靶向PEPT2和FECH增强胶质瘤5-ALA成像质量和光动力学效应的研究

Enhanced 5-ALA-based metabolic imaging and photodynamic therapy efficacy by targeting PEPT2 and FECH..Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumor margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumor identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues. To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyzes the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines. These results suggest that knockdown of FECH is a potential approach to enhancing PpIX fluorescent quality for optimizing the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT..Recently, the report has showed peptide ptransporter 2 (PEPT2)-mediated transport of 5-ALA at the choroid plexus in rat model, however, no data have been indicated concerning the mechanisms of transport of 5-ALA in human gliomas. To understand the mechanisms, whether PEPT2 is present and participates in transport of 5-ALA in gliomas, we focused the PEPT2 in human glioma..The PEPT2 mRNA expression was determined using quantitative RT-PCR (QRT-PCR) in various human astrocytic tumors and normal brain tissue. Cells expressing PEPT2 in normal brain and glioblastoma specimens were identified using immunohistochemistry. PEPT2 transfection with small interference RNA (siRNA）or plasmid were performed to increase intracellular PpIX accumulation in glioma cells. The phototoxicity of ALA-photodynamic therapy (PDT) after transfection with FECH and/or PEPT2 was evaluated by proliferation, invasion, migration assay and cell apoptosis analysis..Our study is manipulated to show the functional characterization of PEPT2 in transport of 5-ALA in gliomas, and enhance 5-ALA-based fluorescent metabolic quality and ALA-PDT efficacy by targeting PEPT2 and FECH.

5-氨基乙酰丙酸（5-ALA）荧光引导胶质瘤手术提高了术中肿瘤识别，但肿瘤的侵润边界往往呈现弱荧光，加大了术者对肿瘤边界的识别难度。我们前期研究明确了亚铁螯合酶（FECH）在5-ALA代谢机制过程中的作用，实现了靶向下调FECH表达提高5-ALA荧光成像质量和光动力学效应的目的。有报道表明位于鼠脑脉络丛顶端膜的寡肽转运蛋白 (PEPT2)可将5-ALA从脑脊液转运至血液内，但胶质瘤细胞5-ALA跨膜转运机制不明。本研究拟探索PEPT2在5-ALA转运过程中的作用，检测不同级别胶质瘤组织PEPT2表达，证实其表达和5-ALA荧光激发的关系；探索野生型胶质瘤细胞PEPT2表达和5-ALA荧光强度的潜在关系；靶向干扰PEPT2表达之后，检测5-ALA荧光强度以及胶质瘤细胞生物学行为的改变，并在此基础上探索联合干扰PEPT2和FECH增强5-ALA荧光成像质量和光动力学效应的可行性。

胶质瘤是中枢神经系统常见的原发性肿瘤。在胶质瘤中，多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）的恶性程度最高。针对GBM侵袭性机制的研究可为开发新的胶质瘤治疗策略提供新的思路，进一步研究胶质瘤细胞侵袭的机制并开发有效的抗侵袭治疗十分必要。而针对胶质瘤侵袭和迁徙的抑制剂则被认为是新型治疗GBM的药物。广泛的证据表明酪氨酸激酶家族及其配体信号系统在神经胶质瘤信号传导中起着重要的作用；但我们对eph/phrin信号系统仍然是不完全理解。这些受体/配体是如何影响神经胶质瘤的趋化行为？在我们系列实验中我们发现在各级别胶质瘤组织中EphB家族成员mRNA水平是明显不同的，且和胶质瘤组织病理学分级相依赖。在195例临床胶质瘤组织样本的临床相关性分析得到EphB基因EphB1表达的水平没有明显差异，仅EphB5表达与组织病理学存在统计学差异。免疫沉降实验揭示了酪氨酸磷酸化EphB1在胶质瘤细胞系中表达很低，被迫表达和自磷酸化EphB1低的细胞系(U251 U87)不影响胶质瘤细胞的迁移和侵袭能力，而在ephrin-B2 / Fc所介导的EphB1磷酸化之后显著减少胶质瘤细胞的迁移和侵袭。过表达ephrin-B2的胶质瘤细胞明显具备了侵袭细胞形态，同时具有高侵袭性；而细胞过EphB1表达之后将上述侵袭性和侵袭形态消失。与此同时，细胞趋化现象的分析显示超表达 EphB1的细胞逆转了ephrin-B2所引起的迁移和侵袭性。这些数据表明EphB1信号以配体依赖的方式抑制神经胶质瘤细胞侵袭和迁徙，同时EphB1基因还可作为恶性神经胶质瘤患者有意义的预后评价指标。. 接下来的我们进行了增强5-ALA荧光强度和PpIX堆积的系列实验：首次研究表明维生素D可以选择性提高胶质瘤细胞内原卟啉IX的荧光强度及ALA介导的光动力治疗效果，这项研究成果有助于使用荧光引导手术术中客观地判定肿瘤组织的边界，为改善荧光引导手术效果提供了新的方向；另外一方面我们也发现小剂量三氧化二砷可以作为有效地提高5-ALA介导胶质瘤荧光切除及光动力治疗的潜在选择手段。