缺氧与高级别脑胶质瘤化疗抵抗和预后的关系及高压氧舱临床疗效的初步研究

The relationships of hypoxia with resistance to chemotherapy and prognosis of high-grade gliomas and study on clinical effect of hyperbaric oxygen therapy

摘要:

目的探究缺氧与人脑胶质瘤细胞化疗抵抗及患者预后的关系及高压氧舱在高级别脑胶质瘤患者中应用的疗效分析。方法体外使用氯化钴诱导人脑胶质瘤细胞U251 缺氧状态，蛋白印迹法（Western blot）检测细胞中缺氧标志物缺氧诱导因子-1α（HIF-1α）的变化，噻唑蓝（MTT）染色法检测U251 细胞替莫唑胺（TMZ）的半抑制浓度（IC50）的变化。逆转录PCR（RT-PCR）检测患者高级别脑胶质瘤组织中HIF-1α的表达特征，比较与正常脑组织及低级别脑胶质瘤组织表达的差异，临床上采用Kaplan-Meier 生存分析法分析高压氧舱治疗与高级别脑胶质瘤患者预后的相关性，同时分析患者HIF-1α的表达与未行高压氧舱治疗的患者预后的相关性。结果体外成功诱导U251 细胞的缺氧状态，Western blot结果示U251-ol 的HIF-1α表达水平高于对照细胞（U251-control），MTT结果显示，U251-ol细胞TMZ 的IC50 值高于U251-control（P <0.05）。RT-PCR 结果示高级别脑胶质瘤HIF-1α的表达高于正常脑组织及低级别脑胶质瘤组织（P <0.05）。Kaplan-Meier生存分析结果示行高压氧舱治疗的高级别脑胶质瘤患者2年内预后优于未行高压氧舱治疗的患者（P <0.05），但长期随访差异无统计学意义（P >0.05）。未行高压氧舱治疗的高级别脑胶质瘤患者中，高表达HIF-1α的患者相比低表达的患者预后不良，差异有统计学意义（P <0.05）。结论缺氧可促进脑胶质瘤细胞化疗抵抗的形成，且不利于患者预后，高压氧舱能有效改善脑胶质瘤组织的缺氧状况，有效提高患者短期预后情况，是值得推广的辅助治疗方式。

Abstract:

Objective To explore the relationships of hypoxia with resistance to chemotherapy of human glioma cells and prognosis of high-grade gliomas, and to analyze the curative effect of hyperbaric oxygen therapy in high-grade glioma patients. Methods In vitro, cobalt chloride was used to induce hypoxia in human glioma cell line U251, Western blot assay was used to detect the expression of hypoxia inducible factor -1α(HIF-1α), which is the marker of hypoxia. thiazolyl blue (MTT) assay was used to detect the changes of half inhibitory concentration (IC50) of Temozolomide (TMZ) in the U251 cells. Fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the expression character of HIF-1α in the high -grade glioma tissues, compared with that of normal brain tissue and low -grade glioma tissue.Kaplan-Meier survival analysis was used to analyze the correlation between hyperbaric oxygen therapy and prognosis of patients with high-grade gliomas, and the correlation between the expression of HIF-1α and prognosis of patients without hyperbaric oxygen therapy. Results In vitro, the hypoxia of U251 cells was induced successfully. Western blot assay showed that HIF-1α of the hypoxia of U251 cells (U251-ol) was significantly higher than that of control cells (U251-control). MTT assay showed that IC50 of TMZ in the U251-ol line was significantly higher than that in U251-control (P < 0.05). Results of RT-PCR showed that the expression of HIF-1α in high-grade gliomas was significantly higher than that in normal brain tissue and low -grade gliomas. Kaplan -Meier survival analysis showed that the prognosis of patients with hyperbaric oxygen therapy was better than that of patients without hyperbaric oxygen therapy in the high-grade gliomas patients in two years (P < 0.05), but there was no significantly statistical difference in the long term (P >0.05). The high-grade gliomas patients with high expression of HIF-1α who did not receive hyperbaric oxygen therapy had a poorer prognosis compared with the low-grade gliomas patients (P < 0.05). Conclusions Hypoxia can promote resistance to chemotherapy of glioma cells, and is not conducive to the prognosis. The hyperbaric oxygen theraphy can effectively reverse hypoxia in glioma tumor tissue, and improve the prognosis of patients.