线粒体质量控制在外源化学物引起坏死性凋亡中的作用

坏死性凋亡是一种可调控的、半胱氨酸天冬氨酸蛋白酶(Cysteine aspartic acid specific protease,Caspase)非依赖的程序性细胞死亡,相较于细胞凋亡,其主要特点是没有核固缩和气球样变,而出现的是线粒体肿胀和三磷酸腺苷(Adenosine triphosphate,ATP)耗竭、胞质肿胀和空泡形成,其最终引起质膜破裂和通透性改变,释放损伤相关分子,如白细胞介素1α、高国家自然科学基金(81573181,81472997);;福建省自然科学基金(2015J01344

蛋白磷酸酶2A调控细胞周期发挥抗肿瘤作用的研究进展

蛋白磷酸酶2A(PP2A)是真核细胞内广泛表达的丝氨酸/苏氨酸蛋白磷酸酶家族的主要成员,参与细胞周期、增殖分化及凋亡等过程中信号通路的去磷酸化调控。研究表明,PP2A不同亚基表达调控与人群肿瘤风险存在关联性;PP2A与细胞周期相关激酶相互作用,二者调控平衡的研究有助于致癌机制的探讨和靶向PP2A抗肿瘤作用的探索。本文对PP2A调控细胞周期的研究进行综述,着重阐述PP2A-B亚基经由细胞周期因子依赖性激酶1(CDK1)调控细胞周期发挥抑癌作用的机制,为肿瘤的化学预防和靶向干预提供理论依据。国家自然科学基金(81172705,81472997,81573181);; 973计划前期研究专项(2014CB560710);; 福建省自然科学基金项目(2014J 01372,2015J01344);; 厦门大学山海基金项目(2013SH007

蛋白磷酸酶2A在线粒体质量控制中的作用研究进展

蛋白磷酸酶2A(PP2A)是真核细胞内广泛表达的丝/苏氨酸蛋白磷酸酶家族成员之一,通过去磷酸化作用参与外源物诱导细胞毒性损伤和致癌过程中关键调控蛋白的可逆磷酸化,进而抑制绝大多数丝/苏氨酸激酶的活性。近年来,PP2A在线粒体质量控制(MQC)中的作用逐渐被认识,广泛参与线粒体生物发生、线粒体动态、线粒体氧化还原平衡和线粒体自噬等重要生物学过程,对拓展MQC相关蛋白翻译后修饰在以线粒体为核心枢纽的信号流中的研究具有重要意义。本文重点阐述PP2A介导的去磷酸化机制在MQC调控中的作用,为从翻译后修饰层面阐明外源物诱导线粒体关联性损伤和致癌作用或肿瘤相关疾病的分子机制、以及为人群暴露和危害标志物的筛查和靶向干预提供依据。国家自然科学基金(81573181,81773465,81874272

外源物诱导肝脏局部先天免疫反应的线粒体调控机制

肝脏作为体内代谢外源因素暴露最重要的器官之一,具有独特的血窦结构和丰富的免疫细胞亚群,构成了肝脏局部免疫微环境。其中,肝脏的先天免疫细胞既可通过清除病原体、抗原提呈作用参与宿主防御,还通过与肝实质细胞的相互作用参与外源因素介导的急性免疫反应、肝细胞毒性转归、慢性肝损伤以及肝致癌作用。线粒体作为细胞应激的关键靶细胞器,是整合肝脏局部免疫信号的分子互作平台,既通过线粒体质量控制精细调控细胞内的分子事件,也通过线粒体损伤相关分子模式介导不同细胞间通讯和交互作用调控免疫微环境的稳态。本文通过对参与肝脏局部免疫反应的先天免疫细胞亚群组成及其介导免疫反应级联的综述,阐述不同外源物诱导肝脏局部免疫的线粒体相关调控机制,为探讨经由肝脏局部免疫反应生物标志筛查和靶向干预以预防和控制肝脏损伤提供线索

