异甘草素对SHG44人脑胶质瘤干细胞放疗敏感性的影响及机制研究
发布时间：2018-08-29 14:25
【摘要】：目的:研究不同浓度的异甘草素对胶质瘤干细胞放疗敏感性的影响及作用机制,为异甘草素作为放疗增敏剂治疗人脑胶质瘤提供依据。方法:1.使用超低吸附悬浮细胞培养板6孔板,通过无血清条件培养法从SHG44人脑胶质瘤贴壁细胞中提取和纯化胶质瘤干细胞,通过CD133、Nestin联合鉴定悬浮细胞球的干细胞特性,通过Bcl-2鉴定悬浮细胞球的胶质瘤细胞源性;2.运用CCK-8试剂盒检测不同浓度异甘草素和不同剂量X射线的单独和联合运用下胶质瘤干细胞的增殖活性,并在显微镜下观察和记录胶质瘤干细胞悬浮球的成球情况。通过细胞活性和干细胞悬浮球在形态上的抑制情况评估异甘草素的放疗增敏效果;3.运用Western blotting技术检测不同浓度异甘草素作用下Notch1信号通路相关蛋白的表达情况,以Notch1信号通路特异性阻断剂DAPT作为阳性对照。检测异甘草素预处理后胶质瘤干细胞对不同剂量X射线照射后NF-κB和凋亡关键蛋白caspase-3的表达情况,以此探讨异甘草素对胶质瘤干细胞X射线放疗增敏的作用机制。结果:1.无血清条件培养能够有效地提取和纯化人脑胶质瘤干细胞悬浮球,免疫荧光染色CD133和Nestin表达阳性说明悬浮细胞球具有干细胞特性,Bcl-2表达阳性说明悬浮细胞球的肿瘤源性;2.72 h后,胶质瘤干细胞的活性被异甘草素所抑(P0.05)。异甘草素处理9天后,胶质瘤干细胞的成球能力被抑制,20?M浓度以上的异甘草素能够明显降低胶质瘤干细胞成球的数目和干细胞球的直径(P0.01)。在72 h内,胶质瘤干细胞的细胞活性并不随X射线的照射剂量的增加而有所改变。在9天的胶质瘤干细胞成球过程中,虽然X射线能够一定程度上减小所形成的悬浮细胞球的直径(P0.01),但是,成球的数目并没有因为X射线的照射而下降,表现出X射线的耐受;3.本研究发现10?M的异甘草素虽然直接抑制胶质瘤干细胞生长的作用有限,但在8 Gy较高剂量X射存在的情况下,能够增加胶质瘤放疗的敏感性(P0.05),表现为所形成的干细胞球数目下降和直径下降。20?M的异甘草素杀伤胶质瘤干细胞的作用明显,且在4 Gy的X射线照射时就有较强的增敏效果,在8 Gy的X射线下联合杀伤胶质瘤干细胞的能力进一步提高(P0.05)。而40?M以上的异甘草素抑制胶质瘤干细胞增殖的作用较为明显,在联合X射线干预下,主要表现出直接抑制增殖的作用;4.Notch1(Notch1信号通路受体)、RBPJK(Notch1信号通路的转录调节因子)和Hes1(Notch1信号通路的靶基因)的表达在异甘草素组和DAPT组比对照组显著下降(P0.05),呈一定的剂量依赖效应。没有X射线干预的情况下,异甘草素组和对照组的P-NF-κB表达量均较低。4 Gy的X射线照射后,异甘草素组P-NF-κB比对照组高(P0.05),且X射线照射后6 h出就开始表达增高,24h进一步升高(P0.05)。没有X射线干预的情况下,异甘草素组的cleaved caspase-3表达比对照组高(P0.05),随着时间推移没有明显变化。在4 Gy的X射线照射后,未加异甘草素组的cleaved caspase-3表达比未放射的对照组明显增高(P0.05),但随着时间的推移未见明显变化。而4 Gy的X射线照射后,异甘草素预处理组在6 h内与未放射组相比,cleaved caspase-3表达没有明显改变,而24 h后,cleaved caspase-3表达开始升高(P0.05)。结论:1.人脑胶质瘤干细胞对放疗具有较强的抵抗,20?M异甘草素能够有效抑制胶质瘤干细胞的增殖,同时提高胶质瘤干细胞的放疗敏感性,此浓度的异甘草素作用24 h后再用X射线照射,能够抑制胶质瘤干细胞球的形成和减小干细胞球的直径;2.异甘草素能够抑制Noch1信号通路的表达,可能通过改变下游基因的表达增加胶质瘤干细胞的放疗敏感性。在X射线照射后,异甘草素能够上调NF-κB和caspase-3的表达,参与X射线造成的胶质瘤干细胞损伤过程。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
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