高氧促进胶质瘤干细胞分化致化疗增敏的体外研究
发布时间：2018-08-11 21:22
【摘要】：背景和目的:胶质瘤作为颅内的高度恶性肿瘤因其侵袭性的生长方式以及对放化疗的高度不敏感,使得胶质瘤患者即使采取严格的手术切除加术后标准放化疗后,仍极易复发,导致患者预后严重不良。既往研究认为在许多肿瘤内存在为数不多、具有多向分化潜能且对肿瘤的发生发展起关键作用的特殊细胞亚群,称之为肿瘤干细胞,及肿瘤干细胞学说。胶质瘤作为颅内最常见的恶性肿瘤,也存在相应的肿瘤干细胞,即胶质瘤干细胞;与所有肿瘤干细胞相似,胶质瘤干细胞也具有自我更新、无限增殖及多向分化能力。基于以上特性,胶质瘤干细胞在术后放化疗抵抗中起着非常的关键作用。所以现阶段肿瘤干细胞学说认为胶质瘤内存在的干细胞是导致肿瘤复发、化疗耐药和治疗失败的主要原因。因此对胶质瘤治疗的研究重点应着重于胶质瘤干细胞清除,或提高药物对胶质瘤干细胞的敏感性等方面。回顾文献我们发现高氧治疗胶质瘤后可明显减缓肿瘤的生长速度,肿瘤细胞凋亡率也显著高于常氧组。临床研究中对高级别的胶质瘤患者采用高压氧联合放化疗治疗,结果显示高压氧联合放化疗可大幅度的提高患者的生存期,近期研究也表明高氧可以增强多种化疗药物对胶质瘤的敏感性,进而延长患者的生存期。然而,高氧如何表现为化疗增敏效应,目前尚缺乏相关实验证据。众所周知的是在大多数实体肿瘤内存在一个特殊微环境,即缺氧微环境,既往研究证实缺氧微环境的存在与肿瘤干细胞增殖、自我更新和维持干细胞干性密切相关;本课题组前期实验中也已证明体外缺氧环境下普通胶质瘤细胞可逆分化形成胶质瘤干细胞,从而进一步增强肿瘤干细胞的干性。与之相反,高氧作为目前纠正胶质瘤缺氧微环境的有效方法,是否可以通过阻遏胶质瘤细胞在缺氧微环境中的逆分化进程或者促进胶质瘤干细胞分化而致其化疗增敏?因此,为证明这一科学假说,我们拟通过体外实验,采用体外诱导培养、免疫荧光、qRT-PCR、Western-blot等方法予以验证,并通过细胞周期、IC50测定、细胞凋亡等检测予以辅助证明,从而为高氧联合化疗治疗胶质瘤并改善其预后提供新的理论依据,并对胶质瘤的综合治疗提供新的思考方向。实验方法:第一部分:(1)胶质瘤干细胞的体外诱导培养:U87、GL261胶质瘤细胞置于37℃、5%CO_2、含有EGF(20 ng/m L)、b FGF(20 ng/m L)、2%B27的无血清DMEM/F12培养基中培养;(2)诱导培养的胶质瘤干细胞生物学观察:干细胞培养基诱导胶质瘤细胞5 d后行图像采集,并观察其生物学形态。(3)免疫荧光检测:取经干细胞培养基诱导5 d且生长状态佳的U87、GL261胶质瘤细胞球,固定后检测胶质瘤干细胞蛋白(CD133、Nestin、MGMT)荧光强弱情况。(4)细胞周期检测:普通GL261、U87胶质瘤细胞与GL261、U87胶质瘤细胞球在常氧条件下分别行细胞周期检测。(5)凋亡检测:常氧下普通GL261、U87胶质瘤细胞与GL261、U87胶质瘤细胞球分别应用TMZ处理24h后行流式凋亡检测。第二部分:(1)胶质瘤干细胞标记蛋白(CD133、Nestin)、分化蛋白(GFAP)、耐药蛋白(MGMT)RNA表达检测:胶质瘤干细胞在不同氧浓度条件(高氧95%O_2、常氧21%O_2)处理后行qRT-PCR检测;(2)肿瘤干细胞标记蛋白(CD133、Nestin)、分化蛋白(GFAP)、耐药蛋白(MGMT)蛋白表达检测:胶质瘤干细胞在不同氧浓度条件(高氧95%O_2、常氧21%O_2)分别处理24h、48h后行Western-blot检测;(3)IC50值测定:胶质瘤干细胞在不同氧浓度条件下(高氧95%O_2、常氧21%O_2)预处理后添加替莫唑胺(TMZ)干预24h行CCK-8测定,测量其吸光度值,并计算其IC50值;(4)凋亡检测:胶质瘤干细胞在不同氧浓度条件下(高浓度氧95%O_2、常氧21%O_2)预处理后添加替莫唑胺干预,行流式细胞仪凋亡检测。结果:(1)U87、GL261胶质瘤细胞置于37℃、5%CO_2、含有EGF(20 ng/m L)、bFGF(20 ng/m L)、2%B27的无血清DMEM/F12培养基中诱导培养5d后发现U87、GL261胶质瘤细胞悬浮生长,且绝大部分聚集成球,形态学上表现出肿瘤干细胞聚集生长成球的特点。(2)干细胞培养基培养U87、GL261胶质瘤细胞5 d后免疫荧光检测表明,U87、GL261胶质瘤细胞高表达干细胞样标记分子CD133、Nestin和耐药标记蛋白MGMT。(3)在常氧条件下周期检测结果显示,与普通细胞相比,GL261、U87胶质瘤细胞球周期阻滞于G0/G1期(P0.05)。(4)凋亡结果显示在常氧条件下,与普通细胞相比,TMZ处理的GL261、U87胶质瘤细胞球凋亡明显减少(P0.05)。(5)与常氧相比,qRT-PCR显示高氧处理的U87、GL261胶质瘤干细胞24 h后其胶质瘤干细胞样标记蛋白(CD133、Nestin)mRNA表达量降低(P0.05),分化标记蛋白GFAPm RNA表达量增高(P0.05),耐药标记蛋白MGMTmRNA表达量降低(P0.05)。(6)Western blot结果显示与各自时间点常氧组相比,高氧干预24 h、48 h的U87、GL261胶质瘤干细胞低表达干细胞标记蛋白(CD133、Nestin),并促进分化标记蛋白GFAP表达;进一步检测发现耐药标记蛋白MGMT呈降低趋势。且不同高氧处理时间的胶质瘤干细胞标记蛋白和分化蛋白表达量也有明显差异,表现为与高氧处理24h的胶质瘤干细胞相比较,高氧处理48h的胶质瘤干细胞低表达干细胞标记蛋白(CD133、Nestin),高表达胶质瘤分化标记蛋白GFAP。(7)胶质瘤干细胞不同氧浓度(高氧95%O_2、常氧21%O_2)预处理后添加替莫唑胺干预,IC50检测提示高浓度氧组IC50值显著低于常氧组(P0.05)。(8)胶质瘤干细胞不同氧浓度(高氧95%O_2、常氧21%O_2)预处理后添加替莫唑胺干预,流式凋亡结果提示高氧组凋亡率显著高于常氧组(P0.05)。结论:高氧可阻断缺氧引起的胶质瘤细胞逆分化进程并能通过抑制胶质瘤干细胞干性表达并促进其分化而致化疗增敏,表现为高氧处理胶质瘤干细胞后低表达肿瘤干细胞标记蛋白和耐药标记蛋白、高表达肿瘤分化标记蛋白;高氧联合应用替莫唑胺(TMZ)处理胶质瘤干细胞后表现为低增值、高凋亡特性。
[Abstract]:BACKGROUND AND OBJECTIVE: Glioma, as a highly malignant intracranial tumor, is highly sensitive to radiotherapy and chemotherapy because of its invasive growth pattern, which makes it easy for glioma patients to relapse even after strict surgical resection and postoperative standard radiotherapy and chemotherapy, leading to severe adverse prognosis. Glioma is one of the most common intracranial malignancies, and there are also corresponding tumor stem cells, that is, glioma stem cells; similar to all tumor stem cells, glioma stem cells Based on