低氧微環(huán)境下替莫唑胺對腦膠質(zhì)瘤細(xì)胞凋亡及對BCL-2表達(dá)的影響
發(fā)布時間：2018-07-17 06:28
【摘要】：第一部分:低氧微環(huán)境下替莫唑胺對腦膠質(zhì)瘤細(xì)胞增殖與凋亡的影響[目的]TMZ如今被當(dāng)作第一線藥物用來治療腦膠質(zhì)瘤。MGMT是一種DNA修復(fù)酶,可以對烷化劑藥物造成的DNA損傷進(jìn)行修復(fù),也會進(jìn)一步使腫瘤細(xì)胞產(chǎn)生耐藥性。按照腦膠質(zhì)瘤細(xì)胞表達(dá)MGMT的高低,腦膠質(zhì)瘤細(xì)胞可以分為MGMT高表達(dá)細(xì)胞和MGMT低表達(dá)細(xì)胞。其中T98G細(xì)胞為高表達(dá)細(xì)胞,U87細(xì)胞為低表達(dá)細(xì)胞。此部分研究通過模擬腫瘤體內(nèi)低氧微環(huán)境,選擇高濃度替莫唑胺干預(yù)T98G細(xì)胞,低濃度替莫唑胺干預(yù)U87細(xì)胞,培養(yǎng)后檢測增殖及凋亡情況。[方法]將T98G細(xì)胞及U87細(xì)胞設(shè)為常氧對照組(21%02)、常氧+替莫唑胺實(shí)驗(yàn)組(21%02+TMZ)、0.5%低氧+替莫唑胺實(shí)驗(yàn)組(0.5%O2+TMZ)和5%低氧+替莫唑胺實(shí)驗(yàn)組(5%02+TMZ),細(xì)胞貼壁后,T98G加1mmolTMZ,U87加0.1mmolTMZ培養(yǎng)72h;倒置顯微鏡下觀察生長情況;利用MTS法檢測吸光度即增值情況;AnnexinV/PI相關(guān)處理染色之后,利用流式細(xì)胞儀檢測其凋亡情況。[結(jié)果]MGMT高表達(dá)的T98G細(xì)胞在倒置顯微鏡下的生長情況:常氧實(shí)驗(yàn)組形狀細(xì)長;0.5%低氧實(shí)驗(yàn)組排列分散、突起不明顯;5%低氧實(shí)驗(yàn)組形狀細(xì)長、突起不明顯。T98G細(xì)胞吸光度:常氧對照組(0.64±0.05);常氧實(shí)驗(yàn)組(0.43±0.12);0.5%低氧實(shí)驗(yàn)組(0.61±0.06);5%低氧實(shí)驗(yàn)組(0.40±0.04)。0.5%低氧實(shí)驗(yàn)組吸光度即增殖率高于常氧實(shí)驗(yàn)組(P0.05)。T98G細(xì)胞凋亡率:常氧對照組(19.57±1.61);常氧實(shí)驗(yàn)組(37.83±9.46);0.5%低氧實(shí)驗(yàn)組(34.63±0.98);5%低氧實(shí)驗(yàn)組(21.10±3.58)。5%低氧實(shí)驗(yàn)組凋亡率低于常氧實(shí)驗(yàn)組(P0.05)。MGMT低表達(dá)的U87細(xì)胞在倒置顯微鏡下的生長情況:常氧實(shí)驗(yàn)組形狀細(xì)長:0.5%低氧實(shí)驗(yàn)組形狀細(xì)長;5%低氧實(shí)驗(yàn)組形狀細(xì)長、排列分散。U87細(xì)胞吸光度:常氧對照組(0.56±0.05);常氧實(shí)驗(yàn)組(0.44±0.07);0.5%低氧實(shí)驗(yàn)組(0.51 ±0.05);5%低氧實(shí)驗(yàn)組(0.35±0.06)。0.5%低氧實(shí)驗(yàn)組吸光度即增殖率高于常氧實(shí)驗(yàn)組(P0.05)。U87細(xì)胞凋亡率:常氧對照組(3.25±1.22);常氧實(shí)驗(yàn)組(8.10± 1.10);0.5%低氧實(shí)驗(yàn)組(6.64±0.89);5%低氧實(shí)驗(yàn)組(4.84±1.78)。5%低氧實(shí)驗(yàn)組凋亡率低于常氧實(shí)驗(yàn)組(P0.05)。[結(jié)論]適度的低氧微環(huán)境可以減弱TMZ對腦膠質(zhì)瘤細(xì)胞的影響,產(chǎn)生耐藥性,抵抗化療作用。第二部分:低氧微環(huán)境下替莫唑胺對腦膠質(zhì)瘤細(xì)胞BCL-2表達(dá)的影響[目的]在梯度低氧微環(huán)境下培養(yǎng)MGMT高表達(dá)的T98G細(xì)胞及MGMT低表達(dá)的U87細(xì)胞。給予預(yù)先確定的替莫唑胺濃度處理后,檢測bcl-2的表達(dá),用以驗(yàn)證低氧微環(huán)境下腦膠質(zhì)逃避TMZ的凋亡誘導(dǎo)是通過上調(diào)bcl-2表達(dá)實(shí)現(xiàn)的。[方法]通過三氣培養(yǎng)箱建立梯度低氧微環(huán)境;將腦膠質(zhì)瘤細(xì)胞T98G細(xì)胞及U87細(xì)胞設(shè)為常氧對照組(21%O2)、常氧+替莫唑胺實(shí)驗(yàn)組(21%O2+TMZ)、0.5%低氧+替莫唑胺實(shí)驗(yàn)組(0.5%O2+TMZ)和5%低氧+替莫唑胺實(shí)驗(yàn)組(5%O2+TMZ),細(xì)胞貼壁后,T98G細(xì)胞加1mmolTMZ,U87細(xì)胞加0.1mmolTMZ培養(yǎng)72h,通過western blot訪法檢測各組bcl-2表達(dá)情況,以此來驗(yàn)證低氧微環(huán)境下替莫唑胺對bcl-2的影響。[結(jié)果]檢測到bcl-2在腦膠質(zhì)瘤細(xì)胞T98G中的表達(dá)量由高到低依次為:常氧對照組、0.5%低氧實(shí)驗(yàn)組、5%低氧實(shí)驗(yàn)組、常氧實(shí)驗(yàn)組。檢測到bcl-2在腦膠質(zhì)瘤細(xì)胞U87中的表達(dá)量由高到低依次為:常氧對照組、0.5%低氧實(shí)驗(yàn)組、5%低氧實(shí)驗(yàn)組、常氧實(shí)驗(yàn)組。[結(jié)論]適度低氧微環(huán)境可以減弱TMZ的作用,通過上調(diào)bcl-2在腦膠質(zhì)瘤細(xì)胞中的的表達(dá),產(chǎn)生耐藥性;低氧微環(huán)境與MGMT可能是產(chǎn)生耐藥的的獨(dú)立或者協(xié)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41
本文編號：2129400
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41
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