發(fā)布時間：2018-07-05 04:34

  本文選題：微管靶向藥物 + 抗腫瘤作用機制�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：腫瘤是當(dāng)今世界的一大難題,腫瘤發(fā)病率、死亡人數(shù)持續(xù)走高,其治療仍迫切需要更有效的藥物。微管蛋白抑制劑是一類作用于微管蛋白并破壞其動態(tài)平衡,從而阻止腫瘤細(xì)胞增殖的抗癌藥物,在臨床多種實體腫瘤治療中處于重要的地位。新型微管靶向抗腫瘤藥物因具有明確的分子靶向性、強效的抗腫瘤以及抑制并破環(huán)腫瘤新生血管形成的作用,成為抗腫瘤藥物研究的新熱點。WX-123-27,WX-127-07, WX-132-18B是由本所通過普篩發(fā)現(xiàn)的三個具有極強抗腫瘤活性的新型分子,初步的實驗表明該類化合物可能作用于微管蛋白[1]。 本課題采用本課題組建立的基于高內(nèi)涵分析(HCA)的抗腫瘤藥物藥效及機制研究的技術(shù)平臺,通過系統(tǒng)、高效的體外實驗,確定上述三種新化合物的抗腫瘤活性及作用的分子機制,以便在藥物發(fā)展早期階段全面了解這些活性分子的藥理學(xué)作用特征,為該類化合物的進一步發(fā)展奠定基礎(chǔ)。 在實驗研究中以已知的微管靶向藥物為陽性對照藥物,采用SRB法在HepG2, HeLa, A549, HLF及HUVEC細(xì)胞上檢測了受試化合物的細(xì)胞增殖抑制活性；采用流式細(xì)胞術(shù)檢測周期阻滯作用；通過高內(nèi)涵細(xì)胞毒性、細(xì)胞形態(tài)、細(xì)胞凋亡的多參數(shù)分析以及與腫瘤相關(guān)的細(xì)胞信號通路(包括2個GPCRs,6個核受體、2個生長因子受體和激酶、7個細(xì)胞周期及DNA損傷、6個炎性及應(yīng)激和8個腫瘤非相關(guān)的通路)的篩查分析受試化合物的細(xì)胞效應(yīng)特征；最后采用秋水仙堿/熒光長春堿的微管蛋白競爭抑制實驗和胰酶消化微管蛋白實驗在分子水平確證受試化合物的微管蛋白作用位點。 實驗結(jié)果如下：WX-123-27, WX-127-07, WX-132-18B在HepG2, HeLa, A549, HLF及HUVEC細(xì)胞上均具有顯著的細(xì)胞增殖抑制活性,WX-123-27的IC50分別為6.83±0.15nM,6.72±0.19nM,6.78±0.31nM,5.51±0.11nM,5.37±0.20nM, WX-127-07的IC50分別為4.47±0.05nM,5.18±0.08nM,4.90±0.19nM,4.10±0.16nM,5.04±0.08nM,WX-132-18B的IC50分別為1.03±0.02nM,1.01±0.01nM,1.03±0.06nM,0.86±0.02nM,0.76±0.04nM,作用強度顯著強于秋水仙堿(IC50分別為21.17±1.22nM,14.19±0.53nM,43.80±1.64nM,145.89±10.97nM,27.67±1.79nM)和長春新堿(IC50分別為16.51±0.36nM,16.76±0.33nM,27.80±2.75nM,43.80±1.48nM,9.15±0.78nM),在HepG2, HeLa, HLF細(xì)胞上作用顯著強于紫杉醇(IC50分別為10.68±0.61nM,12.86±0.25nM,102.07±15.17Nm),在A549, HUVEC上作用與紫杉醇相當(dāng)(IC50分別為4.81±0：61nM,3.04±0.12nM)；高內(nèi)涵細(xì)胞多參數(shù)分析顯示,與紫杉醇濃度依賴性地誘發(fā)A549細(xì)胞微管聚合不同,三種受試化合物與長春新堿、秋水仙堿相似,濃度依賴地誘發(fā)A549細(xì)胞的微管解聚,紫杉醇、長春新堿、秋水仙堿、WX-123-27、WX-127-07、 WX-132-18B使微管含量(以亮度和面積的乘積為指標(biāo))改變的EC50分別為75.84nM,293.2nM,105.5nM,25.91nM,17.31nM,9.43nM,即三種受試化合物的微管效應(yīng)顯著強于三種對照藥物；與三種陽性對照藥物相似,三種受試化合物誘發(fā)A549細(xì)胞在G2/M期阻滯,正常對照組細(xì)胞在Go/Gi, S, G2/M期的分布分別為71.74%,21.95%,6.31%,經(jīng)紫杉醇、長春新堿、秋水仙堿、WX-123-27、WX-127-07、WX-132-18B作用后,G0/G1期比例明顯減少,分別為18.17%,11.63%,19.15%,7.41%,5.62%,3.74%,而G2/M期比例明顯增高,分別為62.90%,71.33%,63.61%,80.35%,72.06%,83.80%；同樣,與三種陽性對照藥物相似,受試化合物使HepG2細(xì)胞核膜通透性增加、誘導(dǎo)細(xì)胞早凋；在較高的濃度下,誘導(dǎo)輕度的Rad51顆粒的形成(約為陽性藥喜樹堿的1/4),但未對篩查的其它30種信號通路有影響。在微管蛋白位點的競爭抑制實驗中,WX-123-27, WX-127-07, WX-132-18B濃度依賴地抑制秋水仙堿與微管蛋白的結(jié)合(IC50分別為1.75±0.54μM,1.28±0.08μM,0.47±0.10μM),僅WX-123-27, WX-127-07在高濃度對熒光長春堿與微管蛋白的結(jié)合表現(xiàn)出一定的抑制作用,但顯著弱于長春新堿,也不能排除高濃度非特異的抑制作用；微管蛋白經(jīng)胰酶消化后的SDS蛋白電泳實驗顯示W(wǎng)X-123-27、WX-127-07、WX-132-18B與微管蛋白作用后的酶解產(chǎn)物與秋水仙堿作用較為相似而不同于長春新堿,進一步證明受試化合物主要作用于微管的秋水仙堿結(jié)合位點。 綜合以上的研究結(jié)果可以看出,受試化合物WX-123-27, WX-127-07, WX-132-18B具有較強的體外抗腫瘤細(xì)胞增殖活性及抗血管內(nèi)皮細(xì)胞增殖活性,其抗腫瘤作用機制符合微管靶向藥物的作用特點,三種受試化合物主要選擇性作用于微管秋水仙堿位點,是具有強抗細(xì)胞增殖活性的新型微管解聚劑。
