【作者】 王彬；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 脑胶质瘤是颅内最常见的原发性恶性脑肿瘤,占全部原发性恶性肿瘤的1.6%,其中又以IV级胶质瘤-----多形性胶质母细胞瘤的恶性程度最高。脑胶质瘤的治疗以外科切除为主,但由于其呈浸润性生长,肉眼难以分辨其明确边界,肿瘤常难以彻底切除。尽管近年来在胶质瘤的综合治疗上取得了不少的进步,但预后仍然很差,肿瘤在治疗原位的复发仍是一个比较普遍的现象。接受了外科手术、术后放疗及化疗综合治疗手段的患者平均生存期为14.6月。脑胶质瘤的临床治疗缺乏突破性进展的一个重要原因很可能是忽视了该病的细胞起源。为了取得实质上的进步,我们需要进一步了解胶质瘤对现有治疗措施抵抗性的机制以及初次治疗后复发的原因。目前关于在胶质瘤放疗后复发这个普遍的现象中起关键作用的是哪种细胞正逐渐成为一个热门话题。先前的观点认为肿瘤细胞术后残留是复发的根源。随着肿瘤干细胞学说的提出,认为一小部分因恶性转化而获得了自我更新能力的干细胞或前体细胞,可能是决定肿瘤的发生、发展、侵袭、转移和对治疗敏感与否的关键细胞。最近国内外一些学者陆续从不同级别的胶质瘤和体外长期培养的胶质瘤细胞株中分离培养出CD133阳性的肿瘤干细胞,发现它们在体外培养条件下可以自我更新,具有多向分化潜能,连续移植均能够稳定地获得与亲本肿瘤表型一致的移植瘤。因而被认为在决定胶质瘤的起源、维持胶质瘤的恶性表型等方面起着十分重要的作用。以上述研究理论为依据,本课题提出脑胶质瘤干细胞如在其它恶性肿瘤组织中的肿瘤干细胞一样,在恶性胶质瘤组织中大多处于静止状态,具有明显的辐射抵抗性,X射线、γ射线等电离辐射对其几乎不起作用。因而,成为胶质瘤放疗后原位复发重要原因之一的假说。为了验证上述假说并阐明脑胶质瘤干细胞在当前胶质瘤放射治疗中的辐射抵抗性,在本课题的第一部分,我们采用CD133标记脑胶质瘤干细胞,以免疫磁珠法从恶性胶质瘤细胞株U251和新鲜人恶性胶质瘤标本中分离出CD133阳性细胞并进行体外培养,通过免疫荧光技术检测干细胞标志物CD133、Nestin,诱导分化后检查分化细胞标志物β-TubulinⅢ、GFAP、MBP的表达以及电镜超微结构观察和裸鼠移植致瘤实验对其干细胞特性加以鉴定,得到以下结果证明我们分离所得到的CD133阳性细胞是脑胶质瘤干细胞:(1)CD133阳性的细胞能够自我更新并连续形成亚克隆;(2)保持着未分化状态,具有多向分化潜能;(3)CD133阳性的细胞裸鼠移植后能够产生与亲本肿瘤表型一致的移植瘤,而CD133阴性细胞不具有上述所有特性。因而, CD133阳性的脑胶质瘤干细胞产生明显的肿瘤致瘤性。在第二部分的实验中,我们使用60Coγ射线照射体外培养的脑胶质瘤干细胞、自身存在于U251、人恶性胶质瘤标本中的脑胶质瘤干细胞,以对比研究它们的放射敏感性。采用TUNEL原位末端标记技术检测凋亡、Annexin V-FITC检测凋亡率,流式细胞仪及裸鼠移植实验分别检测辐射前后细胞周期及致瘤性的变化,并得到如下结果:1:体外培养的脑胶质瘤干细胞的细胞周期分布为G0-G1期占51.65±2.75%,G2-M期23.35±1.25%,S期24.15±2.35%。它们生长、代谢旺盛,对电离辐射敏感,不同剂量的60Coγ射线可诱导凋亡。大于2Gy以上的各照射组凋亡率与对照组相比有显著性差异(P﹤0.05)。细胞移植实验发现:当体外培养的脑胶质瘤干细胞接受2Gy以上剂量照射以后不再具有致瘤性;2:U251细胞株和人恶性胶质瘤标本细胞株在体外培养条件下生长、代谢旺盛,对60Coγ射线也表现出与体外培养的脑胶质瘤干细胞相似的辐射敏感性。但明显的区别在于:当U251和人恶性胶质瘤标本细胞株接受14Gy以下剂量照射后行裸鼠移植仍具有致瘤性;3:不同剂量的60Coγ射线照射U251和人恶性胶质瘤标本细胞株后,应用免疫磁珠分选其中的CD133阳性和CD133阴性细胞并行Annexin V-FITC染色显示:CD133阴性的脑胶质瘤细胞对电离辐射敏感,各照射组凋亡率与对照组相比存在显著性差异(P﹤0.05),裸鼠移植实验均不具有致瘤性;而CD133阳性的脑胶质瘤干细胞则表现为在14 Gy以下,各照射组凋亡率与对照组相比无显著性差异(P﹥0.05),裸鼠移植实验仍然具有致瘤性。4:流式细胞仪检测U251和人恶性胶质瘤标本细胞株中的CD133阳性细胞所占比例及细胞周期发现:自身存在于U251和人恶性胶质瘤标本细胞株中的脑胶质瘤干细胞所占比例不高,范围在9.60---12.18%之间,但其大多处于G0-G1期,约占87.4±4.9%,而S期占6.97±2.73 %,G2-M期占6.66±3.25%。与同时位于其中的CD133-胶质瘤细胞的细胞周期(G0-G1期占57.35±0.35 %, G2-M期18.17±7.83%, S期24.55±7.45%)相比,为不活跃的细胞周期,相对处于静止状态。总之,通过本实验的研究,结果表明:体外培养的脑胶质瘤干细胞处于活跃的细胞周期中,生长代谢旺盛。它们对电离辐射敏感,不同剂量的60Coγ射线可诱导凋亡,低剂量的电离辐射可以破坏脑胶质瘤干细胞的致瘤性。而自身存在于U251和人恶性胶质瘤标本细胞株中的对维持胶质瘤恶性表型起关键作用的脑胶质瘤干细胞则大多处于G0/G1期,抗拒电离辐射。尽管以60Coγ射线为代表的电离辐射可诱导大量CD133阴性的脑胶质瘤细胞凋亡,但却难以杀死CD133阳性的脑胶质瘤干细胞,可能成为胶质瘤经过放射治疗后复发的重要原因之一。更多还原

【Abstract】 Brain tumors account for 1.6% of all primary cancers. The majority of brain tumor are gliomas, and the most malignant form of glioma is grade IV, which is commonly known as glioblastoma multiforme. This highly aggressive tumor develops either from primary gliblastoma multiforme or as the result of the malignant progression from a low grade gliomas. Most importantly, glioblastoma multiforme is characterized by a diffuse tissue-distribution pattern, with extensive dissemination of the tumor cells within the brain that hinders complete surgical resection. Despite intensive multi-modality treatment including tumor resection, post-operative radiotherapy and frequently adjuvant chemotherapy, the prognosis of malignant glioma continues to be poor. Recurrences occur almost exclusively in the brain, most commonly at the site of initial tumor presentation. The median survival when surgical resection, radiotherapy and chemotherapy are combined is 14.6 months.One reason for the lack of clinical advances is ignorance