脂联素对巨噬细胞ATP结合盒转运子A1表达及其功能的影响

目的探讨脂联素对巨噬细胞ATP结合盒转运子A1(ATP binding cassette transporter A1,ABCA1)及其上游调控因子肝脏X受体α(Liver X receptorα,LXRα)表达的影响及其在胆固醇逆转运(Reverse cholesterol transport,RCT)和抗动脉粥样硬化(Atherosclerosis,AS)中可能的作用机制。方法以不同浓度的脂联素(0、1、5、10μg/ml)体外培养巨噬细胞RAW264.7 24 h,RT-PCR法检测细胞中ABCA1和LXRα基因mRNA的转录水平,Western blot法检测细胞中ABCA1和LXRα蛋白的表达水平,闪烁计数法检测细胞内胆固醇的流出情况。结果脂联素能显著上调RAW264.7细胞中ABCA1和LXRα基因mRNA的转录水平及蛋白的表达水平(P<0.05),且呈浓度依赖性;脂联素能浓度依赖性地增加细胞内胆固醇的流出(P<0.05)。结论脂联素可通过LXRα途径上调巨噬细胞ABCA1基因的转录和翻译水平,促进胆固醇逆转运,延缓AS的发生、发展。     

阿昔莫司对RAW 264.7细胞三磷酸腺苷结合盒转运子A1表达及其调控的影响

目的研究阿昔莫司对巨噬细胞RAW264.7三磷酸腺苷结合盒转运子A1(ATP binding cassette transporterA1,ABCA1)及其上游调控因子肝脏X受体α(Liver X receptor,LXRα)的影响,探讨其促进胆固醇逆转运(Reversecholesterol transport,RCT)、抗动脉粥样硬化(Atherosclerosis,AS)的可能机制。方法体外培养RAW264.7细胞,将细胞分为空白对照组(不含阿昔莫司)和不同浓度的阿昔莫司干预组(分别含5、10、25μg/ml阿昔莫司),作用24 h后,采用RT-PCR法检测各组细胞中ABCA1和LXRα基因mRNA的转录水平;Western blot法检测各组细胞中ABCA1和LXRα蛋白的表达;闪烁计数法检测各组细胞内胆固醇的流出。结果阿昔莫司呈浓度依赖性地增加RAW264.7细胞中ABCA1和LXRα基因mRNA的转录水平和蛋白的表达水平(P<0.05或P<0.01)及细胞内胆固醇的流出率(P<0.01)。结论阿昔莫司可通过LXRα途径上调巨噬细胞ABCA1的表达,促使细胞内胆固醇流出,从而延缓AS的发生发展。     

Development of Tetrachlorophthalimides as Liver X Receptor β (LXRβ)‐Selective Agonists

Liver X receptor (LXR) agonists are candidates for the treatment of atherosclerosis via induction of ABCA1 (ATP‐binding cassette A1) gene expression, which contributes to reverse cholesterol transport (RCT) and to cholesterol efflux from the liver and intestine. However, LXR agonists also induce genes involved in lipogenesis, such as SREBP‐1c (sterol regulatory binding element protein 1c) and FAS (fatty acid synthase), thereby causing an undesirable increase in plasma and hepatic triglyceride (TG) levels. Recent studies indicate that LXRα contributes to lipogenesis in liver, and selective LXRβ activation improves RCT in mice. Therefore, LXRβ‐selective agonists are promising candidates to improve atherosclerosis without increasing plasma or hepatic TG levels. However, the ligand‐binding domains in the two LXR isoforms α/β share high sequence identity, and few LXR ligands show subtype selectivity. In this study we identified a tetrachlorophthalimide analogue as an LXRβ‐selective agonist. Structural development led to (E)‐4,5,6,7‐tetrachloro‐2‐(2‐styrylphenyl)isoindoline‐1,3‐dione ( 24 a ), which shows potent and selective LXRβ agonistic activity in reporter gene assays. In binding assays, compound 24 a bound to LXRβ preferentially over LXRα. It also induced the expression of ABCA1 mRNA but not SREBP‐1c mRNA in cells. Compound 24 a appears to be a promising lead compound for therapeutic agents to treat atherosclerosis without the side effects induced by LXRα/β dual agonists.     

