青藤堿通過ROS誘發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致骨肉瘤凋亡并抑制骨肉瘤增殖、侵襲、轉(zhuǎn)移
發(fā)布時(shí)間：2018-09-08 12:54
【摘要】：骨肉瘤是最常見的原發(fā)骨的惡性腫瘤.歸功于新輔助化療的實(shí)行及外科手術(shù)技術(shù)的提升,目前骨肉瘤的10年生存率到達(dá)65%。而然,近二十年來,骨肉瘤的治療鮮有有效的進(jìn)展。對(duì)于復(fù)發(fā)、轉(zhuǎn)移的骨肉瘤患者來說,他們的長(zhǎng)期生存率仍讓人不能滿意。因此我們迫切的期望能找到一種新型、有效,具有潛在骨肉瘤治療意義的藥物。青藤堿,一種傳統(tǒng)治療關(guān)節(jié)炎的生物堿中藥提取物,被證明具有多種抗腫瘤、抑制腫瘤侵襲的活性。然而它對(duì)于骨肉瘤的治療療效及可能通過的分子機(jī)制,我們?nèi)匀徊磺宄�。在本次研究�?通過單細(xì)胞克隆實(shí)驗(yàn)、細(xì)胞周期流式細(xì)胞儀檢測(cè)及周期相關(guān)蛋白檢測(cè),我們發(fā)現(xiàn)青藤堿具有通過抑制骨肉瘤克隆形成能力、引起骨肉瘤細(xì)胞s期周期阻滯而發(fā)揮抑制骨肉瘤增殖的作用。我們的前期實(shí)驗(yàn)發(fā)現(xiàn),青藤堿對(duì)骨肉瘤具有直接殺傷作用。高濃度青藤堿通過引起ROS大量活化從而誘導(dǎo)骨肉瘤細(xì)胞發(fā)生誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激,最終導(dǎo)致骨肉瘤細(xì)胞的凋亡。那么青藤堿是否具有抑制骨肉瘤侵襲、轉(zhuǎn)移的作用呢。通過劃痕實(shí)驗(yàn)、franswell實(shí)驗(yàn)、明膠酶譜、ELISA、蛋白電泳等豐富的實(shí)驗(yàn)手段,我們發(fā)現(xiàn)低濃度作用的青藤堿可以通過抑制CXCR4-STAT3軸調(diào)節(jié)下游MMP-2、 MMP-9、RANKL、VEGF等信號(hào)靶點(diǎn)表達(dá),從而抑制RANKL調(diào)控的破骨細(xì)胞異�；罨瘜�(dǎo)致的骨溶解及VEGF誘導(dǎo)腫瘤新生血管形成,進(jìn)而發(fā)揮抑制骨肉瘤侵襲、轉(zhuǎn)移的作用。上述大量體外實(shí)驗(yàn)為我們提供大量證據(jù)解釋了青藤堿對(duì)骨肉瘤可能的作用機(jī)制。更重要的是,在體內(nèi)動(dòng)物實(shí)驗(yàn)中,通過建立骨肉瘤異位皮下模型及脛骨原位腫瘤模型,我們驗(yàn)證了青藤堿在體內(nèi)的抗腫瘤作用及抑制骨肉瘤侵襲、轉(zhuǎn)移的作用。青藤堿的治療還能明顯緩解患鼠患肢骨癌痛,改善患鼠的生活治療。據(jù)此大量的體內(nèi)外證據(jù)表明,青藤堿具有骨肉瘤治療的潛能。在體內(nèi)外抑制骨肉瘤增殖,不僅發(fā)揮著直接抗骨肉瘤作用,而且通過抑制骨肉瘤侵襲、轉(zhuǎn)移具有間接的抗骨肉瘤功效。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R738.1
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R738.1
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