【作者】 齐玲；

【导师】 李才； 郝春海；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 胶质母细胞瘤(glioblastoma)或称多形性胶质母细胞瘤(glioblastoma multiforme,GBM),在胶质瘤中恶性程度最高,预后极差,目前各种治疗方法的效果均不理想。肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand, TRAIL)是肿瘤坏死因子(TNF)超家族中的一员,它能诱导多种肿瘤细胞发生凋亡,而对正常组织和细胞无明显毒性作用,这一特性使之成为肿瘤治疗研究中的热点。然而,在脑肿瘤研究中已证明,绝大部分GBM对TRAIL诱导凋亡发生抵抗,但其抵抗的分子机制目前尚不清楚。本实验采用体内实验与体外实验相结合的方法,应用细胞培养、酶联免疫吸附试验、流式细胞术、免疫磁珠分选、免疫荧光化学、Western blot及在体动物实验等技术,研究GBM单克隆细胞株对TRAIL诱导凋亡的敏感性,观察TRAIL凋亡蛋白表达及经TRAIL诱导各细胞株发生凋亡级联裂解反应。建立GBM肿瘤干细胞研究体系,研究GBM干细胞对TRAIL诱导凋亡的敏感性及TRAIL凋亡蛋白的表达情况。本研究旨在探讨GBM及其干细胞抵抗TRAIL诱导凋亡的分子机制,为脑肿瘤的治疗提供实验依据。本研究结果表明,GBM单克隆细胞株经TRAIL处理后可诱导凋亡,敏感细胞株半胱氨酸蛋白酶-8(Caspase-8)表达增高;而抵抗细胞株Caspase-8表达降低或不表达。敏感细胞株经TRAIL作用后引起Caspase-8、-3、-9及DFF-45裂解,抵抗细胞株不发生裂解。研究证明GBM干细胞对TRAIL诱导凋亡的敏感性降低,表达Caspase-8量减少;加入顺铂后GBM干细胞对TRAIL敏感性明显增加;加入Caspase-8抑制剂Z-IETD、Caspases广泛抑制剂Z-VAD后,可抑制GBM干细胞对TRAIL诱导的凋亡反应。这些结果表明,在GBM及其干细胞抵抗TRAIL诱导凋亡研究中,Caspase-8表达起重要作用,与TRAIL诱导凋亡抵抗有一定相关性,为脑肿瘤的治疗提供了新的理论依据。更多还原

【Abstract】 Glioblastoma, the most common primary brain tumor in adults,the median survival was no more than 2 years. The glioblastoma is the topographically diffuse nature of the disease, invasive glioblastoma cells activate cellular programs that show a decreased proliferation rate and a relative resistance to apoptosis, which may contribute to chemotherapy and radiation resistance. The current treatments are not very effective. Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） triggers apoptosis in many human tumor cells, and it is almost nontoxic to normal tissues and cells. So TRAIL acted as a key of anti-drugs for tumor. TRAIL-induced apoptosis occurs through DR4 and DR5, FADD recruits Caspase-8 to the receptors for the assembly of a death-inducing signaling complex （DISC）,and then actived a serial of Caspases cleavage reactions through Caspase-8, finally actived cleavage of DFF-45. The researcher certified that most of glioblastoma is resistant with TRAIL, still resistant when added chemotherapy drugs, otherwise people want to know the key and clear the resistance mechanism of TRAIL-induced apoptosis in glioblastoma cells and stem cells.The brain tumor stem cells is the special sub-group which is one of the hot pot in cancer research, it is more important about two methods of isolated BTSC. Reynolds and Weiss set up a new technique by isolated the neural stem cells through the neurosphere assay（NSA）in 1992, and that got a large number of stable neural stem cells （NSC） in culture and then the NSA is popular in BTSC. After that Singh’s group purified BTSC by CD133 magnetic sorting in 2004, it can get a huge number of BTSC at one time. The researcher certified that neural stem cells are resistant to TRAIL induced apoptosis, but we don’t know whether not the same situation happened in BTSC, what is the machnism of resistance reason to TRAIL, and maybe the BTSC is resistant to TRAIL, that leading to glioblastoma resistance with TRAIL. In our experiments, we cultured SC326 and SC189, selected 12 monoclones from each of them, set up GSC326, GSC189 by neurospheres and CD133 magnetic sorting, and then identified them with several techniques: NSA; immunofluorecent stained with NSC marker CD133, nestin and differentiation marker GFAP,β-TubulinⅢ; tumorigenic mouse experiment. The phosphate acid detected monoclones sensitivity of TRAIL, MTS checked the cell death of BTSC to TRAIL. The expression of apoptosis proteins, Caspases and DFF-45 cleavage were detected by Western blot. The aim is study the mechanism of resistance to TRAIL-induced the apoptosis in glioblastoma cells and glioblastoma stem cells.Detected the sensitivity of GBM monoclones to TRAIL-induced apoptosis:1. The 24 monoclones were obtained from the two primary glioblastoma cultures, examined the cell death by acid phosphate assay after they treated with TRAIL, The results showed that the cell death of monoclones were different, SC326 was sensitive to TRAIL, and most of the monoclones in SC326 were sensitive; SC326 was resistant cell line to TRAIL, so most of the monoclones in SC189 were resistant.2. The monoclones expressed the apoptosis proteins: we checked the antibodieds of FADD, Bid, Bak, Bax, Bcl-2, Bcl-x/l, Smac, Caspase-9, DR5, DR4, they are similar in SC326, but the expression of Caspase-8 is very different, the exprestion of SC326- 9, 11, 12 increased. SC189 got the same results, most important point is the expression of Caspase-8, SC189- 11, 15, 17 expressed high level Caspase-8; the expression of SC189-8, 9, 10, 13 is low , or no expression.3. We cultured SC326- 9, 11, 12, 4, 14, 16 and SC189-11, 15, 17, 8, 9, 10, 13, treated with TRAIL, then used Western blot detected the cleavage of TRAIL-induced apoptosis. SC326-4, 14, 