PPARγ信号转导通路在内脂素诱导泡沫细胞形成中的作用机制

本文旨在观察过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)信号转导通路在内脂素(visfatin)调控人单核细胞株THP-1源性巨噬细胞ATP结合盒转运蛋白A1(ATP binding cassette transporter A1,ABCA1)和酰基辅酶A:胆固醇酰基转移酶1(acyl-CoA:cholesterol acyltransferase1,ACAT1)表达中的作用,探讨内脂素诱导泡沫细胞形成的机制和途径。THP-1单核细胞经佛波酯诱导分化为巨噬细胞,随机分组,给予不同浓度的内脂素和PPARγ激动剂罗格列酮(rosiglitazone)进行干预,分别运用RT-PCR和Western blot法检测各组细胞PPARγ、ABCA1及ACAT1 mRNA和蛋白表达,酶荧光学法检测细胞内总胆固醇(total cholesterol,TC)和游离胆固醇(free cholesterol,FC)含量,TC与FC之差为胆固醇酯(cholesterol ester,CE)含量。结果显示,内脂素呈浓度依赖性增加THP-1源性巨噬细胞内FC和CE含量,下调巨噬细胞PPARγ mRNA和蛋白的表达,同时下调其下游的靶基因ABCA1 mRNA和蛋白的表达,上调ACAT1 mRNA和蛋白的表达;而罗格列酮呈浓度依赖性地抑制内脂素所诱导的上述效应。上述结果提示,内脂素可能通过PPARγ信号转导通路下调ABCA1表达,上调ACAT1表达,使细胞内FC流出减少,CE合成增加,从而诱导泡沫细胞的形成。这为研究内脂素致动脉粥样硬化的机制提供了新的理论依据。     

ABCA1和PPARγ与糖尿病动脉粥样硬化关系的研究

目的：研究ATP结合盒转运体A1（ABCA1）在多种糖尿病特有因素刺激下在巨噬细胞中的表达,以及PPARγ激动剂干预后其表达的变化,探讨ABCA1及PPARγ在糖尿病大血管并发症发展中的作用机制。从而为研究糖尿病大血管并发症的发生机制及防治提供一定的理论依据。方法：以巨噬细胞为研究对象,体外模拟糖尿病状态,分别以高葡萄糖、高胰岛素和糖基化终末产物刺激巨噬细胞,检测细胞中ABCA1表达的变化;以PPARγ激动剂预处理巨噬细胞后,再以上述因素刺激细胞,分别检测巨噬细胞中ABCA1的表达并比较。结果：高葡萄糖、高胰岛素和糖基化终末产物（AGE）可作为独立因素,导致细胞中ABCA1表达减少（P〈0.05）。PPARγ激动剂预处理后,ABCA1表达量增加（P〈0.05）。结论：糖尿病状态下,一些糖尿病特有的刺激因素如：高葡萄糖、高胰岛素和糖基化终末产物等作为独立因素使ABCA1表达减少,可能是糖尿病患者动脉粥样硬化发生率较非糖尿病人群增高的原因。PPARγ激动剂干预后,糖尿病状态下ABCA1的表达增加,这提示我们应用PPARγ激动剂可能延缓糖尿病患者动脉硬化进展。     

曲格列酮对THP-1巨噬细胞源性泡沫细胞ABCA1表达及胆固醇流出的影响

目的:研究曲格列酮对THP-1巨噬细胞源性泡沫细胞ABCA1表达及胆固醇流出的影响。方法:采用液体闪烁计数法测定曲格列酮处理后THP-1巨噬细胞源性泡沫细胞胆固醇流出。用RT-PCR和Western blotting的方法检测曲格列酮处理后THP-1巨噬细胞源性泡沫细胞ABCA1 mRNA水平和蛋白质水平的变化。结果:经曲格列酮处理后,THP-1巨噬细胞源性泡沫细胞的胆固醇流出具有时间依赖性的增加,从0h的1．82%上升到24h的7．61%。在mRNA水平和蛋白质水平ABCA1也均随着曲格列酮作用时间增加而表达上调。结论:曲格列酮能增加THP-1巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出。     

