高表达ABCG2基因对人原发性肝癌细胞SP表型的影响

目的研究高表达ABCG2基因对人原发性肝癌细胞SP表型的影响。方法构建pMSCVpuro-ABCG2逆转录病毒质粒,并转染至病毒包装细胞PT67中,将获得的病毒上清液感染人原发性肝癌non-SP(Side population)细胞,筛选稳定感染的细胞系non-SP-ABCG2和non-SP-puro,RT-PCR法检测细胞中ABCG2基因的转录水平,Western blot法检测细胞中ABCG2蛋白的表达水平,显微镜观察细胞形态的变化,Hoechst33342染色分析细胞的SP表型。结果逆转录病毒质粒pMSCVpuro-ABCG2经双酶切及测序证实构建正确;non-SP-ABCG2细胞中ABCG2基因的转录和蛋白表达水平均较空载体pMSCVpuro转染的细胞non-SP-puro明显提高;non-SP-ABCG2细胞的形态发生改变,成梭形生长,偶见小三角样细胞,细胞生长缓慢,细胞间排列不紧密,并出现了SP表型。结论 ABCG2基因的高表达与细胞SP表型的形成具有一定的相关性,并能影响细胞的基本形态。     

稳定表达丙型肝炎病毒HLA-A2限制性多表位基因C1R-AAD细胞克隆的建立

目的建立稳定表达丙型肝炎病毒(HCV)HLA-A2限制性多表位基因的C1R-AAD细胞克隆。方法合成人泛素基因(Ub)和HCVHLA-A2限制性多表位基因(Mep),分别克隆入原核表达质粒pRSET-A,构建多表位抗原基因的原核表达质粒pRSET-Ub-Mep。用BamHⅠ和HindⅢ双酶切质粒pRSET-Ub-Mep,得到复合多表位基因Ub-Mep,亚克隆入真核表达载体pcD-NA3.1(-),构建重组真核表达质粒pcDNA3.1(-)-Ub-Mep,转染至C1R-AAD细胞,G418压力筛选后,通过有限稀释法获得稳定表达Ub-Mep融合蛋白的C1R-AAD细胞克隆,采用RT-PCR法检测稳定转染细胞中Ub-Mep基因mRNA的转录;间接免疫荧光法和Westernblot法检测Ub-Mep蛋白在稳定转染细胞中的表达。结果重组真核表达质粒pcDNA3.1(-)-Ub-Mep经酶切鉴定证明构建正确;在稳定转染的C1R-AAD细胞中可检测到Ub-MepmRNA和蛋白水平的表达。结论已建立了稳定表达HCVHLA-A2限制性多表位抗原的C1R-AAD细胞克隆,为进一步研究HLA-A2限制性多表位基因诱导的细胞免疫应答建立了靶细胞。     

大鼠结缔组织生长因子基因miRNA表达质粒的构建及其稳定转染大鼠肝星状细胞系的建立

目的构建大鼠结缔组织生长因子(Connective tissue growth factor,CTGF)基因miRNA表达质粒,并建立稳定转染大鼠肝星状细胞(Hepatic stellate cell,HSC)系。方法根据大鼠CTGF基因mRNA序列,设计并合成3对寡聚单链DNA X191-1、X191-2和X191-3及1对阴性对照序列DNA X191-4,将4对寡聚单链DNA退火成双链后,分别与载体pcDNA6.2-GW/EmGFP-miR连接,构建CTGF基因miRNA重组表达质粒,分别转染HSC-T6细胞,荧光显微镜观察细胞的转染效率,RT-PCR检测转染细胞中CTGF基因mRNA的转录水平;取干扰效率最高的重组质粒及阴性对照质粒,分别转染HSC-T6细胞,经杀稻瘟菌素持续加压筛选。结果经测序鉴定,重组表达质粒构建正确,插入片段的碱基序列与设计相符;细胞的瞬时转染效率约为50%;3组干扰质粒转染的HSC-T6细胞中,CTGF基因mRNA的转录水平明显低于空白对照组(P<0.01),其中X191-2质粒对CTGF基因转录的干扰效率最高;获得了稳定转染的HSC-T6细胞。结论成功构建了CTGF基因miRNA表达质粒,并获得了稳定转染的肝星状细胞系,为进一步研究肝纤维化的形成机制及其治疗奠定了基础。     

