亚洲免费av电影一区二区三区,日韩爱爱视频,51精品视频一区二区三区,91视频爱爱,日韩欧美在线播放视频,中文字幕少妇AV,亚洲电影中文字幕,久久久久亚洲av成人网址,久久综合视频网站,国产在线不卡免费播放

        ?

        雙亞芐基哌啶與蛋白酶體抑制劑下調(diào)ADRM1表達(dá)誘導(dǎo)HL60細(xì)胞凋亡

        2019-10-30 02:50:24鄭曉輝王玉孝隋華秀
        中國當(dāng)代醫(yī)藥 2019年22期
        關(guān)鍵詞:凋亡存活率

        鄭曉輝 王玉孝 隋華秀

        [摘要]目的 探討雙亞芐基哌啶(RA190)與蛋白酶體抑制劑(MG132)對HL60細(xì)胞的作用及機制。方法 以終濃度0.1、0.2、0.3、0.4、0.5、0.8、1.5、1.6、3.2 μmol/L RA190或MG132處理HL60細(xì)胞為實驗組,不加藥HL60細(xì)胞為對照組,無細(xì)胞完全培養(yǎng)基為空白組,各組培養(yǎng)24 h,CCK8法檢測各組細(xì)胞存活率。以1 μmol/L RA190或MG132處理24 h HL60細(xì)胞為實驗組,雙染法流式細(xì)胞術(shù)分析各組細(xì)胞凋亡率;Western blot檢測細(xì)胞內(nèi)黏附調(diào)節(jié)分子1(ADRM1)表達(dá)水平。結(jié)果 HL60細(xì)胞傳代培養(yǎng)1年后,MG132實驗組的細(xì)胞存活率高于1年前,也高于RA190實驗組;RA190與MG132實驗組的凋亡率顯著高于對照組;RA190實驗組的早期凋亡率顯著高于MG132實驗組;此時,RA190與MG132實驗組內(nèi)ADRM1蛋白表達(dá)灰度值比低于對照組,差異均有統(tǒng)計學(xué)意義(P<0.05)。結(jié)論 傳代培養(yǎng)時間延長可提高HL60細(xì)胞對MG132的耐藥性;RA190與MG132均可下調(diào)HL60細(xì)胞內(nèi)ADRM1蛋白表達(dá),誘導(dǎo)其凋亡;RA190的誘凋亡效果優(yōu)于MG132。

        [關(guān)鍵詞]雙亞芐基哌啶;HL60細(xì)胞;蛋白酶體抑制劑;黏附調(diào)節(jié)分子1;存活率;凋亡

        [中圖分類號] R329.2+5? ? ? ? ? [文獻(xiàn)標(biāo)識碼] A? ? ? ? ? [文章編號] 1674-4721(2019)8(a)-0004-04

        [Abstract] Objective To investigate the effect and mechanism of Bis-Benzylpiperidine (RA190) and proteasome inhibitor (MG132) on HL60 cells. Methods HL60 cells were treated with the final concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.5, 1.6 and 3.2. μmol/L RA190 or MG132 as the experimental group, and HL60 cells control group without medication and blank group with acellular complete medium were designed, each group was cultured for 24 hours. CCK8 assay was used to detect the cell survival rate. Apoptosis rate of HL60 cells treated with 1 μmol/L RA190 or MG132 for 24 hours as experimental group was detected by flow cytometry with double staining. Western blot was used to detect the expression of intracellular adhesion regulating molecule 1 (ADRM1). Results One year after subculture of HL60 cells, the cell survival rate of MG132 experimental group was higher than that of one year ago, and also higher than that of RA190 experimental group; the apoptotic rate of RA190 and MG132 experimental group was significantly higher than that of control group, and the early apoptotic rate of RA190 experimental group was significantly higher than that of MG132 experimental group, at this time, the gray value ratio of ADRM1 protein expression in RA190 and MG132 experimental group was lower than that of control group, and the differences were statistically significant (P<0.05). Conclusion Prolonged subculture time can increase the drug resistance of HL60 cells to MG132; both RA190 and MG132 can down-regulate ADRM1 protein level in HL60 cells, induce cell apoptosis. RA190 is superior to MG132 in inducing apoptosis effect.

