully applied to cultivate primary astrocytic glioma cells and ependymoma cells.There are different cell growth activity in different levels of tumor.Astrocytic glioma cells of gradeⅢandⅣcan survive and to go down to posterity for 4 or 6 generations(about 28 days).Ependymoma cells of gradeⅡandⅢcan survive and to go down to posterity for 4 or 5 generations(about 25 days).3.Observatining in morphology:in innverted microscope,cell morphologys is diverse and in different sizes.The astrocytic glioma cells' appearance is star,triangle,diamond and fusiform.The higher the level of tumor,the shorter time of cell adherence and the stronger growth.While,ependymoma cells' appearance is just like rose-knot.Vary in sizes,cytoplasm-rich,non-branch-like protrusions.4.Immunocytochemistry examination:astrocytic glioma cells:GFAP and nestin are positive,CollagenⅣis negative.Ependymoma cells:GFAP and CD99 are positive,EMA is negative.These tests and cell appearances demonstrate that the cells we cultured were astrocytoma cells and ependymoma cells.5.The experimental results with Boyden chamber:with the increases of calcium ions concentration in vitro,the number trans-membrane cells of the astrocytic glioma cells of gradeⅢandⅣand ependymoma cells of gradeⅡandⅢin primary culture has the tendency which is decreased after the first increased.In contrast,With the increased concentration of magnesium ions,the transmembrane cell number is decreasing.Compared with astrocytic glioma cells of gradeⅢ,the number of gradeⅣcells traversed Matrigel was increased obviously in experiment group(P＜0.05).The same as gradeⅢependymoma cells with which compared gradeⅡgroup(P＜0.05).6.Samples were analysed by gelatin zymography:with the different concentration of the calcium and magnesium ions in vitro,there were 4 or 5 bands in astrocytic gliomas of gradeⅢandⅣand ependymoma cells of gradeⅡandⅢin primary culture.Mloecular weight of MMP-2,intermediate MMP-2,proMMP-2,MMP-9 and proMMP-9 were 62 kD,64 kD,72kD,82 kD and 92kD,respectively.With the increase of calcium concentration,MMP-2 levels lowered after the first increase;and with the increase of magnesium concentration,MMP-2 content is decreasing.There was no significant difference among the calcium groups (P＞0.05),but the difference the magnesium ions groups was statistically significant(P＜0.05).Conclusions1.By using mechanical dispersion & trypsin digestion method,human astrocytic glioma cells and ependymoma cells are cultured successfully and a convenient and effective method is established in gliomas primary culture.2.The forms of astrocytic glioma cells are different from the ependymoma cells.The former are look like star,triangular cells,and so on.But the latter is looks like rose-knot.3.The original malignant glioma cells' invasiveness can affected by the Changes in calcium and magnesium ions in vitro:With the increase of calcium concentration, the invasiveness of glioma cells is lowered after the first increase;and with the increase of magnesium concentration,the invasiveness is decreasing. |
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