放射増敏剂CpG ODN107通过TLR9-ERK/JNK通路诱导脑胶质瘤自噬性死亡的作用机制研究

The resistance of glioma cell to radiation is the major reason for radiotherapy failure, so the study of radiosensitizer is fully necessary. Our previous study showed that CpG ODN107 was a novel, safe, efficient radiosensitizer, and its effect may be related with autophagic cell death induced by the inhibition of mTOR via MAPK pathway. But the target or signal transduction pathway is unclear. Firstly, different degree of malignancy of glioma cells will be applied to further study autophagic cell death induced by CpG ODN107 in combination with irradiation in vitro and in vivo. Next, up or down-regulated TLR9 expression, or siRNA technology to interfere the expression of JNK and ERK, then the radiosensitizing effect and the change of downstream signal molecules will be detect by colony formation assay, confocal laser, et al. As mentioned above, we will elucidate the role of TLR9 and its downstream ERK/JNK pathway in autophagic cell death induced by CpG ODN107 in combination with irradiation. The results will contribute to elucidating the relationship between CpG ODN, autophagic cell death and radiosensitization, then a new theory and target of radiosensitizer will be developed.

脑胶质瘤的放射抵抗是导致放疗失败的重要原因，研究放射増敏剂具有重要意义。本课题前期研究发现CpG ODN107是一个新型、安全、高效的放射増敏剂，其作用可能与通过MAPK通路抑制mTOR表达而诱导细胞自噬性死亡有关。但其作用靶点与具体的调控通路不清。本项目以不同恶性程度的脑胶质瘤为研究对象，在体内外实验中进一步明确CpG ODN107联合射线对细胞自噬的影响；然后分别上调或下调TLR9表达，siRNA技术分别或联合阻断MAPK中ERK、JNK表达后，采用克隆实验、激光共聚焦等技术再次观察放射増敏作用、自噬的变化以及下游信号通路的改变。通过以上实验明确TLR9是否为CpG ODN107的作用靶点，以及是否通过TLR9下游的ERK/JNK通路抑制mTOR从而诱导细胞自噬性死亡。研究结果有助于阐明CpG ODN、自噬性死亡、放射增敏作用三者的关系，有可能为放射增敏剂的研发提供新的理论与新的靶点。

背景：手术切除联合放疗是治疗脑胶质瘤的标准方法，但是由于脑胶质瘤对射线不敏感或敏感性差，因此研制安全、高效的放射增敏剂并阐明其作用机制、靶点，对提高脑胶质瘤的治愈率具有重要意义。.目的：探讨含12-bp的CpG寡脱氧核苷酸(CpG ODN107, CpG 107)诱导脑胶质瘤细胞自噬性死亡而发挥放射增敏作用的分子机制。.结果：1. 明确了CpG ODN107+IR可明显增加U87细胞中自噬体的形成，增加自噬标志性蛋白LC3 II/LC3 I与Beclin-1的表达，降低p62的表达，U87细胞中GFP-LC3融合蛋白聚积成绿色荧光斑点也明显增多。同时，CpG ODN107+IR也可明显增加U251和CHG-5细胞中LC3 II/LC3 I的表达。.2. 明确了CpG ODN107+IR可诱导裸鼠原位移植瘤中自噬体的形成，降低p62的表达。.3. 作用机制研究结果阐明了CpG ODN107+IR可显著增加U87、U251和CHG-5中TLR9 和 p-ERK表达，降低p-mTOR水平；采用siRNA干扰TLR9后，CpG ODN107+IR不能显著增加p-ERK和LC3 II/LC3 I的表达和降低p-mTOR和p62的表达；3-MA和U0126分别预处理U87细胞后，CpG ODN107放射增敏作用被逆转，而Rapamycin预处理U87细胞后，CpG ODN107放射增敏作用进一步增强。.结论：体内外结果表明CpG ODN107+IR能增强脑胶质瘤细胞自噬水平，其作用机制为CpG ODN107作用于TLR9，通过TLR9-ERK途径抑制mTOR从而诱导脑胶质瘤细胞自噬性死亡，本研究为放射增敏剂的研发提供了新的理论与新的靶点。