The effect of dehydroepiandrosterone on lipogenic gene mRNA expression in cultured primary chicken hepatocytes

The effects of dehydroepiandrosterone (DHEA) on lipogenic gene mRNA expression were examined using cultured primary chicken hepatocytes. Cell samples were equilibrated to culture conditions for 24 h and then exposed to DHEA (1, 10 and 100 µM) dissolved in DMSO. The expression of sterol regulatory element‐binding protein‐1 (SREBP‐1) mRNA in cultured primary hepatocytes did not vary with time, except for a significant decrease in gene expression when cells were treated with 10 or 100 µM DHEA for either 1 or 2 h. A similar tendency toward decreased gene expression was evident for acetyl CoA carboxylase. The expression of peroxisome proliferator‐activated receptor α (PPARα) and carnitine palmitoyl transferase I mRNA was dramatically enhanced subsequent to treatment with 10 or 100 µM DHEA for periods of time from 6 to 48 h, while there was a decrease in the expression of PPARα in the presence of 10 µM DHEA after 1 and 2 h of exposure, and in the presence of 100 µM DHEA after 2 h of exposure. Hepatocytes treated with 10 or 100 µM DHEA contained more mitochondria and mitochondria with a higher electron density than did untreated hepatocytes. Furthermore, cell survival was significantly inhibited by treatment with 100 µM DHEA at 24, 48 and 72 h. In contrast, 1 µM DHEA administration significantly increased cell survival after 72 h; however, 10 µM DHEA treatment had no pronounced effect on cell survival. Overall, the results reported here indicate that DHEA accelerates lipid catabolism by direct regulation of hepatic gene expression and by induction of changes in hepatocyte mitochondria.     

人肺癌ILK基因siRNA重组表达质粒的构建及其对A549细胞增殖的影响

目的构建人肺癌整合素连接激酶(Integrin-linked kinase,ILK)基因siRNA重组表达质粒,并检测其对人肺腺癌A549细胞增殖的影响。方法人工合成靶向ILK基因的siRNA干扰序列,克隆至载体pGenesil-1中,构建重组表达质粒pGenesil-1-ILK,利用脂质体转染A549细胞,经G418稳定筛选后,采用RT-PCR和Western blot检测细胞中ILK基因mRNA的转录水平及蛋白的表达水平,MTT法检测细胞的增殖活力。结果重组表达质粒pGenesil-1-ILK经SalⅠ单酶切及测序证明构建正确,其转染A549细胞后,细胞ILK基因mRNA的转录水平和蛋白的表达水平均明显降低(P<0.05),且细胞的增殖能力也显著降低(P<0.05)。结论成功构建了人ILK基因siRNA重组表达质粒,ILK基因沉默可显著抑制A549细胞增殖,为肺癌靶向基因治疗的研究提供了新的思路。     

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。     

RNA干扰技术对MCF-7细胞Aurora-A基因表达的影响

目的构建针对Aurora-A基因的特异性小干扰RNA(siRNA)真核表达载体,并探讨其对人乳腺癌细胞株MCF-7中Aurora-A基因表达的抑制作用。方法将具有短发夹结构的2条DNA序列,经退火形成互补双链,再克隆至载体pGCsi-U6/Neo/GFP中,转化大肠杆菌DH5α,提取质粒进行序列测定。并通过脂质体法转染至MCF-7细胞中,48h后观察转染效率,并采用RT-PCR及Westernblot法检测siRNA对MCF-7细胞Aurora-A基因mRNA及蛋白表达的影响。结果测序鉴定证实目的寡核苷酸片段已被克隆至pGCsi-U6/Neo/GFP载体中,Aurora-A-siRNA质粒转染MCF-7细胞48h后转染效率约为60%,与对照组比较,Aurora-A-siRNA质粒转染的细胞Aurora-A基因mRNA和蛋白的表达均明显下降。结论已成功构建了Aurora-A-siRNA真核表达质粒,其能明显抑制MCF-7细胞Aurora-A基因的表达,为进一步研究Aurora-A基因的功能奠定了基础,并可能为肿瘤的生物学治疗提供新的方法。     

Protein Synthesis in Sub‐Micrometer Water‐in‐Oil Droplets

Water‐in‐oil (w/o) emulsions are used as a cellular model because of their unique cell‐like architecture. Previous works showed the capability of eukaryotic‐cell‐sized w/o droplets (5–50 μm) to support protein synthesis efficiently; however data about smaller w/o compartments (<1 μm) are lacking. This work focuses on the biosynthesis of the enhanced green fluorescent protein (EGFP) inside sub‐micrometric lecithin‐based w/o droplets (0.8–1 μm) and on its dependence on the compartments’ dynamic properties in terms of solute exchange mechanisms. We demonstrated that protein synthesis is strongly affected by the nature of the lipid interface. These findings could be of value and interest for both basic and applied research.     

