【摘要】：背景和目的:膠質(zhì)瘤作為顱內(nèi)的高度惡性腫瘤因其侵襲性的生長方式以及對放化療的高度不敏感,使得膠質(zhì)瘤患者即使采取嚴格的手術(shù)切除加術(shù)后標準放化療后,仍極易復發(fā),導致患者預(yù)后嚴重不良。既往研究認為在許多腫瘤內(nèi)存在為數(shù)不多、具有多向分化潛能且對腫瘤的發(fā)生發(fā)展起關(guān)鍵作用的特殊細胞亞群,稱之為腫瘤干細胞,及腫瘤干細胞學說。膠質(zhì)瘤作為顱內(nèi)最常見的惡性腫瘤,也存在相應(yīng)的腫瘤干細胞,即膠質(zhì)瘤干細胞;與所有腫瘤干細胞相似,膠質(zhì)瘤干細胞也具有自我更新、無限增殖及多向分化能力�；谝陨咸匦�,膠質(zhì)瘤干細胞在術(shù)后放化療抵抗中起著非常的關(guān)鍵作用。所以現(xiàn)階段腫瘤干細胞學說認為膠質(zhì)瘤內(nèi)存在的干細胞是導致腫瘤復發(fā)、化療耐藥和治療失敗的主要原因。因此對膠質(zhì)瘤治療的研究重點應(yīng)著重于膠質(zhì)瘤干細胞清除,或提高藥物對膠質(zhì)瘤干細胞的敏感性等方面�；仡櫸墨I我們發(fā)現(xiàn)高氧治療膠質(zhì)瘤后可明顯減緩腫瘤的生長速度,腫瘤細胞凋亡率也顯著高于常氧組。臨床研究中對高級別的膠質(zhì)瘤患者采用高壓氧聯(lián)合放化療治療,結(jié)果顯示高壓氧聯(lián)合放化療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強多種化療藥物對膠質(zhì)瘤的敏感性,進而延長患者的生存期。然而,高氧如何表現(xiàn)為化療增敏效應(yīng),目前尚缺乏相關(guān)實驗證據(jù)。眾所周知的是在大多數(shù)實體腫瘤內(nèi)存在一個特殊微環(huán)境,即缺氧微環(huán)境,既往研究證實缺氧微環(huán)境的存在與腫瘤干細胞增殖、自我更新和維持干細胞干性密切相關(guān);本課題組前期實驗中也已證明體外缺氧環(huán)境下普通膠質(zhì)瘤細胞可逆分化形成膠質(zhì)瘤干細胞,從而進一步增強腫瘤干細胞的干性。與之相反,高氧作為目前糾正膠質(zhì)瘤缺氧微環(huán)境的有效方法,是否可以通過阻遏膠質(zhì)瘤細胞在缺氧微環(huán)境中的逆分化進程或者促進膠質(zhì)瘤干細胞分化而致其化療增敏?因此,為證明這一科學假說,我們擬通過體外實驗,采用體外誘導培養(yǎng)、免疫熒光、qRT-PCR、Western-blot等方法予以驗證,并通過細胞周期、IC50測定、細胞凋亡等檢測予以輔助證明,從而為高氧聯(lián)合化療治療膠質(zhì)瘤并改善其預(yù)后提供新的理論依據(jù),并對膠質(zhì)瘤的綜合治療提供新的思考方向。實驗方法:第一部分:(1)膠質(zhì)瘤干細胞的體外誘導培養(yǎng):U87、GL261膠質(zhì)瘤細胞置于37℃、5%CO_2、含有EGF(20 ng/m L)、b FGF(20 ng/m L)、2%B27的無血清DMEM/F12培養(yǎng)基中培養(yǎng);(2)誘導培養(yǎng)的膠質(zhì)瘤干細胞生物學觀察:干細胞培養(yǎng)基誘導膠質(zhì)瘤細胞5 d后行圖像采集,并觀察其生物學形態(tài)。(3)免疫熒光檢測:取經(jīng)干細胞培養(yǎng)基誘導5 d且生長狀態(tài)佳的U87、GL261膠質(zhì)瘤細胞球,固定后檢測膠質(zhì)瘤干細胞蛋白(CD133、Nestin、MGMT)熒光強弱情況。(4)細胞周期檢測:普通GL261、U87膠質(zhì)瘤細胞與GL261、U87膠質(zhì)瘤細胞球在常氧條件下分別行細胞周期檢測。(5)凋亡檢測:常氧下普通GL261、U87膠質(zhì)瘤細胞與GL261、U87膠質(zhì)瘤細胞球分別應(yīng)用TMZ處理24h后行流式凋亡檢測。