牛血清白蛋白单克隆抗体的筛选与双抗体夹心ELISA检测方法的建立

目的筛选高效抗牛血清白蛋白(BSA)单克隆抗体细胞株,并建立BSA抗原检测的双抗体夹心ELISA法。方法分别采用辛酸-硫酸铵法和葡萄球菌A蛋白亲和层析法纯化单抗,并进行抗体类型、腹水效价、特异性和相对亲和力测定;应用纯化的单抗建立BSA双抗体夹心ELISA检测法,并进行初步应用。结果筛选出4株可稳定分泌抗BSA单抗的杂交瘤细胞株,抗体类型为IgG,抗体滴度均可达10-6,特异性良好,相对亲和力较高。建立的双抗体夹心ELISA法线性范围为1.25～20ng/ml,R2>0.98,灵敏度为1.25ng/ml。结论已筛选出高效抗BSA的单抗,并建立了BSA双抗体夹心ELISA检测方法。     

Cigarette Smoke Affects ABCAl Expression via Liver X Receptor Nuclear Translocation in Human Keratinocytes

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.     

Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-Like Synoviocytes

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5–9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.     

Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.     

Polyunsaturated Fatty Acids and Modulation of Cholesterol Homeostasis in THP-1 Macrophage-Derived Foam Cells

Transformation of macrophages to foam cells is determined by the rates of cholesterol uptake and efflux. This study uses a real time RT-PCR technique to investigate the role of conjugated linoleic acid (CLA), α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) in the regulation of the ATP-binding cassette A1 (ABCA1) and liver X receptor α (LXR) genes, which are involved in cholesterol homeostasis. Accordingly, these fatty acids significantly reduced the total, free and esterified cholesterols within the foam cells. While the expression of the ABCA1 and LXRα genes was increased in the presence of the pharmacological LXRα ligand, T0901317, their mRNA expression was not significantly affected by CLA, ALA and EPA. These results suggest that although polyunsaturated fatty acids have an effect on cholesterol homeostasis, they cannot change the expression of the ABCA1 and LXRα genes. Alternatively, several other genes and proteins may be involved.     

WuTac血药浓度双抗体夹心定量ELISA检测方法的建立及其临床应用

目的建立WuTac血药浓度双抗体夹心定量ELISA检测方法,并进行初步临床应用。方法建立双抗体夹心ELISA法,定量检测血清中WuTac浓度,筛选最佳抗体包被浓度和酶标抗体稀释度,绘制标准曲线,并对该方法进行验证;应用该方法对36名健康志愿者单次注射不同剂量WuTac(0.05、0.1和0.2mg/kg)前后14个时间点的临床血清标本进行检测。结果最佳抗体包被浓度为0.2μg/ml,最佳酶标抗体稀释度为1∶15000,标准曲线的线性相关系数r≥0.99,线性范围为3.9～125ng/ml。高浓度WuTac标准品的变异系数小于15%;准确性为99.05%±5.00%(92.43%～110.02%);特异性良好。临床血清检测结果表明,WuTac在0.05～0.2mg/kg范围内呈线性药代动力学特征。结论已建立了WuTac血药浓度双抗体夹心定量ELISA检测方法,该方法的灵敏度、精密性和准确性均符合我国生物制品药代动力学研究的要求。     

Danshensu Promotes Cholesterol Efflux in RAW264.7 Macrophages

Contemporary research suggests that macrophage foam cell and cholesterol efflux defect play pivotal role in atherogenesis. We reported on the heretofore unknown therapeutic effect of Danshensu (DSS) in reducing intracellular cholesterol level and unraveled the mechanism of DSS promotes cholesterol efflux. Oxidized low‐density lipoprotein stimulation of Raw264.7 cells into foam cells, which were treated with DSS and co‐treated with Simvastatin and Rosiglitazone. PPARγ, ABCA1, ABCG1, SR‐BI, CD36, and LXR‐α mRNA were quantified by Real‐Time PCR. Western blotting was used to determine protein expression of PPARγ, ABCA1 and CD36. Cellular cholesterol handling was studied by measurement of intracellular lipid droplets concentration and cholesterol efflux. DSS significantly reduced scavenger receptor CD36 and its orthologue SR‐BI. In addition, DSS stimulated the upregulation of cellular cholesterol exporters ABCA1 and ABCG1 to reduce intracellular lipid accumulation. DSS can reduce lipid deposition in Raw264.7 foam cells by balancing CD36 and ABCA1 protein expression.     

