【作者】 王俊玲；

【导师】 杨胜利； 郑乃刚；

【作者基本信息】 郑州大学 ， 医学免疫学， 2011， 硕士

【摘要】 背景与目的食管癌的发生发展是多基因、多阶段、多途径共同作用的结果,但是其确切机制至今尚不完全清楚。槲皮素(quercetin)是具有多种生物活性的植物类黄酮的最有效成分之一。国内外关于槲皮素生物学作用的研究很多,它具有抗肿瘤、抗炎症、抗过敏等生物学功能。丁酸钠(sodium butyrate, SB)是一种短链脂肪酸类的组蛋白脱乙酰化酶抑制物,是人体最基本的生理性去乙酰化酶抑制因子。众多研究证实,SB对多种肿瘤细胞具有抑制增殖、诱导分化和促进凋亡的作用。本研究拟应用纳米脂质体槲皮素(nanoliposomal quercetin, nLQ)和SB,观察其单独和联合用药分别对人食管癌EC9706细胞增殖和凋亡的影响,对抑癌基因p16、p21walf1的表达的影响以及对DNMT1, HDAC1, NF-κB, Cyclin D1, Caspase 3和E-cadherin表达的调节。旨在探讨nLQ和SB在人食管癌EC9706细胞增殖和凋亡中相互作用及其作用机理。IP-10是干扰素-γ(IFN-γ)诱导产生的分子量为10KDa的蛋白质,具有前炎症因子(pro-inflammatory factor)和趋化因子(chemokine)的效应。有研究证实,IP-10不但吸引炎症细胞趋向病灶,而且尚可促进角朊细胞增生。本研究检测nLQ和IP-10单独和联合作用后对人食管癌EC9706细胞增殖的影响,以初步揭示nLQ和IP-10在人食管癌EC9706细胞增殖中相互作用及其作用机制。方法1.MTT方法检测细胞增殖的抑制率(1)检测nLQ和SB单独、联合应用对EC9706细胞增殖的影响实验设4组：nLQ组(20μmol/L,40μmol/L,60μmol/L); SB组(1mmol/L, 2mmol/L,4mmol/L);联合药物组(40μmol/L nLQ+2mmol/L SB);不加药物的空白对照(Control)组。各组细胞培养48h。(2)检测nLQ和IP-10单独、联合应用对EC9706细胞增殖的影响实验设4组：nLQ组(20μmol/L,40μmol/L,60μmol/L); IP-10组(10ng/ml, 20ng/ml,40ng/ml);联合药物组(40μmol/L nLQ+20ng/ml IP-10);不加药物的空白对照(Control)组。各组细胞培养48h。2.细胞凋亡(TUNEL)检测实验分四组,nLQ组(40μmol/L)、SB组(2mmol/L)、联合组(40μmol/L nLQ+2mmol/L SB)及不加药物的空白对照(Control)组。4%多聚甲醛固定各组细胞标本,5μg/ml蛋白酶K 37-C消化8min,4%多聚甲醛后固定5min,各标本加TdT孵育液(0.6μl TdT,0.6μl biotion-16-dUTP,16μl TdT缓冲液)共17.2μl,4-C湿盒过夜。次日加pH7.5的Tris-Cl 1:500稀释碱性磷酸酶标记的链霉亲和素(SA-AP),37℃孵育20min, BCIP/NBT显色,37℃孵育30min,镜下观察,水洗终止反应。同时设以PBS代替TdT孵育液的阴性对照。3.免疫细胞化学单染或双染色(1)标本分两大组①.nLQ和SB处理组：实验分4小组,nLQ组(40μmol/L)、SB组(2mmol/L)、联合组(40μmol/L nLQ+2mmol/L SB)和对照(Control)组。②.nLQ与IP-10组：实验分4小组,nLQ组(40μmol/L)、IP-10组(20ng/ml)、联合组(40μmol/L nLQ+20ng/ml IP-10)和对照(Control)组。(2)免疫细胞化学双染色各组细胞标本经4%多聚甲醛常规固定、吹干及Triton X-100透化处理,用0.3%H202和5mM左旋咪唑先后处理以抑制内源性过氧化物酶(HRP)和内源性碱性磷酸酶(AP),热修复5min,以正常山羊血清封闭,加一抗工作液：p16单抗与DNMT1多抗,p16单抗与HDAC1多抗,p21walf1单抗与DNMT1多抗,p21walf1单抗与HDAC1多抗,4℃过夜。次日以HRP-抗小鼠IgG 37℃孵育30min,DAB底物显棕色,洗标本后再加AP-抗兔IgG或HRP-抗羊IgG,37℃孵育30min,AP底物的NBT-BCIP显蓝色或HRP的底物AEC显红色。同时进行PBS代替一抗的空白对照处理。(3)免疫细胞化学单染色①. nLQ、SB单独和联合处理的细胞标本常规固定、吹干及透化,应用SP试剂盒,检测NF-κB, Cyclin D1, E-cadherin及Caspase 3的表达。同时进行PBS代替一抗的空白对照处理。①. nLQ、IP-10单独和联合处理的细胞标本常规固定、吹干及透化,应用SP试剂盒,检测NF-κB及Cyclin D1的表达。同时做PBS代替一抗的空白对照处理。4.免疫印迹对于nLQ、SB单独和联合处理的细胞,nLQ、IP-10单独和联合处理的细胞,分别用Cellytic M提取总蛋白质,检测蛋白浓度,进行SDS-PAGE电泳,转膜,对DNMT1, NF-κB, HDAC1, Cyclin D1, p21 walf1和p16(以β-Actin作为内参照)进行免疫印迹定性及半定量检测。5.统计学分析采用SSPS16.0软件进行统计学分析,对各组细胞标本免疫化学双染信号计数的比值,以卡方检验进行分析,各组间比较时以α=0.0083为显著性水准；应用图像分析仪扫描各免疫细胞化学单染色信号和免疫印迹图像条带的灰度值,各印迹信号数值为各主带扫描灰度值与各β-Actin扫描灰度值的比值,正态分布的数据以均数±标准差(x±s)表示,以单因素方差分析进行比较,α=0.05为显著性水准。结果1.MTT实验(1)nLQ及SB对EC9706细胞的增殖呈现抑制效应,且抑制呈剂量依赖性,各组间皆有显著性差异(P<0.01)。且联合组与单40μmol/L nLQ或单2mmol/LSB组相比作用更强(P<0.01)。(2)IP-10对EC9706细胞呈现促增殖的剂量依赖性(P<0.01),nLQ拮抗IP-10的促生长作用,但联合组与对照组相比,差异无统计学意义(P>0.05)。2.凋亡检测(TUNEL)人食管癌EC9706细胞中,TUNEL凋亡信号呈蓝紫色,凋亡细胞中发生胞核固缩,或呈新月状位于细胞周缘,部分凋亡细胞尚可见凋亡小体。C组,nLQ组,SB组和nLQ+SB组细胞凋亡率分别为4.3%,23.6%,22.3%,43.8%。与对照组相比,nLQ组,SB组和联合组的细胞凋亡率差异均有统计学意义(P<0.01),且联合组与nLQ组或SB组相比差异显著(P<0.01)。3.免疫细胞化学染色结果(1)nLQ和SB处理的细胞①.与对照组相比,各药物组p16和p21walfl表达的棕色信号均增强,DNMT1/HDAC1表达的蓝色/红色信号均减弱(P<0.0083)；且联合药物组比单nLQ和单SB组效应均显著(P<0.0083)。