發(fā)布時(shí)間：2018-01-06 08:15

  本文關(guān)鍵詞：新型人工合成鈉離子通道阻斷劑抑制前列腺癌生長(zhǎng)的體內(nèi)體外研究　出處：《第三軍醫(yī)大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 前列腺癌 電壓門(mén)控-鈉離子通道 鈉離子通道胺類(lèi)配體 細(xì)胞周期阻滯 侵襲 移植瘤模型

【摘要】：背景和目的:前列腺癌(Prostate cancer)在全球男性中是發(fā)病率第二的腫瘤,2012年有1,100,000例新發(fā)病例,占男性全部癌癥患者的15%。發(fā)達(dá)國(guó)家男性人口占全球男性的17%,但其前列腺癌發(fā)病率最高,占全部前列腺癌患者的2/3。雖然亞洲地區(qū)前列腺癌發(fā)病率較低,但呈現(xiàn)逐年增加的趨勢(shì)。早期前列腺癌通常沒(méi)有癥狀,但發(fā)展到晚期常會(huì)引起一系列癥狀,包括排尿困難(尿量減少,排尿次數(shù)增多)、血尿、勃起功能障礙等;前列腺癌進(jìn)一步進(jìn)展易發(fā)生骨轉(zhuǎn)移,癌癥轉(zhuǎn)移到骨后會(huì)引起臀部、背部、胸部或其他地方的疼痛;當(dāng)腫瘤壓迫脊髓還可能引起腿或足部虛弱麻木、膀胱或者腸失控等。由于前列腺癌的危險(xiǎn)因素包括年齡、種族、地理、家族史和基因改變等多不可改變,且具有復(fù)雜的分子通路,化療被認(rèn)為是治療前列腺癌的主要治療方法。因此,找到高效低毒的放化療藥物對(duì)于前列腺癌的治療顯得至關(guān)重要。電壓門(mén)控-鈉離子通道(Voltage-gated sodium channels,VGSCs)與人類(lèi)多種惡性腫瘤有關(guān),VGSCs在癌癥進(jìn)程中的各個(gè)階段都發(fā)揮著重要的作用,與腫瘤細(xì)胞黏附、增殖、遷移、侵襲和轉(zhuǎn)移等相關(guān)。研究顯示,VGSCs的激動(dòng)劑能促進(jìn)癌癥細(xì)胞的轉(zhuǎn)移,VGSCs的阻斷劑能抑制細(xì)胞增殖、遷移和侵襲等。VGSCs已經(jīng)成為癌癥治療的新靶標(biāo)。因此,重慶醫(yī)科大學(xué)藥學(xué)院實(shí)驗(yàn)室以VGSCs通道蛋白為靶標(biāo),運(yùn)用3D定量構(gòu)效關(guān)系結(jié)構(gòu)模擬軟件建立藥物結(jié)合模型,設(shè)計(jì)并人工合成了一系列具有VGSCs通道蛋白結(jié)合活性的小分子配體-新型鈉離子通道胺類(lèi)配體(Sodium channels amine ligands,SCALs)共15種,分別命名為S0103,S0132,S0135,S0142,S0143,S0152,S0154,S0156,S0158,S0160,S0161,S0163,S0165,S0205,S0307。這一系列小分子配體試圖通過(guò)與VGSCs的結(jié)合,封阻該通道,從而發(fā)揮鈉離子通道阻斷劑的作用抑制前列腺癌轉(zhuǎn)移。本研究以15種SCALs作為篩選化合物,以前列腺癌作為研究對(duì)象,評(píng)估人工合成的SCALs對(duì)前列腺癌生長(zhǎng)的影響。VGSCs的通道蛋白Nav1.6和Nav1.7在前列腺癌PC3和LNCa P細(xì)胞中大量表達(dá),其m RNA表達(dá)水平比正�；蛟錾那傲邢俳M織高6-27倍(p0.05),它們可能是潛在的前列腺癌診斷指標(biāo)和治療靶點(diǎn)。本研究運(yùn)用S0154和S0161對(duì)PC3細(xì)胞進(jìn)行干預(yù),通過(guò)Western blotting實(shí)驗(yàn)檢測(cè)細(xì)胞中Nav1.6和Nav1.7蛋白的表達(dá),從而觀察SCALs對(duì)鈉離子通道蛋白的影響。腫瘤細(xì)胞發(fā)生遠(yuǎn)處轉(zhuǎn)移是一個(gè)復(fù)雜的生理過(guò)程,需要減少細(xì)胞黏附、增加細(xì)胞能動(dòng)性和侵襲能力、蛋白酶解、以及抵抗凋亡等。VGSCs阻斷劑多通過(guò)抑制腫瘤細(xì)胞的侵襲和轉(zhuǎn)移發(fā)揮抗腫瘤效應(yīng)�；|(zhì)金屬蛋白酶類(lèi)(Matrix metalloproteinases,MMPs)是與癌癥轉(zhuǎn)移密切相關(guān)的細(xì)胞內(nèi)信號(hào)分子,腫瘤細(xì)胞分泌MMPs,通過(guò)降解細(xì)胞基底膜進(jìn)而遷移和侵襲到其他組織,實(shí)現(xiàn)癌癥的轉(zhuǎn)移。在一大類(lèi)MMPs中關(guān)于MMP-2和MMP-9的研究較為深入,MMP-2或MMP-9過(guò)表達(dá)時(shí)轉(zhuǎn)移的發(fā)生率增加,抑制MMP-2或者M(jìn)MP-9的表達(dá)轉(zhuǎn)移發(fā)生率減少。本研究運(yùn)用S0154和S0161對(duì)PC3細(xì)胞進(jìn)行干預(yù),通過(guò)檢測(cè)細(xì)胞中MMP-2和MMP-9蛋白的表達(dá),從分子水平上觀察SCALs對(duì)PC3細(xì)胞轉(zhuǎn)移能力的影響。同時(shí),我們還運(yùn)用BALB/c,nu/nu裸鼠建立了PC3細(xì)胞移植瘤模型,用S0154或S0161(以DMSO為對(duì)照)干預(yù)裸鼠移植瘤小鼠,通過(guò)監(jiān)測(cè)其體重變化以及移植瘤大小,進(jìn)而評(píng)估S0154和S0161在體內(nèi)對(duì)PC3移植瘤生長(zhǎng)的影響。實(shí)驗(yàn)方法:1.運(yùn)用MTT實(shí)驗(yàn)初篩15種SCALs對(duì)3株前列腺癌細(xì)胞(PC3、LNCa P、DU145)生長(zhǎng)能力的影響;2.運(yùn)用鈉離子探針實(shí)驗(yàn)檢測(cè)經(jīng)S0154和S0161處理的PC3細(xì)胞內(nèi)Na+的表達(dá)情況,進(jìn)行Western blotting技術(shù)檢測(cè)干預(yù)后PC3細(xì)胞中Nav1.6和Nav1.7蛋白的表達(dá);3.運(yùn)用流式細(xì)胞術(shù)檢測(cè)經(jīng)化合物干預(yù)后PC3細(xì)胞周期,運(yùn)用Western blotting技術(shù)檢測(cè)周期相關(guān)蛋白的表達(dá)情況;4.運(yùn)用流式細(xì)胞術(shù)檢測(cè)經(jīng)S0154和S0161干預(yù)后PC3細(xì)胞凋亡情況;5.