酿酒酵母残留蛋白ELISA定量检测方法的建立

目的建立酿酒酵母残留蛋白(Host cell protein,HCP)ELISA定量检测方法,为重组乙型肝炎疫苗(酿酒酵母)及相关生物制品的质量控制提供依据。方法制备酿酒酵母全细胞蛋白参考品,以其为抗原,免疫家兔,采用辛酸-硫酸铵沉淀法纯化抗体,SDS-PAGE分析纯度,免疫双向扩散法测定抗体效价,Western blot法分析抗体的特异性。以制备的抗酿酒酵母多克隆抗体作为包被抗体,HRP标记的抗酿酒酵母多克隆抗体作为检测抗体,建立酿酒酵母抗原双抗体夹心ELISA定量检测方法;梯度稀释酿酒酵母蛋白抗原参考品,并绘制标准曲线,确定该方法的线性范围及检测限,并进行特异性、准确度、精密度及适用性验证。结果共制备3批酿酒酵母抗原蛋白,其平均含量分别为1.61、1.64和1.63 mg/ml;纯化的IgG抗体纯度为91.7%,抗体效价为1∶64,能与酿酒酵母蛋白多数条带结合;酿酒酵母蛋白抗原浓度在6.25～200 ng/ml的范围内,线性关系良好(R2>0.99),最低检测限为6.25 ng/ml;用建立的方法检测小牛血清、人血白蛋白、MEM培养基、Vero细胞上清蛋白、Vero细胞乙型脑炎疫苗及PHK细胞狂犬疫苗,均无交叉反应,特异性良好;检测不同浓度的抗原回收率为97.6%～107.00%,变异系数均<10%,最低定量检测限为25 ng/ml;检测3批酿酒酵母重组乙型肝炎疫苗生产各工序中酿酒酵母蛋白的残留量,能有效反映杂蛋白去除率。结论成功建立了酿酒酵母HCP双抗体夹心ELISA定量检测方法,该方法特异性强,精密度及准确度良好,可用于生物制品中酿酒酵母HCP的检测。     

Differential Regulation of ABCA1 and Macrophage Cholesterol Efflux by Elaidic and Oleic Acids

Trans fatty acid consumption is associated with an increased risk of coronary heart disease. This increased risk has been attributed to decreased levels of HDL cholesterol and increased levels of LDL cholesterol. However, the mechanism by which trans fatty acid modulates cholesterol transit remains poorly defined. ATP-binding cassette transporter A1 (ABCA1)-mediated macrophage cholesterol efflux is the rate-limiting step initiating apolipoprotein A-I lipidation. In this study, elaidic acid, the most abundant trans fatty acid in partially hydrogenated vegetable oil, was shown to stabilize macrophage ABCA1 protein levels in comparison to that of its cis fatty acid isomer, oleic acid. The mechanism responsible for the disparate effects of oleic and elaidic acid on ABCA1 levels was through accelerated ABCA1 protein degradation in cells treated with oleic acid. In contrast, no apparent differences were observed in ABCA1 mRNA levels, and only minor changes were observed in Liver X receptor/Retinoic X receptor promoter activity in cells treated with elaidic and oleic acid. Efflux of both tracers and cholesterol mass revealed that elaidic acid slightly increased ABCA1-mediated cholesterol efflux, while oleic acid led to decreased ABCA1-mediated efflux. In conclusion, these studies show that cis and trans structural differences in 18 carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation.     