the above characteristics, glioma stem cells play a very important role in the resistance to postoperative radiotherapy and chemotherapy. Therefore, the current cancer stem cell theory believes that glioma stem cells are the main cause of tumor recurrence, chemotherapy resistance and treatment failure. In retrospective literature, we found that hyperoxia treatment of gliomas significantly slowed down the growth rate of tumors, and the apoptosis rate of tumor cells was significantly higher than that of normoxia group. Hyperbaric oxygen combined with radiotherapy and chemotherapy has shown that hyperbaric oxygen combined with radiotherapy and chemotherapy can significantly improve the survival of patients. Recent studies have also shown that hyperoxia can enhance the sensitivity of a variety of chemotherapy drugs to gliomas, thereby prolonging the survival of patients. However, how hyperoxia manifests itself as a chemosensitizing effect is still lacking. It is well known that there is a special microenvironment in most solid tumors, i.e. hypoxic microenvironment. Previous studies have confirmed that the existence of hypoxic microenvironment is closely related to the proliferation, self-renewal and maintenance of stem cell dryness of tumor stem cells. Previous experiments in our group have also proved that normal glioma cells are exposed to hypoxia in vitro. On the contrary, whether hyperoxia, as an effective way to correct the hypoxic microenvironment of glioma, can induce chemotherapy sensitization by inhibiting the reversible differentiation of glioma cells in hypoxic microenvironment or promoting the differentiation of glioma stem cells? In order to prove this scientific hypothesis, we intend to use in vitro experiments, induction culture, immunofluorescence, qRT-PCR, Western-blot and other methods to verify the hypothesis, and through cell cycle, IC50 determination, apoptosis and other tests to support the proof, so as to provide a new theoretical basis for hyperoxia combined chemotherapy treatment of glioma and improve its prognosis. Experimental methods: Part 1: (1) In vitro induction and culture of glioma stem cells: U87, GL261 glioma cells were cultured in a serum-free DMEM/F12 medium containing EGF (20 ng/m L), B FGF (20 ng/m L) and 2% B27 at 37, 5% CO_2, EGF (20 ng/m L), B FGF (20 ng/m L), and 2% B27; (2) Biological view of induced glioma stem cells Immunofluorescence assay: U87, GL261 glioma cell spheres which were induced by stem cell culture medium for 5 days and in good growth condition were taken out, and the fluorescence intensity of glioma stem cell protein (CD133, Nestin, MGMT) was detected after immobilization. The cell cycle of GL261, U87 glioma cells and GL261, U87 glioma cell spheres were detected under normal oxygen conditions. (5) Apoptosis detection: normal GL261, U87 glioma cells and GL261, U87 glioma cell spheres were treated with TMZ for 24 hours and then flow cytometry was used to detect apoptosis. (2) Glioma stem cell marker protein (CD133, Nestin), Differentiation Protein (GFAP), Resistance Protein (MGMT) RNA Expression Detection: Glioma Stem Cells in different oxygen concentration conditions (95% O 2, 21% O 2) after treatment for qRT-PCR detection; (2) Tumor Stem