低氧微环境下替莫唑胺对脑胶质瘤细胞凋亡及对BCL-2表达的影响

发布时间：2018-07-17 06:28

【摘要】：第一部分:低氧微环境下替莫唑胺对脑胶质瘤细胞增殖与凋亡的影响[目的]TMZ如今被当作第一线药物用来治疗脑胶质瘤。MGMT是一种DNA修复酶,可以对烷化剂药物造成的DNA损伤进行修复,也会进一步使肿瘤细胞产生耐药性。按照脑胶质瘤细胞表达MGMT的高低,脑胶质瘤细胞可以分为MGMT高表达细胞和MGMT低表达细胞。其中T98G细胞为高表达细胞,U87细胞为低表达细胞。此部分研究通过模拟肿瘤体内低氧微环境,选择高浓度替莫唑胺干预T98G细胞,低浓度替莫唑胺干预U87细胞,培养后检测增殖及凋亡情况。[方法]将T98G细胞及U87细胞设为常氧对照组(21%02)、常氧+替莫唑胺实验组(21%02+TMZ)、0.5%低氧+替莫唑胺实验组(0.5%O2+TMZ)和5%低氧+替莫唑胺实验组(5%02+TMZ),细胞贴壁后,T98G加1mmolTMZ,U87加0.1mmolTMZ培养72h;倒置显微镜下观察生长情况;利用MTS法检测吸光度即增值情况;AnnexinV/PI相关处理染色之后,利用流式细胞仪检测其凋亡情况。[结果]MGMT高表达的T98G细胞在倒置显微镜下的生长情况:常氧实验组形状细长;0.5%低氧实验组排列分散、突起不明显;5%低氧实验组形状细长、突起不明显。T98G细胞吸光度:常氧对照组(0.64±0.05);常氧实验组(0.43±0.12);0.5%低氧实验组(0.61±0.06);5%低氧实验组(0.40±0.04)。0.5%低氧实验组吸光度即增殖率高于常氧实验组(P0.05)。T98G细胞凋亡率:常氧对照组(19.57±1.61);常氧实验组(37.83±9.46);0.5%低氧实验组(34.63±0.98);5%低氧实验组(21.10±3.58)。5%低氧实验组凋亡率低于常氧实验组(P0.05)。MGMT低表达的U87细胞在倒置显微镜下的生长情况:常氧实验组形状细长:0.5%低氧实验组形状细长;5%低氧实验组形状细长、排列分散。U87细胞吸光度:常氧对照组(0.56±0.05);常氧实验组(0.44±0.07);0.5%低氧实验组(0.51 ±0.05);5%低氧实验组(0.35±0.06)。0.5%低氧实验组吸光度即增殖率高于常氧实验组(P0.05)。U87细胞凋亡率:常氧对照组(3.25±1.22);常氧实验组(8.10± 1.10);0.5%低氧实验组(6.64±0.89);5%低氧实验组(4.84±1.78)。5%低氧实验组凋亡率低于常氧实验组(P0.05)。[结论]适度的低氧微环境可以减弱TMZ对脑胶质瘤细胞的影响,产生耐药性,抵抗化疗作用。第二部分:低氧微环境下替莫唑胺对脑胶质瘤细胞BCL-2表达的影响[目的]在梯度低氧微环境下培养MGMT高表达的T98G细胞及MGMT低表达的U87细胞。给予预先确定的替莫唑胺浓度处理后,检测bcl-2的表达,用以验证低氧微环境下脑胶质逃避TMZ的凋亡诱导是通过上调bcl-2表达实现的。[方法]通过三气培养箱建立梯度低氧微环境;将脑胶质瘤细胞T98G细胞及U87细胞设为常氧对照组(21%O2)、常氧+替莫唑胺实验组(21%O2+TMZ)、0.5%低氧+替莫唑胺实验组(0.5%O2+TMZ)和5%低氧+替莫唑胺实验组(5%O2+TMZ),细胞贴壁后,T98G细胞加1mmolTMZ,U87细胞加0.1mmolTMZ培养72h,通过western blot访法检测各组bcl-2表达情况,以此来验证低氧微环境下替莫唑胺对bcl-2的影响。[结果]检测到bcl-2在脑胶质瘤细胞T98G中的表达量由高到低依次为:常氧对照组、0.5%低氧实验组、5%低氧实验组、常氧实验组。检测到bcl-2在脑胶质瘤细胞U87中的表达量由高到低依次为:常氧对照组、0.5%低氧实验组、5%低氧实验组、常氧实验组。[结论]适度低氧微环境可以减弱TMZ的作用,通过上调bcl-2在脑胶质瘤细胞中的的表达,产生耐药性;低氧微环境与MGMT可能是产生耐药的的独立或者协同因素。
[Abstract]:The first part: the effect of temozolomide on the proliferation and apoptosis of glioma cells in low oxygen microenvironment [Objective]TMZ is now used as a first line drug to treat glioma.MGMT as a DNA repair enzyme, which can repair the DNA damage caused by alkylating agents, and also make the tumor cell resistant to one step. According to glioma The level of cell expression of MGMT, glioma cells can be divided into MGMT high expression cells and MGMT low expression cells. The T98G cells are high expression cells and U87 cells are low expression cells. This part studies the intervention of T98G cells with high concentration of temozolomide by simulating the low oxygen microenvironment in the tumor, and the intervention of U87 cells by the low concentration of temozolomide. [Methods] the proliferation and apoptosis were detected. [Methods] T98G cells and U87 cells were set as normal oxygen control group (21%02), normoxic + temozolomide experimental group (21%02+TMZ), 0.5% hypoxia + temozolomide experimental group (0.5%O2+TMZ) and 5% hypoxia + temozolomide experimental group (5%02+TMZ). After cell adherence, T98G plus 1mmolTMZ, U87 plus 0.1mmolTMZ culture 72h; inverted microscopy. The growth situation was observed under the microscope; the MTS method was used to detect the absorption of the absorbance. After the AnnexinV/PI related treatment, the apoptosis was detected by flow cytometry. [results the growth of the T98G cells with high expression of]MGMT in the inverted microscope: the shape of the experimental group of atmospheric oxygen was slender; the 0.5% hypoxia experimental group was arranged and dispersed, the protuberance was not obvious; 5% low The shape of the oxygen test group was slender, the protuberance was not obvious.T98G cell absorbency: