Effect of peroxisome proliferator-activated receptor γ agonist rosiglitazone on intestinal injury in rats undergoing orthotopic autologous liver transplantation by inhibiting inflammatory response

AIM: To investigate the effect of rosiglitazone, a peroxisome proliferators-activated receptor γ(PPARγ) agonist, on the expression of PPARγ, the activation of NF-κB and intestine injury in the rats undergoing orthotopic autologous liver transplantation(OALT).METHODS: Sprague-Dawley male rats were randomly divided into 4 groups:control group, sham group, OALT group and rosiglitazone(0.3 mg/kg, iv) pretreatment(ROS+OALT) group. The OALT model was established, and the intestinal tissues were collected 8 h after the liver reperfusion. The intestinal tissue sections were stained to visualize the damage. The expression of PPARγ and NF-κB in the tissues, the concentrations of diamine oxidase(DAO) and fatty acid-binding protein 2(FABP2) in the serum and the concentration of TNF-α and IL-6 in the tissues were measured.RESULTS: Compared with sham group, the intestinal mucosa of the rats showed obvious pathological injury after liver reperfusion in OALT group and ROS group, the Chiu,s scores of intestinal mucosa was significantly higher, and the serum concentrations of DAO and FABP2 increased(P<0.05). After rosiglitazone pretreatment, the injury of intestinal mucosa of the rats was alleviated, the Chiu,s scores was lower and the serum concentrations of DAO and FABP2 decreased(P<0.05), the PPARγ expression was obviously up-regulated in the intestinal tissues, the nuclear translocation of NF-κB was reduced and the concentrations of IL-6 and TNF-α were decreased.CONCLUSION: During perioperative period of OALT in rats, the inflammatory responses are obvious. Furthermore, obvious intestinal injury occurs. PPARγ agonist rosiglitazone obviously up-regulates PPARγ expression and inhibits the inflammation in the intestines, thus protecting against intestinal injury in rats undergoing OALT.     

T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

苯并噻二唑甲酸三氟乙酯诱导仙客来抗枯萎病的研究

为了明确新型化合物苯并噻二唑甲酸三氟乙酯对土传病害的诱导抗病作用,本文研究了该化合物诱导仙客来对枯萎病的抗病效果。通过叶面喷施苯并噻二唑甲酸三氟乙酯可诱导仙客来对枯萎病的抗病效果,在仙客来移栽缓苗后7d叶面喷施该化合物,不同浓度对枯萎病的抗病效果不同,其中100mg.L-1的处理,在诱导后25、30和35d的抗病效果分别为82.90%、58.59%和64.17%;在仙客来成株枯萎病发病初期用该化合物诱导,抗病效果仍可达63.91%,显著高于噁霉灵处理（47.63%）。该研究将为寻求通过叶面喷雾施药防治园艺作物土传病害的新途径起到参考作用。     

Prospective clinical trial evaluating spironolactone in Doberman pinschers with congestive heart failure due to dilated cardiomyopathy

Introduction/objectivesWhether the aldosterone antagonist spironolactone has beneficial survival effects in dogs with dilated cardiomyopathy (DCM) is not known. The primary objective of the study was to evaluate the effect of spironolactone, when added to conventional therapy, on survival time in Doberman pinschers with congestive heart failure (CHF) due to DCM.AnimalsSixty-seven client-owned Doberman pinschers with CHF due to DCM.Materials and methodsThe trial design was prospective, randomized, blinded, and placebo controlled. Dogs were randomized to receive 50–75 mg of spironolactone twice daily (n = 34) or a placebo (n = 33), in addition to standard CHF therapy. Follow-up visits were targeted every one–six weeks until endpoint. Quality-of-life questionnaire and physical examination were performed at every visit, while renal biochemistry, ECG, echocardiography, and thoracic radiography were reassessed as needed. The primary endpoint was time to cardiac death, defined as death or euthanasia from CHF or sudden death.ResultsMedian time to primary endpoint in the spironolactone group (183 days) was not statistically significantly different than that for the placebo group (124 days) (P = 0.254). The development of atrial fibrillation (AF) was significantly less frequent in the spironolactone group (n = 7) than the placebo group (n = 15, P = 0.037).ConclusionsWhile median time to cardiac death in the spironolactone group was not statistically significantly different than that in the placebo group, adding spironolactone to conventional therapy resulted in reduced occurrence of AF.     