大豆异黄酮激活PPARγ抑制人乳腺癌MCF-7细胞增殖

目的：探讨两种大豆异黄酮主要成分染料木黄酮（genistein,GEN）和大豆苷元（daidzein,DAI）抑制人乳腺癌MCF-7细胞增殖的作用与过氧化物酶体增殖激活物受体γ（peroxisome proliferators-activated receptorγ,PPARγ）信号途径的关系。方法：采用免疫细胞化学染色方法观察MCF-7细胞的PPARγ表达情况,PPARγ介导的荧光素酶报告基因检测大豆异黄酮和PPARγ配体罗格列酮（rosiglitazone,ROS）对MCF-7细胞PPARγ的激活作用,MCF-7细胞分别经8×10-5mol/L GEN、DAI和1×10-5mol/L的ROS单独或联合1×10-5mol/L的PPARγ特异性抑制剂GW9662联合处理24、48和72 h后,用CCK-8法检测细胞增殖。结果：MCF-7细胞存在有PPARγ表达,GEN、DAI呈剂量依赖性增强报告基因荧光素酶活性,且这种作用可被GW9662明显阻断;GEN、DAI和ROS呈时间依赖性明显抑制MCF-7细胞增殖（P〈0.05）,而GW9662可以显著削弱GEN、DAI和ROS对MCF-7细胞的增殖抑制作用（P〈0.05）。结论：大豆异黄酮可通过激活乳腺癌MCF-7细胞的PPARγ信号途径抑制其增殖。     

研究: 肺炎衣原体抑制LXRα-ABCA1途径促进巨噬细胞脂质蓄积

为探讨肝X受体α (LXRα)-三磷酸腺苷结合盒转运体A1 (ABCA1)途径在肺炎衣原体 (C. pneumoniae)促巨噬细胞脂质蓄积中的作用和机制,以THP-1巨噬细胞源性泡沫细胞为模型,采用高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量,液体闪烁计数器检测细胞内胆固醇流出,RT-PCR检测ABCA1和LXRα mRNA的表达,蛋白质印迹检测ABCA1和LXRα的蛋白质表达;使用LXRα的特异性激动剂T0901317对细胞进行预处理,再观察上述指标的变化．结果显示,C. pneumoniae可促进THP-1巨噬细胞源性泡沫细胞内总胆固醇、游离胆固醇和胆固醇酯含量增加,抑制胆固醇外流,降低细胞ABCA1和LXRα的表达;使用ABCA1激动剂8-溴-环磷酸腺苷预处理细胞或LXR激动剂T0901317预处理细胞后,可明显减弱C. pneumoniae对THP-1细胞ABCA1的表达抑制,促进细胞胆固醇流出,降低细胞内胆固醇的含量．结果提示,C. pneumoniae促进巨噬细胞脂质蓄积及胆固醇流出障碍,其机制可能与LXRα-ABCA1途径有关．     

干扰素-γ对THP-1巨噬细胞源性泡沫细胞胆固醇流出和ABCA1表达的影响

以THP-1巨噬细胞源性泡沫细胞为研究对象,观察干扰素-γ(IFN-γ)对THP-1巨噬细胞源性泡沫细胞胆固醇流出和三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响.以便探讨IFN-γ在动脉粥样硬化发生发展中的作用.采用液体闪烁计数器检测细胞内胆固醇流出, 高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量.运用逆转录-多聚酶链反应和蛋白质印迹分别检测ABCA1 mRNA与ABCA1蛋白质的表达, 采用流式细胞术检测细胞平均ABCA1荧光强度.发现IFN-γ引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯呈时间依赖性增加, 而ABCA1 mRNA和蛋白质表达、细胞平均ABCA1荧光强度以及apoA-1介导的胆固醇流出呈时间依赖性减少, 细胞内胆固醇增多.结果表明IFN-γ抑制THP-1巨噬细胞源性泡沫细胞ABCA1表达及细胞内胆固醇流出,同时增加细胞内胆固醇聚积.     