②.与对照组相比,各药物组的E-cadherin和Caspase 3的免疫反应性(immunohistochemical reactivity, IR)信号增强,Cyclin D1和NF-κB的IR信号减弱(P<0.01)；且联合药物组比各单药物组效应更显著(P<0.01)。(2)nLQ与IP-10处理的细胞①.与对照组相比,单nLQ组p16和p21walf1表达的棕色信号均增强,DNMT1和HDAC1表达均的蓝色/红色信号减弱(P<0.0083),单IP-10组的p16和p21walf1表达均减弱,DNMT1和HDAC1表达均增强(P<0.0083)。nLQ+IP-10组与单IP-10组相比差异显著(P<0.0083)。②.与对照组相比,nLQ组细胞的NF-κB和Cyclin D1的IR信号均减弱,(P<0.01),而IP-10组的IR信号均增强(P<0.01)。nLQ+IP-10组与单IP-10组相比差异显著(P<0.01)。4.免疫印迹(1)nLQ和SB处理组：与对照组相比,各药物组HDAC1, NF-κB, Cyclin D1的印迹信号减弱,p21walf1和p16的免疫印迹信号增强,差异均显著(P<0.01)；且联合加药组与单加药组之间比较差异亦显著(P<0.01)；关于DNMT1的印迹信号,nLQ组和联合组的均减弱(P<0.01),SB组的差异无统计学意义(P>0.05)。(2)nLQ与IP-10处理组：与对照组相比,IP-10组HDAC1, NF-κB, Cyclin D1的印迹信号增强,p21walf1和p16的印迹信号减弱(P<0.01)；nLQ组HDAC1,NF-κB, Cyclin D1的印迹信号减弱,p21walf1和p16的印迹信号增强(P<0.01)。DNMT1的印迹信号,IP-10组的差异不显著p>0.05),nLQ组的减弱有统计学意义(P<0.01)。且IP-10组与联合药物组组之间比较差异亦具有统计学意义(P<0.01)。结论1.nLQ和SB单独和联合应用对人食管癌EC9706细胞增殖均有抑制作用,且二者联合抑制作用更显著。二者均可通过下调DNMT1和HDAC1的活性而上调抑癌基因p16、p21walf1的表达；还可下调NF-κB, Cyclin D1,上调E-cadherin、Caspase 3的表达；提示两者通过上述机制而抑制癌细胞增殖、诱导癌细胞凋亡和逆转癌细胞的恶性,且两者的联合效应更显著。2.IP-10呈现促EC9706细胞增殖的效应。其机制可能是IP-10可通过上调DNMT1和HDAC1的活性而下调抑癌基因p16、p21walf1的表达,上调NF-κB, Cyclin D1表达,进而促进癌细胞增殖。而nLQ对IP-10呈现的拮抗效应。nLQ对IP-10的抑制效应可被视为其抑制肿瘤细胞增殖和促进其凋亡的机制之一。更多还原

【Abstract】 Background and AimThe initiation and progression of esophageal cancer is the result of multiple factors, multiple genes and multiple pathways coaction. Quercetin is one of most effective flavonoid polyphenols. It exhibits anti-allergic, anti-inflammatory and anti-diabetic effects, and has been proposed to be a potential anti-neoplastic agent. Sodium butyrate (SB) as the HDAC inhibitor is an example of the class of short-chain fatty acids and it is a basic human physiological deacetylase inhibitor. Many studies have confirmed that SB can inhibit a variety types of tumor cells proliferation, induce cells differentiation and promote cells apoptosis. The present study was designed to oberseved the effects of nanoliposomal quercetin(nLQ), SB and nLQ+SB on EC9706 cells proliferation and apoptosis, and was detected to regulate the expressions of p16, p21walf1, DNMT1, HDAC, NF-κB, Caspase 3, Cyclin D1 and E-cadherin. Furthermore, it was to explore the interaction mechanism between nLQ and SB. The human cytokine interferon-inducible protein 10 (IP-10) is a member of the-C-X-C- chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon-γ(IFN-γ). The reaserch was to detected the effects of nLQ, IP-10 and nLQ+IP-10 on EC9706 cell proliferation, and to explore the interaction mechanism between nLQ and IP-10.Methods1. Growth suppression rate detected by MTT assay(1) The cultured EC9706 cells were divided into four groups:①. nLQ group:20μmol/L,40μmol/L,60μmol/L.②. SB group:1mmol/L,2mmol/L,4mmol/L.③. Combined group:40μmol/L nLQ+2mmol/L SB.④. Control group:treated with no any drug for 48h.(2) The cultured EC9706 cells were divided into four groups①. nLQ group:20μmol/L,40μmol/L,60μmol/L.