[Abstract]:Tumor is a major problem in the world today. The incidence of cancer and the number of deaths continue to rise, and the treatment still needs more effective drugs. Microtubulin inhibitors are an anticancer drug that acts on microtubule protein and destroys its dynamic balance, thus preventing the proliferation of tumor cells. It is important in the treatment of many clinical solid tumors. The new microtubule targeting antitumor drug has become a new hot spot in antitumor drug research because of its specific molecular targeting, strong anti-tumor and inhibition and the formation of neovascularization of tumor ring tumor,.WX-123-27, WX-127-07, WX-132-18B, a new molecule with three extremely strong anti-tumor activities discovered by this institute. Preliminary experiments show that these compounds may act on tubulin [1]..
In this subject, we set up a technical platform based on high intension analysis (HCA) based on high intension analysis (HCA) for anti-tumor drug efficacy and mechanism. Through systematic and efficient in vitro experiments, the molecular mechanism of the antitumor activity and action of the above three new compounds is determined in order to understand the pharmacology of these active molecules at the early stage of drug development. The characteristics of the study lay the foundation for further development of the compounds.
In the experimental study, using the known microtubule targeting drug as the positive control drug, the cell proliferation inhibition activity of the tested compounds was detected on HepG2, HeLa, A549, HLF and HUVEC cells by SRB, and the flow cytometry was used to detect the cycle arrest, and the multi parameter analysis of the high intension cytotoxicity, cell morphology and apoptosis was carried out. And the cell signaling pathways associated with tumor related cells (including 2 GPCRs, 6 nuclear receptors, 2 growth factor receptors and kinases, 7 cell cycle and DNA damage, 6 inflammatory and stress and 8 unrelated pathways) screening and analyzing the cell effects of the tested compounds; the most later microtubulin competing with colchicine / fluorescent Changchun base Competition inhibition experiments and trypsin digestion of tubulin experiments confirmed the tubulin site of the tested compounds at molecular level.
