本文關(guān)鍵詞：新型Hedgehog抑制劑Bxl-7-15的抗腫瘤作用及機制研究　出處：《華東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Hedgehog Bxl-7-15 耐藥 抗腫瘤 髓母細胞瘤

【摘要】：背景:Hedgehog信號通路在哺乳動物生長發(fā)育中有著關(guān)鍵作用,主要調(diào)控胚胎中樞神經(jīng)系統(tǒng)中神經(jīng)管的形成。該通路異常會引起發(fā)育異常,更是會誘導(dǎo)基底細胞癌等惡性腫瘤的發(fā)生。抑制Hedgehog信號通路可以治療相關(guān)惡性腫瘤,目前已有兩個靶向Hedgehog的藥物維莫德吉和索尼吉步上市用于治療基底細胞癌。然而已上市藥物在臨床階段就出現(xiàn)了嚴重的毒副作用和耐藥問題,亟需研究新型的Hedgehog信號通路抑制劑以克服。由此,我們實驗室與復(fù)旦大學(xué)藥學(xué)院趙偉利/董肖椿課題組合作,根據(jù)已有的Hedgehog抑制劑的化學(xué)結(jié)構(gòu)設(shè)計合成了一系列小分子化合物,以期研發(fā)新型的靶向Hedgehog抗腫瘤藥物。目的:本課題旨在對根據(jù)已有的Hedgehog抑制劑的化學(xué)結(jié)構(gòu)設(shè)計合成的61個小分子化合物進行篩選,對其中Hedgehog信號通路活性抑制能力最強的化合物Bxl-7-15進行機制研究,于Ptch+/-p53+/-轉(zhuǎn)基因小鼠自發(fā)的髓母細胞瘤模型上進一步評價其抑瘤效果。方法：1.構(gòu)建雙螢光素酶檢測模型(Shh Light Ⅱ細胞),對61個小分子化合物進行篩選,以獲取具有顯著Hedgehog信號通路抑制作用的化合物。2.構(gòu)建熒光環(huán)巴胺結(jié)合競爭模型,通過熒光顯微觀察以及流式細胞術(shù)驗證篩選出的Hedgehog信號通路抑制劑Bxl-7-15的作用位點。3.在Shh-Light Ⅱ細胞中轉(zhuǎn)染突變型Smo基因(D473H)構(gòu)建維莫德吉耐藥模型,以驗證化合物Bxl-7-15的抗耐藥作用。4.通過雜交轉(zhuǎn)基因小鼠獲取Ptch+/-p53+/-轉(zhuǎn)基因小鼠,以構(gòu)建自發(fā)髓母細胞瘤模型,并通過X-Gal實驗驗證獲得的腫瘤。5.用Ptch+/-p53+/-轉(zhuǎn)基因小鼠自發(fā)的髓母細胞瘤模型評價化合物Bxl-7-15的體內(nèi)外抗腫瘤作用。結(jié)果:1.61個候篩化合物中,Bxl-7-15具有最強的Hedgehog信號通路抑制能力(IC50=2.33 nM)。2.化合物Bxl-7-15結(jié)合于Smo蛋白。3.化合物Bxl-7-15可以抑制維莫德吉耐藥細胞株的Hedgehog信號通路。4.獲得Ptch+/-p53+/-轉(zhuǎn)基因小鼠及其自發(fā)的腦部腫瘤,同時腫瘤用X-Gal檢測為髓母細胞瘤。5.化合物Bxl-7-15具有顯著的體內(nèi)抑制髓母細胞瘤生長活性。結(jié)論:本研究應(yīng)用雙熒光素酶檢測模型篩選出了具有顯著Hedgehog信號通路抑制活性的化合物Bxl-7-15。Bxl-7-15通過靶向Smo蛋白可以顯著抑制Hedgehog信號通路活性,并且有助于克服上市藥物維莫德吉的耐藥問題。本研究成功構(gòu)建了Ptch+/-p53+/-小鼠自發(fā)髓母細胞瘤模型,并于模型上檢測到Bxl-7-15對髓母細胞瘤有顯著的抑制作用。我們的研究結(jié)果表明,Bxl-7-15是非常有潛力的靶向Hedgehog信號通路的抗腫瘤先導(dǎo)化合物,值得進一步研發(fā)以克服現(xiàn)已上市藥物的耐藥性以及毒副作用問題。同時本研究建立的雙熒光素酶檢測模型和轉(zhuǎn)基因小鼠自發(fā)腫瘤模型可以用于篩選評價Hedgehog抑制劑,為研發(fā)新型的Hedgehog抑制劑奠定了藥理學(xué)實驗基礎(chǔ)。
[Abstract]:Background: Hedgehog signaling pathway in mammalian growth and development plays a key role in the formation of neural tube, the main regulation of embryonic nervous system. This pathway can cause developmental abnormalities, is induced by malignant tumor basal cell carcinoma et al. Inhibition of Hedgehog signaling pathway can be related to treatment of malignant tumor, there are two target to drug Vimal Deji Hedgehog and SONY geb listed for the treatment of basal cell carcinoma. However, drugs already on the market in the clinical stage had toxic side effects and drug resistance is serious, need to study Hedgehog signaling pathway inhibitors to overcome. Thus, our laboratory and Fudan University school medicine Zhao Weili / Dong Xiaochun research group, according to a series of small molecular compounds were synthesized by the chemical structure of the existing design of Hedgehog inhibitors, in order to develop new anticancer drugs targeting Hedgehog Purpose: the purpose of this topic. Based on the chemical structure of Hedgehog inhibitors of the existing design of 61 small molecule compounds were screened, of which Hedgehog signaling pathway inhibition strongest compounds Bxl-7-15 mechanism, medulloblastoma model in Ptch+/-p53+/- transgenic mice spontaneously to evaluate its antitumor effect. 