| Background and objective:Glioma as a high degree of intracranial malignant tumolors and because of its aggressive growth mode and the high insensitivity of radiotherapy and chemotherapy,making the recurrence of glioma after surgery and causing poor prognosis.In recent years,many studies suggest that a small numolber of special cells which have multi-directional differentiation potential and play a key role exist in many tumolors,known as cancer stem cells,and cancer stem cell theory.Glioma as the most common malignant t umolor in the brain,also has cancer stem cells,namely glioma stem cells.and Similialy to all tumolor stem cells,glioma stem cells have the ability of self-renewal,infinite proliferation and multi-directional differentiation.Based on the above characteristics,glioma stem cells play a key role in chemotherapy resistance.So at this stage of the stem cell theory holds that stem cells existing in the glioma is the main reason which leads to t umolor recurrence,chemotherapy resistance and failure treatment.Therefore,the focus of research on glioma treatment should be on the removal of glioma stem cells,or to increase the sensitivity of glioma stem cells to drugs.After reviewing articles,we found that hyperoxia treatment of glioma can significantly slow the growth rate of tumolor,tumolor cell apoptosis rate was significantly higher than the normoxia group.In the clinical study of high-grade glioma patients with hyperbaric oxygen combined with radiotherapy and chemotherapy,the results show that hyperbaric combined with radiotherapy and chemotherapy can significantly improve the survival of patients.Recent studies have also shown that hyperoxia can enhance the sensitivity of a variety of chemotherapy drugs on glioma,and thus extend the survival of patients.However,how high oxygen perform as enhanced sensitization of chemotherapy,there still aret lack of relevant experimental evidence currently.Previous studies have shown that the presence of hypoxic microenvironment in most solid tumolors is closely related to the proliferation of tumolor stem cells,self-renewal and maintenance of stem cell characteristics.In our previous experiment,under hypoxic microenvironment in vitro,the glioma cells can reverse differentiation to form glioma stem cells,and further enhance the stem cell characteristics In contrast,whether hyperoxia can be an effective method for correcting hypoxia microenvironment in gliomas and promoting glioma stem cell differentiation and chemotherapy sensitization still need further study.Therefore,in order to prove this scientific hypothesis,we intend to use experiments in vitro,using inducing culture in vitro,immunofluorescence,qRT-PCR,Western-blot and other methods to verify,and through cell cycle,IC50 determination,apoptosis and other tests to prove.Thus we can provide a new theoretical basis for the treatment of glioma with hyperoxia combined with chemotherapy and to improve its prognosis,and provide a new way of thinking for the comprehensive treatment of glioma.Methods:The first part:?1?The inducing culture of glioma stem cells in vitro: U87 and GL261 glioma cells were incubated at 37 ?,5% CO2,containing EGF?20 ng / m L?,b FGF?20 ng / m L?,2% B27 in serumol-free DMEM / F12 mediumol;?2?induced culture of glioma stem cells biological observation:aquire images of stem cell culture-induced glioma cells after 5 days of incubation,and observe the biological morphology.?3?Immunofluorescence assay: use the U87 and GL261 glioma cells which were cultured with stem cells culture medi umol for 5 days and present a good growth status,cultured with immobilized cell culture.The glioma stem cell protein expression?CD133,Nestin and MGMT?was detected by immunofluorescence assay after immobilization.?4?Cell cycle detection: GL261,U87 glioma cells and GL261,U87 glioma cell spheres were detected by cell cycle in normoxic conditions respectively.?5?Apoptosis detection: normal GL261,U87 glioma cells and GL261,U87 glioma cell spheres were treated with TMZ for 24 h after flow cytometry.The second part:?1?marked protein in glioma stem cells?CD133,Nestin?,differentiation