脂多糖通过核因子-κB途径下调泡沫细胞ATP结合盒转运体A1的表达

脂多糖(LPS)介导的免疫炎症反应与动脉粥样硬化(As)的发生密切相关,ATP结合盒转运体A1(ABCA1)促进细胞内胆固醇流出,具有抗As作用.观察了LPS对THP-1巨噬细胞源性泡沫细胞ABCA1表达及胆固醇流出的影响,并探讨TLR4/NF-κB信号途径和LXRs在此过程中的作用.THP-1巨噬细胞源性泡沫细胞经不同浓度LPS处理或者用LPS作用不同时间,以LPS单独或用NF-κB抑制剂对甲苯磺酰-L-苯丙氨酸氯甲基甲酮(TPCK)预处理细胞后再加入LPS处理.RT-PCR检测ABCA1、TLR4和LXRα mRNA的表达,Western blot检测ABCA1、LXRα及核内NF-κBp65蛋白的表达,液体闪烁计数器检测细胞内胆固醇流出,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量.结果表明,LPS呈浓度和时间依赖性抑制ABCA1的表达,而增加TLR4 mRNA和核内NF-κBp65蛋白的表达,LPS使泡沫细胞内胆固醇流出减少,细胞总胆固醇、游离胆固醇与胆固醇酯增加,TPCK预处理后,LPS的这种作用被部分抑制,LXRα的表达不受LPS和TPCK的影响.这一结果提示,TLR4/NF-κB信号...     

一种新型PPARγ激动剂对人肾癌细胞增殖抑制的实验研究

目的:探讨新型过氧化物酶体增殖激活物受体(PPARγ)激动剂DH9对人肾癌细胞OS-RC-2的增殖抑制作用。方法:予以不同浓度的DH9及罗格列酮作用OS-RC-2细胞12 h、24 h和48 h,荧光素酶活性检测比较两种药物的PPARγ激动效应;MTT法检测细胞增殖情况;流式细胞术观察细胞周期;Annexin V-FITC/PI双染色流式细胞术测定细胞凋亡率;Western blot检测细胞内Bax及Bcl-2等蛋白的变化。结果:不同浓度的DH9与罗格列酮相比,对PPARγ的激动效应DH9明显低于罗格列酮,增殖抑制作用优于罗格列酮(P0.05),并呈现明显的浓度、时间依赖性;加入PPARγ抑制剂GW9662前后DH9的增殖抑制作用差异无统计学意义(P0.05);DH9作用细胞48小时后,G0/G1期细胞比例明显增加(P0.05),S期细胞明显减少(P0.05)。DH9可诱导细胞凋亡,伴随Bcl-2表达的减少以及Bax表达的增加。结论:OS-RC-2细胞中,DH9的增殖作用明显优于罗格列酮,且是通过PPARγ非依赖途径实现;DH9能将OS-RC-2细胞阻滞在G0/G1期,并通过影响Bcl-2和Bax蛋白表达促进细胞凋亡。     

ERK1/2 mediates lung adenocarcinoma cell proliferation and autophagy induced by apelin-13

The aim of this study was to investigate the role of apelin in the cell proliferation and autophagy of lung adenocarcin- oma. The over-expression of APJ in lung adenocarcinoma was detected by immunohistochemistry, while plasma apelin level in lung cancer patients was measured by enzyme-linked immunosorbent assay. Our findings revealed that apelin-13 significantly increased the phosphorylation of ERK1/2, the expression of cyclin D1, microtubule-associated protein 1 light chain 3A/B （LC3A/B）, and beclinl, and con- fwmed that apelin-13 promoted A549 cell proliferation and induced A549 cell autophagy via ERK1/2 signaling. More- over, there are pores on the surface of human lung adeno- carcinoma cell line A549 and apelin-13 causes cell surface smooth and glossy as observed under atomic force micros- copy. These results suggested that ERK1/2 signaling pathway mediates apelin-13-induced lung adenocarcinoma cell proliferation and autophagy. Under our experimental condition, autophagy associated with 3-methyladenine was not involved in cell proliferation.     

Strigolactone-Regulated Hypocotyl Elongation Is Dependent on Cryptochrome and Phytochrome Signaling Pathways in Arabidopsis