進(jìn)行Transwell小室實(shí)驗(yàn),檢測(cè)S0154和S0161對(duì)PC3細(xì)胞侵襲能力的影響,Western blotting技術(shù)檢測(cè)MMP-2和MMP-9蛋白的表達(dá);6.采用劃痕實(shí)驗(yàn)檢測(cè)S0154和S0161對(duì)PC3細(xì)胞遷移能力的影響;7.建立PC3前列腺癌移植瘤模型,研究S0154和S0161對(duì)裸鼠移植瘤生長(zhǎng)情況的影響。實(shí)驗(yàn)結(jié)果:1.15種人工合成的SCALs中,S0154和S0161抑制前列腺癌細(xì)胞株P(guān)C3、DU145和LNCa P生長(zhǎng)的作用最為顯著,IC50值分別在10.51-26.60μM(S0154)、5.07-11.92μM(S0161)之間;2.S0154和S0161增加了PC3細(xì)胞內(nèi)Na+的濃度,抑制PC3細(xì)胞中鈉離子通道蛋白Nav1.6和/或Nav1.7的表達(dá);3.S0154和S0161通過(guò)下調(diào)G2/M期的關(guān)鍵蛋白CDK1和Cyclin D1的表達(dá),從而誘導(dǎo)PC3細(xì)胞周期阻滯于G2/M期;4.20μM的S0154能促進(jìn)PC3細(xì)胞發(fā)生凋亡,但S0161對(duì)PC3細(xì)胞凋亡作用不明顯;5.S0154和S0161下調(diào)PC3細(xì)胞基質(zhì)金屬蛋白酶類(lèi)中MMP-2和/或MMP-9蛋白的表達(dá);同時(shí)顯著抑制PC3細(xì)胞的侵襲能力(Transwell小室實(shí)驗(yàn)),與對(duì)照組相比,2.5μM時(shí)即存在顯著性差異,10μM的S0154能抑制95%的細(xì)胞侵襲,10μM的S0161能完全抑制PC3細(xì)胞的侵襲能力;6.劃痕實(shí)驗(yàn)結(jié)果顯示,S0154和S0161能抑制PC3細(xì)胞的遷移能力,但作用效果不如抑制侵襲明顯;7.S0154和S0161能顯著抑制PC3移植瘤裸鼠中腫瘤的生長(zhǎng),其中S0161干預(yù)2周,實(shí)驗(yàn)結(jié)束時(shí)移植瘤的生長(zhǎng)被抑制了50.4%,但其安全性不如S0154。實(shí)驗(yàn)結(jié)論:在15種SCALs中,S0154和S0161主要通過(guò)抑制CDK1和Cyclin D1蛋白表達(dá)誘導(dǎo)G2/M期周期阻滯,從而發(fā)揮抑制前列腺癌在體內(nèi)外生長(zhǎng)的作用;通過(guò)下調(diào)MMP-2和/或MMP-9表達(dá)抑制腫瘤細(xì)胞侵襲進(jìn)而抑制其轉(zhuǎn)移活性。S0154和S0161可抑制腫瘤細(xì)胞生長(zhǎng)和侵襲,可抑制前列腺癌移植瘤的生長(zhǎng),是潛在的前列腺癌化療藥。
[Abstract]:Background and objective: prostate cancer (Prostate cancer) in the world of men is second incidence of tumors, 1100000 new cases in 2012, developed countries accounted for 15%. of all cancers in male male population accounted for 17% of the male world, but the incidence of prostate cancer has the highest total prostate cancer patients with 2/3. while in Asia the incidence of prostate cancer is low, but increased year by year. Early prostate cancer often has no symptoms, but to the development of advanced and often cause a series of symptoms, including dysuria (urine volume decreased, urination, hematuria), erectile dysfunction; prostate cancer prone to further progression of bone metastasis, cancer metastasis to bone may cause the hips, back, chest or other parts of the pain; when the tumor compression of the spinal cord may cause leg or foot numbness weakness, bladder or bowel control due to the risk of prostate cancer. Factors including age, ethnicity, geography, family history and genetic changes can not change, and has a complex molecular pathways, chemotherapy is considered to be the main method for the treatment of prostate cancer. Therefore, to find efficient and low toxicity of chemotherapy in the treatment of prostate cancer is very important. The voltage-gated sodium channel (Voltage-gated - sodium channels, VGSCs) associated with a variety of human malignant tumors, VGSCs at each stage in the process of cancer plays an important role, and tumor cell adhesion, proliferation, migration, invasion and metastasis. Research shows that VGSCs agonist can promote the metastasis of cancer cells, VGSCs blockers can