Effects of Chinese yellow wine on homocysteine-induced dysfunction in rat endothelial progenitor cells

AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.     

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

Sox2 is one important marker of pluripotent stem cells, a study found that neural stem cells with high expression of Sox2 as donor cells showed higher reprogramming ability in nuclear transplantation.In this study, through enhancing exogenous Sox 2 gene expression of sheep bone marrow mesenchymal stem cells, in order to raise their reprogramming ability, and improve the efficiency of somatic cell cloning in animal.Total RNA was extracted from sheep testicular tissue, and with this template, Sox2 cDNA sequence was amplified and inserted into the eukaryotic expression vector pEGFP-N1 to build a recombinant vector pEGFP-N1-Sox2.The vector was transfected into the sheep bone marrow mesenchymal stem cells by liposome method, and through G418 and fluorescence screening to obtain and amplify monoclone.DNA sequencing showed that sheep Sox 2 gene CDS sequence was obtained, and recombinant plasmid was successfully constructed.Identification of fluorescence confirmed that stable sheep bone marrow mesenchymal stem cell lines transfected with Sox2 were established.This study obtained the sheep bone marrow mesenchymal stem cell lines with high expression of Sox2, and provided a new idea for raising reprogramming efficiency in the process of somatic cell cloning.     

低水平镉暴露对破骨细胞分化的影响

为研究低水平镉(Cd)暴露对破骨细胞(osteoclast,OC)分化的影响,试验以RAW264.7细胞(单核巨噬细胞系)为材料,在巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)存在的条件下,用不同浓度Cd处理4 d;利用CCK-8法检测破骨细胞及其前体细胞活性变化,抗酒石酸酸性磷酸酶(TRAP)染色试验观察破骨细胞生成,激光共聚焦显微镜观察破骨细胞形态变化,蛋白免疫印迹(Western blot)技术和荧光定量聚合酶链式反应(qRT-PCR)检测破骨细胞标志性蛋白及其mRNA水平。结果显示,随着Cd浓度升高,细胞活力受到明显的抑制(P<0.01),并呈浓度-效应关系;与对照组相比,破骨细胞产生的数目和面积均显著或极显著下降(P<0.05或P<0.01);2、5μmol·L^-1 Cd处理组破骨细胞封闭带的形成均受到抑制;2和5μmol·L^-1 Cd处理组破骨细胞特异性蛋白及其mRNA表达量均显著或极显著下降(P<0.05或P<0.01),并呈剂量依赖性。结果表明,低微摩尔水平镉暴露能够抑制破骨细胞的分化。     

鸭LXRα基因过表达对原代肝细胞及PC3细胞脂质代谢的效应研究

旨在探究鸭LXRα基因对脂肪代谢的表达调控机制,本试验以pEGFP-N3为载体,构建携带HIS标签的LXRα基因真核过表达载体,并将过表达载体分别转染鸭原代肝细胞及PC3细胞,使用鸭HIS标签试剂盒测定LXRα基因在肝细胞及PC3细胞中的过表达量,分析该基因分别在两种细胞中过表达与各脂质指标含量的相关性,进而探究其对鸭肝细胞及PC3细胞脂质代谢的调控作用。试验结果显示,LXRα基因在两种细胞中过表达与三酰甘油含量(triglyceride,TG)及高密度脂蛋白(high density lipoprotein,HDL)含量均呈极显著正相关关系(P<0.01),与总胆固醇(total cholesterol,TC)含量及低密度脂蛋白含量(low density lipoprotein,LDL)则均呈极显著负相关关系(P<0.01)。本研究揭示了LXRα基因过表达对TG和HDL的合成代谢有重要的正向调控作用,对TC和LDL具有反向调控作用,且在这两种类型的细胞中调控机制相似。这一研究结果将对今后深入研究LXRα基因在脂质代谢网络通路中的作用及解决鸭体脂肪过度沉积等问题奠定理论基础。     

马立克病肿瘤组织内HSP60表达及生物学作用初步研究

本试验旨在研究HSP60与马立克病肿瘤发生、发展之间的相关性。通过人工感染,建立鸡马立克病肿瘤模型,定期剖杀,利用病理组织学和免疫组织化学方法,检测HSP60与肿瘤细胞定位之间的相关性;设计HSP60 RNA干扰序列,构建重组慢病毒,转染MSB-1细胞,利用流式细胞技术,探索降低HSP60转录表达对MSB-1细胞凋亡水平的影响。结果显示：HSP60在肿瘤细胞的细胞质内强表达;成功构建了HSP60 RNA干扰慢病毒,且5147序列干扰效果最佳;5147序列慢病毒转染48 h时,与对照序列组和空白对照组相比,HSP60转录、表达水平极显著降低（P<0.01）,MSB-1细胞凋亡水平极显著升高（P<0.01）。在马立克病肿瘤发生、发展过程中,HSP60组织细胞定位与肿瘤细胞具有明显的相关性,降低HSP60表达水平能够导致MSB-1细胞凋亡升高,说明HSP60对肿瘤细胞的存活具有重要的生物学作用。     

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine–leucine peptide

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)‐Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4‐day‐old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two‐step method of collagenase‐I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2 mM and 2 mM as well as with the Gly‐Leu peptide concentration of 0.2 mM and 2 mM. The experiment lasted for 5 days. The results showed that TOR, S6K1 and 4E‐BP1 mRNA expressions in the medium with Leu concentration of 2 mM were significantly higher than that in 0.2 mM group (p < 0.05). There was no difference between the medium with Gly‐Leu concentration of 2 mM and 0.2 mM on the TOR, S6K1 and 4E‐BP1 mRNA expressions (p > 0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E‐BP1 mRNA expressions of skeletal muscle satellite cells, but Gly‐Leu peptide has no effect on them.     

Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf‐ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B‐Raf and C‐Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf‐ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.     

Thiamine deficiency affects glucose transport and β‐oxidation in rats

Thiamine is recognized as a cofactor for many enzymes involved in intermediary metabolism responsible for energy production. Animal model of thiamine deficiency (TD) included direct evaluation of glucose uptake by estimation of ³H‐deoxyglucose transport across red blood cells membranes and β‐oxidation of fatty acids in isolated leucocytes. Feeding of animals with the thiamine‐deficient diet (0.018 mg/kg diet) for 30 days resulted in disturbances in energy production. The thiamine intake was limited not only by vitamin B₁ deficiency in the diet, but also by time‐dependent drop of feed consumption by rats fed this diet. At the end of experiment, diet consumption in this group of rats was 52% lower than in the control group. This was accompanied by low glucose uptake by erythrocytes of rats suffering vitamin B₁ deficiency for longer time. At the end of experimental period, glucose uptake was over 2 times lower in TD erythrocytes than in control RBC. Such drop of energy production was not compensated by delivery of energy from fatty acid degradation. In leucocytes from TD rats, the β‐oxidation was also suppressed. Observed significant decrease of serum insulin from 2.25 ± 0.25 ng/ml (day 0) to 1.94 ± 0.17 ng/ml (day 30) might have significant impact on observed energy production disorders. The results from this study indicate that the thiamine deficiency significantly reduces feed intake and causes modest abnormalities in glucose and fatty acid utilization.