the normal oxygen control group (0.64 + 0.05), the normal oxygen experimental group (0.43 + 0.12), 0.5% hypoxia experimental group (0.61 + 0.06), and 5% hypoxia experimental group (0.40 + 0.04).0.5% hypoxia experimental group was higher than the normal oxygen experimental group (P0.05).T98G cell apoptosis rate: normal oxygen control group (19.57 + 1.61); The experimental group (37.83 + 9.46) and 0.5% hypoxic experimental group (34.63 + 0.98), 5% hypoxia experimental group (21.10 + 3.58).5% hypoxia experimental group was lower than normal oxygen experimental group (P0.05).MGMT low expression of U87 cell growth under the inverted microscope: the shape of the normal oxygen experiment group was slender in the 0.5% hypoxia experimental group; the shape of the 5% hypoxia experimental group was slender and the shape of the 5% hypoxia experimental group was slender The absorbance of scattered.U87 cells: normal oxygen control group (0.56 + 0.05), normal oxygen experimental group (0.44 + 0.07), 0.5% hypoxia experimental group (0.51 + 0.05), and 5% hypoxia experimental group (0.35 + 0.06).0.5% hypoxia experimental group (0.35 + 0.06) the proliferation rate was higher than that of normal oxygen experimental group (P0.05).U87 cell apoptosis rate: normal oxygen control group (3.25 + 1.22); normal oxygen experimental group (8.10 + 1.10); 5% hypoxic experimental group (6.64 + 0.89), 5% hypoxia experimental group (4.84 + 1.78).5% hypoxia experimental group was lower than normal oxygen experimental group (P0.05). [Conclusion] moderate hypoxia environment can weaken the effect of TMZ on glioma cells, produce resistance and resist chemotherapy. Second parts: temozolomide on glioma cells BCL-2 under low oxygen microenvironment. The effect [Objective] to cultivate MGMT high expression T98G cells and MGMT low expression U87 cells in the gradient hypoxia microenvironment. The expression of Bcl-2 was detected after the predefined temozolomide concentration treatment, and the expression of Bcl-2 was detected in the hypoxic microenvironment to verify that the apoptosis induction of the glial escape TMZ was achieved through the up regulation of bcl-2 expression. [Methods] pass through three T98G cells and U87 cells of glioma cells were set up as normal oxygen control group (21%O2), oxygen + temozolomide experimental group (21%O2+TMZ), 0.5% hypoxia + temozolomide experimental group (0.5%O2+TMZ) and 5% hypoxia + temozolomide experimental group (5%O2+ TMZ), and T98G cells plus 1mmolTMZ, U87 cells plus 0.1mmolTM after cell adherence. 72h was cultured by Z, and the expression of Bcl-2 in each group was detected by Western blot. In order to verify the effect of temozolomide on Bcl-2 in low oxygen microenvironment. [results] the expression of Bcl-2 in glioma cells T98G was detected from high to low in order of the normal oxygen control group, 0.5% hypoxia experimental group, 5% hypoxia experimental group, and normal oxygen experimental group. The expression of U87 in glioma cells from high to low was followed by: normoxic control group, 0.5% hypoxia experimental group, 5% hypoxia experimental group, and aero oxygen experimental group. [Conclusion] moderate hypoxia microenvironment can weaken the effect of TMZ, and increase the expression of Bcl-2 in brain glioma cells and produce drug resistance; low oxygen microenvironment and MGMT may produce drug resistance. Independent or synergetic factors.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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