重组戊型肝炎病毒抗原ELISA检测方法的建立

目的建立重组戊型肝炎病毒(Hepatitis E virus,HEV)抗原双抗体夹心ELISA定量检测方法,用于HE疫苗中HEV抗原含量的检测。方法以重组HEV病毒样颗粒作为免疫原,免疫母鸡,制备抗HEV-IgY多克隆抗体,纯化后作为包被抗体,HRP标记的抗HEV单克隆抗体作为检测抗体,建立HEV抗原双抗体夹心ELISA定量检测方法,并对其进行验证。用建立的方法检测3批HE疫苗成品的HEV抗原含量,计算HEV抗原对氢氧化铝的吸附率。结果建立的ELISA方法线性范围为2～128 ng/ml,最低定量限为2 ng/ml;该方法检测冻干甲肝减毒活疫苗、重组人白介素-2(IL-2)、乙型肝炎表面抗原(HBsAg)、人血白蛋白、小牛血清及健康人血清均无交叉反应;该方法检测3个浓度的HEV抗原内部参考品的回收率在95.13%～104.50%之间,试验内及试验间变异系数均<15%;包被抗HEV-IgY的酶标板于37℃放置5 d,其检测HEV抗原内部参考品的A450值及敏感性均未发生显著变化;3批HE疫苗成品中HEV抗原对氢氧化铝的吸附率均大于95%,符合相关质控要求。结论已成功建立了HEV抗原双抗体夹心ELISA定量检测方法,可用于HEV抗原含量的检测。     

炎症对高脂状态下肺泡Ⅱ型上皮细胞脂质蓄积的影响

目的观察高脂状态下肺泡Ⅱ型上皮细胞(Alveolar typeⅡepithelial cells,ATⅡ)株A549细胞的脂质蓄积,并探讨炎症对高脂状态下A549细胞脂质蓄积的影响及可能的作用机制。方法将A549细胞分为4组:对照组(不加药物)、高脂组(100μg/ml LDL)、炎症组(300 ng/ml LPS)、联合处理组(100μg/ml LDL+300 ng/ml LPS),处理24 h后,油红O染色观察各组细胞内脂质蓄积情况;Real-time PCR检测固醇调节元件结蛋白2(Sterol regulatory elememnt binding proteins 2,SREBP2)、HMGCoA(3-Hydroxy-3-methlglutary 1 coezyme A)还原酶和低密度脂蛋白受体(Low-density lipoprotein receptor,LDLr)基因mRNA转录水平;Western blot法检测SREBP2、LDLr和三磷酸腺酐结合盒转运体A1(ATP-binding cassette sub-family A member 1,ABCA1)蛋白表达水平。结果联合处理组细胞内脂质蓄积较对照组、高脂组及炎症组严重;与对照组相比,高脂组的SREBP2、HMGCoA还原酶、LDLr基因mRNA转录水平反馈性下调(0.54±0.09)、(0.51±0.06)及(0.34±0.06)倍(P<0.05);联合处理组各基因mRNA转录水平下调程度低于高脂组;SREBP2及LDLr蛋白表达水平的变化趋势与基因转录水平基本一致;与对照组相比,高脂组ABCA1蛋白的表达反馈性上调(2.78±0.38)倍(P<0.01);联合处理组ABCA1蛋白表达上调无高脂组明显。结论炎症可加重高脂状态下肺泡Ⅱ型上皮细胞内的脂质蓄积,其机制可能与炎症干扰SREBP2-LDLr/HMGCoA还原酶通路介导的细胞内脂质稳态有关。     

人催乳素化学发光免疫分析试剂盒的制备及验证

目的制备人催乳素(hPRL)化学发光免疫分析(CLIA)试剂盒,并进行验证。方法采用2株不同结合位点的抗hPRL单克隆抗体,1株用于包被微孔板,另1株用于标记HRP,建立双抗体夹心检测系统,配合化学发光底物组装成试剂盒,并进行各项技术指标的验证。结果试剂盒最佳定量范围在5～100ng/ml,标准曲线的相关系数r=0.9999,灵敏度为0.49ng/ml,平均回收率为100.9%,试验内和试验间变异系数分别小于6.3%和10.9%。试剂盒有效期可达1年,特异性良好,出现Hook效应的样品浓度为1500ng/ml。与进口化学发光试剂盒和放射免疫分析试剂盒对比检测84份临床标本的结果呈高度相关,相关系数分别为0.9537和0.9486。结论已成功制备灵敏、特异的hPRLCLIA试剂盒,可用于人催乳素的临床检测。     