        [Key words] Bis-Benzylidine Piperidon; HL60 cell; Proteasome inhibitor; Adhesion regulating molecule 1; Survival rate; Apoptosis

        惡性腫瘤自主生長、逃避凋亡、侵襲及轉(zhuǎn)移的特征是其內(nèi)部基因突變或表達(dá)異常所致。靶向異?;蛑委熓钱?dāng)前研究熱點。ADRM1蛋白是26S蛋白酶體上的泛素受體,介導(dǎo)NF-κB、TGF-β1受體等底物蛋白降解,在肺癌、肝癌、急性白血病等多種癌細(xì)胞中呈過表達(dá);其表達(dá)下調(diào)顯著促進癌細(xì)胞凋亡[1-7]。因此,ADRM1可能成為腫瘤治療靶點。

        傳統(tǒng)的蛋白酶體抑制劑(MG132)可抑制泛素蛋白酶體活性,抵消17β-雌激素對原代神經(jīng)元內(nèi)Cav1.2蛋白下調(diào)作用[8],抑制病毒復(fù)制[9],誘導(dǎo)Burkitt淋巴瘤、急性髓系白血病(acute myeloid leukemia,AML)、肝癌等癌細(xì)胞凋亡[6,10-12],協(xié)同促進全反式維甲酸誘導(dǎo)GTF2I-RARA融合基因陽性的HL60細(xì)胞分化[13];還可通過Smad信號通路抑制TGF-β誘導(dǎo)的炎癥反應(yīng),抑制腎間質(zhì)纖維化[14]。近幾年發(fā)現(xiàn)的靶向ADRM1藥物雙亞芐基哌啶(RA190)可快速觸發(fā)多泛素蛋白累積,誘導(dǎo)對硼替佐米耐藥的多發(fā)性骨髓瘤細(xì)胞凋亡,抑制卵巢癌、肝癌等多種癌細(xì)胞增殖[3,15-20]。那么,RA190與MG132在AML中作用效果如何?本實驗擬通過觀察髓系白血病細(xì)胞株HL60在RA190或MG132作用下的存活率和凋亡率,比較兩藥對HL60細(xì)胞的作用及與ADRM1蛋白表達(dá)的相關(guān)性,為臨床采用靶向ADRM1治療AML方案奠定基礎(chǔ)。

        1 材料與方法

        1.1 主要材料與儀器

        人急性粒細(xì)胞白血病部分分化型細(xì)胞HL60為本實驗室保存;MG132(Sigma),RA190、CCK8(MCE),Western blot抗體(CST公司),RPMI 1640(Corning),胎牛血清(Gemini);酶標(biāo)儀、高電流電泳儀PowerPac HC(Bio-rad公司);流式細(xì)胞儀EPICSXL (貝克曼庫爾特公司);垂直電泳槽VE-180(上海天能科技有限公司);快速Western Blot儀器MA01821(Millipore)。

        1.2 方法

        1.2.1 CCK8法? 設(shè)3復(fù)孔,在96孔板中接種100 μl細(xì)胞懸液,終濃度1×105/ml,設(shè)不同濃度RA190與MG132藥物實驗組(終濃度:0.1、0.2、0.3、0.4、0.5、0.8、1.5、1.6、3.2 μmol/L)、對照組(不加藥)和空白組(只含完全培養(yǎng)基),于37℃、5% CO2培養(yǎng)箱培養(yǎng)24 h后,加入10 μl CCK-8溶液,孵育3 h;酶標(biāo)儀測定450/630 nm處吸光度。實驗重復(fù)3次。細(xì)胞存活率=(實驗組-空白組)A450/630值/(對照組-空白組)A450/630值×100%。

