【摘要】：目的:研究不同濃度的異甘草素對(duì)膠質(zhì)瘤干細(xì)胞放療敏感性的影響及作用機(jī)制,為異甘草素作為放療增敏劑治療人腦膠質(zhì)瘤提供依據(jù)。方法:1.使用超低吸附懸浮細(xì)胞培養(yǎng)板6孔板,通過無血清條件培養(yǎng)法從SHG44人腦膠質(zhì)瘤貼壁細(xì)胞中提取和純化膠質(zhì)瘤干細(xì)胞,通過CD133、Nestin聯(lián)合鑒定懸浮細(xì)胞球的干細(xì)胞特性,通過Bcl-2鑒定懸浮細(xì)胞球的膠質(zhì)瘤細(xì)胞源性;2.運(yùn)用CCK-8試劑盒檢測(cè)不同濃度異甘草素和不同劑量X射線的單獨(dú)和聯(lián)合運(yùn)用下膠質(zhì)瘤干細(xì)胞的增殖活性,并在顯微鏡下觀察和記錄膠質(zhì)瘤干細(xì)胞懸浮球的成球情況。通過細(xì)胞活性和干細(xì)胞懸浮球在形態(tài)上的抑制情況評(píng)估異甘草素的放療增敏效果;3.運(yùn)用Western blotting技術(shù)檢測(cè)不同濃度異甘草素作用下Notch1信號(hào)通路相關(guān)蛋白的表達(dá)情況,以Notch1信號(hào)通路特異性阻斷劑DAPT作為陽性對(duì)照。檢測(cè)異甘草素預(yù)處理后膠質(zhì)瘤干細(xì)胞對(duì)不同劑量X射線照射后NF-κB和凋亡關(guān)鍵蛋白caspase-3的表達(dá)情況,以此探討異甘草素對(duì)膠質(zhì)瘤干細(xì)胞X射線放療增敏的作用機(jī)制。結(jié)果:1.無血清條件培養(yǎng)能夠有效地提取和純化人腦膠質(zhì)瘤干細(xì)胞懸浮球,免疫熒光染色CD133和Nestin表達(dá)陽性說明懸浮細(xì)胞球具有干細(xì)胞特性,Bcl-2表達(dá)陽性說明懸浮細(xì)胞球的腫瘤源性;2.72 h后,膠質(zhì)瘤干細(xì)胞的活性被異甘草素所抑(P0.05)。異甘草素處理9天后,膠質(zhì)瘤干細(xì)胞的成球能力被抑制,20?M濃度以上的異甘草素能夠明顯降低膠質(zhì)瘤干細(xì)胞成球的數(shù)目和干細(xì)胞球的直徑(P0.01)。在72 h內(nèi),膠質(zhì)瘤干細(xì)胞的細(xì)胞活性并不隨X射線的照射劑量的增加而有所改變。在9天的膠質(zhì)瘤干細(xì)胞成球過程中,雖然X射線能夠一定程度上減小所形成的懸浮細(xì)胞球的直徑(P0.01),但是,成球的數(shù)目并沒有因?yàn)閄射線的照射而下降,表現(xiàn)出X射線的耐受;3.本研究發(fā)現(xiàn)10?M的異甘草素雖然直接抑制膠質(zhì)瘤干細(xì)胞生長的作用有限,但在8 Gy較高劑量X射存在的情況下,能夠增加膠質(zhì)瘤放療的敏感性(P0.05),表現(xiàn)為所形成的干細(xì)胞球數(shù)目下降和直徑下降。20?M的異甘草素殺傷膠質(zhì)瘤干細(xì)胞的作用明顯,且在4 Gy的X射線照射時(shí)就有較強(qiáng)的增敏效果,在8 Gy的X射線下聯(lián)合殺傷膠質(zhì)瘤干細(xì)胞的能力進(jìn)一步提高(P0.05)。而40?M以上的異甘草素抑制膠質(zhì)瘤干細(xì)胞增殖的作用較為明顯,在聯(lián)合X射線干預(yù)下,主要表現(xiàn)出直接抑制增殖的作用;4.Notch1(Notch1信號(hào)通路受體)、RBPJK(Notch1信號(hào)通路的轉(zhuǎn)錄調(diào)節(jié)因子)和Hes1(Notch1信號(hào)通路的靶基因)的表達(dá)在異甘草素組和DAPT組比對(duì)照組顯著下降(P0.05),呈一定的劑量依賴效應(yīng)。沒有X射線干預(yù)的情況下,異甘草素組和對(duì)照組的P-NF-κB表達(dá)量均較低。4 Gy的X射線照射后,異甘草素組P-NF-κB比對(duì)照組高(P0.05),且X射線照射后6 h出就開始表達(dá)增高,24h進(jìn)一步升高(P0.05)。沒有X射線干預(yù)的情況下,異甘草素組的cleaved caspase-3表達(dá)比對(duì)照組高(P0.05),隨著時(shí)間推移沒有明顯變化。在4 Gy的X射線照射后,未加異甘草素組的cleaved caspase-3表達(dá)比未放射的對(duì)照組明顯增高(P0.05),但隨著時(shí)間的推移未見明顯變化。而4 Gy的X射線照射后,異甘草素預(yù)處理組在6 h內(nèi)與未放射組相比,cleaved caspase-3表達(dá)沒有明顯改變,而24 h后,cleaved caspase-3表達(dá)開始升高(P0.05)。結(jié)論:1.人腦膠質(zhì)瘤干細(xì)胞對(duì)放療具有較強(qiáng)的抵抗,20?M異甘草素能夠有效抑制膠質(zhì)瘤干細(xì)胞的增殖,同時(shí)提高膠質(zhì)瘤干細(xì)胞的放療敏感性,此濃度的異甘草素作用24 h后再用X射線照射,能夠抑制膠質(zhì)瘤干細(xì)胞球的形成和減小干細(xì)胞球的直徑;2.異甘草素能夠抑制Noch1信號(hào)通路的表達(dá),可能通過改變下游基因的表達(dá)增加膠質(zhì)瘤干細(xì)胞的放療敏感性。在X射線照射后,異甘草素能夠上調(diào)NF-κB和caspase-3的表達(dá),參與X射線造成的膠質(zhì)瘤干細(xì)胞損傷過程。
[Abstract]:AIM: To study the effect of isoliquiritigenin on the radiosensitivity of glioma stem cells and its mechanism, and to provide evidence for isoliquiritigenin as a radiosensitizer in the treatment of human glioma. METHODS: 1. The human glioma adherent cells were extracted from SHG44 glioma adherent cells by using ultra-low adsorption suspension cell culture plate with 6 holes and serum-free conditioned culture method. Glioma stem cells were extracted and purified, and the stem cell characteristics of suspension cell spheres were identified by CD133 and Nestin, and the glioma cell lines were identified by Bcl-2. The proliferation activity of glioma stem cells under different concentrations of isoliquiritin and different doses of X-ray was detected by CCK-8 kit, and the proliferation activity of glioma stem cells was observed under microscope. Microscopic observation and recording of the formation of glioma stem cell suspension spheres. The radiosensitization effect of isoglycyrrhizin was evaluated by cell activity and morphological inhibition of the cell suspension spheres. 