YM155通过抑制生存素表达对骨肉瘤生物学行为的影响

发布时间：2018-05-07 09:32

本文选题：骨肉瘤 + 生存素　；参考：《吉林大学》2016年博士论文

【摘要】：鉴于生存素和其抑制剂YM155在肿瘤行为学中的重要意义,本课题结合体内和在体研究,通过免疫学、基因和分子生物学方法,对YM155在骨肉瘤治疗中的应用进行深入研究。本研究分为3部分:第一部分:生存素在骨肉瘤中的表达及其临床意义研究生存素是发现的凋亡抑制因子家族的一个成员,在成熟组织中不表达,但能够在肿瘤细胞中特异性高表达,因此引起人们的广泛关注。本研究收集住院手术31例骨肉瘤患者的瘤组织和瘤周正常组织,经q RT-PCR和免疫组织化学染色检测生存素的表达。结果显示,与正常组织相比,骨肉瘤中生存素m RNA和蛋白表达均显著增高(p0.01)。本研究还同时通过q RT-PCR和Westernblot检测了3种骨肉瘤细胞系细胞(Saos-2,MG63和U2OS)和1种成骨细胞系细胞h HFOB1.19中生存素的表达。结果显示,Saos-2、MG63和U2OS细胞中生存素m RNA和蛋白水平均明显高于h HFOB1.19细胞(p0.01)。为进一步研究生存素的临床意义,本研究对所有瘤标本进行了Ki67评分并研究其与生存素表达的相关性,结果显示生存素高表达组Ki67评分明显高于生存素低表达组(p0.01),并且生存素高表达组患者生存时间要明显短于生存素低表达组患者。这些结果表明,生存素在骨肉瘤患者体内表达上调,并与肿瘤恶性程度以及疾病预后相关,提示生存素可作为骨肉瘤临床治疗的潜在基因靶点。在第一部分研究中,我们揭示了生存素在骨肉瘤发生发展中的重要作用。YM155是生存素的一种特异性抑制剂,在体外和动物模型中对多种肿瘤的生长和转移均具有较强的抑制作用,但其对骨肉瘤生物学行为的作用尚未完全阐明。在本部分研究中,我们发现YM155刺激可明显抑制骨肉瘤细胞系Saos-2细胞和MG63细胞在体外的增殖能力和成克隆能力。同时在体实验也证实,在MG63细胞注射至裸鼠皮下制作的荷瘤模型中,YM155刺激可明显抑制肿瘤内生存素的表达,并且导致骨肉瘤瘤体的缩小和重量的减轻。通过Transwell实验检测发现,YM155处理后的Saos-2细胞和MG63细胞穿过基质胶和滤膜的数目明显较对照组减少,并呈剂量依赖性。并且,经YM155刺激后,YM155刺激可明显增加Saos-2细胞和MG63细胞细胞中Caspase 3,8和10的比率,两种细胞凋亡数目明显增多。这些研究结果说明YM155可在体内和体外抑制骨肉瘤细胞的生长、抗凋亡、迁移及侵袭能力,可作为生存素靶向治疗的新型治疗药物应用于骨肉瘤临床治疗。第三部分:YM155对骨肉瘤细胞阿霉素化疗敏感性的影响第二部分研究结果表明YM155可抑制骨肉瘤细胞增殖和侵袭能力,并促进其凋亡。对化疗药物抵抗是骨肉瘤临床治疗中极为棘手的难点之一,在本部分内容中我们进一步研究YM155对骨肉瘤细胞阿霉素化疗敏感性的影响。本研究采用DOX浓度递增法建立骨肉瘤耐药细胞模型MG63/DOX。与MG63细胞相比,MG63/DOX细胞株内生存素蛋白和m RNA表达明显增高,阿霉素对其细胞活性的抑制作用明显减弱。我们对MG63/DOX细胞在阿霉素、YM155以及YM155联合阿霉素刺激下细胞增殖、克隆和凋亡的情况进行检测。结果显示,相同浓度的阿霉素可明显抑制MG63细胞的增殖能力,但其对第二部分:YM155对骨肉瘤细胞增殖、凋亡以及侵袭能力的影响MG63/DOX的增殖的抑制作用明显减弱。YM155可抑制MG63/DOX细胞的增殖,与阿霉素合用后对细胞增殖的抑制能力明显增强。并且,单一阿霉素不能抑制MG63/DOX细胞的成克隆数目,但YM155可抑制MG63/DOX细胞的成克隆数目,与阿霉素合用后,这种抑制能力明显增强。通过PI/Annexin染色后流式细胞检测发现,单一阿霉素不能诱导MG63/DOX细胞的凋亡,但YM155可诱导MG63/DOX的轻度凋亡,阿霉素与YM155合用后,可诱导MG63/DOX细胞明显凋亡。进一步研究发现,YM155可降低两种细胞中p-Akt、p-PI3K和Mcl-1的表达,并呈剂量依赖性,但不同剂量的YM155对Bcl-2和XIAP表达均无明显影响。这些结果提示,YM155可能通过抑制PI3K/Akt途径,增强阿霉素对骨肉瘤细胞的细胞毒性和促凋亡能力,有望作为骨肉瘤化疗联合药物应用于临床治疗。
[Abstract]:In view of the importance of Survivin and its inhibitor YM155 in the study of tumor behavior, this subject combines in vivo and in vivo studies and studies the application of YM155 in the treatment of osteosarcoma by immunological, gene and molecular biology methods. This study is divided into 3 parts: the first part: the expression of Survivin in osteosarcoma and its clinical meaning The study of survivin is a member of the found apoptosis inhibitory factor family, which is not expressed in the mature tissue, but can be highly expressed in the tumor cells. Therefore, people have received extensive attention. This study collected the tumor tissue of 31 patients with osteosarcoma in hospitalized surgery and the common tissue of the tumor Zhou Zheng, with Q RT-PCR and immunohistochemical staining. The expression of survivin was detected. The results showed that the expression of survivin m RNA and protein in osteosarcoma increased significantly compared with normal tissues (P0.01). The expression of 3 osteosarcoma cell line cells (Saos-2, MG63 and U2OS) and 1 kinds of osteoblast cell line cells were also detected by Q RT-PCR and Westernblot. The results showed that the expression of Survivin in osteosarcoma cell line cells was also detected. The levels of survivin m RNA and protein in Saos-2, MG63 and U2OS cells were significantly higher than h HFOB1.19 cells (P0.01). In order to further study the clinical significance of survivin, this study conducted a Ki67 score on all tumor specimens and studied the correlation with the expression of survivin. The results showed that the Ki67 score in the high expression group was significantly higher than that of the subsurvivin. The results showed that the expression of survivin was up regulated in the patients with osteosarcoma and was associated with the malignancy and prognosis of the tumor, suggesting that survivin could be used as a potential gene target for the clinical treatment of osteosarcoma in the first part of