of the cellular origin of this disease, which delays the application of molecular analyses to treatment and impairs the ability to anticipate tumor behavior reliably. To make ture progression in cancer therapy, we need to better understand what derives tumor resistance and mediates tumor recurrence after initial successful therapy. Now there is increasing awareness about which cells may play the critical role in the recurrence after post-operative radiotherapy of gliblastoma multiforme. The previous opinion towards the source cells of recurrence was the residual cancer cells after surgical resection, but now as the cancer stem-cell hypothesis proposes that a minority of transformed stem cells, or progenitors with acquired self-renewal properties, are the source of tumor-cell renewal and thereby determine a tumor’s behavior, including proliferation, progression and response to therapy, and recently several groups have discovered that brain cancer stem cells can self-renew under clonal conditions, differentiate into neurons and glia-like cells as well as abnormal cells with mixed phenotypes and CD133 positive cells transplantation at low density could readily form new tumors in immunodeficient mice. So the CD133 positive brain glioma stem cells were considered as a critical cell and should be responsible for the initiation and maintenance of malignant gliomas. Taking the knowledge above as a foundation, in this paper, we hypothesized that CD133 positive cells, representing the small population of brain glioma stem cells, are resistant to current gliblastoma multiforme therapies and play a very important role in tumor recurrence after radiotherapy.In order to verificate this hypothesis and to address the mechanism of brain glioma stem cells showing strong resistance to radiotherapy, we used CD133 as a surface marker to isolate CD133 positive and CD133 negative cells by Magnetic cell sorting from human malignant glioma cell line U251 and the primary glioblastoma multiforme culture cell lines in part 1 of our research and charactered as stem cells identified by stem cells surface markers and differentiated cells surface markers, ultrastructure observing with electron microscope and engrafting to nude mice for tumorigenesis test. Several lines of evidence support that the CD133 positive cells we have isolated from the cell lines above were brain glioma stem cells:(a) The isolated CD133 positive cells can self-renew and proliferate to generate contiguous sub-spheres; (b) The cells keep undifferentiated feature, have multipotency and can differentiate into multi-lineage progenies; (c) The isolated CD133 positive cells can produce brain tumors in nude mice, and the tumors are the phenocopy of the original tumor from which they were derived, however, CD133 negative cells can’t form tumors. So the tumorigenicity obviously depend on CD133 positive brain glioma stem cells.To evaluate the radiosensitivity of CD133+ cells which were lived alone in vitro and CD133+ cells which were existed in U251 and the glioblastoma multiforme specimens, 60Co irradiation was carried out to deliver different doses for CD133+ cells lived alone in vitro, and for U251 and the glioblastoma multiforme specimens in part 2 of our research. Flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling technique (TUNEL), Annexin V-FITC staining and nude mice transplantation were employed to examine cell cycle, apoptosis and tumorigenicity for the cells all of the above before and after 60Co irradiation respectively.The results were the following:1 The CD133 positive brain glioma stem cells lived alone in vitro were in active cell cycle, and G0-G1 phase was 51.65±2.75%,G2/M phase was 23.35±1.25%,S phase was 24.15±2.35%。