钙敏感受体通过胱硫醚-γ-裂解酶 抑制巨噬细胞泡沫化的机制研究

摘要:目的　探讨高表达的钙敏感受体（CaR）是否能通过增强胱硫醚-γ -裂解酶（CSE）的表达来促进H2S的分泌进而抑制巨噬细胞向泡沫细胞的转化。方法　敏感硫电极法检测细胞中H2S含量的变化;油红O染色检测阳性细胞的相对含量;HPLC测定细胞内胆固醇含量;ELISA法检测细胞因子白细胞介素-10（IL-10）、巨噬细胞游走抑制因子（MIF）和肿瘤坏死因子-α（TNF-α）的分泌情况;Western blot法检测各组细胞中CaR、CSE、CD36和ACAT-1的表达。结果　与空白对照组比较GdCl3组和NaHS组均能明显增加细胞H2S的分泌,而NPS2390 组明显降低细胞H2S的相对含量;GdCl3和NaHS组阳性细胞率明显降低,细胞内 胆固醇酯（CE）、游离胆固醇（FC）和 总胆固醇（TC）含量也均明显下降,而 NPS2390 组阳性细胞率明显增多,细胞内CE、FC和TC含量也均明显增加;GdCl3和NaHS组细胞上清中TNF-α和MIF含量均明显降低,IL-10的含量明显增多,而NPS2390组细胞培养的上清中TNF-α和MIF的含量均明显增加,IL-10 的含量明显降低;GdCl3和NaHS组CD36和ACAT-1的蛋白表达均明显降低,而 NPS2390组 CD36和 胆固醇酰基转移酶（ACAT-1）的蛋白表达均明显增强;GdCl3组CaR和CSE的蛋白表达均明显增加,而NPS2390组CaR和CSE的蛋白表达均明显被抑制;GdCl3组CSE的蛋白表达明显增加,而CSEsiRNA组和GdCl3+ CSEsiRNA组CSE的蛋白表达均明显被抑制;GdCl3 组H2S的相对含量明显增加,而GdCl3+CSE siRNA组和CSE siRNA组H2S的相对含量均明显降低。结论　CaR可以通过增强巨噬细胞内CSE的表达来促进H2S的分泌,进而抑制巨噬细胞向泡沫细胞的转化。     

硫化氢对肝硬化胶原蛋白生成的影响

观察硫化氢(H2S)对肝硬化胶原蛋白形成的影响,探明其在肝硬化形成过程中的作用。方法采用四氯化碳(CCl4)复合因素法建立肝硬化大鼠模型。选取Wistar大鼠20只,按数字表法随机分成正常对照组6只、肝硬化遭模组(造模组)7只和肝硬化造模并加硫化氢钠(NaHS)组(观察组)7只,对观察组进行腹腔注射NaHS 1.4 μ...     

NaHS抑制宫颈癌细胞增殖的初步机制

目的:探讨NaHS对子宫颈癌细胞增殖的影响及其机制。方法:首先将子宫颈癌HeLa细胞分成对照组和不同剂量的NaHS处理组,并采用MTT法检测细胞存活率和流式细胞仪检测细胞凋亡率。使用不同浓度的格列本脲预先处理HeLa细胞30 min,再用400μmol/L的NaHS处理24 h,探讨NaHS抑制子宫颈癌细胞增殖的初步机制。结果:不同剂量的NaHS处理组能明显抑制HeLa细胞增殖而促进HeLa细胞凋亡;格列本脲预先处理可以部分阻断这种作用。结论:NaHS抑制子宫颈癌细胞增殖可能与ATP敏感性钾通道有关。     