Cell Marker Protein (CD133, Nestin), Differentiation Protein (GFAP), Resistance Protein (MGMT) protein expression detection: Glioma Stem Cells in different oxygen concentration conditions (hyperoxia 9) 5% O_2 and 21% O_2 were treated for 24 hours and 48 hours respectively, and then Western-blot was used to detect the IC50 value. (3) IC50 value: Glioma stem cells were pretreated with TMZ at different oxygen concentrations (95% O_2, 21% O_2) and CCK-8 was measured after 24 hours of intervention, and the absorbance value was measured, and the IC50 value was calculated. (4) Apoptosis detection: Glioma stem cells were treated with different oxygen concentrations (TMZ). Results: (1) U87, GL261 glioma cells were cultured in a serum-free DMEM/F12 medium containing EGF (20 ng/m L), bFGF (20 ng/m L) and 2% B27 for 5 days, and U87 and GL261 glioma cells were suspended in the medium containing EGF (20 ng/m L), 5% CO_2, 5% EGF (20 ng/m L). (2) Immunofluorescence assay showed that U87, GL261 glioma cells expressed high levels of stem cell-like markers CD133, Nestin and MGMT after 5 days of culture in stem cell medium. The results of phase detection showed that GL261 and U87 glioma cell cycle arrest was at G0/G1 phase (P After 24 hours, the expression of CD133, Nestin mRNA was decreased (P 0.05), the expression of GFAPm RNA was increased (P 0.05), and the expression of MGMT mRNA was decreased (P 0.05). (6) Western blot showed that U87, GL261 gliomas were drier and finer after 24 hours, 48 hours of hyperoxia than those of normoxia at the corresponding time points. The expression of stem cell marker protein (CD133, Nestin) was low, and the expression of differentiation marker protein GFAP was promoted. Further detection showed that MGMT was decreased, and the expression of marker protein and differentiated protein of glioma stem cells were also significantly different in different hyperoxia treatment time, which showed the phase of glioma stem cells treated with hyperoxia for 24 hours. In comparison, 48 h hyperoxia treated glioma stem cells showed low expression of stem cell marker protein (CD133, Nestin) and high expression of glioma differentiation marker protein (GFAP). (7) Pretreatment of glioma stem cells with different oxygen concentrations (95% O_2, 21% O_2) plus temozolomide intervention, IC50 detection showed that the IC50 value of hyperoxia group was significantly lower than that of normoxia group (P 0.05). The results of flow cytometry showed that the apoptotic rate in hyperoxia group was significantly higher than that in normoxia group (P Chemotherapy sensitization was induced by hyperoxia, which resulted in low expression of tumor stem cell marker protein and drug resistance marker protein, and high expression of tumor differentiation marker protein.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
本文编号：2178271