Alpha-Lipoic Acid Alleviates Acute Inflammation and Promotes Lipid Mobilization During the Inflammatory Response in White Adipose Tissue of Mice

Recently, white adipose tissue has been shown to exhibit immunological activity, and may play an important role in host defense and protection against bacterial infection. Αlpha‐lipoic acid (α‐LA) has been demonstrated to function as an anti‐inflammatory and anti‐oxidant agent. However, its influence on the inflammatory response and metabolic changes in white adipose tissue remains unknown. We used male C57BL/6 mice as models to study the effect of α‐LA on the inflammatory response and metabolic changes in white adipose tissue after stimulation with lipopolysaccharide (LPS). The non‐esterified fatty acid content was measured by an automatic biochemical analyzer. The expression of inflammation‐, lipid‐ and energy metabolism‐related genes and proteins was determined by quantitative real‐time polymerase chain reaction and western blotting. The results indicated that α‐LA significantly decreased the epididymis fat weight index and the non‐esterified fatty acid content in plasma compared with the control group. LPS significantly increased the expression of inflammation genes and α‐LA reduced their expression. The LPS‐induced expression of nuclear factor‐κB protein was decreased by α‐LA. Regarding lipid metabolism, α‐LA significantly counteracted the inhibitory effects of LPS on the expression of hormone‐sensitive lipase gene and protein. α‐LA evidently increased the gene expression of fatty acid transport protein 1 and cluster of differentiation 36. Regarding energy metabolism, α‐LA significantly increased the expression of most of mitochondrial DNA‐encoded genes compared with the control and LPS group. Accordingly, α‐LA can alleviate acute inflammatory response and this action may be related with the promotion of lipid mobilization in white adipose tissue.     

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing mRNA and other nucleic acids,especially large mRNA.Cas9 protein is the nuclease part of the powerful gene-editing tool,CRISPR/Cas9 system,Cas9 mRNA is particularly large,thus presents a big challenge for delivery.We assembled a multilayered biodegradable nanocarrier to load Cas9 mRNA inside to protect Cas9 mRNA from degradation.We used a microfluidic chip to synthesize a small,positively charged,and degradable core to attract negatively charged Cas9 mRNA.The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome.The multilayered nanocarriers elevated the delivery efficiency of Cas9 mRNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers.In addition,the multilayered nanocarriers do not require reduced serum medium for transfection.When using the standard complete medium for transfection,the multilayered nanocarriers could increase the expression of Cas9 mRNA by over 15 folds compared to commercially used non-viral carriers.The co-delivery of Cas9 mRNA and sgRNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers.Based on the higher transfection efficiency of Cas9 mRNA/sgRNA than commercially used non-viral carriers,these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 mRNA/sgRNA and other nucleic acids.     

A Recombinant Collagen–mRNA Platform for Controllable Protein Synthesis

We have developed a collagen–mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin‐healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell‐free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase–mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.     

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。     

蛇毒类凝血酶基因真核表达载体的构建

目的 构建大连蛇岛蝮蛇毒类凝血酶(Gloshedobin,GSB)基因真核表达载体,试图借助IL-2的分泌通路,使GSB分泌至胞外而达到溶栓和防栓的目的。方法 应用PCR重组技术,将GSB的5’端和3’端分别与IL-2基因的信号肽和poly A尾相连接,将此重组基因克隆入逆转录病毒载体 pLNCX,以脂质体介导转染人外用血淋巴细胞,并以绿色荧光蛋白(GFP)基因作为报告基因检测转染效率、确定DNA和脂质体的比例。转染24h后从mR-NA、DNA和蛋白质水平检测GSB的表达情况。结果 mRNA和DNA水平均有外源基因GSB的表达;SDS-PAGE显示在相对分子质量 30 000左右出现一条带,且转染上清具有精氨酸酯酶活性。结论 构建的真核表达载体可表达具有活性的GSB,为GSB基因治疗血栓的体内研究打下基础。     