NLS-RARα基因沉默对HL-60细胞增殖及分化的影响

目的探讨抑制NLS-RARα基因表达对急性早幼粒细胞白血病(Acute promyeolic leukemia,APL)细胞株HL-60增殖及分化的影响。方法将针对NLS-RARα基因的shRNA真核表达质粒(干扰组)和阴性对照质粒(阴性对照组)采用脂质体法转染HL-60细胞,并设未转染组,经G418筛选出稳定转染的细胞,采用MTT法检测细胞的增殖活力;RT-PCR和QRT-PCR法检测细胞中NLS-RARα基因mRNA的转录水平;Western blot法检测细胞中NLS-RARα蛋白的表达水平;流式细胞术检测细胞表面分化抗原CD11b的表达及细胞周期的分布。结果与阴性对照组和未转染组比较,干扰组HL-60细胞的增殖活力、NLS-RARα基因mRNA的转录水平和蛋白的表达水平均明显下降(P<0.05);CD11b的表达明显升高(P<0.05);G1、G2期细胞比例明显增加,S期细胞比例明显减少(P<0.05)。结论抑制NLS-RARα基因的表达可抑制HL-60细胞增殖,促进其分化。本实验为进一步研究APL发生发展的机制及APL的分子诊断和靶向治疗新途径奠定了基础。     

JTV1基因真核表达质粒的构建及其在K562细胞中的表达

目的 构建JTV1基因真核表达质粒并稳定转染人白血病细胞系K562,检测转染细胞中JTV1基因mRNA和蛋白的表达水平及其对K562细胞增殖的影响。方法从人外周血单个核细胞中克隆JTV1基因,并将其插入pcDNA3.1表达载体中,构建真核重组表达质粒pcDNA3.1-JTV1,经脂质体介导转染K562细胞,采用RT-PCR和Western blot法鉴定转染细胞中JTV1基因mRNA和蛋白的表达水平;MTT法检测JTV1稳定表达对K562细胞增殖的影响。结果重组表达质粒pcDNA3.1-JTV1经双酶切及测序证实,目的基因已插入质粒中;人JTV1基因能在K562细胞中稳定表达;JTV1具有抑制K562细胞增殖的作用。结论已成功构建了JTV1基因真核表达质粒,并获得了稳定表达人JTV1基因的K562细胞克隆,为进一步研究人JTV1基因的功能及其与白血病细胞增殖及凋亡的相关性提供了细胞模型。     

The RelB-BLNK Axis Determines Cellular Response to a Novel Redox-Active Agent Betamethasone during Radiation Therapy in Prostate Cancer

Aberrant levels of reactive oxygen species (ROS) are potential mechanisms that contribute to both cancer therapy efficacy and the side effects of cancer treatment. Upregulation of the non-canonical redox-sensitive NF-kB family member, RelB, confers radioresistance in prostate cancer (PCa). We screened FDA-approved compounds and identified betamethasone (BET) as a drug that increases hydrogen peroxide levels in vitro and protects non-PCa tissues/cells while also enhancing radiation killing of PCa tissues/cells, both in vitro and in vivo. Significantly, BET increases ROS levels and exerts different effects on RelB expression in normal cells and PCa cells. BET induces protein expression of RelB and RelB target genes, including the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), in normal cells, while it suppresses protein expression of RelB and MnSOD in LNCaP cells and PC3 cells. RNA sequencing analysis identifies B-cell linker protein (BLNK) as a novel RelB complementary partner that BET differentially regulates in normal cells and PCa cells. RelB and BLNK are upregulated and correlate with the aggressiveness of PCa in human samples. The RelB-BLNK axis translocates to the nuclear compartment to activate MnSOD protein expression. BET promotes the RelB-BLNK axis in normal cells but suppresses the RelB-BLNK axis in PCa cells. Targeted disruptions of RelB-BLNK expressions mitigate the radioprotective effect of BET on normal cells and the radiosensitizing effect of BET on PCa cells. Our study identified a novel RelB complementary partner and reveals a complex redox-mediated mechanism showing that the RelB-BLNK axis, at least in part, triggers differential responses to the redox-active agent BET by stimulating adaptive responses in normal cells but pushing PCa cells into oxidative stress overload.     