The experimental results are as follows: WX-123-27, WX-127-07 and WX-132-18B have significant inhibitory activity on cell proliferation on HepG2, HeLa, A549, HLF and HUVEC cells. WX-123-27 IC50 is 6.83 + 0.15nM, 6.72 + 0.19nM, 5.51 +, 5.37 +, 5.18, 4.90, 4.10 +, 4.10 +, 4.10 +. 0.16nM, 5.04 + 0.08nM, WX-132-18B IC50 were 1.03 + 0.02nM, 1.01 + 0.01nM, 1.03 + 0.06nM, 0.86 + 0.02nM, 0.76 + 0.04nM, and the action intensity was significantly stronger than colchicine (IC50 respectively 21.17 +, 14.19 + 0.53nM, 43.80 +, 145.89 +, 27.67 +) and vincristine. 75nM, 43.80 + 1.48nM, 9.15 + 0.78nM), the effect on HepG2, HeLa, HLF cells is stronger than Taxol (IC50 is 10.68 + 0.61nM, 12.86 + 0.25nM, 102.07 + 15.17Nm). In A549, the action of the HUVEC is equivalent to Taxol (4.81 + 3.04, 3.04 +), and the high concentration cell multi parameter analysis shows that the concentration dependence of taxol concentration The microtubule polymerization of A549 cells was different. The three tested compounds were similar to vincristine and colchicine, and the concentration dependent induced microtubule depolymerization of A549 cells. Paclitaxel, vincristine, colchicine, WX-123-27, WX-127-07, WX-132-18B changed the EC50 content of microtubules (the product of brightness and area as the index of 75.84nM, 293.2nM, respectively). 105.5nM, 25.91nM, 17.31nM, 9.43nM, that is, the microtubule effect of three kinds of tested compounds is significantly stronger than that of three control drugs; it is similar to three positive control drugs. Three kinds of subjects induce A549 cells to block in the G2/M phase, and the distribution of the normal control group in Go/Gi, S, and G2/M are 71.74%, 21.95%, 6.31%, after paclitaxel, vincristine, autumn. After the action of Narcissus, WX-123-27, WX-127-07 and WX-132-18B, the proportion of G0/G1 phase decreased significantly, which were 18.17%, 11.63%, 19.15%, 7.41%, 5.62%, 3.74%, and the G2/M period increased significantly, respectively, 62.90%, 71.33%, 63.61%, 80.35%, 72.06%, 83.80%; similarly, similar to the three positive control drugs, the tested compound increased the nuclear membrane permeability of HepG2 cells, Induction of cell early withering; at a higher concentration, the induction of mild Rad51 particles (about 1/4 of the positive drug camptothecin) was not affected by the other 30 signaling pathways of screening. In the competition inhibition test of the microtubule loci, the concentration of WX-123-27, WX-127-07, and WX-132-18B inhibited the combination of colchicine and microtubule protein (I C50 was 1.75 + 0.54 mu M, 1.28 + 0.08 mu M, 0.47 + 0.10 M), only WX-123-27, WX-127-07 showed a certain inhibition effect on the combination of fluorescent Changchun alkali and microtubule protein at high concentration, but it was significantly weaker than vincristine, and it could not exclude high concentration and nonspecific inhibition effect. The microtubulin protein electrophoresis after trypsin digestion showed that the electrophoresis of SDS protein electrophoresis showed that the microtubulin was digested by trypsin. The enzymatic hydrolysates of WX-123-27, WX-127-07, WX-132-18B and microtubule were similar to colchicine, but were different from vincristine, which further demonstrated that the tested compounds were mainly used in the colchicine binding site of microtubules.
The above results can be seen that the experimental compounds WX-123-27, WX-127-07, WX-132-18B have strong anti-tumor cell proliferation activity and anti vascular endothelial cell proliferation activity in vitro. The anti-tumor mechanism of the compound is consistent with the role of microtubule targeting drugs. The three kinds of tested compounds are mainly selective in microtubule colchicine. The site is a new microtubule depolymerization agent with strong anti proliferative activity.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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