[Abstract]:BACKGROUND AND OBJECTIVE: Glioma, as a highly malignant intracranial tumor, is highly sensitive to radiotherapy and chemotherapy because of its invasive growth pattern, which makes it easy for glioma patients to relapse even after strict surgical resection and postoperative standard radiotherapy and chemotherapy, leading to severe adverse prognosis. Glioma is one of the most common intracranial malignancies, and there are also corresponding tumor stem cells, that is, glioma stem cells; similar to all tumor stem cells, glioma stem cells Based on the above characteristics, glioma stem cells play a very important role in the resistance to postoperative radiotherapy and chemotherapy. Therefore, the current cancer stem cell theory believes that glioma stem cells are the main cause of tumor recurrence, chemotherapy resistance and treatment failure. In retrospective literature, we found that hyperoxia treatment of gliomas significantly slowed down the growth rate of tumors, and the apoptosis rate of tumor cells was significantly higher than that of normoxia group. Hyperbaric oxygen combined with radiotherapy and chemotherapy has shown that hyperbaric oxygen combined with radiotherapy and chemotherapy can significantly improve the survival of patients. Recent studies have also shown that hyperoxia can enhance the sensitivity of a variety of chemotherapy drugs to gliomas, thereby prolonging the survival of patients. However, how hyperoxia manifests itself as a chemosensitizing effect is still lacking. It is well known that there is a special microenvironment in most solid tumors, i.e. hypoxic microenvironment. Previous studies have confirmed that the existence of hypoxic microenvironment is closely related to the proliferation, self-renewal and maintenance of stem cell dryness of tumor stem cells. Previous experiments in our group have also proved that normal glioma cells are exposed to hypoxia in vitro. On the contrary, whether hyperoxia, as an effective way to correct the hypoxic microenvironment of glioma, can induce chemotherapy sensitization by inhibiting the reversible differentiation of glioma cells in hypoxic microenvironment or promoting the differentiation of glioma stem cells? In order to prove this scientific hypothesis, we intend to use in vitro experiments, induction culture, immunofluorescence, qRT-PCR, Western-blot and other methods to verify the hypothesis, and through cell cycle, IC50 determination, apoptosis and other tests to support the proof, so as to provide a new theoretical basis for hyperoxia combined chemotherapy treatment of glioma and improve its prognosis. Experimental methods: Part 1: (1) In vitro induction and culture of glioma stem cells: U87, GL261 glioma cells were cultured in a serum-free DMEM/F12 medium containing EGF (20 ng/m L), B FGF (20 ng/m L) and 2% B27 at 37, 5% CO_2, EGF (20 ng/m L), B FGF (20 ng/m L), and 2% B27; (2) Biological view of induced glioma stem cells Immunofluorescence assay: U87, GL261 glioma cell spheres which were induced by stem cell culture medium for 5 days and in good growth condition were taken out, and the fluorescence intensity of glioma stem cell protein (CD133, Nestin, MGMT) was detected after immobilization. The cell cycle of GL261, U87 glioma cells and GL261, U87 glioma cell spheres were detected under normal oxygen conditions. (5) Apoptosis detection: normal GL261, U87 glioma cells and GL261, U87 glioma cell spheres were treated with TMZ for 24 hours and then flow cytometry was used to detect apoptosis. (2) Glioma stem cell marker protein (CD133, Nestin), Differentiation Protein (GFAP), Resistance