神經膠母細胞瘤細胞株之神經分化研究

Abstract

神經膠母細胞瘤(glioblastoma)是一種很常見且非常惡性的人體原發性腦瘤，現今療法無法有效控制神經膠母細胞瘤且預後並不樂觀。丙氧鳥苷(ganciclovir, GCV)是一種抗病毒藥物，在丙氧鳥苷/泡疹病毒胸腺嘧啶激酶(Herpes simplex virus thymidine kinase, HSVtk)系統(GCV/HSVtk system)中，作為治療各種癌症的基因療法。由於此系統具有選擇性毒殺表現HSVtk細胞的能力，在轉譯醫學研究領域之細胞療法中，此系統可應用於毒殺未分化之幹細胞。因此，我們認為可藉由調整細胞增生及分化方式，GCV/HSVtk system具有作為治療神經膠母細胞瘤的潛力。本研究中我們主要目的有二：(1)建立以GFAP啟動子調控，且可選擇性毒殺神經膠母細胞瘤之GCV/HSVtk system；(2)並探討此系統促進神經膠母細胞瘤神經分化之能力。 我們以CMV啟動子(cytomegalovirus promoter)調控HSVtk與綠色螢光蛋白(EGFP)作為對照組質體，以GFAP啟動子(glial fibrillary acidic protein promoter)調控者則作為實驗組質體，並將這兩種質體分別轉殖入非神經膠細胞株NIH-3T3纖維母細胞和Neuro 2A神經細胞，以及神經膠母細胞瘤細胞株U251和U87中。經G418藥物篩選後，建立出五株能表現HSVtk的細胞株：N2A-pCMV-HSVtk、N2A-pGFAP-HSVtk、U251-pCMV-HSVtk、U251-pGFAP-HSVtk與U87-pGFAP-HSVtk，並藉由西方墨點法進行HSVtk產物分析。由MTT細胞存活實驗我們訂定了GCV濃度5 μg/ml 作為本研究後續使用之濃度，而在GFAP啟動子調控下只有U251-pGFAP-HSVtk以及U87-pGFAP-HSVtk細胞株會被GCV毒殺，證實了GFAP啟動子能專一性毒殺神經膠母細胞瘤且神經細胞不受其影響。在神經膠母細胞瘤細胞株被GCV處理的七天過程中，經藥物處理後第三天細胞會啟動細胞凋亡，經藥物處理後第五天由Hoechst/PI染色中能觀察到死亡細胞。 下一部份的實驗著重於分析GCV是否能促進神經膠母細胞瘤細胞株進行神經分化，因此我們使用III-tubulin, GFAP, nestin進行免疫螢光染色來鑑定存活的細胞種類。在以GCV處理U251-pGFAP-HSVtk七天的過程中，nestin在細胞中的表現量增加；在除去GCV使細胞生長三天後，我們獲得一群具有腫瘤幹細胞特性的細胞株且表現很強的nestin螢光；GCV能殺死表現GFAP強烈螢光的神經膠母細胞瘤，在以GCV處理第五天之後能找到具神經細胞特性且表現III-tubulin之細胞。同樣的細胞分化情形在對照組細胞株U251-pCMV-HSVtk以及另一實驗組細胞株U87-pGFAP-HSVtk也獲得驗證。 由本研究之結果，我們證實由GFAP 啟動子所調控之GCV/HSVtk系統確實能選擇性毒殺可表現GFAP之神經膠母細胞瘤，且此系統可誘導這些細胞進行神經分化。

Glioblastoma is the most common and most malignant primary brain tumor with poor prognosis. Ganciclovir/herpes simple virus thymidine kinase (GCV/HSVtk system), originally comes from an antiviral drug ganciclovir (GCV), has been applied as a clinical gene therapy aimed at the treatment of various types of cancers. Since this system allows selective elimination of HSVtk positive cells, it also has been applied to ablate undifferentiated stem cells in potential cell therapy for translational medicine research. Therefore, GCV/HSVtk system could be a candidate for glioblastoma treatment by regulating cell proliferation and differentiation. In the present study, we aimed to (1) establish a GFAP promoter driven GCV/HSVtk system to selectively ablate glioblastoma and (2) verify its possibility of neural differentiation of glioblastoma under the GCV challenge. pCMV-HSVtk-P2A and pGFAP-HSVtk-P2A were cloned and transfected into both non-glial (NIH-3T3 and N2A) and glioblastoma cell lines (U251 anU87) to verified the promoter specificity of GFAP in glioblastoma cell lines. N2A-pGFAP-HSVtk, N2A-pCMV-HSVtk, U251-pGFAP-HSVtk, U251-pCMV-HSVtk, and U87-pGFAP-HSVtk stable clones were established after G418 selection and were analyzed by Western blot. MTT assay was applied to obtain an optimal concentration at 5 μg/ml GCV for glioblastoma ablation. Under GFAP promoter control, U251 and U87 stable clones exhibited GCV sensitivity while N2A remained non-reactive. During GCV treatment, cells undergone apoptosis at the 3rd day of treatment and dying cells were identified after the 5th day of treatment by PI/Hoechst staining. In order to further analyze the ability of neural differentiation in these glioblastoma stable clones, the immunocytochemistry ofβIII-tubulin, GFAP, and nestin was performed to characterize the cell status during GCV treatment. Increasing of nestin expression level was observed in the survival cells of U251-pGFAP-HSVtk stable clone during 7-days of GCV treatment. A cancer stem cell-like cell population could be enriched from survival cells, which were recovered after GCV withdrawn. Lower expression level of GFAP was detected in survival cells after GCV treatment. βIII-tubulin positive neuron-like cells could be identified after the 5th day of GCV treatment. Similarly, expression patterns of, GFAP, and nestin were also obtained from positive control cell line U251-pCMV-HSVtk and another experimental stable clone of U87-pGFAP-HSVtk with the same treatment of GCV. In conclusion, we established a GFAP promoter controlled GCV/HSVtk system that could successfully ablate GFAP-positive glioblastoma cells whereas non-glial cells remained intact and our data suggested that the neural differentiation of glioblastoma cells could be promoted after the challenge of GCV/HSVtk system.