军曹鱼MHC-Ⅱα基因全长cDNA的克隆及其组织表达分析

本研究根据其他鱼类MHC-Ⅱα基因的保守序列设计兼并引物,运用同源克隆和末端快速扩增方法扩增了军曹鱼MHC-Ⅱα基因的全长cDNA序列,并对cDNA及其氨基酸序列进行了分析,比较了军曹鱼和其他物种的MHC-Ⅱα氨基酸序列的差异,分析了军曹鱼MHC-Ⅱα基因的组织分布及经LPS刺激后头肾组织中MHC-Ⅱα基因的表达变化。结果表明,军曹鱼MHC-Ⅱα cDNA全长998 bp,包括53 bp的5′末端非编码区（5′UTR）、234 bp的3′末端非编码区（3′UTR）及711 bp的开放阅读框（ORF）,编码236个氮基酸,其蛋白质分子质量约为25.94 ku,等电点为4.39;军曹鱼MHC-Ⅱα蛋白质序列具有一些重要的特征,包括前导肽、α1、α2、CP/TM/CYT区和保守的半胱氨酸等;军曹鱼MHC-Ⅱα与鼠、人及其它鱼类的氨基酸同源性在25.0%～69.5%之间。Real-time PCR检测结果显示,MHC-Ⅱα基因在正常军曹鱼组织中均有表达,但其表达量在各种组织中存在差异,其中较强表达于头肾、鳃,中等程度表达于脾脏、肠,在心脏、脑、肌肉中表达较弱;经LPS刺激后,头肾中MHC-Ⅱα基因表达下调。     

武定鸡红细胞免疫功能研究及其意义

应用红细胞Ｃ３ｂ受体花环（Ｃ３ｂＲＲ）和免疫复合物花环（ＩＣＲ）试验,证实武定鸡红细胞表面存在补体Ｃ３ｂ受体（Ｃ３ｂｒｃｃｃｐｔｏｒ,Ｃ３ｂＲ）,表明红细胞免疫系统（Ｒｃｄｃｃｌｉｍｍｕｎｅｓｙｓｔｅｍ,ＲＣＩＳ）的概念亦适用于这种动物。武定鸡的ＲＣＩＳ有其独特之处,文中对武定鸡红细胞Ｃ３ｂＲＲ和ＩＣＲ分别代表红细胞表面Ｃ３ｂ“空位”和“占位”状态的含义做出了新的解释。     

金黄色葡萄球菌α-溶血素的研究进展

目的α-溶血素在金黄色葡萄球菌(金葡菌)肺炎的感染过程中起着非常重要的作用,可作为治疗金葡菌肺炎的新靶标。因此,研究百里香酚对金葡菌α-溶血素表达的影响具有重要意义。方法测定最小抑菌质量浓度(MIC)和生长曲线并进行了溶血试验、蛋白免疫印迹分析、荧光定量PCR以及细胞毒性试验。结果百里香酚对金葡菌USA300的MIC为256 μg/mL,亚抑菌质量浓度的百里香酚(32 μg/mL)可下调金葡菌hla和RNAIII的转录水平,降低α-溶血素的表达,对溶血素介导的肺癌上皮细胞A549损伤具有保护作用。结论百里香酚具有成为新型抗金葡菌感染药物的潜力。     

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal ([Ca²⁺]_i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP₃R1), the channel responsible for Ca²⁺ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP₃R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP₃R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP₃R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34^cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP₃R1 phosphorylation, although inactivation of p34^cdc2 kinase by roscovitine dramatically reduced IP₃R1 phosphorylation. Neither inhibitor affected total expression of IP₃R1. Altogether, our results show that IP₃R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.     