the first part of the group (P0.01). In the study, we revealed the important role of Survivin in the development of osteosarcoma,.YM155 is a specific inhibitor of survivin, which has a strong inhibitory effect on the growth and metastasis of various tumors in vitro and in animal models, but its role in the biological behavior of osteosarcoma has not been fully elucidated. In this part of the study, I We found that YM155 stimulation significantly inhibited the proliferation and cloning ability of osteosarcoma cell line Saos-2 cells and MG63 cells in vitro. In vivo experiments also confirmed that YM155 stimulation could significantly inhibit the expression of intratumoral survivin in the tumor model of MG63 cells injected subcutaneously in nude mice, and resulted in the reduction of osteosarcoma tumor. The Transwell test showed that the number of Saos-2 cells and MG63 cells passing through matrix glue and filter membrane after YM155 treatment was significantly less than that of the control group, and was dose-dependent. And, after YM155 stimulation, YM155 stimulation could significantly increase the ratio of Caspase 3,8 and 10 in Saos-2 cells and MG63 cell cells, and two kinds of cells. These results suggest that YM155 can inhibit the growth, anti apoptosis, migration and invasion of osteosarcoma cells in vivo and in vitro, and can be used as a new therapeutic drug for survivin targeting therapy in osteosarcoma clinical treatment. The third part: the effect of YM155 on the chemosensitivity of osteosarcoma cells with adriamycin. The results show that YM155 can inhibit the proliferation and invasion of osteosarcoma cells and promote its apoptosis. Chemotherapeutic resistance is one of the most difficult problems in the clinical treatment of osteosarcoma. In this part, we further study the effect of YM155 on the chemosensitivity of osteosarcoma cells with adriamycin. This study uses DOX concentration increasing method. To establish an osteosarcoma resistant cell model MG63/DOX., the expression of survivin protein and m RNA in MG63/DOX cell lines was significantly higher than that of MG63 cells, and the inhibitory effect of adriamycin on its cell activity was significantly weakened. The proliferation, cloning and apoptosis of MG63/DOX cells in adriamycin, YM155 and YM155 combined with adriamycin were carried out. The results showed that the same concentration of adriamycin could significantly inhibit the proliferation of MG63 cells, but the effect of YM155 on the proliferation, apoptosis and invasion of osteosarcoma cells was significantly reduced by the inhibitory effect of MG63/DOX on the proliferation of MG63/DOX, and the inhibition of the proliferation of MG63/DOX cells by.YM155, and the inhibition of cell proliferation after adriamycin was combined with adriamycin. The single doxorubicin could not inhibit the number of MG63/DOX cells, but YM155 inhibited the number of clones in MG63/DOX cells. After the combination of adriamycin, the inhibition ability was significantly enhanced. After PI/Annexin staining, flow cytometry found that single doxorubicin could not induce the apoptosis of MG63/DOX cells, but YM155 It can induce mild apoptosis of MG63/DOX, and adriamycin can induce apoptosis in MG63/DOX cells after combined use of adriamycin and YM155. Further studies have found that YM155 can reduce the expression of p-Akt, p-PI3K and Mcl-1 in two cells, and is dose-dependent, but the different doses of YM155 have no significant effect on the expression of Bcl-2 and XIAP. These results suggest that YM155 may pass through Inhibition of PI3K/Akt pathway enhances the cytotoxicity and pro apoptotic ability of adriamycin to osteosarcoma cells, and is expected to be used as a combination chemotherapy for osteosarcoma.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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