α1-酸性糖蛋白单克隆抗体的制备及检测方法的建立

目的制备α1-酸性糖蛋白(α1-Acid glycoprotein,α1-AGP)单克隆抗体(McAb),建立α1-AGP的ELISA检测方法,用于检测人体血液、尿液及各种组织液中α1-AGP的水平。方法用高氯酸沉淀人血清中α1-AGP,经离子交换层析和高压液相层析纯化后,免疫BALB/c小鼠,建立特异性抗α1-AGP单克隆抗体杂交瘤细胞株,制备并鉴定McAbs,分析各株分泌抗体的效价、特异性、位阻群、类及亚类。制备针对α1-AGP的双抗体夹心ELISA检测试剂盒,分析其稳定性、灵敏度和特异性,并进行临床评价。结果建立了9株特异性抗α1-AGP单克隆抗体杂交瘤细胞株,均分泌IgG抗体,所有抗体的轻链均为κ链。根据位阻分析结果,将9个细胞株分泌的抗体分为5个位阻群,腹水效价在1×10-4~1×10-7之间,且均有较好特异性。制备的α1-AGP双抗体夹心ELISA试剂盒灵敏度达2ng/ml,且检测结果与α1-AGP含量呈现良好的线性关系(r=0.9916),并与其他血清蛋白无交叉反应,试剂盒置37℃放置3d及4℃放置2个月,稳定性良好。用该试剂盒检测96份正常人血样,其α1-AGP平均值为0.43~1.32mg/ml;105份Ⅱ型糖尿病患者血样,其平均值为0.88~2.35mg/ml,经统计学分析,两者差异有显著意义。结论已成功制备出α1-AGP McAb,并建立了针对α1-AGP的双抗体夹心ELISA,制备的试剂盒可用于临床检测。     

Effect of Compounds Affecting ABCA1 Expression and CETP Activity on the HDL Pathway Involved in Intestinal Absorption of Lutein and Zeaxanthin

The antioxidant xanthophylls lutein and zeaxanthin are absorbed from the diet in a process involving lipoprotein formation. Selective mechanisms exist for their intestinal uptake and tissue‐selective distribution, but these are poorly understood. We investigated the role of high‐density lipoprotein (HDL), apolipoprotein (apo) A1 and ATP‐binding cassette transporter (ABC) A1 in intestinal uptake of lutein in a human polarized intestinal cell culture and a hamster model. Animals received dietary lutein and zeaxanthin and either a liver X receptor (LXR) agonist or statin, which up‐ or down‐regulate intestinal ABCA1 expression, respectively. The role of HDL was studied following treatment with the cholesteryl ester transfer protein (CETP) modulator dalcetrapib or the CETP inhibitor anacetrapib. In vitro, intestinal ABCA1 at the basolateral surface of enterocytes transferred lutein and zeaxanthin to apoA1, not to mature HDL. In hamsters, plasma lutein and zeaxanthin levels were markedly increased with the LXR agonist and decreased with simvastatin. Dalcetrapib, but not anacetrapib, increased plasma and liver lutein and zeaxanthin levels. ABCA1 expression and apoA1 acceptor activity are important initial steps in intestinal uptake and maintenance of lutein and zeaxanthin levels by an HDL‐dependent pathway. Their absorption may be improved by physiological and pharmacological interventions affecting HDL metabolism.     

测定α_1型干扰素的ELISA夹心法

应用ELISA夹心法测定rHuIFNα1含量，其灵敏度为1．56ng／ml，批内CV4.2％，批间CV7．8％。应用此方法测定上海生物制品研究所生产的rHuIFNα1（20μg／支）两批含量分别为19．5μg／支及21.9μg／支。