The effect of low glucose and high lactic acid on the survival of Hela

目的:探讨低糖高乳酸环境对宫颈癌细胞生存的影响,以及对EGFR-mTOR通路的调节作用。方法:将Hela细胞培养于常糖(葡萄糖10mmol/L); 、低糖(葡萄糖3mmol/L)、高乳酸(葡萄糖10mmol/L,乳酸2.5mmol/L)、低糖高乳酸(葡萄糖3mmol/L,乳酸2.5mmol/; L)4种环境下,CCK-8法测定Hela细胞的生长抑制率,流式细胞术测定细胞周期。荧光实时定量PCR法检测EGFR和mTOR; mRNA水平表达。结果:与常糖组相比,高乳酸组的细胞抑制率显著升高(P<0.01),48h细胞G_1/G_0期比例显著升高(P<0.01),细胞; 凋亡率、EGFR和mTOR; mRNA表达水平均无变化。与常糖组相比,低糖组的细胞抑制率显著升高(P<0.01),48h细胞凋亡率显著降低(P<0.01),细胞各周期比例变化; 与常糖组无差异,EGFR表达水平降低(P<0.05)。与常糖组相比,低糖高乳酸组的细胞抑制率显著升高(P<0.01),但低于低糖组(P<0.01; )和高乳酸组(P<0.05),48h G_1/G_0期比例显著升高(P<0.01),细胞诱导凋亡率显著升高(P<0.01),EGFR和mTOR; mRNA表达水平均显著升高(P<0.01)。结论:Hela细胞在低糖高乳酸环境中的存活状况好于单纯低糖和单纯高乳酸环境,且伴随着EGFR和mTO; R基因表达水平上升。Objective:To explore the effect of the low glucose and high lactic acid; environment of survival of cervical carcinoma cells,and the regulation; of EGFR-mTOR signal way.Methods:Hela cells were cultured in four; conditions:normal glucose (glucose 10mmol/L),low glucose (glucose; 3mmol/L),high lactic acid (lactic acid 2.5mmol/L) and low glucose add; high lactic acid (glucose 3mmol/L,lactic acid 2.5mmol/L).Growth; inhibition rate of Hela cell was determined by CCK-8.Flow cytometry; (FCM)were performed to evaluate the cell cycle.The expression levels of; EGFR and mTOR mRNA were detected using real-time quantitative polymerase; chain reaction (real-time PCR).Results:Compared with those in regular; sugar environment,the cell growth inhibition rates were significantly; increased in high lactic acid environment(P<0.01).At 48h,the rate of; G_1/G_0 was significantly increased (P<0.01),while the apoptosis rate of; the cells and the expressions of EGFR and mTOR mRNA had no; change.Compared with those in regular sugar environment,the cell growth; inhibition rates were significantly increased in high lactic acid; environment,(P<0.01),while lower than those in low glucose; environment(P<0.01) and in high acid environment(P<0.05).The rates of; G_1,G_2 and S phase had no change.The expressions of EGFR mRNA was; reduced (P<0.05).The cell growth inhibition rates were significantly; increased in high lactic acid add low glucose environment (P<0.01).At; 48h,the apoptosis rate and the rate of G_1/G_0 were significantly; increased (P<0.01),while the EGFR and mTOR expression levels were also; increased (P<0.05).Conclusion:Hela in the low glucose add high lactic; acid survives better than those in low glucose and in high lactic acid; environment,while the expression of EGFR and mTOR is increased.泉州市科技计划立项项目; 国家面上项

低糖高乳酸环境下吉非替尼诱导HeLa细胞EGFR-TKI耐药的研究

目的探讨低糖高乳酸微环境对表皮生长因子酪氨酸激酶抑制剂(EGFR-TKI)抑制HeLa细胞的影响和可能机制。方法HeLa细胞在常糖(葡萄糖10; mmol/L)和低糖高乳酸(葡萄糖3 mmol/L +乳酸2.5 mmol/L)环境下培养,并分别给予2.67; mumol/L吉非替尼干预。采用CCK-8法测定HeLa细胞生长抑制率,流式细胞术检测细胞周期,荧光定量RT-PCR检测EGFR和mTOR; mRNA水平的表达。结果与常糖组比较,低糖乳酸组24 h和72 h细胞抑制率均显著升高(P < 0.01),48; h细胞抑制率略高于常糖组。与吉非替尼阴性组比较,常糖+吉非替尼组和低糖乳酸+吉非替尼组在24、48、72 h三个时点细胞抑制率均显著上升(P <; 0.01)。与常糖组相比,低糖乳酸组48 h细胞诱导凋亡率显著上升(P <; 0.01),低糖乳酸+吉非替尼组细胞诱导凋亡率较低糖乳酸组显著降低(P < 0.01)。与常糖组比较,低糖乳酸组存活细胞的EGFR和mTOR; mRNA表达水平上调(P < 0.05)。常糖+吉非替尼组的EGFR和mTOR mRNA水平均上调(P <; 0.05)。与低糖乳酸组比较,低糖乳酸+吉非替尼组的EGFR和mTOR mRNA上调水平有显著差异(P <; 0.01)。结论高乳酸低糖环境下吉非替尼可大幅度上调存活HeLa细胞EGFR和mTOR表达水平,可能是诱导HeLa细胞抵抗EGFR-TKI的机制; 。泉州市第一医院青年科研; 泉州市科技计划项目; 国家面上项

