本文選題：骨肉瘤 + 生存素��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：鑒于生存素和其抑制劑YM155在腫瘤行為學(xué)中的重要意義,本課題結(jié)合體內(nèi)和在體研究,通過免疫學(xué)、基因和分子生物學(xué)方法,對(duì)YM155在骨肉瘤治療中的應(yīng)用進(jìn)行深入研究。本研究分為3部分:第一部分:生存素在骨肉瘤中的表達(dá)及其臨床意義研究生存素是發(fā)現(xiàn)的凋亡抑制因子家族的一個(gè)成員,在成熟組織中不表達(dá),但能夠在腫瘤細(xì)胞中特異性高表達(dá),因此引起人們的廣泛關(guān)注。本研究收集住院手術(shù)31例骨肉瘤患者的瘤組織和瘤周正常組織,經(jīng)q RT-PCR和免疫組織化學(xué)染色檢測(cè)生存素的表達(dá)。結(jié)果顯示,與正常組織相比,骨肉瘤中生存素m RNA和蛋白表達(dá)均顯著增高(p0.01)。本研究還同時(shí)通過q RT-PCR和Westernblot檢測(cè)了3種骨肉瘤細(xì)胞系細(xì)胞(Saos-2,MG63和U2OS)和1種成骨細(xì)胞系細(xì)胞h HFOB1.19中生存素的表達(dá)。結(jié)果顯示,Saos-2、MG63和U2OS細(xì)胞中生存素m RNA和蛋白水平均明顯高于h HFOB1.19細(xì)胞(p0.01)。為進(jìn)一步研究生存素的臨床意義,本研究對(duì)所有瘤標(biāo)本進(jìn)行了Ki67評(píng)分并研究其與生存素表達(dá)的相關(guān)性,結(jié)果顯示生存素高表達(dá)組Ki67評(píng)分明顯高于生存素低表達(dá)組(p0.01),并且生存素高表達(dá)組患者生存時(shí)間要明顯短于生存素低表達(dá)組患者。這些結(jié)果表明,生存素在骨肉瘤患者體內(nèi)表達(dá)上調(diào),并與腫瘤惡性程度以及疾病預(yù)后相關(guān),提示生存素可作為骨肉瘤臨床治療的潛在基因靶點(diǎn)。在第一部分研究中,我們揭示了生存素在骨肉瘤發(fā)生發(fā)展中的重要作用。YM155是生存素的一種特異性抑制劑,在體外和動(dòng)物模型中對(duì)多種腫瘤的生長(zhǎng)和轉(zhuǎn)移均具有較強(qiáng)的抑制作用,但其對(duì)骨肉瘤生物學(xué)行為的作用尚未完全闡明。在本部分研究中,我們發(fā)現(xiàn)YM155刺激可明顯抑制骨肉瘤細(xì)胞系Saos-2細(xì)胞和MG63細(xì)胞在體外的增殖能力和成克隆能力。同時(shí)在體實(shí)驗(yàn)也證實(shí),在MG63細(xì)胞注射至裸鼠皮下制作的荷瘤模型中,YM155刺激可明顯抑制腫瘤內(nèi)生存素的表達(dá),并且導(dǎo)致骨肉瘤瘤體的縮小和重量的減輕。通過Transwell實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn),YM155處理后的Saos-2細(xì)胞和MG63細(xì)胞穿過基質(zhì)膠和濾膜的數(shù)目明顯較對(duì)照組減少,并呈劑量依賴性。并且,經(jīng)YM155刺激后,YM155刺激可明顯增加Saos-2細(xì)胞和MG63細(xì)胞細(xì)胞中Caspase 3,8和10的比率,兩種細(xì)胞凋亡數(shù)目明顯增多。這些研究結(jié)果說明YM155可在體內(nèi)和體外抑制骨肉瘤細(xì)胞的生長(zhǎng)、抗凋亡、遷移及侵襲能力,可作為生存素靶向治療的新型治療藥物應(yīng)用于骨肉瘤臨床治療。第三部分:YM155對(duì)骨肉瘤細(xì)胞阿霉素化療敏感性的影響第二部分研究結(jié)果表明YM155可抑制骨肉瘤細(xì)胞增殖和侵襲能力,并促進(jìn)其凋亡。對(duì)化療藥物抵抗是骨肉瘤臨床治療中極為棘手的難點(diǎn)之一,在本部分內(nèi)容中我們進(jìn)一步研究YM155對(duì)骨肉瘤細(xì)胞阿霉素化療敏感性的影響。本研究采用DOX濃度遞增法建立骨肉瘤耐藥細(xì)胞模型MG63/DOX。與MG63細(xì)胞相比,MG63/DOX細(xì)胞株內(nèi)生存素蛋白和m RNA表達(dá)明顯增高,阿霉素對(duì)其細(xì)胞活性的抑制作用明顯減弱。我們對(duì)MG63/DOX細(xì)胞在阿霉素、YM155以及YM155聯(lián)合阿霉素刺激下細(xì)胞增殖、克隆和凋亡的情況進(jìn)行檢測(cè)。結(jié)果顯示,相同濃度的阿霉素可明顯抑制MG63細(xì)胞的增殖能力,但其對(duì)第二部分:YM155對(duì)骨肉瘤細(xì)胞增殖、凋亡以及侵襲能力的影響MG63/DOX的增殖的抑制作用明顯減弱。YM155可抑制MG63/DOX細(xì)胞的增殖,與阿霉素合用后對(duì)細(xì)胞增殖的抑制能力明顯增強(qiáng)。并且,單一阿霉素不能抑制MG63/DOX細(xì)胞的成克隆數(shù)目,但YM155可抑制MG63/DOX細(xì)胞的成克隆數(shù)目,與阿霉素合用后,這種抑制能力明顯增強(qiáng)。通過PI/Annexin染色后流式細(xì)胞檢測(cè)發(fā)現(xiàn),單一阿霉素不能誘導(dǎo)MG63/DOX細(xì)胞的凋亡,但YM155可誘導(dǎo)MG63/DOX的輕度凋亡,阿霉素與YM155合用后,可誘導(dǎo)MG63/DOX細(xì)胞明顯凋亡。進(jìn)一步研究發(fā)現(xiàn),YM155可降低兩種細(xì)胞中p-Akt、p-PI3K和Mcl-1的表達(dá),并呈劑量依賴性,但不同劑量的YM155對(duì)Bcl-2和XIAP表達(dá)均無明顯影響。這些結(jié)果提示,YM155可能通過抑制PI3K/Akt途徑,增強(qiáng)阿霉素對(duì)骨肉瘤細(xì)胞的細(xì)胞毒性和促凋亡能力,有望作為骨肉瘤化療聯(lián)合藥物應(yīng)用于臨床治療。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R738.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

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本文編號(hào)：1856391