inhibit cell proliferation. The migration and invasion of.VGSCs has become a new target for cancer therapy. Therefore, the laboratory Medical University Of Chongqing School of medicine in VGSCs channel protein as the target, the use of 3D quantitative structure-activity relationship model structure The software to set up the drug binding model, designed and synthesized a series of small molecules with VGSCs channel protein ligand binding activity of novel sodium channel amine ligand (Sodium channels amine ligands, SCALs) a total of 15 species, which were named S0103, S0132, S0135, S0142, S0143, S0152, S0154, S0156, S0158. S0160, S0161, S0163, S0165, S0205, S0307. series of small molecule ligands by combining with VGSCs, blocking the channel, so as to play the role of sodium ion channel blockers inhibit the metastasis of prostate cancer. In this study, 15 kinds of SCALs as screening compounds, with prostate cancer as the research object, a lot of artificial expression evaluation the synthesis of SCALs on the growth of prostate cancer.VGSCs channel protein Nav1.6 and Nav1.7 PC3 and LNCa P in prostate cancer cells, the expression level of the m RNA 6-27 times higher than normal or hyperplastic prostate tissue (P0.05), which May be a potential prostate cancer diagnostic markers and therapeutic targets. This study uses S0154 and S0161 intervention in PC3 cells, the expression of Nav1.6 and Nav1.7 protein by Western blotting assay in cells, to observe the effect of SCALs on sodium channel protein. The tumor cells had distant metastasis is a complex physiological process. The need to reduce cell adhesion, invasion and increase of cell activity ability, proteolysis, and resistance to apoptosis by inhibiting.VGSCs blocker multiple tumor cell invasion and metastasis play an anti-tumor effect. Matrix metalloproteinases (Matrix, metalloproteinases, MMPs) is closely related to the intracellular signaling molecules and cancer metastasis, tumor cell MMPs secretion. Through the degradation of basement membrane and cell migration and invasion to other organizations, realize the transfer of cancer. About MMP-2 and MMP-9's research is in a large class of MMPs Further, the transfer of over expression of MMP-2 or MMP-9 increased incidence, inhibit the expression of MMP-2 or MMP-9 reduced the incidence of metastasis. This study uses S0154 and S0161 to intervene by PC3 cells, the expression of MMP-2 and MMP-9 protein was detected in cells, observe the effect of SCALs on metastasis of PC3 cells at the molecular level. At the same time. We also use BALB/c, nu/nu established PC3 cell xenografts in nude mice model with S0154 or S0161 (DMSO control) intervention xenograft in nude mice, by monitoring the changes of body weight and tumor size, S0154 and S0161 and evaluate the in vivo effects of PC3 on the growth of transplanted tumor. Methods: 1. using MTT at the beginning of experiment screen 15 SCALs of 3 strains of prostate