青藤碱通过ROS诱发内质网应激导致骨肉瘤凋亡并抑制骨肉瘤增殖、侵袭、转移

发布时间：2018-09-08 12:54

【摘要】：骨肉瘤是最常见的原发骨的恶性肿瘤.归功于新辅助化疗的实行及外科手术技术的提升,目前骨肉瘤的10年生存率到达65%。而然,近二十年来,骨肉瘤的治疗鲜有有效的进展。对于复发、转移的骨肉瘤患者来说,他们的长期生存率仍让人不能满意。因此我们迫切的期望能找到一种新型、有效,具有潜在骨肉瘤治疗意义的药物。青藤碱,一种传统治疗关节炎的生物碱中药提取物,被证明具有多种抗肿瘤、抑制肿瘤侵袭的活性。然而它对于骨肉瘤的治疗疗效及可能通过的分子机制,我们仍然不清楚。在本次研究中,通过单细胞克隆实验、细胞周期流式细胞仪检测及周期相关蛋白检测,我们发现青藤碱具有通过抑制骨肉瘤克隆形成能力、引起骨肉瘤细胞s期周期阻滞而发挥抑制骨肉瘤增殖的作用。我们的前期实验发现,青藤碱对骨肉瘤具有直接杀伤作用。高浓度青藤碱通过引起ROS大量活化从而诱导骨肉瘤细胞发生诱导内质网应激,最终导致骨肉瘤细胞的凋亡。那么青藤碱是否具有抑制骨肉瘤侵袭、转移的作用呢。通过划痕实验、franswell实验、明胶酶谱、ELISA、蛋白电泳等丰富的实验手段,我们发现低浓度作用的青藤碱可以通过抑制CXCR4-STAT3轴调节下游MMP-2、 MMP-9、RANKL、VEGF等信号靶点表达,从而抑制RANKL调控的破骨细胞异常活化导致的骨溶解及VEGF诱导肿瘤新生血管形成,进而发挥抑制骨肉瘤侵袭、转移的作用。上述大量体外实验为我们提供大量证据解释了青藤碱对骨肉瘤可能的作用机制。更重要的是,在体内动物实验中,通过建立骨肉瘤异位皮下模型及胫骨原位肿瘤模型,我们验证了青藤碱在体内的抗肿瘤作用及抑制骨肉瘤侵袭、转移的作用。青藤碱的治疗还能明显缓解患鼠患肢骨癌痛,改善患鼠的生活治疗。据此大量的体内外证据表明,青藤碱具有骨肉瘤治疗的潜能。在体内外抑制骨肉瘤增殖,不仅发挥着直接抗骨肉瘤作用,而且通过抑制骨肉瘤侵袭、转移具有间接的抗骨肉瘤功效。
[Abstract]:Osteosarcoma is the most common primary malignant tumor of bone. Thanks to neoadjuvant chemotherapy and improved surgical techniques, the current 10-year survival rate for osteosarcoma is 65. However, the treatment of osteosarcoma has made little progress in the past 20 years. In patients with recurrent and metastatic osteosarcoma, their long-term survival rate remains unsatisfactory. Therefore, we urgently hope to find a new, effective, potential treatment of osteosarcoma drugs. Sinomenine, a traditional Chinese medicine extract of alkaloids for arthritis, has been shown to have a variety of anti-tumor and anti-tumor activity. However, its therapeutic efficacy and possible molecular mechanisms for osteosarcoma remain unclear. In this study, we found that sinomenine has the ability to inhibit osteosarcoma clone formation by single cell cloning, cell cycle flow cytometry and cyclin detection. Osteosarcoma cell cycle arrest plays a role in inhibiting the proliferation of osteosarcoma. Our previous experiments have shown that sinomenine has direct killing effect on osteosarcoma. High concentration of sinomenine induces endoplasmic reticulum stress and apoptosis of osteosarcoma cells by activating ROS. Does sinomenine inhibit the invasion and metastasis of osteosarcoma? Through scratch test, franswell test, gelatinase spectrum analysis and protein electrophoresis, we found that low concentration of sinomenine can regulate the expression of downstream MMP-2, MMP-9,RANKL,VEGF and other signal targets by inhibiting the CXCR4-STAT3 axis. Thus the osteolysis induced by abnormal activation of osteoclasts regulated by RANKL and angiogenesis induced by VEGF could be inhibited and the invasion and metastasis of osteosarcoma could be inhibited. These in vitro experiments provide us with a great deal of evidence to explain the possible mechanism of sinomenine on osteosarcoma. More importantly, in vivo animal experiments, we established an ectopic subcutaneous model of osteosarcoma and an in situ tumor model of tibia, we verified the anti-tumor effect of sinomenine in vivo and the inhibition of invasion and metastasis of osteosarcoma. Sinomenine can also significantly alleviate the pain of bone cancer in the limbs of rats and improve the living conditions of the mice. According to the evidence in vivo and in vitro, sinomenine has the potential to treat osteosarcoma. Inhibiting the proliferation of osteosarcoma in vivo and in vitro not only plays a direct role in anti-osteosarcoma, but also has indirect anti-osteosarcoma effect by inhibiting the invasion and metastasis of osteosarcoma.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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