摘要
目的分析CREB基因参与胶质瘤U251细胞系多药耐药的形成及可能存在的机制。方法免疫组化法检测各级别脑胶质瘤标本中CREB水平差异;分布诱导法建立胶质瘤U251耐药细胞系U251/TR;Crispr/cas9特异性敲除耐药株的CREB基因,流式细胞分析术(FCM)检测药物干预下细胞凋亡情况;Western blotting、rt-PCR检测ABCG2、MGMT、MRP及P-gp基因表达水平。结果成功培养出稳定的脑胶质瘤耐药细胞系U251/TR,耐药倍数为7。特异性敲除耐药株CREB基因获得U251/RC细胞系,TMZ干预下U251/RC凋亡水平远高于U251/TR。Western及rt-PCR测定结果显示,U251/RC的ABCG2、MGMT、MRP及P-gp表达水平均明显降低。结论 CREB通过多种机制参与调控下游ABCG2、MGMT、MRP及P-gp的表达水平,参与胶质瘤细胞多药耐药性的产生与发展。
Objective To investigate the role of CREB gene in the formation of muhidrug resistance in glioma U251 cell line and its possible mechanism. Methods The expression of CREB in different gliomas was detected with immunohistochemistry. The glioma U251 drug resistant cell lines U251/TR were obtained with distributed induction method. Crispr/cas9 specific knockout the CREB gene from U251/TR. Flow cytometry （FCM） was used to detect the apoptosis of cells under drug intervention. The expression of ABCG2, MGMT, MRP and P- gp were detected with Western blotting and rt-PCR. Results The stable glioma cell line was successfully cultured with resistance index of 7. The expression of U251 / RC was significantly higher in U251 / RC cell line than that in U251 / RC cell line. Western and rt-PCR results showed that the expression levels of ABCG2, MGMT, MRP and P-gp in U251 / RC were significantly decreased. Conclusion CREB regulates the expression of down- stream ABCG2, MGMT, MRP and P-gp by various mechanisms, and participates in the production of multi- drug resistance in glioma cells.
出处
《医学研究与教育》
CAS
2017年第2期1-10,共10页
Medical Research and Education
基金
河北省自然科学基金资助项目(20170187)