截肢后大鼠血浆及肝肾组织中硫化氢／胱硫醚-γ-裂解酶含量的变化

目的探讨截肢后大鼠血浆及肝、肾等远隔器官组织中内源性硫化氢(hydrogensulfide,H2S)的含量变化规律,以期找到硫化氢在创伤与修复过程中的作用机制。方法通过手术制备大鼠左后肢截肢模型,将雄性Wistar大鼠按手术时间随机分为9组:正常对照组(7只);手术后1、2、4、6、12、24、48、72h组(各7只)。测定血浆中H2S含量及肝、肾组织中CSE活性,分析其随时间变化规律。光镜观察各组组织形态学变化。结果与正常对照组相比,手术后大鼠血浆H2S含量逐渐降低,6h达最低值39.286±6.526(P<0.01),此后逐渐升高,48h后基本恢复正常。肝、肾组织CSE活性变化大体趋势与血浆相同,但下降幅度及到达最低值时间不同,肝在术后12h最低为166.162±10.878(P<0.01),肾在术后6h最低为60.664±7.574(P<0.01)。肝、肾组织出现一定程度组织损伤。结论H2S是一种新的内源性介质,在体内广泛存在,可能参与了组织损伤与修复的一系列病理生理过程。     

Acquired radioresistance of cancer and the AKT/GSK3β/cyclin D1 overexpression cycle

Fractionated radiotherapy (RT) is widely used in cancer therapy for its advantages in the preservation of normal tissues. However, repopulation of surviving tumor cells during fractionated RT limits the efficacy of RT. In fact, repopulating tumors often acquire radioresistance and this is the major cause of failure of RT. We have recently demonstrated that human tumor cells acquire radioresistance when exposed to fractionated radiation (FR) of X-rays every 12 hours for 1 month. The acquired radioresistance was associated with overexpression of cyclin D1, a result of a series of molecular changes; constitutive activation of DNA-PK and AKT with concomitant down-regulation of glycogen synthase kinase-3β (GSK3β) which results in suppression of cyclin D1 proteolysis. Aberrant cyclin D1 overexpression in S-phase induced DNA double strand breaks which activated DNA-PK and established the vicious cycle of cycling D1 overexpression. This overexpression of cyclin D1 is responsible for the radioresistance phenotype of long-term FR cells, since this phenotype was completely abrogated by treatment of FR cells by the API-2, an AKT inhibitor or by a Cdk4 inhibitor. Thus, targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway can be an efficient modality to suppress acquired radioresistance of tumor cells. In this article, I overview the newly discovered molecular mechanisms underlying acquired radioresistance of tumor cells induced by FR, and propose a strategy for eradication of tumors using fractionated RT by overcoming tumor radioresistance.     

叶黄素对人肝癌细胞HepG2的抑制作用及其机制研究

目的研究叶黄素对人肝癌细胞的抑制作用并探讨其可能机制。方法以HepG2人肝癌细胞株和L02正常人肝细胞株为研究对象,设叶黄素低、中、高剂量组(20、40、80 mol/L)和溶剂对照组。常规细胞培养后进行细胞计数、MTT法测定细胞存活率、DCFH-DA法测定ROS活性、HPLC法测定ATP含量、RT-PCR测定Bax mRNA及p53mRNA表达水平。结果 HepG2细胞经叶黄素干预处理后,低、中、高剂量组的细胞存活率显著降低(P<0.05),并且随干预时间的增加而持续降低,ROS水平和ATP含量显著降低(P<0.05);高剂量组的Bax mRNA表达和中、高剂量组的p53 mRNA表达显著增加(P<0.05)。而L02细胞经叶黄素干预处理后,细胞存活率和ATP含量均无显著性变化。结论叶黄素能抑制人肝癌细胞的生长,其机制可能与叶黄素清除细胞内ROS、抑制肝癌细胞ATP生成及上调凋亡基因Bax和p53 mRNA的表达有关。[营养学报,2012,34(4):332-335]     

The C60-fullerene porphyrin adducts for prevention of the doxorubicin-induced acute cardiotoxicity in rat myocardial cells