Seedling development including hypocotyl elongation is a critical phase in the plant life cycle. Light regula- tion of hypocotyl elongation is primarily mediated through the blue light photoreceptor cryptochrome and red/far-red light photoreceptor phytochrome signaling pathways, comprising regulators including COP1, HY5, and phytochrome- interacting factors （PIFs）. The novel phytohormones, strigolactones, also participate in regulating hypocotyl growth. However, how strigolactone coordinates with light and photoreceptors in the regulation of hypocotyl elongation is largely unclear. Here, we demonstrate that strigolactone inhibition of hypocotyl elongation is dependent on cryp- tochrome and phytochrome signaling pathways. The photoreceptor mutants cry1 cry2, phyA, and phyB are hyposensi- tive to strigolactone analog GR24 under the respective monochromatic light conditions, while cop1 and pifl pif3 pif4 pif5 （pifq） quadruple mutants are hypersensitive to GR24 in darkness. Genetic studies indicate that the enhanced respon- siveness of cop1 to GR24 is dependent on HY5 and MAX2, while that of pifq is independent of HY5. Further studies demonstrate that GR24 constitutively up-regulates HY5 expression in the dark and light, whereas GR24-promoted HY5 protein accumulation is light- and cryptochrome and phytochrome photoreceptor-dependent. These results suggest that the light dependency of strigolactone regulation of hypocotyl elongation is likely mediated through MAX2-dependent promotion of HY5 expression, light-dependent accumulation of HY5, and PIF-regulated components.     

Salt Stress in Arabidopsis： Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant＇s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase （MAPK） cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein （LTP）-related hybrid proline-rich protein （HyPRP）, as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azil are hypersensitive to salt stress, while AZIl-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZIl-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.     

Adenine Phosphoribosyl Transferase 1 is a Key Enzyme Catalyzing Cytokinin Conversion from Nucleobases to Nucleotides in Arabidopsis

In plants, the cytokinin metabolic processes, including cytokinin biosynthesis, interconversion, inactivation, and degradation, play critical roles in the regulation of cytokinin homeostasis and plant development. Purine meta- bolic enzymes have been implied to catalyze the cytokinin interconversion in previous works. In this study, we report that Adenine Phosphoribosyl Transferase 1 （APT1） is the causal gene of the high-dose cytokinin-resistant mutants. APT1 catalyzes the cytokinin conversion from free bases to nucleotides, and is functionally predominant among the five members of the Arabidopsis Adenine Phosphoribosyl Transferase family. Loss of APT1 activity in plants leads to excess accumulation of cytokinin bases, thus evoking myriad cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression. Thus, our study defines APT1 as a key metabolic enzyme participating in the cytokinin inactivation by phosphoribosylation.     

A Crucial Role of the RGS Domain in Trans- Golgi Network Export of AtRGS1 in the Protein Secretory Pathway

The secretory pathway is responsible for the transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to their destinations via the Golgi/trans-Golgi network （TGN）, Cargo proteins at each sta- tion are actively sorted by specific sorting signals on the cargo and the corresponding coat complexes. Here, we used the Arabidopsis regulator of G-protein signaling （AtRGS1）, which contains an N-terminal potentially sensing glucose seven-transmembrane domain and a C-terminal RGS domain, as a model to uncover sorting motifs required for its cell surface expression. Expression of wild-type and truncated or mutated AtRGS1 fluorescent fusion proteins identified two cysteine residues in the extracellular N-terminus that are essential for endoplasmic reticulum exit and/or correct folding of AtRGS1. The linker between the seven-transmembrane and RGS domains contains an endoplasmic reticulum export signal, whereas the C-terminus is dispensable for the plasma membrane expression of AtRGS1. Interestingly, deletion of the RGS domain results in Golgi/TGN localization of the truncated AtRGS1. Further analysis using site-directed mutagen- esis showed that a tyrosine-based motif embedded in the RGS domain is essential for Golgi/TGN export of AtRGS1. These results reveal a new role for the RGS domain in regulating AtRGS1 trafficking from the Golgi/TGN to the plasma membrane and explain the interaction between the seven-transmembrane and RGS domains.     

Novel Natural Allelic Variations at the Rht-1 Loci n Wheat

Plant height is an important agronomic trait. Dramatic increase in wheat yield during the ＂green revolution＂ is mainly due to the widespread utilization of the Reduced height （Rht）-1gene. We analyzed the natural allelic variations of three homoeologous loci Rht-A1, Rht-B1, and Rht-D1 in Chinese wheat （Triticum aestivum L.） micro-core collections and the Rht-B1/D1 genotypes in over 1,500 bred cultivars and germplasms using a modified EcoTILLING. We identified six new Rht-A1 allelic variations （Rht-Alb-g）, eight new Rht-B1 allelic variations （Rht-Blh-o）, and six new Rht-D1 allelic variations （Rht-Dle-j）. These allelic variations contain single nucleotide polymorphisms （SNPs） or small insertions and deletions in the coding or uncoding regions, involving two frame-shift mutations and 15 missenses. Of which, Rht-Dle and Rht-Dlh resulted in the loss of interactions of GID1-DELLA-GID2, Rht-Blicould increase plant height. We found that the Rht-Blh contains the same SNPs and 197 bp fragment insertion as reported in Rht-Blc. Further detection of Rht-Blh in Tibet wheat germplasms and wheat relatives indicated that Rht-Blc may originate from Rht-Blh. These results suggest rich genetic diversity at the Rht-1 loci and provide new resources for wheat breeding.     