Pregnane X receptor involves in the effect of aflatoxin B1 on necroptosis in human normal L02 liver cells

目的初步探讨孕烷X受体(PXr)对黄曲霉毒素b1(Afb1)诱导肝细胞dnA损伤和坏死性凋亡的影响。方法采用已构建的PXr高表达l02-PXr和空白载体对照l02-P b细胞;实时荧光定量PCr(Q rT-PCr)检测细胞nr1I2和CyP3A4 M rnA水平改变;蛋白免疫印迹(WESTErn blOTTIng)检测细胞内PXr和坏死性凋亡下游效应自噬分子lC3-Ⅰ和lC3-Ⅱ蛋白相对表达含量;双核微核试验(CbMn)检测细胞遗传损伤情况;采用噻唑蓝(MTT)法测定Afb1对细胞活性抑制影响;利用坏死性凋亡抑制剂nEC-1构建坏死性凋亡抑制的细胞模型,验证Afb1诱导的坏死性凋亡的效应。结果与l02-P b细胞相比,l02-PXr细胞nr1I2 M rnA和PXr蛋白显著上调(均P<0.001)。Afb1显著地诱导l02-P b和l02-PXr细胞中CyP3A4 M rnA上调(均P<0.05),在l02-PXr细胞中的效应更为明显。与对照组相比,Afb1在5~30μMOl/l呈剂量反应关系诱导l02-P b和l02-PXr细胞的微核率增高(均P<0.05),l02-PXr细胞更为明显;同时,Afb1明显地诱导两株细胞的核芽率和核桥率,但随Afb1剂量增高都有下降趋势。细胞活性随Afb1浓度(1.875~120μMOl/l)增加呈剂量反应关系抑制(均P<0.05);且相对于l02-P b细胞,l02-PXr细胞对Afb1处理48 H诱导的细胞活性抑制作用更为敏感(P<0.05)。nEC-1可显著性抑制Afb1诱导的l02-PXr细胞活性抑制率(P<0.05),然而却不能降低Afb1诱导l02-P b细胞活性抑制率。此外,Afb1显著性诱导l02-P b和l02-PXr坏死性凋亡下游lC3-Ⅱ的上调(均P<0.05);且与l02-P b细胞相比,nEC-1对Afb1诱导的l02-PXr细胞活性抑制和lC3-Ⅱ上调的抑制效果更为明显(P<0.05)。结论 PXr参与Afb1诱导人肝细胞dnA损伤介导的坏死性凋亡,与PXr促进Afb1诱导CyP3A4基因上调有关。Objective To investigate the effects of pregnane X receptor(PXR) over expression on aflatoxin B1(AFB1)-induced DNA damage and necroptosis in human normal liver L02 cells.Methods The established cells models of stable transfection of over expression PXR(L02-PXR) and null vector p Babe-puro(L02-p B) were used.The background levels of NR1I2 m RNA and PXR protein, and the expression of AFB1-induced CYP3A4 m RNA and LC3-I / LC-3II protein were determined by the real time PCR(q RT-PCR) and Western blotting, respectively.The cytokinesis-block micronucleus(CBMN)assay was adopted to evaluate the genotoxicity.The cell viability inhibition rate was determined by MTT assay, after treatment with different doses of AFB1.The inhibition models of necroptosis were established by treatment with necroptosis inhibitor Nec-1.Results The expression of NR1I2 m RNA and PXR protein in L02-PXR cells were higher than that in L02-p B cells(all P<0.001).The level of CYP3A4 m RNA was significantly up regulated in L02-p B and L02-PXR cells by treatment with AFB1(all P<0.05).Compared with control group(Ctrl), MN frequencies in L02-p B and L02-PXR cells were significantly increased by treatment with AFB1 in a dose-dependent manner(all P <0.05), especially, in L02-PXR cells.Meanwhile, NBD and NBP frequencies were significantly increased by treatment with AFB1.However, AFB1 with a higher dose induced downward trends in frequencies of NBD and NBP.Moreover, the inhibition rate of cell viability was increased after treatment with AFB1(1.875~120 μmol / L) in a dose-dependent manner(all P <0.05); specifically, the inhibitory effects of AFB1-treatment after 48 h were significantly stronger in L02-PXR cells than in L02-p B cells(P <0.05).Interestingly, necroptosis inhibitor Nec-1 could inhibit AFB1-induced cell death in L02-PXR cells(P<0.05).On the contrary, Nec-1 could not prevent L02-p B cells from death by treatment with AFB1.In addition, the expression of necroptotic LC3-II, a classical marker of autophagy, was significantly increased by treatment with AFB1 in two cell lines(all P <0.05).Notably, pre-treatment with Nec-1 was able to block the inducement of necroptotic LC3-II in a more efficiently way in L02-PXR cells than in L02-p B cells(P <0.05).Conclusion PXR involved in the effect of AFB1 on necroptosis by DNA damage mediation in human liver cells;specifically, the up regulation of CYP3A4 gene may relate to the AFB1-induced DNA damage.国家自然科学基金(81172705;81072334); 广东省自然科学基金(S2011020002769); 福建省自然科学基金(2014J01372