1.: the construction of dual luciferase detection model (Shh Light cells), the 61 small molecular compounds were screened to obtain significant inhibition of the Hedgehog signaling pathway of compound.2. was constructed with cyclopamine competition model by fluorescence microscopy and Bxl-7-15 Hedgehog signal pathway inhibitor of flow cytometry to verify the screening of site.3. transfer dye mutant Smo gene in Shh-Light II cells (D473H) to build dimensional Mo resistant model to verify the combined DEGI. Bxl-7-15.4. for the anti drug resistant function of Ptch+/-p53+/- transgenic mice by hybridization of transgenic mice to construct spontaneous medulloblastoma model and anti tumor obtained by X-Gal experiments with tumor.5. Ptch+/-p53+/- transgenic mice spontaneously evaluate medulloblastoma model compounds Bxl-7-15 in vitro and in vivo. Results: 1.61 when the compounds. Hedgehog signal pathway has the strongest inhibition ability of Bxl-7-15 (IC50=2.33 nM).2. Bxl-7-15 compounds bind to Smo protein.3. compound Bxl-7-15 could inhibit the mo de resistant cell lines - Hedgehog signaling pathway.4. Ptch+/-p53+/- transgenic mice and spontaneous brain tumor, and tumor medulloblastoma.5. compound Bxl-7-15 has significant inhibitory activity in medulloblastoma cell tumor growth was determined by X-Gal. Conclusion: the application of dual luciferase assay screening model The Hedgehog signaling pathway has significant inhibitory activity of compound Bxl-7-15.Bxl-7-15 can significantly inhibit the activity of Hedgehog signaling pathway by Smo protein to target, and help to overcome the drug resistance problem listed Vimal Deji. This study successfully constructed Ptch+/-p53+/- mice spontaneous medulloblastoma model, and model to detect Bxl-7-15 significantly inhibited for medulloblastoma. Our results suggest that Bxl-7-15 is targeting anti-tumor lead compounds of the Hedgehog signaling pathway has great potential, worthy of further research to overcome drug resistance has been listed drugs and side effects. At the same time, this study established the dual luciferase detection model of transgenic mice and spontaneous tumor model can be used for screening and evaluation Hedgehog inhibitors, which lays the foundation for the development of new pharmacological inhibitors of Hedgehog.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96
，

本文編號：1386155