皮肤烫伤修复期斯钙素-1基因表达与组织过氧化氢水平变化

目的：检测皮肤烫伤修复期斯钙素-1（STC1）基因表达与组织过氧化氢（H202）水平的变化。方法：在小鼠Ⅱ度烫伤模型上,采用生物化学和RT-PCR方法检测损伤部位组织H2O2含量及STC1 mRNA水平。结果：组织H2O2水平在皮肤烫伤后12h升高,略微下降后继续升高,至烫伤后120h达到最高值;而STC1 mRNA水平始终处于较高水平,分别在烫伤后12h和120h各出现一次高峰。结论：皮肤烫伤修复期组织STC1 mRNA水平与组织H2O2水平变化具有相关性。     

KCTDIO is critical for heart and blood vessel development of zebrafish

KCTD10 is a member of the PDIP1 family, which is highly conserved during evolution, sharing a lot of similarities among human, mouse, and zebrafish. Recently, zebrafish KCTD13 has been identified to play an important role in the early development of brain and autism. However, the specific function of KCTD10 remains to be elucidated. In this study, experiments were carried out to determine the expression pattern of zebrafish KCTD10 mRNA during em- bryonic development. It was found that KCTD10 is a ma- ternal gene and KCTD10 is of great importance in the shaping of heart and blood vessels. Our data provide direct clues that knockdown of KCTDIO resulted in severe pericardial edema and loss of heart formation indicated by morphological observation and crucial heart markers like amhc, vmhc, and cmlc2. The heart defect caused by KCTD10 is linked to RhoA and PCNA. Flk-1 staining revealed that intersomitic vessels were lost in the trunk, although angioblasts could migrate to the midline. These findings could be helpful to better understand the determinants responsible for the heart and blood vessel defects.     

HL-60细胞诱导分化过程中STIM1 siRNA干扰的电转染研究

目的：以感受器相互作用分子（STIMI）为报告基因优化悬浮培养的二甲基亚砜（DMSO）诱导分化的HL-60细胞的电穿孔转染条件。方法：通过控制电压、电容、电阻、细胞状态等转染条件,采用不同条件组合后用电穿孔法将靶向STIMI的siRNA转入悬浮培养的dHL-60细胞,用荧光显微镜观察转染率,蛋白免疫印记方法检测干扰效率。结果：适当提高电压能提高悬浮培养的dHL-60细胞的转染效率,细胞状态不同干扰效率有所差异,DMSO诱导4d的dHL-60细胞在电压295V、电容1180μF、电阻5000的条件下转入STIMI siRNA系列并得到最大干扰效率（约60％）。结论：针对dHL-60细胞这种比较特殊的细胞系来说,电穿孔转染法是一种较好的基因转染方法,通过优化其转染条件,可以提高其对dHL-60细胞的SiRNA干扰效率。     

黄芪多糖对内毒素诱导人单核/巨噬细胞炎症因子表达的影响

目的：研究黄芪多糖对内毒素诱导的人单核/巨噬细胞中多种炎症因子表达的影响。方法：采用体外培养的人单核/巨噬细胞,以100ng/ml内毒素作为刺激因子,应用人炎症因子抗体芯片检测1mg/ml黄芪多糖处理后人单核/巨噬细胞中IL-1β、IL-6、IL-8和金属蛋白酶组织抑制剂-2（TIMP-2）等多种炎症因子表达的变化情况。结果：黄芪多糖可显著抑制内毒素诱导单核细胞分泌IL-1β和IL-6的活性,同时,促进TIMP-2蛋白表达。结论：黄芪多糖对内毒素诱导的人单核/巨噬细胞多种炎症因子表达有调节作用。