缺氧响应特性的脂质-聚合物放疗增敏剂抑制脑胶质瘤增殖的研究

Hypoxia-responsive lipid-poly-radiosensitizer inhibits proliferation of glioma

目的研究提高脑胶质瘤细胞的放疗(radiotherapy,RT)敏感性的药物及其效果。方法制备有缺氧响应特性的脂质-聚合物[ALP-(MIs) n]放疗增敏剂,并检测其性质:(1)用核磁共振(1H-NMR)检测聚甲硝唑[ALP-(MIs) 25]是否合成成功;(2) Nano-ZS粒度/Zeta电位测定仪检测实验组ALP-(MIS) 25和对照组聚乳酸-羟基乙酸共聚物(PLGA)聚合物(AL-PLGA)在常氧和缺氧环境下的粒径变化,透射电镜观察脂质-聚合物的形态。细胞水平检测:用克隆集落形成实验检测ALP-(MIS) 25对C6细胞株放疗的增敏性。动物水平检测:(1)构建稳转荧光素酶的C6细胞原位小鼠脑胶质瘤模型;(2)检测肿瘤内缺氧环境;(3)检测小鼠脑胶质瘤的荧光素酶强度,统计小鼠的生存时间。结果 ALP-(MIS) n的制备及其性质:(1) ALP-(MIS) n合成成功;(2) ALP-(MIS) n和AL-PLGA构建成功,电镜观察示为圆形结构,ALP-(MIS) n直径为(88. 81±0. 98) nm,AL-PLGA直径为(80. 38±1. 07) nm;相比较于常氧环境下,缺氧环境下ALP-(MIS) 25的水粒学直径增大,圆形结构破坏;而AL-PLGA在缺氧环境下水粒学直径和形态结构均未发生改变。克隆集落形成实验结果显示,与PBS+RT组、AL-PLGA+RT组相比,ALP-(MIS) n+RT组的C6细胞株克隆形成率明显下降,差异有统计学意义(P <0. 01)。稳转荧光素酶的C6细胞原位小鼠脑胶质瘤模型构建成功;缺氧探针免疫荧光实验结果显示脑胶质瘤组织缺氧。与PBS组、PBS+RT组、AL-PLGA+RT组相比,ALP-(MIs) 25+RT组小鼠的胶质瘤增殖速度最慢,中位生存期最长,差异有统计学意义(P <0. 001,P <0. 05)。结论 ALP-(MIS) n联合放射治疗在缺氧环境下可有效抑制脑胶质瘤增殖。

Objective To solve the bottleneck problem of improving radiosensitivity of glioma cells.Methods ALP-(MIs)n preparation and characterization:(1)If P-(MIs)25 was synthesized correctly,it was confirmed by 1H-NMR assay.(2)Under normoxic and hypoxic conditions,the Malvern ZetasizerNanoZS was used to measure the mean diameter of ALP-(MIs)25 and AL-PLGA.The morphology of ALP-(MIs)25 and AL-PLGA were observed by TEM.In Vitro analysis: The radiosensitity of ALP-(MIs)25on C6 cells were detected by clone formation assay.In Vivo analysis:(1)Glioma-bearing mice were prepared by intracranialinjection C6/Luci/GFP cells.(2) Detection of hypoxic environment in tumors.(3) Tumor growth of C6 bearing mice were monitored by in vivo real-time fluorescence imaging system to monitor the anti-tumor effect of ALP-(MIs)25.The survival time was calculated from the day of glioma cellinoculation (0 day) to the death day.Results ALP-(MIs)25 preparation and characterization:(1) 1H-NMR spectra assay suggested that P-(MIs)25 was synthesized correctly.(2) The average diameters of ALP-(MIs)25 and AL-PLGA were approximately (88.81±0.98)nm and (80.38±1.07)nm.The TEM images suggested that the AL-PLGA and ALP-(MIs)25 were spherical NPs with a unimodal size distribution under normoxic conditions.In sharp contrast,the conformation of ALP-(MIs)25 was disassembled,and the conformation of AL-PLGA remained unchanged under hypoxic conditions.In Vitro analysis: The results of colony formation assay showed that,compared with PBS+RT group and AL-PLGA+RT group,the plating efficiency ofALP-(Mis)25+RT was significantly decreased in C6cells ( P < 0.01 ),and the sensitization enhancement ratio (SER D0 ) of C6 cells was 1.34.In Vivo analysis:(1) The orthotopic transplantation tumor model was successfully established with C6/Luci/GFP cells.(2) The results of hypoxia probe immunofluorescence showed that hypoxia was present in glioma tissues.(3) Compared with other experimental groups,ALP-(MIs)25+RT group suppressed tumor progression,prolonged survival time of the mice more effectively ( P <0.001, P <0.05).Conclusion ALP-(MIs)25 combined with radiotherapy can effectively inhibit the proliferation of glioma.