16 and SC189-11, 15, 17, they showed the cleavage of Caspase-8, 3, 9 and DFF-45. SC326- 9, 11, 12 and SC189- 8, 9, 10, 13 haven’t the cleavage reaction. TRAIL actived the Caspase-8 in TRAIL-sensitive monoclones and continued to induce a series of Caspases cleavage. Otherwise the TRAIL-resistant monoclones haven’t cleavage. GBM neurospheres were isolated, indentified, purified and detected the sensitivity of TRAIL:1. We set up GSC326, GSC189 neurosphere culture by using the technique of neurosphere assay.2. The neurospheres were checked by sub-neurosphere assay, and for self-renew, proliferation, and neurosphere reformation. It certified that GSC326, GSC189 have the strong characterics of self-renew, proliferation, and neurosphere reformation.3. The neurosphere were stained with NSC marker of CD133, nestin, the results showed positive; stained with differentiated marker, they expressed a low level GFAP, but almost no expresstion ofβ-TubulinⅢ, it certified that GSC326, GSC189 were BTSC.4. We added the differentiation media in the GSC326, GSC189, the neurosphere has the ability of differention, GSC326, GSC189 was positive with differentiation marker ofβ-TubulinⅢand GFAP.5. The cells of neurospheres were injected into the NOD/SCID mice about 6～7 weeks old, they producted the brain tumor, MRI T1 and T2 images showed that the brain has tumor, and Evan blue staining was positive. After isolated the tumor from mice and planted into the SFM, they can form a lot of neurospheres; CD133 and nestin staining was positive.6. We checked the cell death of GSC326, GSC189 neurospheres to TRAIL-induced apoptosis, and results showed that they were more resistant than SC326 and SC189. The cell death can inceased after added in cisplatin. When GSC326 treated with Caspase-8 and Caspases inhibitor, the inhibitor decreased the cell death significantly.7. Western blot detected the apoptosis proteins: DR5, FADD and Caspase-8, Caspase-8 has low expression, maybe this is the resistance reason of TRAIL.in neurosphere.CD133+/- GBM stem cells were checked the sensitivity of TRAIL. 1. We applied to CD133 magenic sorting to isolate and purify BTSC, and then checked the percentage of CD133⁺ by FCM. The percentage of CD133⁺ can catch to 90% after passed two times magnetic column. This technique can get the CD133⁺ cells directly and effectively.2. The capacity of neurosphere formation was checked after sorting: CD133⁺ formed the neurosphere effectively, 100 cells can form 30～40 neurospheres; CD133- has low ability for neurosphere formation, 100 cells formed no more than 5 neurospehres. After treated with TRAIL, GSC326 after sorting decreased the capacity of neurosphere formation, GSC189 was increased a little bit.3. MTS detected the sensitivity of TRAIL in CD133^+/- : GSC326 CD133⁺ was more sensitive to TRAIL, GSC189 CD133⁺ was more resistant; the inhibitor of Caspase-8 and Caspases can decrease the cell death. The cells after sorting, SC326 and SC189 had the same characteristic, they have the similar results of sensitivity of TRAIL, it is possible that this is reason for the michanism of TRAIL resistance.4. Western blot checked the apoptosis proteins: DR5, FADD and Caspase-8, the results showed that the expression was similar, compared with neurosphere they had the same level expression. So we can use two techniques to get the BTSC, but CD133 magnetic sorting system is more effective and direct.The principal innovation of this study:1. It is the first time to study the molecular mechanism of resistance to TRAIL-induced apoptosis in glioblastoma cells and glioblastoma stem cells. And this thesis certified that the expression of Caspase-8 has the direct relationship with sensitivity of TRAIL-induecd apoptosis.2. Compared the neurosphere assay and CD133 magnetic sorting, they both can get BTSC, but the latter is more effective and direct.3. The expresstion of Caspase-8 is low in BTSC, and it regulated the sensitivity of TRAIL-induced apoptosis.In conclusion, we can use TRAIL to treat the brain tumor, but we need to solve TRAIL resistance. This thesis studied on molecular mechanism of resistance to TRAIL-induced apoptosis in glioblastoma cells and glioblastoma stem cells, and it provided a further theoretical basis. After this study, we still need to do the research of the resistant cells how to expess high level Caspase-8. The technique of BTSC will improve the research and provide a new method for cancer treatment. To study the occurrence of brain tumors provides a powerful tool for the future, further study of the occurrence of the normal nervous tissue and brain tumor incidence of the relationship. BTSC also led to identification of neuroscience research in a new era.更多还原