人参皂苷Rh2诱导儿童急性淋巴白血病细胞凋亡与自噬的分子机制研究

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy. It has been reported that activation of PI3K/Akt/mTOR signaling pathway played a crucial role in initiation and progression of leukemia. Meanwhile, the aberrant activation of this pathway can reflect prognosis. The pathway has been a research focus of targeted therapies for leukemia. Our previous studies have found that GRh2 could induce apoptosis through mitochondrial pathway in ALL cells. Meanwhile, it could also induce autophagy and decrease the expression level of p-mTOR. Therefore, we speculate this signal pathway might involve in antitumor mechanism of GRh2, which modulates apoptosis and autophagy in ALL cells. In this study, we use the methods such as flow cytometry, confocal images analysis, shRNA interference and western blot. The potential role of PI3K/Akt/mTOR pathway in GRh2-induced apoptosis and autophagy would be investigated by using the signal pathway inhibitors. Then the interaction between autophagy and apoptosis induced by 20(S)-GRh2 would be evaluated through autophagy inhibitor. Finally, the effect and mechanism of GRh2 on ALL would be examined in the animal model. These findings would illuminate the molecular mechanism of GRh2 for targeted ALL cells from apoptosis and autophagy aspects, and provide a promising strategy for treatment of pediatric leukemia.

急性淋巴细胞白血病(ALL)是儿童最常见的恶性肿瘤。研究表明PI3K/Akt/mTOR信号通路的激活在白血病发生、发展中起关键作用，同时其激活程度也预示患者预后，该通路已成为白血病靶向治疗的研究热点。我们前期研究发现人参皂苷 Rh2(GRh2)诱导ALL细胞凋亡的同时，也能诱导细胞自噬，使p-mTOR蛋白表达减少。因而提出了GRh2通过PI3K/Akt/mTOR通路调控ALL细胞凋亡与自噬，发挥其抗ALL作用的假说。本项目拟采用流式细胞术、激光共聚焦图像分析、shRNA干扰、免疫印迹等研究手段，通过信号通路抑制剂，探讨该通路在GRh2诱导的ALL细胞凋亡和自噬中的作用；通过自噬干预，考察GRh2诱导的凋亡与自噬之间的作用关系；利用ALL动物模型研究GRh2的抗ALL作用与机制。以期从凋亡与自噬角度，全面深层次地揭示GRh2的靶向抗ALL分子机制。为儿童白血病的治疗提供新的思路和实验线索。

急性淋巴细胞白血病（Acute lymphoblastic leukemia, ALL）是儿童最常见的恶性肿瘤。研究表明PI3K/Akt/mTOR信号通路的激活在白血病发生、发展中起关键作用，同时其激活程度也预示患者预后。目前临床使用的化疗药物产生严重的毒副作用以及出现的耐药现象，是影响患者生活质量和白血病治疗失败的重要原因。人参皂苷Rh2（ginsenoside Rh2, GRh2）为近年来从天然传统中草药人参中提取的一种天然活性成分，具有良好的抗肿瘤药理活性，而且对正常细胞无毒副作用。本课题通过细胞活性检测，GRh2对T系Jurkat细胞的细胞活性抑制作用较前B系Reh细胞显著。在细胞和动物水平，结果发现GRh2组的p-PI3K、p-Akt、p-mTOR蛋白表达较空白对照组均有显著降低，证明了GRh2通过PI3K/Akt/mTOR信号通路作用于ALL细胞。在细胞和动物水平，结果发现GRh2组的p-PI3K、p-Akt、p-mTOR蛋白表达较空白对照组均有显著降低，证明了GRh2通过抑制PI3K/Akt/mTOR信号通路作用于ALL细胞。并通过流式细胞术、激光共聚焦、Western blotting等手段，证实GRh2可通过抑制PI3K/Akt/mTOR通路提高Jurkat细胞内的调亡和自噬水平。通过自噬抑制剂和RNA干扰技术，发现自噬抑制剂3MA和shRNA-Atg5抑制自噬后，能够使细胞活性明显下降，促进GRh2诱导的白血病细胞凋亡；而用诱导剂RAPA诱导自噬，能够使细胞活性上升，对GRh2诱导的白血病细胞凋亡有抑制作用。将Jurkat细胞通过尾静脉注射给NOD/SCID小鼠，建立ALL动物模型。结果观察到GRh2能够缓解ALL动物的临床症状，体重逐渐回升。GRh2给药组的白细胞数量较白血病组明显减少。同时通过流式细胞术和免疫组化检测，结果发现与ALL模型组相比，GRh2给药组的小鼠血液和骨髓的中CD3和CD45阳性细胞的明显下降；并且使ALL小鼠脾脏的白血病细胞浸润明显减少。并通过调节脾脏的免疫因子和免疫细胞，增强GRh2的抗白血病作用。本项目在细胞和动物水平阐明了GRh2靶向PI3K/Akt/mTOR信号通路介导的抗ALL细胞作用及分子机制，为儿童白血病的靶向治疗提供了新的思路和实验线索。

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