3. Western blotting was used to detect the expression of Notch1 signaling pathway related proteins in different concentrations of isoglycyrrhizin and Notch1 signaling was performed. The expression of NF-kappa B and Caspase-3 in glioma stem cells pretreated with isoglycyrrhizin at different doses of X-ray irradiation was detected to explore the mechanism of isoglycyrrhizin in enhancing the sensitivity of glioma stem cells to X-ray irradiation. Human glioma stem cell suspension spheres were effectively extracted and purified. Immunofluorescence staining of CD133 and Nestin showed that the suspension cell spheres possessed stem cell characteristics. The positive expression of Bcl-2 indicated that the suspension cell spheres were tumor-derived. After 2.72 hours, the activity of glioma stem cells was inhibited by isoliquiritin (P 0.05). The ability of tumor stem cells to form globules was inhibited, and isoliquiritin above 20? M significantly decreased the number of globules and the diameter of globules (P 0.01). The activity of glioma stem cells did not change with the increase of X-ray irradiation dose within 72 hours. However, X-ray can reduce the diameter of the suspended cell spheres to a certain extent (P 0.01), but the number of the spheres did not decrease due to X-ray irradiation, showing X-ray tolerance; 3. This study found that 10?M isoliquiritigenin has limited direct inhibitory effect on the growth of glioma stem cells, but exists at a higher dose of 8 Gy X-ray irradiation. In this case, it can increase the sensitivity of glioma radiotherapy (P 0.05). The number of stem cell spheres and the diameter of stem cell spheres decreased. 20? M isoglycyrrhizin can kill glioma stem cells obviously, and it can enhance the sensitivity of glioma stem cells when irradiated by 4 Gy X-ray. The ability of combined killing glioma stem cells under 8 Gy X-ray is improved. The effect of isoglycyrrhizin above 40? M on the proliferation of glioma stem cells was more obvious, which was mainly inhibited by X-ray irradiation. 4. Notch1 (Notch1 signaling pathway receptor), RBPJK (transcription regulator of Notch1 signaling pathway) and Hes1 (target gene of Notch1 signaling pathway) were expressed in glioma stem cells. The expression of P-NF-kappa B in isoglycyrrhizin group and control group was lower than that in control group (P 0.05) without X-ray intervention. The expression of P-NF-kappa B in isoglycyrrhizin group was higher than that in control group (P 0.05) after X-ray irradiation (P-NF-kappa B) in isoglycyrrhizin group was lower than that in control group (P-NF-kappa B) without X-ray intervention (P 0.05). The expression of cleaved caspase-3 in the isoliquiritigenin group was higher than that in the control group (P 0.05) without X-ray intervention. The expression of cleaved caspase-3 in the isoliquiritigenin group was significantly higher than that in the non-irradiated group (P 0.05) after 4 Gy X-ray irradiation. The expression of cleaved caspase-3 did not change significantly after 4 Gy X-ray irradiation. However, the expression of cleaved caspase-3 began to increase after 24 hours of irradiation (P 0.05). Conclusion: 1. Human glioma stem cells have strong resistance to radiotherapy, and 20?M isoliquiritin can effectively inhibit the expression of cleaved caspase-3. Proliferation of glioma stem cells and radiosensitivity of glioma stem cells can be improved by isoliquiritigenin, which can inhibit the formation of glioma stem cell spheres and reduce the diameter of stem cell spheres after irradiation with X-ray after 24 hours. 2. isoliquiritigenin can inhibit the expression of Noch1 signaling pathway, possibly by altering the expression of downstream genes. After X-ray irradiation, isoliquiritigenin can up-regulate the expression of NF-kappa B and caspase-3, and participate in the process of X-ray-induced damage to glioma stem cells.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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