This is a fullerene-based low toxic nanocationite designed for targeted delivery of the paramagnetic stable isotope of magnesium to the doxorubicin (DXR)-induced damaged heart muscle providing a prominent effect close to about 80% recovery of the tissue hypoxia symptoms in less than 24 hrs after a single injection (0.03 - 0.1 LD50). Magnesium magnetic isotope effect selectively stimulates the ATP formation in the oxygen-depleted cells due to a creatine kinase (CK) and mitochondrial respiratory chain-focusing "attack" of 25Mg2+ released by nanoparticles. These "smart nanoparticles" with membranotropic properties release the overactivating cations only in response to the intracellular acidosis. The resulting positive changes in the energy metabolism of heart cell may help to prevent local myocardial hypoxic (ischemic) disorders and, hence, to protect the heart muscle from a serious damage in a vast variety of the hypoxia-induced clinical situations including DXR side effects.     

iTRAQ-Based Quantitative Proteomics Reveals the Energy Metabolism Alterations Induced by Chlorogenic Acid in HepG2 Cells

Epidemiological studies have suggested that coffee consumption is associated with a decrease in the risk of developing obesity and diabetes; however, the detailed mechanisms underlying these effects of coffee consumption remain poorly understood. In this study, we examined the effects of chlorogenic acid on energy metabolism in vitro. Hepatocellular carcinoma G2 (HepG2) cells were cultured in a medium containing chlorogenic acid. Chlorogenic acid increased the activity of mitochondrial enzymes, including citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase (MDH), which are involved in the tricarboxylic acid (TCA) cycle. Proteome analysis using the isobaric tags for the relative and absolute quantitation (iTRAQ) method revealed the upregulation of proteins involved in the glycolytic system, electron transport system, and ATP synthesis in mitochondria. Therefore, we propose a notable mechanism whereby chlorogenic acid enhances energy metabolism, including the TCA cycle, glycolytic system, electron transport, and ATP synthesis. This mechanism provides important insights into understanding the beneficial effects of coffee consumption.     

Neuroprotective effects of carnosic acid on neuronal cells under ischemic and hypoxic stress

Abstract

Ischemia/hypoxia induces oxidative stress which is associated with neurodegenerative diseases. The present study investigated protective mechanism of carnosic acid (CA) on ischemia/reperfusion and hypoxia-induced neuronal cell injury. The results showed that CA reduced 52% of the infarct volume from brains under ischemia/reperfusion in vivo and protected the PC12 cells from hypoxic injury in vitro. CA (1.0 µM) enhanced cell viability, prevented lactic dehydrogenase (LDH) release, scavenged reactive oxygen species (ROS), increased superoxide dismutase activity, and attenuated Ca²⁺ release, lipid peroxidation, and prostaglandin E₂ production in hypoxic PC12 cells. In addition, CA also reduced nitric oxide (NO) and interleukine (IL)-1 and IL-6 production from activated BV-2 microglia. Furthermore, its effect on hypoxia-induced mitogen-activated protein kinases (MAPKs) signaling pathway and caspase-3 was examined. Extracellular signal-regulated protein kinases, c-jun NH₂-terminal kinase, and p38 MAPK were activated during hypoxia. CA inhibited MAPKs, caspase-3, and COX-2 activation and correlated well with the diminished LDH release and apoptosis (TUNEL) in PC12 cells under hypoxia. Taken together, CA protected neuronal cells under ischemia/hypoxia through scavenging or reducing of ROS and NO, inhibiting COX-2 and MAPK pathways by anti-inflammatory and anti-oxidative properties.     