cancer cells (PC3, LNCa, P, DU145) affected the growth ability; 2. using the expression of sodium ion probe assay by S0154 and S0161 PC3 cells in Na+, Western blottin G technique to detect the expression of Nav1.6 and Nav1.7 stem cells PC3 protein prognosis; 3. using flow cytometry to detect the compound after the intervention of PC3 cell cycle, the expression of Western blotting detected by cycle related proteins; 4. using flow cytometry to detect the S0154 and S0161 intervention PC3 cell apoptosis; 5. Transwell cell the effect of detection of S0154 and S0161 on the invasion ability of PC3 cells, the expression of Western blotting in detecting MMP-2 and MMP-9 protein; 6. by scratch assay and S0161 S0154 on the migration of PC3 cells; 7. to establish the xenograft model of prostate cancer PC3, effects of S0154 and S0161 on the growth of transplanted tumor in nude mice experiments. Results: 1.15 kinds of synthetic SCALs, S0154 and S0161 inhibit prostate cancer cell lines PC3, DU145 and LNCa P growth is the most significant, IC50 value in 10.51-26.60 M (S0 154), 5.07-11.92 M (S0161); 2.S0154 and S0161 increased the intracellular PC3 concentration of Na+, inhibiting the expression of sodium channel in PC3 cell protein Nav1.6 and / or Nav1.7; 3.S0154 and S0161 through the down-regulation of G2/M protein CDK1 and Cyclin in the key period of D1, thereby inducing PC3 cell cycle arrest in G2/M period; 4.20 M S0154 can promote the apoptosis of PC3 cells, but S0161 has no obvious effect on the apoptosis of PC3 cells; 5.S0154 and S0161 downregulated the expression of matrix metalloproteinases PC3 cells in MMP-2 and / or MMP-9 protein; also significantly inhibited PC3 cell invasion (Transwell assay), compared with the control group. That there is a significant difference between the 2.5 M, 10 M S0154 can inhibit the cell invasion of 95%, 10 M S0161 can completely inhibit the invasion of PC3 cells; 6. scratch experiments showed that S0154 and S0161 can inhibit PC3 cell migration, but the effect of As 7.S0154 and S0161 significantly inhibited the invasion; PC3 can significantly inhibit tumor growth in nude mice, the S0161 intervention for 2 weeks, at the end of the experiment, the tumor growth was inhibited by 50.4%, but its safety is better than S0154. in the experimental conclusion: 15 kinds of SCALs, S0154 and S0161 mainly through inhibition of CDK1 and Cyclin D1 protein the expression of G2/M induced by cycle arrest, inhibit the growth of prostate cancer in vitro and in vivo effect; through down-regulation of MMP-2 and / or MMP-9 expression inhibits tumor cell invasion and metastasis inhibited the activity of.S0154 and S0161 can inhibit the growth and invasion of tumor cells, inhibit the growth of transplanted tumor of prostate cancer, prostate cancer chemotherapy drug potential.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.25

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本文編號(hào)：1387032