摘要
恶性胶质瘤是中枢神经系统最为常见的原发性肿瘤之一,约占颅内肿瘤的45%-50%,具有分化程度差、增殖活性高、呈侵袭性生长的特性,致残致死率高。恶性胶质瘤细胞具有在脑组织内侵袭和迁移的能力,肿瘤与脑组织的真实边界很难确定,不仅为手术和其他辅助治疗带来困难,也是肿瘤播散与复发的主要原因。因此,深入了解恶性胶质瘤侵袭迁移的生物学规律是当前亟待研究的问题。
既往研究发现,恶性胶质瘤在脑实质内的侵袭有两个主要路径：一是肿瘤细胞沿着有髓神经纤维进行迁移,这是恶性胶质瘤主要的侵袭路径；二是肿瘤细胞沿着血管基底膜途径侵袭,新生血管的形成是肿瘤细胞扩散的引导结构。有关恶性胶质瘤细胞侵袭性的分子机制的研究提示恶性胶质瘤的侵袭迁移是肿瘤细胞与宿主细胞及细胞外基质相互作用的复杂过程,涉及黏附分子、酶、信号转导系统、基因调控、血管生成等多方面的因素。
近年来,人们利用CD133、Nestin等标志物成功鉴定和分离出了胶质瘤干细胞,认为胶质瘤干细胞群体中存在侵袭性的胶质瘤干细胞亚群,他们具有高侵袭性,可能是肿瘤迁移和复发的“根源”,为研究恶性胶质瘤侵袭迁移的生物学特性提供了新的思路。本课题将重点研究胶质瘤干细胞标记物CD133在胶质母细胞瘤及瘤周不同部位的表达情况,并分析其与CD34+组织微血管增殖的相关性,以探讨胶质瘤干细胞在恶性胶质瘤中侵袭迁移的生物学规律。本课题研究分三步完成：
1.采用免疫组织化学染色和Western blot法检测CD133在各组标本中的表达情况。两种方法均显示CD133在对照组脑组织中不表达,而在肿瘤和水肿交界区、肿瘤中心区,瘤周水肿区组织中呈阳性表达。各区域间CD133表达的差异具有统计学意义(P<0.05)。光学显微镜下可见,在交界区CD133+细胞分布密集,往往形成以微血管为中心的假菊型团样结构,在中心区CD133+细胞常在微血管附近簇状分布,偶可见CD133+细胞围绕坏死域形成的假栅栏样结构,在水肿区也可见CD133+细胞环绕微血管形成的假菊型团样结构但以散在分布为主,还可见沿着小血管基底膜和髓质神经纤维分布的CD133+细胞。
2.采用免疫组织化学染色法检测CD34+组织微血管在组织标本中的表达。高倍视野下(×200倍)CD34+MVD(个/视野)在肿瘤和水肿交界区、肿瘤中心区、肿瘤周围水肿区、对照组脑组织中分别是31.32±3.97、21.80±2.58、15.28±2.37、4.67±1.53；各组之间的差异具有统计学意义(P<0.05)。与对照组脑组织相比,胶质母细胞瘤及瘤周组织中的微血管不仅有MVD的增加,而且形态结构也有明显的缺陷,多为幼稚血管,管壁仅一层内皮细胞,缺乏基底膜,有些甚至呈裂隙状。
3.利用统计学方法分析CD133+胶质瘤干细胞的分布与组织微血管增殖的相关性。胶质母细胞瘤中CD133的表达水平和CD34+组织微血管密度的分布呈正相关(r=0.928,P<0.05)。
结论
1.胶质瘤干细胞向微血管生成丰富的交界区迁移富集,提示他们和组织微血管之间可能存在密切的“趋向”联系。
2.胶质瘤干细胞在脑实质内也能沿着血管基底膜和髓质神经纤维途径侵袭。
3.切断胶质瘤干细胞和组织微血管之间的“趋向”联系可以干预肿瘤干细胞的迁移。
4.抗肿瘤微血管治疗可能是恶性胶质瘤有效的治疗手段之一。
Malignant gliomas, accounting for 45%-50% of intracranial neoplasm, are the most common and lethal primary brain tumors. Malignant gliomas are poorly differentiated, highly proliferated and aggressive. Being the primary reason for tumor recurrence and dissemination, the ability to invade and spread into normal brain tissue obscures the histological boundary between the malignant glioma and healthy brain, and renders difficulties to surgical removal and other adjuvant therapies. Therefore, insight into the underlying biological features of invasion and migration of malignant glioma continues the urgent problem.