白藜芦醇通过上调沉默信息调节因子1调节香烟提取物诱导心肌线粒体产生活性氧簇的机制

目的探讨沉默信息调节因子1（SIRT1）调节香烟提取物（CSE）诱导心肌线粒体产生活性氧簇（ROS）的机制及白藜芦醇（Res）心肌保护作用。方法 H9C2细胞分5组：C组（对照组）、二甲亚砜组（DMSO溶剂对照组）、CSE组（CSE刺激组）、CSE＋Res组、Res组。采用Taqman探针荧光定量PCR检测各组SIRTI的mRNA表达、Weston Blot检测各组细胞SIRTI蛋白表达、荧光分光光度计检测细胞线粒体膜电位、荧光微量平板法检测不同浓度CSE刺激H9C2细胞产生ROS的变化、特异性荧光探针CM-H2DCFDA检测活细胞内ROS变化。结果与C组相比,CSE组SIRT1mRNA及蛋白质表达显著降低,ROS表达显著增强。与CSE组相比,CSE＋Res组SIRT1mRNA及蛋白质表达升高,ROS表达显著减少。随CSE刺激浓度增加,线粒体膜电位降低,ROS产生增加。提前给予Res可使线粒体膜电位升高,ROS产生减少。结论 Res可通过刺激SIRT1的表达而抑制CSE诱导的心肌细胞线粒体功能受损、ROS的释放,这可能是其吸烟相关慢性阻塞性肺疾病诱导的心脏氧化应激损伤中发挥心脏保护作用的机制之一。     

人肝癌细胞氧化应激模型中热休克蛋白90α对20S蛋白酶体功能的影响

目的观察人肝癌细胞氧化应激模型中细胞内Hsp90α和20S蛋白酶体的表达的变化以及Hsp90α表达下调后对20S蛋白酶体功能的影响;明确热休克蛋白90α对20s蛋白酶体功能的影响。方法以500μMH2O2建立氧化应激HepG2细胞模型;通过ROS探针法观察细胞在不同氧化应激条件下ROS的分布和含量,通过MTT比色法观察氧化应激对细胞存活的影响;以免疫印迹方法检测氧化应激对细胞内Hsp90α及20S蛋白酶体的合成的影响;以pSilencer2.1-U6neo载体,用电穿孔法建立稳定的Hsp90α抑制表达细胞株,通过检测糜蛋白酶活性观察氧化应激和降低Hsp90α表达对蛋白酶体活性的影响。结果氧化应激后细胞内ROS的含量增多,且分布从胞浆向胞核转移;随着作用时间延长,H2O2对HepG2细胞增殖的抑制程度增强;氧化应激导致胞内Hsp90α表达量先增加后降低,20S蛋白酶体表达量明显降低;siRNA干扰组蛋白酶体活性显著下降。结论氧化应激可影响细胞内Hsp90α和20S蛋白酶体的表达并改变其细胞内分布;氧化应激及降低Hsp90α的表达均可影响蛋白酶体的活性;Hsp90α对蛋白酶体功能维持起保护性的作用。     

柠檬酸镱诱导肝癌细胞HepG2凋亡作用及蛋白质表达变化研究

目的 研究稀土配合物柠檬酸镱([YbCit2]3-)诱导人肝癌HepG2细胞株凋亡的作用及其机制.方法 用DMEM培养基培养人肝癌细胞株HepG2细胞,以DMEM无血清培养细胞为对照组,以0.01～5.00 mmol/L[YbCit2]3-无血清培养基培养组为处理组,应用四噻唑蓝(MTT)法检测[YbCit2]3-对HepG2细胞生长的影响;以2.00 mmoL/L[YbCit2]3-无血清培养基培养组为处理组,Hoechst 33258染色法考察[YbCit2]3-对细胞的作用,采用差异蛋白质组学及H2O2、线粒体膜电位检测的方法研究[YbCit2]3-对HepG2细胞作用的可能机制.结果 2.00～5.00 mmol/L浓度范围内处理72 h,[YbCit2]3-能抑制HepG2细胞的生长,半数抑制浓度(IC50)值为(2.46 ±0.23)mmol/L.2.00 mmol/L[YbCit2]3-处理HepG2细胞48、72 h,Hoechst 33258染色检测表明其作用是诱导HepG2细胞凋亡.HepG2细胞经2.00 mmol/L[YbCit2]3-处理72 h后,通过双向电泳分离和质谱鉴定,鉴定出14个差异表达蛋白质点,包括丝切蛋白1、过氧化物氧化还原酶6、S100钙结合蛋白A6、蛋白酶26s非ATP酶亚基13亚型3等在细胞中分别参与抗凋亡、氧化还原、细胞增殖、蛋白降解等过程的蛋白质.2.00 mmol/L[YbCit2]3-分别作用HepG2细胞24、48 h后,细胞线粒体膜电位检测红绿荧光比值对照组为2.45 ±0.28,24 h组为1.56 ±0.23,48 h组为1.16 ±0.18,与对照组比,差异具有统计学意义(F=23.97,P=0.001);胞内H2O2检测荧光强度对照组为20.00 ±2.08,24 h组为40.00 ±5.50,48 h组为48.00±2.03,与对照组相比,差异具有统计学意义(F=48.40,P=0.000),表明[YbCit2]3-作用后线粒体膜电位下降,H2O2产生增加.结论 [YbCit2]3-可通过调节胞内氧化应激水平和线粒体凋亡途径诱导HepG2癌细胞凋亡.     