        1.2.2 Annexin V FLUOS Staining Kit 流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡? 設(shè)2復(fù)孔,在6孔板中接種4 ml細(xì)胞懸液,終濃度1×106/ml,分別以1 μmol/L MG132和 RA190 作用 HL60細(xì)胞24 h。收集細(xì)胞,800 r/min離心5 min,棄上清,預(yù)冷的PBS洗2次,之后按Annexin V FLUOS Staining Kit試劑盒說明書操作,實驗重復(fù)3次。

        1.2.3 Western blot檢測細(xì)胞內(nèi)蛋白表達(dá)? 設(shè)2復(fù)孔,細(xì)胞接種與分組加藥操作同“1.2.2”。收集各組細(xì)胞,PBS洗2次,加入RIPA細(xì)胞裂解液(含1 mmol/L PMSF、1 mmol/L正釩酸鈉、10 mg/L抑肽酶)提取總蛋白,取40 μg總蛋白進行Western blot實驗。操作步驟詳見《分子克隆實驗指南》(4版)。實驗重復(fù)3次。

        1.3 統(tǒng)計學(xué)方法

        數(shù)據(jù)應(yīng)用GraphPad Prism 5軟件處理,正態(tài)分布的計量資料用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,組間比較采用t檢驗,以P<0.05 為差異有統(tǒng)計學(xué)意義。

        2結(jié)果

        2.1不同濃度RA190、MG132作用下HL60細(xì)胞存活率的比較

        2.1.1傳代1年前后HL60細(xì)胞在MG132作用下存活率的比較? 設(shè)終濃度為0.1、0.2、0.3、0.4、0.5、0.8、1.5、1.6、3.2 μmol/L MG132實驗組和不加藥對照組,傳代培養(yǎng)1年前后HL60細(xì)胞存活率見表1。MG132終濃度為0.1 μmol/L時,細(xì)胞存活率無顯著變化;MG132終濃度為0.2~1.5 μmol/L時,同一MG132終濃度下,1年后HL60細(xì)胞存活率均高于1年前,差異有統(tǒng)計學(xué)意義(P<0.05)。MG132對1年后HL60細(xì)胞的半抑制濃度(IC50)約0.88 μmol/L高于1年前的約0.42 μmol/L。

        2.1.2 HL60細(xì)胞在RA190與MG132作用下存活率的比較? 設(shè)終濃度為0.2、0.4、0.8、1.6、3.2 μmol/L RA190與MG132實驗組和不加藥對照組。在0.2~1.6 μmol/L,RA190實驗組各濃度下存活率均低于MG132實驗組,差異有統(tǒng)計學(xué)意義(P<0.05);RA190對HL60細(xì)胞的IC50約0.68 μmol/L低于MG132的約0.88 μmol/L。

        3討論

        從遺傳上看,癌都是由一個失去了增殖控制的細(xì)胞發(fā)展而來;人體每天都有幾十億個細(xì)胞進行分裂,任何一個細(xì)胞都有可能由遺傳成分的改變而癌變。細(xì)胞的惡性轉(zhuǎn)化需要發(fā)生多個遺傳改變,因此腫瘤發(fā)生是一個漸進式的過程,涉及多級反應(yīng)和突變的積累。癌細(xì)胞不斷積累突變,賦予突變細(xì)胞新的特性,使癌細(xì)胞更具危險性,即惡性度提高,從而越來越不受體內(nèi)調(diào)節(jié)機制的控制,并逐漸轉(zhuǎn)移侵襲正常組織。本實驗中,MG132對1年后HL60細(xì)胞的IC50高于1年前,可能是由于HL60細(xì)胞在長時間的分裂過程中積累突變,使細(xì)胞惡性度提高,提高了對MG132的耐藥性,這提示抗腫瘤藥治療效果的臨床前實驗應(yīng)選擇對應(yīng)階段患者的腫瘤細(xì)胞為實驗對象。