protein?GFAP?and drug resistance protein?MGMT?RNA detection: glioma stem cells were cultured in different oxygen concentration conditions?hyperoxide 95% O2,normoxia 21%O2??CD133,Nestin?then conduct qRT-PCR test.?2?marked protein in glioma stem cells?CD133,Nestin?,differentiation protein?GFAP?and drug resistance protein?MGMT?protein detection: protein were detected by Western-blot test after glioma stem cells cultured in different oxygen concentration conditions?hyperoxide 95% O2,normoxia 21%O2?after 24 h and 48 h.?3?IC50 test: glioma stem cells were treated with temozolomide?TMZ?for 10 h after pretreatment with different oxygen concentration?hyperoxia 95% O2,21% O2?for CCK-8 test,aquring their absorbance values and calculate the IC50 value.?4?Apoptosis detection:glioma stem cells were treated with temozolomide after pretreatment with different concentrations of oxygen?95% O2 at high concentration of oxygen,21% O2?,and then conduct flow cytometry apoptosis detection.Results:?1?U87 and GL261 glioma cells were cultured in serumol-free DMEM / F12 mediumol at 37 ?,5% CO2,containing EGF?20 ng / m L?,b FGF?20 ng / m L?and 2% B27.5 days later,U87 and GL261 glioma cells were found to grow suspendly,and most of them were clustered into globules.Morphologically showed tumolor stem cells characteristics of gathering to grow into a ball.?2?Immunofluorescence assay of U87 and GL261 glioma cells after 5 days of culturing with stem cell culture showed that the stem cells marked protein expression of CD133,Nestin and drug resistance protein MGMT were highly expressed in U87 and GL261 glioma cells.?3?The results of cycle test showed that GL261 and U87 glioma stem cells were arrested in G0 / G1 phase?P <0.05?compared with normal cells.?4?Apoptosis showed that the apoptosis of GL261 and U87 glioma stem cells treated with TMZ was significantly lower than that of normal cells under normoxic conditions?P <0.05?.?5?Compared with normoxia condition,after 24 h of treated in hyperoxia,U87 glioma stem cells qRT-PCR results showed that the m RNA expression level of marked protein in glioma stem cells?CD133 and Nestin?was decreased?P <0.05?,the expression of GFAP mRNA was higher?P <0.05?,and the expression of drug resistance protein GFAP m RNA was higher?P <0.05?.?6?Western blot analysis showed that U87 and GL261 glioma stem cells had low expression of stem cell marker protein?CD133,Nestin?after treated with high oxygen fo r 24 h and 48 h compared with normoxia at each time point,and promoted the expression of differentiated marker protein GFAP.Further detection showed that the drug-resistant marker MGMT showed a decreasing trend.The expression of marker protein and drug-resistant protein in glioma stem cells with different oxygen treatment time was also significantly different.Compared with glioma stem cells treated with hyperoxia for 24 h,the glioma stem cells treated with hyperoxia for 48 h showed low expression of ste m cells labeled protein?CD133,Nestin?,high expression differentiation marker protein GFAP.?7?IC50 test showed that glioma stem cells were treated with temozolomide after pretreatment with different oxygen concentration?hyperoxia 95% O2,normoxia 21% O2?,and the IC50 value of the high concentration oxygen group was significantly lower than that of the normoxia group?P <0.05?.?8?Glioma stem cells were treated with temozolomide after pretreatment with different oxygen concentration?hyperoxia 95% O2,normoxia 21% O2?,the flow apoptosis test results of glioma stem cells was significantly higher than that of normoxia group?P <0.05?.Conclusion:Hyperoxia can inhibit the expression of stem cell characteristics in glioma stem cells and promote the differentiation of chemotherapy which can induce higher sensitization,and manifest that glioma stem cells express lower tumolor stem cell marker protein and drug-resistant marker protein,higher expression of tumolor differentiation marker protein after high oxygen treatment.Hyperoxia combined with temozolomide?TMZ?treatment of glioma stem cells showed low proliferation and high apoptosis characteristics. |
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