本文選題：新型合成藥物 + 組蛋白去乙酰化酶抑制劑��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究目的：開發(fā)具有生物活性高、毒性低等特點的新藥一直是腫瘤治療研究領(lǐng)域的熱點,其中分子合成藥物由于能夠克服傳統(tǒng)化療藥物的缺陷而備受關(guān)注。NL-101是一種連接組蛋白去乙�；敢种苿㏒AHA以及烷化劑苯達莫司汀的活性基團而合成的化合物。本文探討這種化合物在體外和體內(nèi)對于白血病細胞的殺傷作用以及相關(guān)分子機制。研究方法：采用MTT法檢測NL-101、SAHA和苯達莫司汀對多種白血病細胞系增殖能力的影響,用SPSS軟件計算半數(shù)抑制劑量(ICso),用CompuSyn軟件計算SAHA和苯達莫司汀的藥物聯(lián)合指數(shù)(CI)；用流式細胞術(shù)分析藥物處理后細胞的周期和凋亡變化；Western blot檢測藥物處理后組蛋白H3乙�；�,以及蛋白凋亡和DNA損傷相關(guān)蛋白包括y-H2AX、PARP、caspase-3、Bax、Bcl-2和Bcl-xL的表達；Ficoll法分離病人原代細胞,瑞氏染色觀察細胞經(jīng)藥物處理后的形態(tài)改變。體內(nèi)實驗部分,通過逆轉(zhuǎn)錄病毒感染構(gòu)建共表達AML1-ETO和C-KIT突變的移植白血病小鼠模型,給予NL-101、苯達莫司汀和SAHA注射治療,動態(tài)監(jiān)測小鼠體重以及體內(nèi)白血病細胞浸潤情況,記錄各組小鼠生存期。研究結(jié)果：在體外實驗中：(1)苯達莫司汀和SAHA在白血病細胞中具有協(xié)同作用。(2)NL-101對多種白血病細胞系的生長有明顯的抑制作用,其中Kasumi-1和NB4細胞的敏感性最為顯著；NL-101對正常細胞系的作用較小,顯示出對腫瘤細胞作用的選擇性；NL-101的抗增殖能力顯著高于苯達莫司汀,與SAHA沒有顯著差異。(3)NL-101導(dǎo)致白血病細胞周期阻滯于S期,以及顯著的細胞凋亡。(4)NL-101的細胞毒性作用在分子水平上表現(xiàn)為激活依賴caspase-3的凋亡通路以及Bcl-2家族促凋亡蛋白；NL-101能增加DNA損傷標(biāo)志蛋白y-H2AX和乙酰化組蛋白H3的表達。(5)NL-101能誘導(dǎo)急性髓系白血病患者的原代細胞發(fā)生DNA損傷和細胞凋亡。在體內(nèi)實驗中：(1)成功建立共表達AML1-ETO和C-KIT突變的移植白血病小鼠模型,造血器官中有大量原始細胞(c-Kit+、Gr1-、CD11b-)浸潤。(2)NL-101能抑制t(8；21)白血病細胞的增殖和浸潤,顯著延長白血病小鼠的生存期,具有比苯達莫司汀和SAHA更強的抗白血病活性。(3)NL-101對小鼠體重沒有顯著影響,小鼠的耐受性良好。研究結(jié)論：由SAHA和苯達莫司汀的活性基團合成的新型藥物NL-101,在體內(nèi)及體外實驗中均具有抑制白血病細胞增殖以及誘導(dǎo)細胞凋亡的生物學(xué)效應(yīng)。針對作用機制的研究顯示NL-101具有抑制組蛋白去乙�；负推茐腄NA結(jié)構(gòu)的雙重活性。
[Abstract]:Objective: to develop new drugs with high bioactivity and low toxicity has been a hot topic in the field of tumor therapy. Among them, molecular synthetic drugs have attracted much attention because of their ability to overcome the defects of traditional chemotherapeutic drugs. NL-101 is a kind of compound synthesized by linking histone deacetylase inhibitor SAHA and the active group of benzamoastine. The cytotoxicity of this compound to leukemia cells in vitro and in vivo and its molecular mechanisms were investigated. Methods: the effects of NL-101 SAHA and bendamoastine on the proliferation of various leukemia cell lines were detected by MTT assay. SPSS software was used to calculate the dose of ICSU, CompuSyn software was used to calculate the combined index of SAHA and bentamoastine, and flow cytometry was used to analyze the changes of cell cycle and apoptosis after drug treatment. Western blot was used to detect the acetylation level of histone H3 after drug treatment. The expression of protein apoptosis and DNA damage related proteins, including the expression of y-H2AXPnPARPncaspase-3, Bcl-2 and Bcl-xL, was detected by Ficoll method. The morphological changes of the cells treated with drugs were observed by Rayleigh staining. In vivo experiment, the transplanted leukemia mice model which co-expressed AML1-ETO and C-KIT mutations was constructed by retrovirus infection. NL-101, bentamoastine and SAHA were injected into mice to dynamically monitor the body weight and infiltration of leukemia cells in vivo. The survival time of mice in each group was recorded. Results: in vitro, the effects of bendamolastine and SAHA on the growth of various leukemia cell lines were significantly inhibited. The sensitivity of Kasumi-1 and NB4 cells was the most significant. NL-101 had little effect on normal cell lines, which showed that the selective action of NL-101 on tumor cells was significantly higher than that of bentamoastine. There was no significant difference between NL-101 and SAHA. NL-101 resulted in cell cycle arrest in S phase. The cytotoxic effects of NL-101 on apoptosis were as follows: activation of caspase-3 dependent apoptotic pathway and increased expression of DNA damage marker protein y-H2AX and acetylated histone H3 in Bcl-2 family. It can induce DNA damage and apoptosis in primary cells of patients with acute myeloid leukemia. In vivo, the mouse model of leukemia transplanted with AML1-ETO and C-KIT mutations was successfully established. A large number of primitive cells, c-Kit Gr1-Gr1-NL-101, could inhibit the proliferation and infiltration of tn821) leukemia cells, and prolong the survival time of leukemia mice. There was no significant effect on body weight of mice with stronger antileukemia activity than bendamoastine and SAHA. The mice had good tolerance. Conclusion: NL-101, a novel drug synthesized from the active groups of SAHA and bentamoastine, has the biological effects of inhibiting the proliferation and inducing apoptosis of leukemia cells in vivo and in vitro. Studies on the mechanism of action showed that NL-101 had dual activities of inhibiting histone deacetylase and destroying the structure of DNA.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R733.7

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