SIRT1 promotes autophagy of pancreatic cancer cells induced by hypoxia via regulating FOXO1/RAB7 signaling pathway

AIM: To investigate the effect of SIRT1 on the autophagy of pancreatic cancer cells under hypoxia condition, and to analyze the underlying mechanism of regulating FOXO1/RAB7 signaling pathway. METHODS: Western blot and immunofluorescence methods were used to determine the expression of SIRT1 in the pancreatic cancer cells. The small interfering RNA targeting SIRT1 and SIRT1 over-expression plasmid were transfected into the pancreatic cancer Panc-1 cells. Confocal microscopy was used to detect the LC3 expression. Western blot was used to analyze the protein levels of LC3, p62 and FOXO1/RAB7 signaling pathway-related molecules. Co-immunoprecipitation was used to detected the protein interaction between SIRT1 and FOXO1. RESULTS: The expression level of SIRT1 in the nucleus of Panc-1 cells was increased under hypoxia condition. Compared with negative control under hypoxia condition, knock-down of SIRT1 expression attenuated the autophagy flux in the pancreatic cancer Panc-1 cells (P<0.05). Over-expression of SIRT1 increased the protein levels of FOXO1 and RAB7. On the contrary, knock-down of SIRT1 expression inhibited the protein levels of FOXO1 and RAB7. The protein interaction between SIRT1 and FOXO1 in the pancreatic cancer cells was observed. CONCLUSION: SIRT1 in pancreatic cancer Panc-1 cells under hypoxia condition is over-expressed in the nucleus. Down-regulation of SIRT1 inhibits autophagy and its mechanism may be related to FOXO1/RAB7 signaling pathway.     

藏绵羊KLF7基因表达特征分析及其过表达对前脂肪细胞增殖分化的影响

【目的】分析藏绵羊Krüppel样因子7(Krüppel-like factor 7,KLF7)基因表达特征,研究过表达该基因对前脂肪细胞增殖及分化的影响。【方法】从藏绵羊脂肪组织中分离前脂肪细胞进行培养及成脂诱导,应用实时荧光定量PCR技术检测KLF7基因在藏绵羊7个组织(大脑、皮下脂肪、肾脏、背最长肌、瘤胃、睾丸和回肠)和前脂肪细胞不同分化阶段(第0、2、4和8天)的mRNA相对表达水平;应用RT-PCR方法从藏绵羊脂肪组织中扩增KLF7基因CDS区序列,并将其连接到pcDNA3.1(+)真核表达载体获得pcDNA3.1-KLF7过表达质粒,转染前脂肪细胞;应用实时荧光定量PCR方法检测脂肪细胞增殖及分化标志基因mRNA表达水平;采用EdU和CCK-8方法分别检测过表达KLF7基因对EdU阳性细胞数和细胞活力的影响;采用油红O染色检测过表达KLF7基因后脂肪细胞脂滴生成量。【结果】KLF7基因在藏绵羊7个组织中均有表达,其中在大脑中的表达量最高,其次为皮下脂肪和肾脏,均显著高于其他组织(P<0.05);诱导分化第2、4和8天脂肪细胞mRNA表达量均显著高于分化前(P＜0.05),且分化第2天表达量最高;pcDNA3.1-KLF7过表达质粒转染前脂肪细胞2 d后显著或极显著抑制增殖标志基因CDK4、CyclinB1和CyclinD1的表达水平(P＜0.05;P＜0.01),极显著降低细胞活力及EdU阳性细胞数量(P＜0.01);pcDNA3.1-KLF7过表达质粒转染前脂肪细胞,诱导分化8 d后,脂肪细胞分化标志基因PPARγ、Glut4和ELOVL6的mRNA相对表达水平显著或极显著下调(P＜0.05;P＜0.01),且脂质沉积极显著减少(P＜0.01),表明过表达KLF7基因可抑制藏绵羊前脂肪细胞增殖及分化。【结论】KLF7基因在藏绵羊多个组织中广泛表达,且大脑、皮下脂肪、肾脏中表达量较高;诱导分化后脂肪细胞表达量显著高于分化前,且分化第2天表达量最高;过表达KLF7基因可抑制藏绵羊前脂肪细胞的增殖及分化。试验结果为阐明藏绵羊脂肪沉积的分子调控机制提供了基础数据。