        RA190與MG132實驗組的早期與晚期凋亡率均高于對照組,RA190實驗組的早期凋亡率高于MG132實驗組,差異均有統(tǒng)計學(xué)意義(P<0.05),這提示RA190與MG132均可促進HL60細(xì)胞凋亡,前者的作用效果優(yōu)于后者。此時,兩實驗組ADRM1蛋白表達(dá)灰度值比低于對照組,說明RA190與MG132通過降低HL60細(xì)胞內(nèi)ADRM1蛋白表達(dá)誘導(dǎo)癌細(xì)胞凋亡,這提示靶向ADRM1蛋白表達(dá)可應(yīng)用于臨床AML的治療。本實驗不足之處在于只用了1株細(xì)胞且未做藥物對ADRM1過表達(dá)或沉默白血病細(xì)胞的作用效果比較,結(jié)果較缺乏說服力。

        [參考文獻(xiàn)]

        [1]Zhao X,Chao Y,Chen P,et al.hRpnl3,a newly identified component of the 19S particle,regulates proliferation,differentiation,and function in the human osteoblast-like cell line MG63:role of hRpnl3 in osteoblasts[J].J Physiol Biochem,2012,68(1):129-139.

        [2]Williams GH,Stoeber K.The cell cycle and cancer[J].J Pathol,2012,226(2):352-364.

        [3]Jiang RT,Yemelyanova A,Xing D,et al.Early and consistent overexpression of ADRM1 in ovarian high-grade serous carcinoma[J].J Ovarian Res,2017,10(1):53.

        [4]Zheng X,Guo Y,Chen Y,et al.Knockdown of adhesion-regulating molecule 1 inhibits proliferation in HL60 cells[J].Acta Haematol,2015,134(2):88-100.

        [5]Chen Y,F(xiàn)u D,Xi J,et al.Expression and clinical significance of UCH37 in human esophageal squamous cell carcinoma[J].Dig Dis Sci,2012,57(9):2310-2317.

        [6]鄭曉輝,黃家福,徐淑娟,等.蛋白酶體抑制劑 MG132 對急性髓系白血病細(xì)胞增殖與凋亡的影響[J].中國藥理學(xué)通報,2019,3(35):327-334.

        [7]Chen H,Gao F,He M,et al.Long-read rna sequencing identifies alternative splice variants in hepatocellular carcinoma and tumor-specific isoforms[J].Hepatology,2019[Epub ahead of print]

        [8]Lai YJ,Zhu BL,Sun F,et al.Estrogen receptor α promotes Cav1.2 ubiquitination and degradation in neuronal cells and in APP/PS1 mice[J].Aging Cell,2019,22:e12961.

        [9]Llamas-González YY,Campos D,Pascale JM,et al.A functional ubiquitin-proteasome system is required for efficient replication of New World Mayaro and Una Alphaviruses[J].Viruses,2019,11(4):E370.

        [10]Lee KH,Lee J,Woo J,et al.Proteasome inhibitor-induced IκB/NF-κB activation is mediated by Nrf2-dependent light chain 3B induction in lung cancer cells[J].Mol Cells,2018,41(12):1008-1015.

        [11]Wang L,Howell MEA,Sparks-Wallace A,et al.p62-mediated selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress[J].PLoS Pathog,2019,15(4):e1007541.

        [12]Kim HN,Park GH,Park SB,et al.Extracts from Sageretia thea reduce cell viability through inducing cyclin D1 proteasomal degradation and HO-1 expression in human colorectal cancer cells[J].J Proteomics,201,10(192):334-345.

        [13]Yan W,Li J,Zhang Y,et al.RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion,and inhibition of the ubiquitin-proteasome pathway contributes to the reversion of ATRA resistance[J].Cancer Cell Int,2019,19:84

        [14]Han L,Zhu B,Chen H,et al.Proteasome inhibitor MG132 inhibits the process of renal interstitial fibrosis[J].Exp Ther Med,2019,17(4):2953-2962.

        [15]Anchoori RK,Karanam B,Peng S,et al.A bis-benzylidine piperidone targeting proteasome ubiquitin receptor RPN13/ADRM1 as a therapy for cancer[J].Cancer Cell,2013,24:791-805.

