基质金属蛋白酶对胶质瘤干细胞侵袭作用的影响

The Effect of Cancer Stem Cells on Glioma Invasion Mechanism Through Matrix Metalloproteinases

目的:分别在体内及体外条件下,观察恶性胶质瘤肿瘤干细胞(Cancer Stem cells,CSCs)的侵袭能力,检测恶性胶质瘤不同细胞亚群的基质金属蛋白酶-2(MMP-2)和-9(MMP-9)的表达,探讨其与CSCs侵袭能力的相关性。方法:应用神经干细胞(neural stem cells,NSCs)培养基体外分离培养原代恶性胶质瘤细胞,应用自我更新分析、免疫细胞化学染色、体内成瘤分析等技术鉴定CSCs细胞亚群。分别在体内、体外条件下,应用细胞侵袭分析技术观察不同肿瘤细胞亚群的侵袭能力,应用实时RT-PCR、Western blot、免疫组织化学染色方法,检测不同肿瘤细胞亚群中MMP-2、MMP-9的表达情况。结果:原代培养的恶性胶质瘤细胞被分离鉴定为CSCs和非肿瘤干细胞(non-CSCs)。胶质瘤CSCs的促侵袭能力明显强于non-CSCs;MMP-2的表达水平在两个细胞亚群间存在显著差异(P<0.0001),且均明显高于MMP-9表达;在胶质瘤CSCs接种的动物脑组织中,MMP-2与MMP-9的表达均呈时间依赖性增高,MMP-9则仅在肿瘤组织中表达,其表达增长率高于MMP-2,且主要定位于肿瘤的边缘。结论:在不同的条件下,胶质瘤CSCs可能是通过MMP-2或MMP-9促进了肿瘤细胞的侵袭及肿瘤的侵袭性生长。特异性抑制胶质瘤CDCs明胶酶分泌,尤其是MMP-9,将为抗肿瘤治疗提供有效的手段。未来针对于CSCs与其它MMPs家族成员及其抑制剂的研究将有利于进一步阐述胶质瘤的侵袭机制,同时也将为抗恶性胶质瘤的治疗提供新的策略。

Objective： Some studies have suggested that a small group of cells within glioma mass have similar features with neural stem cells （NSCs）. This subpopulation isolated from glioma cells has been identified as cancer stem cells （CSCs） and may play a key role in tumor initiation and maintenance. Matrix metalloproteinases （MMPs）, particularly gelatinases （MMP-2 and MMP-9）, which degrades protein components of the extra-cellular matrix and basement membrane seem to be largely involved in the invasion of glioma. The invasion property and gelatinases gene expression of CSCs are still unknown. Methods： We used NSCs medium to isolate spheres from primary cultured tumor cells derived from glioblastoma multiforme （GBM） patients, and identified them as glioma CSCs by using self-renewal assay, immunocytostaining, and in vivo tumorigenesis analysis. Cell invasion system was used to detect the invasive ability of different subpopulations. Rodent brain tumor models were established to simulate malignant glioma in vivo and invasive tumorigenesis of different subpopulations was evaluated. The expression of MMP-2 and MMP-9 mRNA and protein in vitro and in vivo were detected by real-time RT-PCR, Western blot, and immunohistochemistry staining, respectively. Results： Glioma cells were purified and characterized into two different subpopulations-glioma CSCs and non-CSCs. Glioma CSCs had increased capability of invasiveness compared with non-CSCs in vitro. CSCs derived from glioma cells formed tumors after implantation, which are characterized by invasiveness, angiogenesis and proliferation. But very few tumors were generated after implantation with non-CSCs. Glioma CSCs secreted higher level of MMP-2 than that in non-CSCs subpopulation in vitro. The expression levels of both MMP-2 and MMP-9 were increased in tissues of glioma CSCs. But low expression of MMP-9 was detected in adjacent normal brain tissues. Conclusion： Glioma CSCs may enhance tumor invasiveness through gelatinases, and MMP-2 and MMP-9 have distinguishable effects under different circumstances. Targeting gelatinases, especially MMP-9 to may be a new strategy to inhibit the invasiveness of glioma.