Comparison Of Biological Characteristics Among Glioma Stem Cell Lines And Preliminary Studies On Radioresistance Of Human Glioma Stem Cells

PartⅠ:Comparison of biological characteristics among glioma stem cell linesObjective: To investigate and compare the biological characteristics of glioma stem/progenitor cells (GSPCs) among different glioma cell lines.Methods:Human glioma stem/progenitor cell lines SU-2,SU-3 and human glioma cell lines U-87MG, U-251 were cultured in vitro in medium favored for stem cell growth, in medium favored for cell differentiation,respectively. The morphological difference of GSPCs among four glioma cell lines were compared. Differences in expression levels of glioma stem cell markers CD133, Nestin and glial cell differentiation marker GFAP, and the percentage of G0～G1 cells and apoptosis cells were also investigated and compared.Results: Under the culture conditions favored for stem cell growth, the apperances of four GSPCs spheres were different and the sphere-forming times were different, too. The percentages of CD133+, Nestin+and G0～G1 cells in four cell lines revealed with flow cytometry were much higher than those under differentiation culture conditions. Expression level of CD133 gene detected with real-time fluorescent quantitative PCR showed significant increasing in tumor spere than in adherent tumor cells(P<0.01). The percentage of apoptosis cells in tumor spheres was lower. Under stem cell culture conditions, all of the four cell lines were mainly composed of Nestin+ cells, the percentage which varied from 60% to 95% , and the percentage of CD133+ cells varied from 1% to 5%.Conclusions: The basic biological characteristics of GSPCs from different glioma cell lines were close to each other, but the contents of stem cells and the experssion levels of cell surface markers showed certain difference, which deserve further attention when make comparison between different literatures. In the 2D in vitro culture system, the tumor spheres were mainly composed of Nestin+ cells when under stem cell culture condition. PartⅡ: Preliminary studies on the molecular mechanisms of radioresistance of human glioma stem cellsObjective: To study the radioresistance of human glioma stem/progenitor cells and investigate the relevant molecular mechanisms.Methods:Human cerebral glioma stem/progenitor cell lines SU-2, SU-3 and human glioma cell lines U-87MG, U-251 were cultured in vitro under stem cell culture conditions. These glioma stem/progenitor cell lines were irradiated by liner accelerator according to radiation dosages of 5Gy,10Gy and 15Gy. Then using the flow cytometry to quantify the percentage of CD133+,Nestin+,GFAP+ cells. Real-time fluorescent quantitative PCR was performed to detect the CD133 gene expression level at the time of 24h,48h and 72h after radiation. Then expression lvevls of DNA Damage Signaling Pathway were analyzed with relevant PCR array in U-87MG GSPCs before radiation and after 72h at a dosage of 10 Gy radiation.Results: Under radioation dosages ranges between 0～15Gy, the percentage of CD133+ cells and the CD133 expression level increased obviously, but the percentage of Nestin+,GFAP+ cells descended. The result of DNA Damage Signaling Pathway PCR Array showed that the expression levels of BRCA1,DMC1,GTSE1,NBN decreased more than upregulation of two times and GML,IP6K3,N4BP2,PPP1R15A,RAD50,SEMA4A,TREX1,XPA about more than two times.Conclusions: The CD133+ cells are the main cell population to participate the radiationresistance of glioma. The molecular mechanisms maybe enhance the DNA double strands damage, repair capacity and excision repair capacity,and correct the human pclymerase, and down regulation the level of anti-oncogene expression, and to remove the function of P53 in order to diminish cell apoptosis.