Manganese superoxide dismutase: guardian of the powerhouse

The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component.     

RNA干扰her-2基因对骨肉瘤细胞血管内皮生长因子-A和白细胞介素-8表达的影响

目的探讨her-2基因沉默对人骨肉瘤细胞株saos-2中血管内皮生长因子-A(Vascular endothelial growth factor-A,VEGF-A)和白细胞介素-8(Interleukin-8,IL-8)表达的影响。方法构建her-2 shRNA重组表达质粒,转染至骨肉瘤细胞株saos-2,同时设空白对照组和shNC阴性对照组;RT-PCR检测her-2、VEGF-A和IL-8基因mRNA的转录水平;Western blot检测her-2蛋白表达的变化;ELISA法检测细胞培养液中VEGF-A和IL-8的分泌水平。结果构建的her-2 shRNA表达载体能抑制her-2基因的表达,对her-2基因mRNA的转录和蛋白表达的抑制率分别为63.05%和62.59%;转染重组质粒her-2 shRNA后VEGF-A和IL-8基因mRNA的转录水平及蛋白分泌水平均显著降低(P<0.01)。结论 her-2基因沉默后,VEGF-A和IL-8基因mRNA的转录水平和蛋白表达水平明显降低,her-2基因参与了骨肉瘤细胞VEGF-A和IL-8基因的表达调控,提示her-2基因可作为研究骨肉瘤血管生成分子机理的新靶点。     

小鼠微RNA miR-21真核表达质粒的构建及其在小鼠肾小球系膜细胞中的表达

目的构建小鼠微RNA miR-21真核表达质粒,并在小鼠肾小球系膜细胞中表达。方法人工合成小鼠miR-21基因序列,构建miR-21真核表达质粒pGenesil-miR-21。使用脂质体Lipofectamine2000转染小鼠肾小球系膜细胞后,G418筛选,获得稳定转染克隆,提取总RNA,通过实时荧光定量RT-PCR技术检测miR-21的表达。结果经酶切鉴定和测序证实,合成的miR-21基因序列完全正确,并已成功克隆到真核表达质粒pGenesil-1上。重组真核表达质粒转染小鼠肾小球系膜细胞后,筛选出的阳性克隆可稳定高表达miR-21。结论已成功构建miR-21真核表达质粒,并在小鼠肾小球系膜细胞中高效表达,为进一步探讨miR-21的生物学功能奠定了基础。     

Implications of the Wilms’ Tumor Suppressor Wt1 in Cardiomyocyte Differentiation

The Wilms’ tumor suppressor Wt1 is involved in multiple developmental processes and adult tissue homeostasis. The first phenotypes recognized in Wt1 knockout mice were developmental cardiac and kidney defects. Wt1 expression in the heart has been described in epicardial, endothelial, smooth muscle cells, and fibroblasts. Expression of Wt1 in cardiomyocytes has been suggested but remained a controversial issue, as well as the role of Wt1 in cardiomyocyte development and regeneration after injury. We determined cardiac Wt1 expression during embryonic development, in the adult, and after cardiac injury by quantitative RT-PCR and immunohistochemistry. As in vitro model, phenotypic cardiomyocyte differentiation, i.e., the appearance of rhythmically beating clones from mouse embryonic stem cells (mESCs) and associated changes in gene expression were analyzed. We detected Wt1 in cardiomyocytes from embryonic day (E10.5), the first time point investigated, until adult age. Cardiac Wt1 mRNA levels decreased during embryonic development. In the adult, Wt1 was reactivated in cardiomyocytes 48 h and 3 weeks following myocardial infarction. Wt1 mRNA levels were increased in differentiating mESCs. Overexpression of Wt1(-KTS) and Wt1(+KTS) isoforms in ES cells reduced the fraction of phenotypically cardiomyocyte differentiated clones, which was preceded by a temporary increase in c-kit expression in Wt1(-KTS) transfected ES cell clones and induction of some cardiomyocyte markers. Taken together, Wt1 shows a dynamic expression pattern during cardiomyocyte differentiation and overexpression in ES cells reduces their phenotypical cardiomyocyte differentiation.     