Effects of cadmium on promoter methylation and transcriptional level of PPP2R1A gene in hepatocytes

目的分析镉染毒处理肝细胞中蛋白磷酸酶2A(PP2A)-Aα支架亚基基因PPP2r1A启动子区甲基化状态及其转录水平的改变。方法采用永生化人正常肝l02细胞及肝细胞癌HEP g2细胞为研究对象,对其进行以下分组和处理:1低、中和高剂量氯化镉(Cd Cl2)处理组,分别予浓度为20.0、40.0和60.0μMOl/l Cd Cl2处理24 H;2低、中和高剂量5-氮杂-2’-脱氧胞苷(5-AzA-d C)处理组,分别予浓度为2.5、5.0和10.0μMOl/l 5-AzA-d C处理48 H;3 5-AzA-d C组予浓度为5.0μMOl/l的5-AzA-d C处理48 H,Cd Cl2组予浓度为40.0μMOl/l的Cd Cl2处理24 H,(5-AzA-d C+Cd Cl2)组予浓度为5.0μMOl/l的5-AzA-d C预处理48 H后再予浓度为40.0μMOl/l Cd Cl2处理24 H;4 Cd Cl2处理组予浓度为40.0μMOl/l Cd Cl2处理24 H。上述4种分组均设对照组,予等体积生理氯化钠溶液或二甲基亚砜处理。经1~3处理后的细胞采用实时荧光定量聚合酶链反应(PCr)检测PPP2r1A、金属硫蛋白1b(MT1b)和dnA甲基转移酶3A(dnMT3A)的MrnA转录水平(以对照组水平为1.00)。经4处理后的细胞采用亚硫酸氢盐修饰后PCr扩增PPP2r1A启动子区克隆测序检测CP g岛的甲基化情况。结果 l02细胞和HEP g2细胞中,不同剂量Cd Cl2处理组PPP2r1A MrnA转录水平随镉处理剂量增高呈剂量依赖性下降(P0.05)。结论外源化学物Cd Cl2可诱导肝细胞中PPP2r1A转录水平降低,可能与镉能够引起目的基因启动子区甲基化状态改变有关,提示PP2A亚基基因的表观遗传学调控可影响镉诱导的肝细胞功能。Objective To analyze the effects of cadmium on the promoter methylation and transcriptional level of protein phosphatase 2A( PP2A)-Aα supported subunit gene PPP2R1 A gene in hepatocytes.Methods The immortalized human fetal liver cell line L02 and the hepatocellular carcinoma cell line Hep G2 were selected as the research objects: 1 Cells were treated with low-,medium- and high-dose( 20.0,40.0 and 60.0 μmol / L) cadmium chlorid( Cd Cl2) for 24 h.2Cells were treated with low-,medium- and high-dose( 2.5,5.0 and 10.0 μmol / L) 5-aza-2'-deoxycytidine( 5-Aza-d C)for 48 h.3 Cells were given 5.0 μmol / L for 48 h in 5-Aza-d C group,cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h in Cd Cl2 group and cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h after 48 h pretreatment of 5.0 μmol / L 5-Aza-d C in( 5-Aza-d C + Cd Cl2) group.4 Cells were treated with 40.0 μmol/L Cd Cl2 for 24 h in Cd Cl2 group.The above groups were all given the controls with the same volumes of physiological sodium chloride solution or dimethyl sulfoxide.Real-time fluorescent quantitative polymerase chain reaction( PCR) detection was used to detect the mRNA transcriptional levels of PPP2R1 A,Metallothionein 1B( MT1B),DNA methyltransferase 3A( DNMT3A) after treatments 1-3.After treatment4,cloning sequencing was used to detect the Cp G island methylation status of PPP2R1 A promoter after bisulfite sequencing PCR.Results In L02 and Hep G2 cells,the transcriptional levels of PPP2R1 A mRNA in Cd Cl2 group were decreased in a dose-dependent manner( P 0.05).Conclusion It was indicated the Cd Cl2 could lead to the transcription inhibition of PPP2R1 A,and the effect may be related with the change of its promoter methylation status.These data showed epigenetic regulation of PP2 A subunit genes may affect the function of hepatocytes exposed to cadmium.国家自然科学基金(81172705;81072334;81130052); 广东省自然科学基金重点项目(S2011020002769

