胶质瘤干细胞5-ALA成像质量及代谢机制的研究

Gliomas are the most common primary brain tumors with limited therapeutic options, high recurrence rate and mortality. 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 5-ALA imaging quality of glioma usually show vague, 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), ATP-binding cassette, sub-family B, member 6 (ABCB6) and Peptide Transporter 2 (PEPT2) gene, which encodes a key enzyme that catalyzes the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated the enzymes in human glioma cell lines. These results suggest that targeting the enzymes of 5-ALA metabolism to enhance PpIX fluorescent quality is a potential approach for optimizing the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT. The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized perivascular niche within tumors. These niches represent the hallmarks of malignant gliomas and bear analogy to the microenvironments in which physiological stem cells in the brain are found. The key enzyme cues and mechanisms of 5-ALA in GSCs are poorly understood. The challenge now is to better understand the complex mechanisms of transport of 5-ALA in GSCs, which will allow the development of effective strategies to enhance 5-ALA-based metabolic imaging and photodynamic therapy efficacy. The FECH, ABCB6 and PEPT2 mRNA expression was determined using quantitative RT-PCR (QRT-PCR) in GSCs and wild type glioma cell. Cells expressing key enzymes in GSCs and wild type glioma cell were identified using immunohistochemistry. The key enzyme transfection with small interference RNA (siRNA）or plasmid were performed to increase intracellular PpIX accumulation in GSCs. The phototoxicity of ALA-photodynamic therapy (PDT) after transfection with FECH and/or ABCB6 and/or PEPT2 was evaluated by cell apoptosis analysis. Our study is manipulated to show the functional characterization of FECH, ABCB6 and PEPT2 in transport of 5-ALA in GSCs, and enhance 5-ALA-based fluorescent metabolic quality and ALA-PDT efficacy by targeting key enzymes in vitro and in vivo

研究表明胶质瘤干细胞（GSCs）是胶质瘤难治和复发的根源，只有彻底杀灭肿瘤组织微环境中的GSCs才能彻底根除胶质瘤。5-氨基乙酰丙酸（5-ALA）荧光引导胶质瘤手术可以有效提高胶质瘤的术中识别，提高手术切除率并延长患者生存时间，但该技术对GSCs特异性及敏感性却不得而知，详细的GSCs的5-ALA代谢机制不明。我们前期研究明确了亚铁螯合酶、ABCB6以及寡肽转运蛋白在胶质瘤细胞5-ALA代谢过程中的作用，实现了靶向关键酶提高5-ALA荧光成像质量和光动力学效应的目的。本研究拟探索GSCs 是否激发5-ALA荧光以及成像质量，检测GSCs 5-ALA代谢酶的表达，证实其表达和干细胞5-ALA荧光激发的关系及详细机制；靶向干扰关键酶表达之后，检测GSCs 5-ALA荧光强度以及GSCs生物学行为的改变，并在此基础上探索联合靶向增强GSCs 5-ALA荧光成像质量和光动力学效应的可行性。

胶质母细胞瘤是颅内常见的中枢神经系统肿瘤，具有恶性程度高，预后差等特点。针对此治疗难题，我们根据以下三个方面展开了实验研究。1. 非编码RNA在胶质瘤中的作用研究；2. 纳米靶向胶质瘤成像及纳米载药治疗胶质瘤的研究；3. 探寻对胶质瘤有效的治疗药物及胶质瘤化疗增敏研究；通过系列研究，我们发现miRNAs可能是脑胶质瘤病理进程的重要推手。进一步发现胶质瘤细胞中同时抑制差异表达miRNAs的具有强协同作用，为合理利用这类特殊的内源性物质提供了一个极好的切入点，也为脑胶质瘤的防治提供了一个全新的治疗策略；我们采用热分解法合成疏水性四氧化三铁纳米粒子（SPIO NPs），然后使用薄膜水化法将SPIO NPs和疏水性阿霉素经DSPE-PEG 2000包裹，合成载药纳米粒子，最后将荧光分子载入纳米粒子。试验中发现在所有的治疗组当中，多功能纳米粒子组的载瘤模型表现出最明显的抗肿瘤效果，并且没有明显的毒副作用。这些结果表明我们构建的纳米粒子不仅能用于胶质瘤的双模态成像，同时能向胶质瘤位点递送化疗药物，实现诊疗一体化。. 胶质瘤血脑屏障的存在也极大的限制了化疗药物穿透进入中枢神经系统。卡莫司汀（BCNU）是广泛应用于临床的抗肿瘤药物。但是其本身存在的一些难题阻止它在更大的临床范围内发挥作用。因此，亟需探索一种新的针对胶质瘤专门设计的卡莫司汀投递方式。在体内使用BODIPY探针证明T7修饰后的胶束展现出更加明显的累积作用，并且修饰过的胶束在肿瘤中的累积比未修饰的更加明显。同时，靶向治疗组疗效最好，体重降低最少，肿瘤最小，并且明显延长生存时间。 . 接下来，我们努力探索针对胶质瘤化疗的新药物。通过实验发现异新藤黄酸通过激活AMPK-mTOR信号通路抑制胶质瘤的生长，这可能在脑胶质瘤治疗中具有临床应用前景；来自银莲花的活性药理成分Raddeanin A（RA）同样抑制了胶质母细胞瘤的生长，这些药物都为胶质瘤治疗提供了潜在的化疗策略。