基于溶酶体酶代谢途径的CpG ODN细胞内代谢研究

CpG oligodeoxynucleotides (ODNs) are synthetic DNA sequences containing unmethylated cytosine-guanine motifs with potent immunomodulatory effects. Via Toll-like receptor 9 (TLR9)agonism of dendritic cells and B cells, CpG ODNs induce cytokines, activate natural killer cells, and elicit vigorous T-cell responses that lead to significant antitumor effects, including improved efficacy of chemotherapeutic agents. CpG ODNs have been explored for cancer theray, due to their immunostimulatory properties. The pharmacokinetic properties of CpG ODNs were evaluated in blood. It was ignored the relationships between the pharmacokinetic properties of CpG ODNs in cell and pharmacologic actions. CpG ODN107 is a novel radiosensitizer for glioma in our lab. In the present study, the major metabolite of CpG ODN107 was 3′N-1 in blood. In vitro, deoxyribonuclease Ⅱ (DNaseⅡ) effected metabolism of CpG ODN107. On the other hand, CpG ODN107 possessed a radiosensitizing effect via TLR9-mediated NF- κB activation and NO production in the tumor cells. Basic understanding and evaluation of the pharmacokinetic properties of CpG ODN107 in tumor cell is foundational to their appropriate design and application. Firstly, it would be chose CpG ODN107 as model drugs. The Triple Quadrupole mass spectrometer (QQQ-MS) would be applied for the quantification of CpG ODN107 in U87. The enzyme linked immunosorbent assay (ELISA) would be applied for determine the cytokines NO. The pharmacokinetic properties of CpG ODNs were evaluated in cell by mathematical model. The second, The Time of Flight mass spectrometer (TOF-MS )would be applied for the qualitation of CpG ODN107 in U87 with endosome acidify suppressed and hypo-express DNaseⅡby siRNA. The third, it would be chose CpG ODN1826 to validate the metabolism and metabolic enzyme of CpG ODN. CpG ODN1826 is a confessedly effective immunostimulatory B-type CpG ODN for murine. The QQQ-MS would be applied for the quantification of CpG ODN1826 in RAW264.7. The ELISA would be applied for determine the cytokines TNF-α. The pharmacokinetic properties of CpG ODN1826 would be evaluated in RAW264.7 cell by mathematical model. The TOF-MS would be applied for the qualitation of CpG ODN1826 in RAW264.7 with endosome acidify obstacle and hypo-express DNaseⅡby siRNA. The reaserch would revea lthe relationship between the pharmacokinetic properties of CpG ODNs in cell and pharmacologic actions.

CpG ODN具有免疫激活作用，已成为肿瘤免疫治疗研究新热点。目前对CpG ODN的代谢研究多基于血液，而忽视胞内代谢对药效的直接影响。前期课题组对CpG ODN107（简称107）在小鼠体内、外的代谢研究，提示其3′端外切代谢是主要代谢途径，DNaseⅡ发挥作用；而107需入胞后与TLR9结合才能发挥药效。因此，研究107胞内代谢变化，对提高107的序列稳定性、药物开发具有重要指导意义。本项目首先研究107在人脑胶质细胞株U87的胞内代谢，拟采用QQQ-MS定量胞内CpG ODN浓度以及ELISA检测细胞因子释放，以明确胞内变化与细胞因子释放之间的关系，再利用内体酸化障碍细胞和siRNA干扰DNaseⅡ后，研究胞内107代谢方式和代谢酶；最后采用小鼠特异性序列1826在小鼠巨噬细胞株RAW264.7中验证代谢过程。以期揭示胞内CpG ODN代谢方式以及代谢动力学与药理活性的关联。

胞嘧啶-鸟嘌呤寡脱氧核苷酸（cytosine phosphate guanidine oligodeoxynucleotide ,CpG ODN）在肿瘤、过敏性疾病、感染性疾病、遗传性疾病等众多疾病的治疗中具有广阔的应用前景。CpG ODN的临床应用面临很多问题，包括在机体内的稳定性差、不利的药代动力学特点和未知的细胞内变化过程，特别是细胞内CpG ODN的稳定性直接影响其药理作用，研究其细胞内的CpG ODN代谢变化规律，明确该类药物的代谢特征，对阐明药理作用，进行结构改造、剂型设计等新药开发工作都具有重要的意义。本项目首先采用对脑胶质瘤具有放射增敏作用的CpG ODN107作为模型药物，研究了其在人脑胶质细胞株U87的胞内代谢情况与活化细胞的关系，研究采用激光共聚焦定性和QQQ-MS定量胞内CpG ODN浓度以及ELISA法检测细胞活化情况，明确了CpG ODN107胞内浓度与细胞活化的存在线性关系，在48h 时CpG ODN在细胞中均聚集达到峰值，48h后，逐渐下降，而细胞的核因子NF-κB的活化在48h达高峰，随后逐渐下降；而在内体酸化障碍细胞模型和siRNA干扰DNaseⅡ低表达细胞模型中，细胞的核因子NF-κB的活化持续增加，提示CpG ODN107代谢需经内体酸化进入溶酶体，DNaseⅡ参与了CpG ODN107的代谢；同时，小鼠特异性序列CpG ODN1826在小鼠巨噬细胞株RAW264.7中具有同样的结果. 本项研究显示了溶酶体参与了CpG ODN代谢过程，其中DNaseⅡ是其中的代谢酶，其在细胞内代谢影响药理作用。