线粒体关联性内质网膜与细胞Ca2+依赖性死亡的研究进展

线粒体关联性内质网膜（MAM）是线粒体和内质网发生交互作用的功能平台。研究发现有几十种蛋白质富集于两细胞器外膜之间,作为MAM蛋白质组成构成连接结构,功能上与Ca2+信号传导等密切相关,影响生理或外源环境因素诱导的细胞转归和命运结局。文章重点阐述了MAM组成结构和功能调节及其在细胞Ca2+依赖性死亡中的作用。国家自然科学基金(81773465,81573181,81472997);;福建省自然科学基金项目(2015J01344

CRISPR/Cas9敲低环氧合酶2表达对黄曲霉毒素B1诱导肝细胞核DNA损伤与脂质蓄积的影响

为探讨黄曲霉毒素B1(AFB1)诱导的肝细胞核DNA损伤与脂质蓄积的关系,以慢病毒质粒为载体,采用CRISPR/Cas9技术构建含靶向环氧合酶2(COX-2)编码基因(PTGS2)小向导RNA(sgRNA)的重组质粒,经测序验证成功后,制备假病毒感染HepG2细胞,构建稳定敲低COX-2表达的细胞.通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)检测mRNA和蛋白水平,结果显示与HepG2-Cas9-NC对照细胞相比,HepG2-Cas9-PTGS2敲低细胞中PTGS2 mRNA与COX-2蛋白表达水平分别减少至(49.1±1.8)%和(48.1±0.7)%.给予细胞AFB1处理,通过WB和免疫荧光(IF)法检测DNA损伤标志物γH2AX的表达水平,结果显示处理组敲低细胞中,γH2AX蛋白水平和荧光焦点形成数目显著低于对照细胞(p<0.05);进一步检测脂质合成相关指标,发现敲低细胞中PPARγ蛋白、总胆固醇(TC)、总甘油三脂(TG)水平以及油红O染色阳性脂滴的分布密度,均显著低于对照细胞中(p<0.05).在敲低细胞中回补COX-2后给予AFB1处理,γH2AX蛋白水平和脂质分布密度均显著升高(p<0.05).综上,成功构建了HepG2-Cas9-PTGS2敲低细胞,并发现敲低COX-2表达对AFB1诱导的肝细胞核DNA损伤和脂质蓄积有显著抑制作用,为进一步研究靶向干预COX-2在外源化学物诱导肝细胞毒性中的作用机制提供了细胞模型和依据.国家自然科学基金(81573181,81472997,81773465);;福建省自然科学基金(2015J01344