②. IP-10 group:10ng/ml,20ng/ml,40ng/ml.③. Combined group:40μmol/L nLQ+20ng/ml IP-10.④. Control group:treated with no any drug for 48h.2. Apoptotic rateThe cultured respective groups of EC9706 cells were examined with TUNEL assay in control group; nLQ group treated with 40μmol/L; the SB group treated with 2mM; the combined group,40μmol/L nLQ+2 mmol/L SB.3. Immunocytochemistry staining(1) Double immunocytochemical stainingThe monoclonal anti-p16 or anti- p21walf1 combined with polyclonal anti-DNA methyltransferase 1 (DNMT1) or anti-histone deacetylasel (HDAC1) were used as the primary antibodies. The HRP/AP (alkaline phosphatase)-conjugated monoclonal IgG/polyclonal IgG was used as the secondary antibody. Finally, the brown signal developed under the substrate of DAB of HRP; while the red signal, as the substrate of AEC of HRP; meanwhile, the dark blue signal developed under NBT-BCIP as the AP substrate.(2) Single immunocytochemical stainingThe expression level and location in each group of EC9706 cells were immunostained on the 4% paraformaldehyde fixed slides. Respective E-cadherin, Caspase3, NF-κB and Cyclin D1 primary antibodies, followed by the SP kits were incubated on the slides. At the same time the primary antibody substituted for PBS was set as the negative control.4. Western blottingThe total protein was extracted from each group of EC9706 cells and the SDS-PAGE was performed onto the nitrocellulose membrane (NCM). The expressions of DNMT1, HDAC1, NF-κB, Cyclin D1, p21walf1 and p16 were determined by Western blotting (β-actin as internal reference).5. Statistical analysisThe data obtained were statistically treated with SPSS 16 soft ware. The data of double immunocytochemical staining were analyzed with chi-square test, andα=0.0083 was considered as significance level.The data shown as (x+s) of immunocytochemical staining and immunoblotting were analyzed with chi-square test and ANOVA among groups, and a=0.05 was considered as significance level.Results1. MTT assay(1) The cell growth suppression by nLQ and SB respectively showed in dose-dependent manner (P<0.01). The nLQ+SB group was more significant than the single drug group(P<0.01).(2) The cell growth suppression by nLQ or the cell growth facilitation by IP-10 showed in dose-dependent manner(P<0.01). However, there was no significant difference between the control group and nLQ+IP-10 group (P>0.05).2. ApoptosisThe TUNEL apoptotic signal in bluish-violet color showed in the cells. Apoptotic rates of control group, nLQ group, SB group and nLQ+SB group were 4%, 23.6%,22.3%,43.8% respectively.Compard with the control group, the apoptotic rate of each drug group was significant (P<0.01).And there was significant different between the nLQ+SB group and singular drug group(P<0.01).3. Immunohistochemical staining(1) The cells treated with nLQ+SB①. Double immunocytochemical staining:When compared with the control group up-regulation of pl6/p21walfl gene expression in brown signals and down-regulation of DNMT1/HDAC1 in blue/red signals shown in the nLQ group, SB group and nLQ+SB group were significant (P<0.0083). There was significant difference between the nLQ+SB group and singular drug group (P<0.0083).