Lutein Prevents High Fat Diet‐Induced Atherosclerosis in ApoE‐Deficient Mice by Inhibiting NADPH Oxidase and Increasing PPAR Expression

Epidemiological and experimental studies provide supportive evidence that lutein, a major carotenoid, may act as a chemopreventive agent against atherosclerosis, although the underlying molecular mechanisms are not well understood. The main aim of this study was to investigate the effects of lutein on the alleviation of atherosclerosis and its molecular mechanisms involved in oxidative stress and lipid metabolism. Male apolipoprotein E knockout mice (n = 55) were fed either a normal chow diet or a high fat diet (HFD) supplemented with or without lutein for 24 weeks. The results showed that a HFD induced atherosclerosis formation, lipid metabolism disorders and oxidative stress, but noticeable improvements were observed in the lutein treated group. Additionally, lutein supplementation reversed the decreased protein expression of aortic heme oxygenase‐1 and increased the mRNA and protein expressions of aortic nicotinamide‐adenine dinucleotide phosphate oxidase stimulated by a HFD. Furthermore, the decreased mRNA and protein expression levels of hepatic peroxisome proliferator‐activated receptor‐α, carnitine palmitoyltransferase 1A, acyl CoA oxidase 1, low density lipoprotein receptors and scavenger receptor class B type I observed in mice with atherosclerosis were markedly enhanced after treatment with lutein. Taken together, these data add new evidence supporting the anti‐atherogenic properties of lutein and describing its mechanisms of action in atherosclerosis prevention, including oxidative stress and lipid metabolism improvements.     

Conjugation of the 9-kDa Isoform of Granulysin with Liposomes Potentiates Its Cytotoxicity

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni²⁺ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-x_L. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway.     

人CD147基因siRNA真核表达质粒的构建及其对HeLa细胞中CD147基因表达的影响

目的构建针对人CD147基因的siRNA真核表达质粒,并观察其对HeLa细胞中CD147基因表达的影响。方法根据GenBank中登录的人CD147基因序列,设计并合成针对CD147基因的特异性小干扰片段,并将其定向克隆至带有卡那霉素抗性和增强型绿色荧光蛋白的真核表达载体pGenesil-1中,对重组质粒进行酶切分析和DNA序列测定。用脂质体将重组质粒转染至HeLa细胞中,观察其对HeLa细胞CD147基因mRNA及蛋白表达水平的影响。结果酶切鉴定和测序结果表明,3个表达短发夹RNA的质粒及其阴性对照质粒构建正确。3个siRNA真核表达质粒对HeLa细胞CD147基因mRNA转录水平的抑制率分别为32.5%、66.8%和59.1%;对CD147蛋白表达水平的抑制率分别为28.3%、63.5%和56.2%。结论已成功构建针对人CD147基因的siRNA真核表达质粒,其对HeLa细胞CD147基因的表达具有明显的抑制作用,为进一步研究CD147基因的功能奠定了基础。     

Cyclopropaneoctanoic Acid 2‐Hexyl Upregulates the Expression of Genes Responsible for Lipid Synthesis and Release in Human Hepatic HepG2 Cells

Recently we have found cyclopropaneoctanoic acid 2‐hexyl (CPOA2H) in humans and demonstrated its elevated levels in patients with metabolic diseases associated with hypertriglyceridemia. However, it is still unclear whether CPOA2H may influence lipid metabolism in lipogenic tissues. To verify this, HepG2 hepatocytes and 3T3‐L1 adipocytes were cultured with various concentrations of CPOA2H, and then the expressions of genes associated with lipid metabolism were determined. Incubation with CPOA2H at concentrations found in patients with metabolic diseases enhanced the expression of hepatocyte genes associated with lipid synthesis and release, in particular, the fatty acid synthase gene (nearly 20‐fold increase in the mRNA level). In contrast, incubation with CPOA2H caused the downregulation of most adipocyte genes associated with lipid synthesis, whereas the level of leptin mRNA was increased. These findings suggest that CPOA2H may contribute to hypertriglyceridemia in patients with metabolic diseases, upregulating the expression of hepatocyte genes responsible for lipid synthesis and release.     