Reduced secretion of triacylglycerol in CaCo-2 cells transfected with intestinal fatty acid-binding protein

The fatty acid-binding proteins are hypothesized to be involved in cellular fatty acid transport and trafficking. We established CaCo-2 cells stably transfected with intestinal fatty acid-binding protein (I-FABP) and examined how the expression of this protein may influence fatty acid metabolism. I-FABP expression was detectable in I-FABP-transfected cells, whereas parent CaCo-2 cells as well as mock-transfected cells failed to express detectable levels of I-FABP mRNA or protein at any stage of differentiation. For studies of lipid metabolism, cells were incubated with [¹⁴C]oleic acid in taurocholate micelles containing monoolein, and distribution of labeled fatty acid in cellular and secreted lipids was examined. In one transfected cell clone, expressing the highest level of I-FABP, labeled cellular triacylglycerol increased approximately twofold as compared to control cells. The level of intracellular triacylglycerol in two other I-FABP-transfected clones resembled that of control cells. However, secretion of triacylglycerol was markedly reduced in all the I-FABP-expressing cell lines. Our data suggest that increased expression of I-FABP leads to reduced triacylglycerol secretion in intestinal cells.     

人核糖核苷酸酶抑制因子表达下调对膀胱癌T24细胞在体内外发生上皮间质转化的影响

目的探讨人核糖核苷酸酶抑制因子(human ribonuclease inhibitor,hRI)表达下调对膀胱癌T24细胞在体内外发生上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法干涉质粒pGensil-1-RI稳定转染T24细胞,经G418筛选阳性克隆,并设实验组、空质粒对照组及未转染对照组,RT-PCR检测各组细胞中RI基因mRNA的转录水平、细胞免疫荧光和Western blot检测RI蛋白的表达水平;HE染色检测细胞形态的变化;Western blot检测各组细胞中E-cadherin、Twist、Slug和Vimentin等相关蛋白表达水平;将3组细胞注入BALB/c裸鼠皮下,35 d后取瘤并称重;免疫组织化学检测瘤组织中RI、E-cadherin和Vimentin蛋白的表达水平。结果荧光显微镜下观察可见,干涉质粒pGensil-1-RI转染成功;实验组RI基因mRNA的转录水平和蛋白的表达水平较两对照组分别降低了63.31%和64.11%、49.6%和49.5%(P<0.001);实验组细胞呈梭形,核质比增大;与实验相比,两对照组E-cadhein蛋白表达水平分别增加52.76%和46.93%(P<0.001),而Twist、Slug和Vimentin蛋白分别降低了50.49%和54.63%、43.74%和51.55%、60.35%和53.77%(P<0.001);两对照组的瘤重明显低于实验组,分别低84.91%和80.89%(P<0.01),其RI和E-cadherin蛋白表达水平降低,而Vimentin蛋白表达水平则增加。结论沉默RI基因能明显增加T24细胞的转移、侵袭及EMT的能力。     