        [16]Lu X,Zhou C,Li R,et al.Long noncoding RNA AFAP1-AS1 promoted tumor growth and invasion in cholangiocarcinoma[J].Cell Physiol Biochem,2017,42:222-230.

        [17]Song Y,Park PMC,Wu L,et al.Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma[J].Leukemia,2019[Epub ahead of print]

        [18]Anchoori RK,Jiang R,Peng S,et al.Covalent Rpn13-binding inhibitors for the treatment of ovarian cancer[J].ACS Omega,2018,3(9):11 917-11 929.

        [19]Yu GY,Wang X,Zheng SS,et al.RA190,a proteasome subunit ADRM1 inhibitor,suppresses intrahepatic cholangiocarcinoma by inducing NF-κB-mediated cell apoptosis[J].Cell Physiol Biochem,2018,47(3):1152-1166.

        [20]Lu X,Nowicka U,Sridharan V,et al.Structure of the Rpn13-Rpn2 complex provides insights for Rpn13 and Uch37 as anticancer targets[J].Nat Commun,2017,8:15 540.

        (收稿日期:2019-04-24? 本文編輯:許俊琴)

        猜你喜歡
        凋亡存活率
        園林綠化施工中如何提高植樹存活率
        損耗率高達(dá)30%,保命就是保收益!這條70萬噸的魚要如何破存活率困局?
        水產(chǎn)小白養(yǎng)蛙2年,10畝塘預(yù)計年產(chǎn)3.5萬斤,畝純利15000元!存活率90%,他是怎樣做到的?
        海南石斑魚明年或減產(chǎn)40%!魚苗存活率低,成魚賣不起價,石斑魚怎么了?
        普伐他汀對人胰腺癌細(xì)胞SW1990的影響及其協(xié)同吉西他濱的抑瘤作用
        細(xì)胞自噬與人卵巢癌細(xì)胞對順鉑耐藥的關(guān)系
        右美托咪定混合氯胺酮對新生大鼠離體海馬細(xì)胞凋亡的影響
        Livin和Survivin在卵巢癌中的表達(dá)及相關(guān)性研究
        雷帕霉素對K562細(xì)胞增殖和凋亡作用的影響
        科技視界(2016年5期)2016-02-22 19:03:28
        索拉非尼對胃癌細(xì)胞MGC80—3抑制作用實驗研究
        人妻无码人妻有码不卡| 亚洲国产日韩精品一区二区三区 | 中国午夜伦理片| 日韩内射美女人妻一区二区三区| 亚洲另类激情综合偷自拍图| 亚洲精品女优中文字幕| 文字幕精品一区二区三区老狼| 国产午夜福利不卡在线观看| 亚洲在AV极品无码天堂手机版| 亚洲av一区二区国产精品| av免费资源在线观看| 久久久久亚洲av无码专区喷水| 女人做爰高潮呻吟17分钟| 精品的一区二区三区| 亚洲国产成人av毛片大全| 国产大片内射1区2区| 日韩人妻无码一区二区三区久久99| 国产一区二区三区精品久久呦| 少妇高潮免费在线观看| 无码毛片内射白浆视频| 三级在线看中文字幕完整版| 三级国产女主播在线观看| 亚洲成人av在线播放不卡 | 在线涩涩免费观看国产精品| 久久青草伊人精品| 粉嫩的18在线观看极品精品| 国产激情久久久久影院小草| 久久久精品人妻一区二区三区蜜桃 | 无码人妻丰满熟妇区免费| 麻豆一区二区99久久久久| 骚片av蜜桃精品一区| 亚洲精品大全中文字幕| 亚洲日韩激情无码一区| 99久久精品国产一区二区蜜芽 | 深夜福利啪啪片| 中文幕无线码中文字蜜桃| 亚洲国产精品美女久久久| 麻豆精品国产av在线网址| 中文字幕精品久久久久人妻| 国产高清在线91福利| 青青草中文字幕在线播放|