人Makorin环指蛋白1基因shRNA真核表达质粒的构建及鉴定

目的构建人Makorin环指蛋白1(MKRN1)基因shRNA真核表达质粒,并检测其对HEK293细胞MKRN1基因表达的影响。方法设计并合成特异性针对人MKRN1基因的小干扰片段,定向克隆至带有卡那霉素抗性基因和绿色荧光蛋白基因的真核表达载体pGenesil-1中,对重组质粒进行酶切鉴定及DNA序列分析,并用脂质体将其转染到HEK293细胞中,观察其对细胞MKRN1基因mRNA转录和蛋白表达水平的影响。结果经酶切鉴定和序列分析证明,3个人MKRN1基因shRNA真核表达质粒及其阴性对照质粒构建正确,转染HEK293细胞后,3个shRNA真核表达质粒在mRNA水平对细胞MKRN1基因的抑制率分别为52.4%、26.2%和42.9%,在蛋白水平对细胞MKRN1基因的抑制率分别为69.1%、27.9%和50.0%。结论已成功构建了人MKRN1基因的shRNA真核表达质粒,为进一步研究MKRN1基因的功能及其与人端粒酶逆转录酶的关系奠定了基础。     

靶向Pin1基因的shRNA干扰质粒的构建及其对大肠癌SW620细胞Pin1基因的抑制作用

目的构建靶向Pin1基因的短发夹RNA(shRNA)干扰真核表达质粒,并检测其对大肠癌SW620细胞Pin1基因的抑制作用。方法设计合成特异性针对人Pin1基因的寡核苷酸序列,退火后与pGenesil-1载体连接,构建靶向Pin1基因的shRNA真核表达质粒pGenesil-1-Pin1(+),利用脂质体转染大肠癌SW620细胞,以质粒pGenesil-1-Pin(-)转染细胞和未转染细胞为对照。利用荧光显微镜观察转染后细胞表达的绿色荧光蛋白,估算转染效率;应用实时荧光定量PCR和Western blot检测其对SW620细胞Pin1基因mRNA转录及蛋白表达的影响。结果所构建的特异Pin1shRNA真核表达质粒经酶切及测序证明构建正确,转染SW620细胞的转染效率为64%。shRNA对SW620细胞Pin1基因mRNA的抑制率在转染后72h最高,为70.2%,蛋白抑制率为60%。结论已成功构建了靶向Pin1基因的shRNA干扰真核表达质粒,可抑制SW620细胞Pin1基因mRNA的转录及蛋白的表达。     

人MCIR基因在杆状病毒-昆虫细胞表达系统中的表达

目的利用杆状病毒表达系统进行人恶性黑色素瘤A375细胞MCIR基因在Sf9昆虫细胞中的表达。方法以pMD18-T-MCIR为模板，利用PCR方法扩增人MCIR基因，将MCIR基因连接到pfastBacl质粒上，与穿梭载体DH10Bac转座，获得Bacmid-MCIR质粒后，通过脂质体介导，转染Sf9细胞，使其表达重组杆状病毒，经SDS-PAGE检测表达产物，放射受体分析法检测目的蛋白MCIR活性。结果Bacmid-MCIR质粒转染Sf9细胞后的SDS-PAGE图谱，在相对分子质量为35000处有一条新生蛋白带。放射受体分析结果显示表达的蛋白与标记配体特异亲和，具有生物学活性。结论MCIR基因成功在真核细胞中得到表达。     

稳定表达Ag85A蛋白细胞系的建立

目的建立稳定表达Ag85A蛋白的K562真核细胞系,为进一步开展以阳离子脂质体为转运载体的口服疫苗的免疫原性研究奠定基础。方法将已构建的重组质粒pCDNA3.1+/Ag85A包裹阳离子脂质体LipofectamineTM 2000后,转染真核细胞K562,SDS-PAGE和Western blot分析重组Ag85A蛋白的表达。用G418筛选稳定表达Ag85A蛋白的细胞株,流式细胞术和间接免疫荧光技术检测Ag85A蛋白的表达及定位。结果重组质粒pCDNA3.1+/Ag85A转染K562细胞时,与脂质体的最适比例为2μg:2μl;转染细胞表达的Ag85A蛋白相对分子质量约为32000,且具有良好的反应原性。稳定转染3个月后,重组质粒转染的细胞有42.37%的细胞显示FITC标记,且细胞胞浆中及细胞膜上均有Ag85A蛋白的表达。结论已获得稳定表达Ag85A蛋白的K562细胞系,可用于Ag85A蛋白的大量制备及抗体方面的研究。