转化生长因子-β1基因shRNA表达质粒的构建及其对结肠癌细胞SW480侵袭能力的影响

目的构建转化生长因子-β1(Transforming growth factor-β1,TGF-β1)基因短发夹RNA(Short hairpin RNA,shRNA)表达质粒,并观察其对结肠癌细胞SW480侵袭能力的影响。方法设计并合成2对靶向TGF-β1基因的RNA干扰序列和1对阴性对照序列,并构建重组表达质粒pshRNA-TGF-β1a、pshRNA-TGF-β1b和pshRNA-TGF-β1c(阴性对照),以脂质体介导转染SW480细胞,荧光显微镜观察转染情况;RT-PCR和Western blot检测转染细胞中TGF-β1基因mRNA的转录水平和蛋白的表达水平;Transwell侵袭试验检测沉默TGF-β1基因表达对细胞侵袭能力的影响。结果 TGF-β1基因shRNA重组表达质粒经酶切鉴定和测序分析证明构建正确。与空白对照组相比,3种质粒转染的细胞均有较强的荧光表达;pshRNA-TGF-β1a和pshRNA-TGF-β1b组细胞TGF-β1基因mRNA的转录水平和蛋白的表达水平均明显减低(P<0.05);SW480细胞的侵袭能力也明显降低(P<0.05)。结论成功构建了TGF-β1基因shRNA重组表达质粒,其能有效抑制结肠癌细胞SW480中TGF-β1基因的表达,并可显著降低细胞的侵袭能力,为结肠癌的基因治疗提供了新的思路。     

miR-34a过表达对小鼠脑皮质神经干细胞中巢蛋白表达的影响

目的探讨miR-34a(microRNA-34a)过表达对小鼠脑皮质神经干细胞(Neutral stem cell,NSCs)标志物巢蛋白(Nestin)表达的影响。方法通过神经球(Neurosphere)形成试验原代培养ICR胎鼠脑皮质NSCs,在脂质体2000的介导下分别转染miR-34a模拟物(mmu-miR-34a mimics)及其阴性对照(mmu-miR-34a control),另设脂质体对照组(仅转染脂质体)及空白对照组,转染48 h后,采用real time RT-PCR检测各组细胞中miR-34a的表达水平及Nestin基因mRNA的转录水平,Western blot检测Nestin蛋白的表达水平。结果 miR-34a模拟物转染后,显著上调了miR-34a在NSCs中的表达水平(P<0.01);miR-34a的过表达显著下调了NSCs中Nestin基因mRNA转录及蛋白的表达水平(P均<0.01)。结论外源性过表达miR-34a可下调NSCs中Nestin基因mRNA的转录水平及蛋白的表达水平,为NSCs的增殖、分化调控的研究提供了新的理论依据。     

人C-反应蛋白重组真核表达质粒的构建及其对人脐静脉内皮细胞LOX-1和TF表达的影响

目的构建人C-反应蛋白(C-reactive protein,CRP)重组表达质粒pTracer CMV2-CRP,并观察其在人脐静脉内皮细胞(Human umbilical vein endothelical cells,HUVEC)中的表达及其对凝集素样氧化型低密度脂蛋白受体-1(Lectin-type oxidizedLDL receptor-1,LOX-1)、组织因子(Tissue factor,TF)表达的影响。方法以质粒pCR-BluntⅡ-TOPO-CRP为模板,PCR扩增CRP基因CDS序列,克隆至pTracer CMV2载体中,转化感受态大肠杆菌DH5α,构建重组表达质粒pTracer CMV2-CRP,转染HUVEC,设实验组(转染pTracer CMV2-CRP)、阴性对照组(转染pTracer-CMV2)及正常对照组,各组细胞经G418抗性筛选,RT-PCR及Western b1ot检测CRP基因的过表达效应及CRP的表达对HUVEC中LOX-1和TF转录及蛋白水平的影响。结果重组真核表达质粒pTracer CMV2-CRP经双酶切鉴定及测序证明构建正确。实验组细胞中,CRP基因过表达,且LOX-1和TF基因的转录及蛋白水平明显高于正常对照组和阴性对照组(P<0.05)。结论已成功构建人CRP基因重组真核表达质粒pTracerCMV2-CRP,并在HUVEC中过表达CRP,且明显上调HUVEC中LOX-1和TF的表达,为进一步阐述CRP在动脉粥样硬化形成过程中的作用提供了新的实验依据。     