Its’growth and metabolism were vigorous when seeding them in the culture condition of DMEM/F12 combining bFGF, EGF and LIF. The cells were sensitive to 60Coγ- irradiation, exceed 2Gy irradiation could induce apoptosis . Intracranial cell transplantation revealed there was no tumor appear when the cells received exceed 2Gy irradiation.2 As the same actively divided cell lines in vitro, human malignant glioma cell line U251 and the primary glioblastoma multiforme culture cell lines showed the same sensibility to the 60Coγ- irradiation , but there was great difference existed in that they could still form tumors when they received below 14 Gy irradiation.3 After U251 and the glioblastoma multiforme culture cell lines received different dose 60Co irradiation respectively, we utilized Magnetic cell sorting to isolate the CD133+ cells and CD133- cells. Annexin V-FITC staining showed apoptotic rate of CD133- cells obviously increased following irradiation dose increase and there was no tumor appear after intracranial or subcutaneouly transplantation into the nude mice. But towards CD133+ cells, Annexin V-FITC staining showed apoptotic rate slowly increased following irradiation dose increase until the cells received 14 Gy irradiation . The results of CD133+ cells transplantations showed the cells received below 14 Gy irradiation could form tumors respectively within 4 to 10 weeks, and the tumors still be the phenocopy of the original tumor through immunohistochemistry identification.4 After U251 and the glioblastoma multiforme culture cell lines received different dose 60Co irradiation respectively, we utilized Magnetic cell sorting to isolate the CD133+ cells and CD133- cells. From cell cycle distribution we could find the percentage of G0/G1 phase of CD133- cells was 57.35±0.35 %, G2/M phase was 18.17±7.83%, S phase was 24.55±7.45 %. However, the percentage of G0/G1 phase of CD133+ cells was 87.4±4.9 %, G2/M phase was 6.66±3.25%, S phase was 6.97±2.73 %. It indicated that CD133+ cells existed in U251 and the glioblastoma multiforme culture cell lines were in unactive mitotic division status.In our research, we found the most number of CD133 positive cells existed in U251 and the primary glioblastoma multiforme culture cell lines were in G0/G1 phase and were resistant to 60Coγ- irradiation. The maintenance of malignance of human adult gliblastoma multiforme obviously depends on the CD133 positive brain glioma stem cells. Although ionizing radiation could induce a large number of cancer cells (CD133 negative cells) apoptosis or even kill them, but can only show litter influence on CD133 positive cells, and the CD133 positive brain glioma stem cells could still form tumor which recapitulated the phenotypic heterogeneity found in the initial tumor after intracranial or subcutaneouly transplantation. These data suggest that CD133 positive brain glioma stem cells are resistant to current radiotherapy and may represent a cell target for novel malignant glioma therapies.更多还原