| Objective The purposes of research is to detect if rh-Eendostain has an inhibitive effect in the proliferation on human glioma cells.It aims to provide a theoretical basis for the drug screening.Methods (1)Human brain gliomas were cultured under the conditions:at 37℃, P <95% of the air in the DMDM medium. Digesting cells with 0.125% trypsin and 0.01%EDTA, cells were passaged at ratio of 1:3 or 1:4.(2) Glioma cell line,at the concentration of 5～10×104/ml,were inoculated in 96 well culture plates,After 24 hours, the final concentration of 50ng/ml, 100ng/ml, 150ng/ml of rh-endostatin were givened,.Each plate was given control group.0.5～lmg/ml of MTT solution,100ul was added and cells were cultured. The culture supernatant was removed and 150ul of DMSO was added.After 10 mins, the OD value was measured at the length of 490nm wave。(3) Coverslips were given the concentration of 5×106/ml glioma cells.The medium was changed after 2 days,then add rh-endostatin,then fixed by 4% paraformaldehyde buffer for 20 min.Cells light-loaded for 30 minutes under the conditions of 37℃with Fluo-3.With Bio-Red Radiance 2100 laser scanning confocal microscope of Ar ion laser operating system,cells were scaned.(4) Monoplast suspension was harvested and added rh-endostatin. Monoplast suspension was harvested after trypsinization.70% of the cold ethanol fixed 24h. Propidine iodide was added and cells were keeped in a cool dark place. By using FACSCalibur flow cytometry,cells were tested. Results Apoptotic peak was observed in FCM histogram of cells and the apoptotic index was meseaused.Results (1) The morphological response of glioma cells. The control group cells were in good conditions is round cells, short spindle and have polygonal short cell process. Cells are transparent and have a good refractive index. With the increase in drug concentration and effect time,the size of cells were reduced and have an irregular show. Refractive index was reduced. And a small number of cells can be seen floating in the culture medium.At the concentration of 150ng/ml,cells were in bad conditions and a large number of cells were floating in the culture medium. And necrotic cells and debris could be seen. Cells shows that particulate matter.(2) The growth by MTT The inhibitive rate of experimental groups were higher than that of control groups. The OD value of the control group and experimental groups were 0.302±0.012,0.284±0.006,0.218±0.014 and 0.175±0.012 respectively. There is a significance.(3) The Ca2+ concentration of cell with laser scanning confocal microscopy The Ca2+ concentration of the control group and experimental group were 23.21±2.18,37.36±3.05,51.47±3.12 and 64.41±3.01 respectively. (4) The Flow cytometry assay illustrated there is a sub-lipliod apoptotic peak in the experimental groups on the left of peak of G0/G1. G0/G1 phase cells were increased and S phase cells were decreased in cell cycle.Conclusion rh-Eendostain can inhibit the proliferation of human glioma cells and is dose-dependent. The research provides a theoretical basis for the drug screening. |
PDF Full Text Request