Protein (MGMT) RNA Expression Detection: Glioma Stem Cells in different oxygen concentration conditions (95% O 2, 21% O 2) after treatment for qRT-PCR detection; (2) Tumor Stem Cell Marker Protein (CD133, Nestin), Differentiation Protein (GFAP), Resistance Protein (MGMT) protein expression detection: Glioma Stem Cells in different oxygen concentration conditions (hyperoxia 9) 5% O_2 and 21% O_2 were treated for 24 hours and 48 hours respectively, and then Western-blot was used to detect the IC50 value. (3) IC50 value: Glioma stem cells were pretreated with TMZ at different oxygen concentrations (95% O_2, 21% O_2) and CCK-8 was measured after 24 hours of intervention, and the absorbance value was measured, and the IC50 value was calculated. (4) Apoptosis detection: Glioma stem cells were treated with different oxygen concentrations (TMZ). Results: (1) U87, GL261 glioma cells were cultured in a serum-free DMEM/F12 medium containing EGF (20 ng/m L), bFGF (20 ng/m L) and 2% B27 for 5 days, and U87 and GL261 glioma cells were suspended in the medium containing EGF (20 ng/m L), 5% CO_2, 5% EGF (20 ng/m L). (2) Immunofluorescence assay showed that U87, GL261 glioma cells expressed high levels of stem cell-like markers CD133, Nestin and MGMT after 5 days of culture in stem cell medium. The results of phase detection showed that GL261 and U87 glioma cell cycle arrest was at G0/G1 phase (P After 24 hours, the expression of CD133, Nestin mRNA was decreased (P 0.05), the expression of GFAPm RNA was increased (P 0.05), and the expression of MGMT mRNA was decreased (P 0.05). (6) Western blot showed that U87, GL261 gliomas were drier and finer after 24 hours, 48 hours of hyperoxia than those of normoxia at the corresponding time points. The expression of stem cell marker protein (CD133, Nestin) was low, and the expression of differentiation marker protein GFAP was promoted. Further detection showed that MGMT was decreased, and the expression of marker protein and differentiated protein of glioma stem cells were also significantly different in different hyperoxia treatment time, which showed the phase of glioma stem cells treated with hyperoxia for 24 hours. In comparison, 48 h hyperoxia treated glioma stem cells showed low expression of stem cell marker protein (CD133, Nestin) and high expression of glioma differentiation marker protein (GFAP). (7) Pretreatment of glioma stem cells with different oxygen concentrations (95% O_2, 21% O_2) plus temozolomide intervention, IC50 detection showed that the IC50 value of hyperoxia group was significantly lower than that of normoxia group (P 0.05). The results of flow cytometry showed that the apoptotic rate in hyperoxia group was significantly higher than that in normoxia group (P Chemotherapy sensitization was induced by hyperoxia, which resulted in low expression of tumor stem cell marker protein and drug resistance marker protein, and high expression of tumor differentiation marker protein.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
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相关期刊论文 前2条
1 代丽丽;钟士江;;新生小鼠神经干细胞体外培养、分化及鉴定[J];药物评价研究;2016年01期
2 汪攀;赵秀文;兰川;吴南;;缺氧诱导胶质瘤细胞肿瘤干样细胞形成体外初步研究[J];第三军医大学学报;2015年07期
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