新型Hedgehog抑制剂Bxl-7-15的抗肿瘤作用及机制研究

发布时间：2018-01-06 03:40

  本文关键词：新型Hedgehog抑制剂Bxl-7-15的抗肿瘤作用及机制研究　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： Hedgehog Bxl-7-15 耐药 抗肿瘤 髓母细胞瘤

【摘要】：背景:Hedgehog信号通路在哺乳动物生长发育中有着关键作用,主要调控胚胎中枢神经系统中神经管的形成。该通路异常会引起发育异常,更是会诱导基底细胞癌等恶性肿瘤的发生。抑制Hedgehog信号通路可以治疗相关恶性肿瘤,目前已有两个靶向Hedgehog的药物维莫德吉和索尼吉步上市用于治疗基底细胞癌。然而已上市药物在临床阶段就出现了严重的毒副作用和耐药问题,亟需研究新型的Hedgehog信号通路抑制剂以克服。由此,我们实验室与复旦大学药学院赵伟利/董肖椿课题组合作,根据已有的Hedgehog抑制剂的化学结构设计合成了一系列小分子化合物,以期研发新型的靶向Hedgehog抗肿瘤药物。目的:本课题旨在对根据已有的Hedgehog抑制剂的化学结构设计合成的61个小分子化合物进行筛选,对其中Hedgehog信号通路活性抑制能力最强的化合物Bxl-7-15进行机制研究,于Ptch+/-p53+/-转基因小鼠自发的髓母细胞瘤模型上进一步评价其抑瘤效果。方法：1.构建双萤光素酶检测模型(Shh Light Ⅱ细胞),对61个小分子化合物进行筛选,以获取具有显著Hedgehog信号通路抑制作用的化合物。2.构建荧光环巴胺结合竞争模型,通过荧光显微观察以及流式细胞术验证筛选出的Hedgehog信号通路抑制剂Bxl-7-15的作用位点。3.在Shh-Light Ⅱ细胞中转染突变型Smo基因(D473H)构建维莫德吉耐药模型,以验证化合物Bxl-7-15的抗耐药作用。4.通过杂交转基因小鼠获取Ptch+/-p53+/-转基因小鼠,以构建自发髓母细胞瘤模型,并通过X-Gal实验验证获得的肿瘤。5.用Ptch+/-p53+/-转基因小鼠自发的髓母细胞瘤模型评价化合物Bxl-7-15的体内外抗肿瘤作用。结果:1.61个候筛化合物中,Bxl-7-15具有最强的Hedgehog信号通路抑制能力(IC50=2.33 nM)。2.化合物Bxl-7-15结合于Smo蛋白。3.化合物Bxl-7-15可以抑制维莫德吉耐药细胞株的Hedgehog信号通路。4.获得Ptch+/-p53+/-转基因小鼠及其自发的脑部肿瘤,同时肿瘤用X-Gal检测为髓母细胞瘤。5.化合物Bxl-7-15具有显著的体内抑制髓母细胞瘤生长活性。结论:本研究应用双荧光素酶检测模型筛选出了具有显著Hedgehog信号通路抑制活性的化合物Bxl-7-15。Bxl-7-15通过靶向Smo蛋白可以显著抑制Hedgehog信号通路活性,并且有助于克服上市药物维莫德吉的耐药问题。本研究成功构建了Ptch+/-p53+/-小鼠自发髓母细胞瘤模型,并于模型上检测到Bxl-7-15对髓母细胞瘤有显著的抑制作用。我们的研究结果表明,Bxl-7-15是非常有潜力的靶向Hedgehog信号通路的抗肿瘤先导化合物,值得进一步研发以克服现已上市药物的耐药性以及毒副作用问题。同时本研究建立的双荧光素酶检测模型和转基因小鼠自发肿瘤模型可以用于筛选评价Hedgehog抑制剂,为研发新型的Hedgehog抑制剂奠定了药理学实验基础。
[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.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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本文编号：1386155