RNA干扰P115基因对胃癌细胞巨噬细胞移动抑制因子表达的抑制作用

目的构建高尔基体转运蛋白P115基因shRNA表达载体,探讨P115基因沉默对胃癌细胞株BGC-823中巨噬细胞移动抑制因子(Macrophage migration inhibitory factor,MIF)表达的影响。方法设计4对针对P115基因的shRNA序列,构建重组表达质粒,转染高表达P115的胃癌细胞株BGC-823。RT-PCR、Western blot和免疫细胞化学检测P115及MIF的mRNA和蛋白的表达。结果 4个P115基因shRNA质粒经单酶切和测序证实构建正确;转染BGC-823细胞后,均能抑制P115基因的表达,其中以pGPU6/GFP/Neo-shP115-2的沉默效果最好,其对P115基因mRNA表达的抑制率为75.07%,对P115蛋白表达的抑制率为70.97%;转染pGPU6/GFP/Neo-shP115-2后,MIF基因的mRNA及蛋白表达水平均明显降低(P<0.05)。结论 P115基因沉默后,BGC-823细胞MIF的表达明显降低,提示P115可能参与调节胃癌细胞MIF的表达,P115基因可作为研究胃癌发生发展分子机理的新靶点。     

抑癌基因WWOX对Lewis肺癌细胞c-jun蛋白表达及其转录活性的影响

目的研究抑癌基因WWOX对Lewis肺癌细胞c-jun蛋白表达及其转录活性的影响,探讨WWOX基因的抑癌机制。方法采用脂质体转染法将WWOX基因重组真核表达质粒转染Lewis肺癌细胞,RT-PCR和Western blot法检测WWOX基因mRNA的转录水平和蛋白的表达水平;免疫组化法检测WWOX基因转染后Lewis细胞中c-jun蛋白的表达水平;半定量RT-PCR法检测c-jun调控的4种肿瘤相关基因p21、cyclinD1、FasL及VEGF mRNA的转录水平。结果重组真核表达质粒pcDNA4.0/Myc-His-WWOX转染Lewis细胞后,WWOX基因在mRNA和蛋白水平上均得到表达;与未转染细胞和空载体转染细胞相比,WWOX基因转染细胞胞浆中c-jun蛋白的表达量升高,而细胞核中c-jun蛋白的表达量未见明显差异;p21基因mRNA的转录水平升高,cyclinD1、FasL和VEGF基因mRNA的转录水平降低。结论WWOX基因可在Lewis细胞中表达,其转染Lewis肺癌细胞后,不直接调控c-jun蛋白的表达量,但可影响其转录活性。     

RNA干扰BC047440基因表达对HCT-8细胞增殖能力的影响

目的探讨RNA干扰结肠癌相关基因BC047440的表达对结肠癌细胞HCT-8增殖能力的影响。方法将BC047440基因shRNA干扰质粒pGPU6/GFP/Neo-BC047440-331(a1)、pGPU6/GFP/Neo-BC047440-451(a2)、pGPU6/GFP/Neo-BC047440-615(a3)、pGPU6/GFP/Neo-BC047440-756(a4)采用脂质体法转染结肠癌细胞HCT-8,荧光显微镜观察转染效率,荧光定量PCR法检测细胞BC047440基因mRNA的表达水平,MTT法分析细胞的增殖活性。结果细胞的转染效率为53%;干扰质粒a1、a2、a3、a4转染的HCT-8细胞BC047440基因mRNA的表达水平分别下降了36.1%、47.0%、53.8%和63.3%,a4质粒的干扰效率最高;细胞的增殖能力也明显下降。结论 BC047440基因RNA干扰质粒可明显抑制HCT-8细胞BC047440基因mRNA的表达及细胞的增殖,可能成为抑制结肠癌细胞生长的新基因。