②. Single immunocytochemical staining:Immunohistochemical reactivity (IR) appeared as brownish granules. When compared with the control group, the enhanced E-cadherin IR signal and attenuated Cyclin D1 and NF-κB IR signals in the drug group were significant (P<0.01). The nLQ+SB group exhibited more significant than the singular drug group (P<0.01).(2) Cells treated with nLQ and IP-10①. Double immunocytochemical staining:The down-regulated DNA methylation and up-regulated histone acetylation of p16/p21walf1 displayed in the 40μM nLQ group in comparison with the C group (P<0.0083). The epigenetic modification effect induced by IP-10 could be counteracted by that of nLQ+IP-10 group (P<0.0083).②. Single immunocytochemical staining:The immunohistochemical reactivity (IR) appeared as brownish granules. Compared with the control group, the Cyclin D1 and NF-κB IR signals were attenuated in the nLQ group (P<0.05), while those IR signals in the IP-10 group were enhanced (P<0.05). There was no significance between the control group and nLQ+IP-10 group (P>0.05), but There was significance between the IP-10 group and nLQ+IP-10 group4. Immunoblotting(1) The cells treated with nLQ and SB Compared with the control group, the immunoblotting signals of p21walf1 and p16 were enhanced(P<0.01), and those of HDAC1, NF-κB, Cyclin D1 were decreased in each group (P<0.01). There was significant difference between the drugs nLQ+SB group and the single drug group (P<0.01). However, the immunoblotting signals of DNMTlwas not significant in the SB group(P>0.05).(2) The cells treated with nLQ and IP-10 The immunoblotting showed that the expressions of HDAC1, NF-κB and Cyclin D1 were down-regulated by nLQ (P<0.05); while there was contrary effect induced by IP-10. The immunoblotting signals of DNMT1 was not significant in the IP-10 group(P>0.05). There was significant difference between the nLQ+IP-10 group and IP-10 group (P<0.01).Conclusion1. The nLQ or SB can inhibit proliferation of EC9706 cells, and the inhibition effect can be enhanced by nLQ combined with SB. Both of them can up-regulate the expression of p16/p21walf1 via down-regulating the activity of DNMT1 and HDAC1, and down-regulate expression of NF-κB or Cyclin D1. It may suggest that the nanaoliposomal quercetin combined with sodium butyrate may inhibit the cancer cell proliferation and reverse its malignancy.2. IP-10 can promote EC9706 cell proliferation. The mechanism may be that IP-10 can down-regulate the expression of p16/p21walf1 via up-regulating the activity of DNMT1 and HDAC1, and can up-regulate the expression of NF-κB, Cyclin D1. However, The nLQ can counteract the effect of IP-10 in certain extent.更多还原