Lawrence Johnathan E, Patel Ashish S, Rovin Richard A, Belton Robert J, Bammert Catherine E, Steele Christopher J, Winn Robert J
Upper Michigan Brain Tumor Center, Marquette, MI 49855, USA ; Biology Department, Northern Michigan University, Marquette, MI 49855, USA.
Upper Michigan Brain Tumor Center, Marquette, MI 49855, USA ; Purdue University, West Lafayette, IN 47906, USA.
ISRN Surg. 2014 Mar 4;2014:405360. doi: 10.1155/2014/405360. eCollection 2014.
Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.
引言。5-氨基乙酰丙酸（5-ALA）是血红素合成的前体。一种代谢产物，原卟啉IX（PpIX），选择性地积聚在包括胶质母细胞瘤在内的肿瘤组织中。术前给予5-ALA构成了胶质母细胞瘤（GBM）肿瘤荧光引导切除术（FGR）的基础。然而，并非所有胶质瘤都能积聚足够量的PpIX以产生荧光，从而限制了FGR的效用。因此，我们开发了一种检测方法来确定影响GBM中PpIX荧光的细胞和药理学因素。该检测方法利用了一种经过基因工程改造以表达黄色荧光蛋白的GBM细胞系。方法。用人YFP表达载体转染人GBM细胞系U87MG。在用一系列5-ALA剂量处理后，测量PpIX和YFP荧光。计算PpIX与YFP荧光的比率。结果。YFP荧光允许对细胞数量进行定量，并且不干扰5-ALA代谢。PpIX/YFP荧光比率提供了准确的相对PpIX水平，从而能够评估PpIX在组织中的积聚情况。结论。组成型YFP表达与细胞数量密切相关，并允许对PpIX进行定量。仅绝对PpIX荧光并不能提供关于PpIX在细胞内积聚的信息。我们的研究表明，我们的PpIX/YFP比率检测方法可能是一种用于体外5-ALA检测及其在FGR手术期间与其他化合物相互作用的有前景的模型。