Previous studies have shown that there are two main invasive paths for malignant glioma:one is along the myelinated nerve fibers, which is the primary way for glioma cells moving; the other is along the basement membrane of blood vessels. The formation of new blood vessels are the induced structures for tumor cell diffusing. The studies on molecular mechanism of invasiveness of malignant glioma cells suggested that the invasion of malignant gliomas is a complicate and consecutive process undergoing adhesion, degradation and migration multi-step, and involving multi-factor such as cell adhesion, molecules, enzymes, cytokines, gene regulation and angiogenesis, etc.
In recent years, the glioma stem cells are identified and isolated successfully, using bio-markers, such as CD 133 and Nestin. In the glioma stem cells, more invasive subpopulation are thought to exist, which might be the root cause of tumor recurrence and migration. These findings provided a novel clue to investigate biological characteristics of invasion of malignant gliomas. To understand deeply the invasive path of glioma stem cells within the tumor and surrounding tissue, we focused on the expression of CD133, a marker for glioma stem cells, and CD34, a marker for endothelial cells, in different parts of glioblastoma tissue and tumor margin in the present study.The correlation between CD133 positive cells and CD34 positive microvascular proliferation was analyzed. The study including three parts:
1. The expression of CD133 in different parts within the tumor and surrounding tissue was detected by immunohistochemistry and western blotting. No expression of CD133 was found in the control brain tissue. The expression scores of CD133 were 7.26±1.35,5.18±1.12,2.67±0.98 by immunohistochemistry, while 0.79±0.03, 0.38±0.01,0.20±0.04 by western blot, respectively in tumor marginal zones, tumor cores, and edematous zones. The difference of CD133 expression in different groups are significant statistically (P<0.05). Under the microscope, CD133-positive cells densely congregated around the vessels and formed perivascular pseudorosettes in tumor marginal zones. In the core of the tumor, CD 133 positive cells clustered around the vessels and occasionally led to pesudopalisades around necrosis. In the edematous zones, perivascular pseudorosettes of CD133 positive cells scattered and CD133 positive cells were also observed to line up along myelinated nerve fibers and the basement membrane of vessels.
2. The microvessel density (MVD) in different parts within the tumor and surrounding tissue was determined by CD34 immunohistostaining. Under the microscope (magnification×200). CD34+MVD/HP were 31.32±3.97、21.80±2.58、15.28±2.37、4.67±1.53 respectively in tumor marginal zones, tumor cores, edematous areas and the control brain tissues. The difference of the microvessel density in different groups were significant statistically (P<0.05). Compared with in the control brain tissue, besides the increased microvessel density, most of those microvessels in the tumor and tumor margin were immature morphologically, only composed of a single layer of endothelial cells and lost of basement membrane, even with the cleft wall.
3. The correlation was analyzed statistically between the distribution of CD 133 positive glioma stem cells and CD34+ microvascular proliferation. The expression level of CD133+ was positively correlated with CD34+MVD (r=0.928, P<0.05) in glioblastoma.
Conclusion:
1. The glioma stem cells tend to congregate in the tumor margin zones where the microvessels are also enriched. It suggests the glioma stem cells tropism for microvessels.
2. Glioma stem cells would invade along the myelinated nerve fibers and the basement membrane of vessels within the parenchyma of the brain.
3. Blocking Glioma stem cells tropism for microvessels would be the novel strategy to prevent migration of Glioma stem cells.
4. Antiangogenesis therapy might be one of efficient anti-tumor management.
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