Maternal vitamin D deficiency influences long-chain polyunsaturated fatty acids and pregnancy outcome in association with alterations in one-carbon metabolism

Preeclampsia is a pregnancy-specific disorder, leading to maternal and infant morbidity and mortality. Abnormal placentation has been reported in preeclampsia. Nutrients like vitamin D and long-chain polyunsaturated fatty acids (LCPUFA) are known to play a role in placental development. In an animal model, we have previously demonstrated that maternal vitamin D deficiency increases the thromboxane/prostacyclin ratio and contributes to inflammation and vasoconstriction. We hypothesize that maternal vitamin D status influences placental LCPUFA metabolism through alterations in one carbon metabolism in women with preeclampsia. To test this hypothesis, we recruited 69 normotensive control (NC) women and 50 women with preeclampsia. Women with preeclampsia had lower placental protein and mRNA levels of cystathionine-β-synthase (CBS), higher plasma malondialdehyde (MDA) levels and higher levels of arachidonic acid (AA) and total omega-6 fatty acids in the placenta. Women with preeclampsia also demonstrated higher placental mRNA levels of cyclooxygenase-2 (COX-2) as compared to NC women. Maternal 25(OH)D levels were negatively associated with maternal plasma MDA levels. Placental vitamin D receptor (VDR) levels were positively associated with CBS while maternal MDA levels were positively associated with serum levels of thromboxane-B2 (TXB2) levels. Our findings indicate that vitamin D deficiency increases oxidative stress through alterations in one carbon metabolism to influence pro-inflammatory omega-6 metabolic pathway in the placenta. This study demonstrates a possible mechanism through which vitamin D deficiency can result in an imbalance in the LCPUFA metabolites and contribute to placental inflammation and endothelial dysfunction in preeclampsia.     

肝组织工程支架评价方法研究

目的：以人肝细胞系HepG2作为种子细胞,建立一种细胞支架评价方法,为肝组织工程筛选合适支架。方法：将HepG2细胞接种在生物可降解聚合物支架——聚乳酸-乙醇酸共聚物（PLGA）、壳聚糖（3％CS）和丝素（2％SF）上．体外常规培养;采用四唑盐（MTT）比色法、HE染色和尿素氮检测试剂盒对接种在支架上HepG2细胞的生长、分布及功能情况进行检测。结果：培养在3种支架上的HepG2细胞均维持增殖状态,相比之下．丝素支架上的细胞增殖较快,而PLGA和壳聚糖支架上的细胞增殖相对缓慢;培养至第7d时,组织学检测显示丝素支架上的HepG2细胞分布均匀,数量最多;而在PLGA和壳聚糖支架上只能发现少量细胞;细胞功能检测显示丝素和PLGA支架上的尿素合成能力下降缓慢,而壳聚糖支架上的尿素合成能力快速下降。结论：3种支架均具有比较好的生物相容性：相比较而言．丝素更适合作为肝组织工程支架;该方法可用于批量筛选肝组织工程支架。     