第二部分:(1)膠質(zhì)瘤干細胞標記蛋白(CD133、Nestin)、分化蛋白(GFAP)、耐藥蛋白(MGMT)RNA表達檢測:膠質(zhì)瘤干細胞在不同氧濃度條件(高氧95%O_2、常氧21%O_2)處理后行qRT-PCR檢測;(2)腫瘤干細胞標記蛋白(CD133、Nestin)、分化蛋白(GFAP)、耐藥蛋白(MGMT)蛋白表達檢測:膠質(zhì)瘤干細胞在不同氧濃度條件(高氧95%O_2、常氧21%O_2)分別處理24h、48h后行Western-blot檢測;(3)IC50值測定:膠質(zhì)瘤干細胞在不同氧濃度條件下(高氧95%O_2、常氧21%O_2)預(yù)處理后添加替莫唑胺(TMZ)干預(yù)24h行CCK-8測定,測量其吸光度值,并計算其IC50值;(4)凋亡檢測:膠質(zhì)瘤干細胞在不同氧濃度條件下(高濃度氧95%O_2、常氧21%O_2)預(yù)處理后添加替莫唑胺干預(yù),行流式細胞儀凋亡檢測。結(jié)果:(1)U87、GL261膠質(zhì)瘤細胞置于37℃、5%CO_2、含有EGF(20 ng/m L)、bFGF(20 ng/m L)、2%B27的無血清DMEM/F12培養(yǎng)基中誘導培養(yǎng)5d后發(fā)現(xiàn)U87、GL261膠質(zhì)瘤細胞懸浮生長,且絕大部分聚集成球,形態(tài)學上表現(xiàn)出腫瘤干細胞聚集生長成球的特點。(2)干細胞培養(yǎng)基培養(yǎng)U87、GL261膠質(zhì)瘤細胞5 d后免疫熒光檢測表明,U87、GL261膠質(zhì)瘤細胞高表達干細胞樣標記分子CD133、Nestin和耐藥標記蛋白MGMT。(3)在常氧條件下周期檢測結(jié)果顯示,與普通細胞相比,GL261、U87膠質(zhì)瘤細胞球周期阻滯于G0/G1期(P0.05)。(4)凋亡結(jié)果顯示在常氧條件下,與普通細胞相比,TMZ處理的GL261、U87膠質(zhì)瘤細胞球凋亡明顯減少(P0.05)。(5)與常氧相比,qRT-PCR顯示高氧處理的U87、GL261膠質(zhì)瘤干細胞24 h后其膠質(zhì)瘤干細胞樣標記蛋白(CD133、Nestin)mRNA表達量降低(P0.05),分化標記蛋白GFAPm RNA表達量增高(P0.05),耐藥標記蛋白MGMTmRNA表達量降低(P0.05)。(6)Western blot結(jié)果顯示與各自時間點常氧組相比,高氧干預(yù)24 h、48 h的U87、GL261膠質(zhì)瘤干細胞低表達干細胞標記蛋白(CD133、Nestin),并促進分化標記蛋白GFAP表達;進一步檢測發(fā)現(xiàn)耐藥標記蛋白MGMT呈降低趨勢。且不同高氧處理時間的膠質(zhì)瘤干細胞標記蛋白和分化蛋白表達量也有明顯差異,表現(xiàn)為與高氧處理24h的膠質(zhì)瘤干細胞相比較,高氧處理48h的膠質(zhì)瘤干細胞低表達干細胞標記蛋白(CD133、Nestin),高表達膠質(zhì)瘤分化標記蛋白GFAP。(7)膠質(zhì)瘤干細胞不同氧濃度(高氧95%O_2、常氧21%O_2)預(yù)處理后添加替莫唑胺干預(yù),IC50檢測提示高濃度氧組IC50值顯著低于常氧組(P0.05)。(8)膠質(zhì)瘤干細胞不同氧濃度(高氧95%O_2、常氧21%O_2)預(yù)處理后添加替莫唑胺干預(yù),流式凋亡結(jié)果提示高氧組凋亡率顯著高于常氧組(P0.05)。結(jié)論:高氧可阻斷缺氧引起的膠質(zhì)瘤細胞逆分化進程并能通過抑制膠質(zhì)瘤干細胞干性表達并促進其分化而致化療增敏,表現(xiàn)為高氧處理膠質(zhì)瘤干細胞后低表達腫瘤干細胞標記蛋白和耐藥標記蛋白、高表達腫瘤分化標記蛋白;高氧聯(lián)合應(yīng)用替莫唑胺(TMZ)處理膠質(zhì)瘤干細胞后表現(xiàn)為低增值、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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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