The Realtion Of BCL-2with The Biological Propeties Of U251Cell And Its Temozolomide Resistance

Part1:The Expression of BCL-2in Human Glioma and Its Relationship with the Prognosis of the High-Grade GliomaObjective:To investigate the expression level of BCL-2in human glioma and detect the prognostic value of BCL-2in high-grade glioma(HGG) patients.Methods:The glioma brain tissues and the normal brain tissues were collected from50glioma patients and10controls after surgery. The BCL-2expression in the brain tissues was tested through immunohistochemistry. All the HGG patients accepted surgery operation, radiotherapy, and temozolomide. The HGG patients were divided into BCL-2positive group and BCL-2negative group according the BCL-2expression. All the HGG patients were followed up for at least6months and the progression markers such as disease control rate, free survival time, drug safety were evaluated.Result:Immunohistochemistry result showed that BCL-2expression was found in64%(32/50) glioma tissues and0%(0/10) normal brain tissues. The BCL-2positive rate of HGG patients (73.0%,27/37) was higher than low grade glioma(LGG)(38.5%,5/13).In the36HGG patients, the objective response rateand disease control rate the BCL-2positive group were19.2%(5/26) and30.6%(8/26), compared with the BCL-2negative group were60.0%(6/10)and70.0%(7/10).Conclusion:Glioma tissues express BCL-2highly,the BCL-2protein expression and is associated with the malignant degree Glioma;BCL-2may influence the prognosis of HGG Part2:Construction of pEGFP-BCL-2Expression Plasmid and Establishment of IIts Stable Expression CellObjective:To construct the plasmid of pEGFP-BCL-2and observe its expression in U251cells.Methods:The BC1-2cDNA was cloned from U251cells through RT-PCR, and then was put into pEGFP-N1vector. The BCL-2construct and control vector were transfected into U251cells by FUGNE HD and the stable cell line (BCL-2-U251and N1-U251) were selected through G418. The mRNA and protein expression of BCL-2were studied.Results:The BC1-2mRNA expression in control U251cells, N1transfected cells and BCL-2overexpression cells were:0.32±0.01,0.30±0.01,1.21±0.23respecteively, and the BCL-2protein expression were:0.131±0.002,0.012±0.002,4.813±0.5134respectively.Conclusion:The pEGFP-BCL-2has been successfully constructed, and it can stably expressed in U251cells. Part3:The Realtion of the Expression of BCL-2with the Biological Propeties of U251Cell and Its Temozolomide Resistance.Objective:To investigate the effect of upregulation or silence of BCL-2on the the biological propeties of U251cell and its temozolomide resistance.Method:The U251cells were transfect with pEGFP-BCL-2construct or infected by BCL-2-shRNA virus. They were divided into three groups:pEGFP-BCL-2-U251, BCL-2-ShRNA-U251, and U251control. The effect of upregulation or the downregulation of BCL-2on the proliferation of cell, cycle and apoptosis, colony formation and invasion, and its relationship with temozolomide resistance.Results:The proliferation of U251cell was lower in BCL-2-shRNA-U251infected cells than that of the control U251cells, while the overexpression of BCL-2increased the proliferation in pEGFP-BCL-2-U251tranfested cells than that of the control U251cells.The apoptosis and the G2/M ratio was higher in BCL-2-shRNA-U251infected cells than that of the pEGFP-BCL-2-U251tranfested cells and the contrl cells(P<0.05). The S ratio in the pEGFP-BCL-2-U251tranfested cells was higher than that of control and BCL-2-shRNA-U251infected cells(P<0.05). After the treatment with50uM temozolomide for24h, the apoptosis of all the three groups were increased, and G2/M raio was increased in control cells and BCL-2overexpression cells, but was decreased in BCL-2knock down cells(P<0.05). The IC50temozolomide in pEGFP-BCL-2-U251transfected cells, U251control cells, and BCL-2-shRNA-U251were148.0μM,84.3μM and51.6μM respectively.The colony forming rate of these three groups were67.7%±2.5%,65.0%±3.0%and26.6%±2.1%(P<0.05), the Transwell cell number after20h treatment were61.3±3.2,42.0±3.1and25.6±3.7(P<0.05) respectively in these three group. Conclusion:The expression of BCL-2is related with the malignant degree of U251cells, and also indecrease temozolomide resistance.