香烟烟气与纳米二氧化钛对人支气管上皮细胞的遗传损伤研究

目的探讨香烟烟气与纳米二氧化钛(nano-TiO2)单独及联合染毒对人支气管上皮细胞株(16-HBE)DNA断裂及染色体畸变的影响。方法将状态良好的16-HBE单独及混合暴露于香烟烟气提取物(cigarette smoke extract,CSE)和nano-TiO2,其终浓度分别为0、0mg/L,50、0mg/L,75、0mg/L,100、0mg/L,0、10mg/L,50、10mg/L,75、10mg/L,100、10mg/L。24h后,采用四甲基偶氮唑盐(MTT)法检测HBE的存活率,以单细胞凝胶电泳检测DNA损伤程度,以微核试验检测染色体畸变。结果除10mg/L nano-TiO2单独染毒组以外,各染毒组的存活率较对照组降低,彗星率(Comet%)较对照组升高,有统计学意义(P0.05);75、100mg/L CSE单独染毒组及各联合染毒组的彗星尾长(Ltail)较对照组升高,有统计学意义(P0.05);100mg/L CSE单独染毒组及100mg/L CSE+10mg/L nano-TiO2联合染毒组的尾部DNA含量(TailDNA%)较对照组升高,有统计学意义(P0.05);100mg/L CSE单独染毒组及75mg/L CSE+10mg/L nano-TiO2,100mg/L CSE+10mg/L nano-TiO2联合染毒组的尾矩(TM)、Olive尾矩(OTM)均较对照组升高,有统计学意义(P0.05);除10mg/L nano-TiO2单独染毒组以外,各染毒组微核率均较对照组升高,有统计学意义(P0.01)。各指标析因分析结果均未显示CSE及nano-TiO2联合染毒有交互作用。CSE与HBE的彗星率和微核率均呈正相关(P0.01)。结论香烟烟气诱导的HBE DNA断裂和染色体畸变随染毒剂量增加而加重;尚未见香烟烟气和nano-TiO2对HBE的遗传损伤具有交互作用。     

Modification of low dose radiation induced radioresistance by 2-deoxy-D-glucose in Saccharomyces cerevisiae: mechanistic aspects

Use of 2-deoxy-D-glucose (2-DG) in combination with radiotherapy to radio-sensitize the tumor tissue is undergoing clinical trials. The present study was designed to investigate the effect of 2-DG on radiation induced radioresistance (RIR) in normal cells. The sub-lethal radiation dose to the normal cells at the periphery of target tumor tissue is likely to induce radioresistance and protect the cells from lethal radiation dose. 2-DG, since, enters both normal and tumor cells, this study have clinical relevance. A diploid respiratory proficient strain D7 of S. cerevisiae was chosen as the model system. In comparison to non-pre-irradiated cultures, the cultures that were pre-exposed to low doses of UVC (254 nm) or (60)Co-gamma-radiation, then maintained in phosphate buffer (pH 6.0, 67 mM), containing 10 mM glucose (PBG), for 2-5 h, showed 18-35% higher survivors (CFUs) after subsequent exposure to corresponding radiation at lethal doses suggesting the radiation induced radioresistance (RIR). The RIR, in the absence of 2-DG, was associated with reduced mutagenesis, decreased DNA damage, and enhanced recombinogenesis. Presence of 2-DG in PBG countered the low dose induced increase in survivors and protection to DNA damage. It also increased mutagenesis, altered the recombinogenesis and the expression of rad50 gene. The changes differed quantitatively with the type of radiation and the absorbed dose. These results, since, imply the side effects of 2-DG, it is suggested that new approaches are needed to minimize the retention of 2-DG in normal cells at the time of radiation exposure.