硒蛋白与肾脏疾病的研究进展

硒是人体所必需的一种微量元素,硒蛋白是硒在机体内存在的主要功能形式,在维持机体的正常生理功能,以及肿瘤、免疫和代谢相关疾病等的发生发展过程中发挥着重要作用。近年来的研究提示硒蛋白参与了多种肾脏疾病的病理生理过程。我们对硒蛋白在肾缺血再灌注损伤、糖尿病肾病、慢性肾脏病及肾癌等肾脏疾病中的可能作用及机制,以及补硒治疗的最新进展进行综述,旨在为肾脏疾病的发病机制研究和诊治提供新的思路。     

磁场和磁处理水对鸡和小鼠离体肝线粒体ATP生成的影响研究

用Ｒａｔｈｂｕｎ定磷法研究磁场和磁处理水对鸡和小鼠离体肝线粒体活性的影响。结果表明，磁场和磁处理水可明显提高肝线粒体活性，与对照组相比，都有显著的差异，提示磁场和磁处理水对离体肝线粒体的氧化磷酸化，即对ＡＴＰ的生成有明显的促进作用。     

大鼠心肌梗死后心肌炎症与骨髓干细胞归巢于缺血心肌的关系

目的:分析大鼠急性心肌梗死后心肌组织炎症与骨髓干细胞归巢于缺血心肌的关系。方法:实验于2005-05在西安交通大学动物实验中心完成。①选取清洁级健康雄性SD大鼠128只,取8只大鼠作为正常对照组,剩余120只大鼠随机数字表法分为单纯粒单细胞集落刺激因子组、地塞米松干预组、假手术组,40只/组。②单纯粒单细胞集落刺激因子组、地塞米松干预组建立急性心肌梗死模型,假手术组在左前降支挂线不结扎,正常对照组不进行任何手术。③单纯粒单细胞集落刺激因子组、地塞米松干预组、假手术组大鼠于造模前连续5d皮下注射粒单细胞集落刺激因子50μg/(kg·d)。地塞米松干预组于造模后1h一次性给予地塞米松500μg/kg肌注。④各组大鼠于造模后1,3,5,10d免疫组化观察心肌组织中ckit阳性细胞数及肿瘤坏死因子的表达。造模后28d进行梗死灶组织形态学观察。结果:128只SD大鼠全部进入结果分析。①术后28d各组心肌梗死灶组织形态学观察结果:单纯粒单细胞集落刺激因子组心内膜下有完整的心肌结构,心肌细胞排列整齐,中层由从梗死区边缘伸向斑痕区的心肌组织和结缔组织组成;地塞米松干预组心内膜下仅有散在的心肌细胞,心内膜与心外膜之间的结缔组织稀薄,中层未见有心肌组织;假手术组心脏结构完整未见瘢痕组织。②术后不同时间点各组ckit多克隆抗体特异性免疫组化染色结果:术后1,3,5d各时相点,以单纯粒单细胞集落刺激因子组ckit 细胞密度最大,地塞米松干预组ckit 细胞明显受到抑制(P<0.01);术后10d两组梗死区周边均未发现ckit 细胞。假手术组术后各时间点均未见ckit 细胞。③术后不同时间点各组肿瘤坏死因子的表达情况:地塞米松干预组术后1,3,5,10d各时间点肿瘤坏死因子表达均显著低于单纯粒单细胞集落刺激因子组(P<0.01),而假手术组未发现有肿瘤坏死因子表达。结论:心肌损伤后的炎症反应是骨髓干细胞归巢的先决条件,炎症抑制后限制了骨髓干细胞归巢。提示骨髓干细胞归巢于缺血心肌是机体潜在的修复机能。     

HPLC测定桑叶中绿原酸、芦丁和木犀草苷含量

目的建立同时测定桑叶中绿原酸、芦丁和木犀草苷含量的方法。方法样品用70％乙醇溶液提取,采用Phenomenex LunaC18色谱柱（4．6mm×250mm,5μm）分离;流动相：0．1％磷酸-乙腈,梯度洗脱;流速：1：0mL·min^-1;检测波长：348nm。结果绿原酸、芦丁、木犀草苷分别在3．048～152．4,1．059～52．96,1．437-71．85μg·mL一内具有良好的线性关系;加标回收率分别为100．4％,98.3％,98．7％;RSD分别为1．7％,2．0％,1．9％（n=9）。结论该方法简单易行,准确性和重复性好,可用于桑叶的质量控制。     

ZD7288抑制急性内脏痛大鼠痛觉敏化

观察超极化激活环核苷酸门控阳离子通道（HCN通道）的特异性阻断剂ZD7288对急性内脏痛大鼠痛觉敏化的影响。方法：选用成年雄性SD大鼠,通过结肠内注射1％醋酸1mL,建立急性内脏痛模型;免疫组织化学法检测HCN2在模型大鼠腰骶段背根神经节及胸腰段与腰骶段脊髓背角的表达;通过腹壁撤退反射评分和腹外斜肌放电测量,观察模型大鼠鞘内分别给予50与100nmol／LZD7288后内脏痛觉敏化是否发生改变。结果：HCN2在模型大鼠腰骶段背根神经节及胸腰段与腰骶段脊髓背角的表达均较对照大鼠增强（P〈0．05）。鞘内注射50～100nmol／LZD7288可以剂量依赖性降低急性内脏痛模型大鼠的腹壁撤退反射评分和腹外斜肌放电幅值（P〈0．05）。结论：ZD7288可抑制急性内脏痛大鼠的痛觉敏化,而背根神经节和脊髓的HCN2通道可能在其发病中起作用。     

Crude extract of Rheum palmatum L induced cell death in LS1034 human colon cancer cells acts through the caspase‐dependent and ‐independent pathways

Crude extract of Rheum palmatum L (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub‐G1 phase (apoptosis), the activities of caspase‐8, ‐9, and ‐3, reactive oxygen species (ROS) and Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). DNA damage, nuclei condensation, protein expression, and translocation were examined by Comet assay, 4′‐6‐diamidino‐2‐phenylindole (DAPI) staining, Western blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration‐ and time‐dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl‐2 protein ratio and CERP promoted the activities of caspase‐8, ‐9, and ‐3. Both ROS and Ca²⁺ levels were increased by CERP but the compound decreased levels of ΔΨ_m in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G, cytochrome c, and GADD153 to induce apoptosis through mitochondrial‐dependent pathway. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 969–980, 2014.     

Directed-Evolution Analysis of Human Cytochrome P450 2A6 for Enhanced Enzymatic Catalysis

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (k_cat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated K_m values and therefore catalytic efficiencies (k_cat/K_m) were not improved. The increase in K_m values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.     

HPLC法同时测定腹泻康片中秦皮甲素和秦皮乙素的含量

目的建立同时测定腹泻康片中秦皮甲素和秦皮乙素含量的方法。方法采用高效液相色谱法,色谱柱:Kromasil C18柱(4.6 mm×250 mm,5μm);流动相:乙腈-0.1%磷酸(8∶92);检测波长:334 nm;流速:1 m L·min-1;柱温:30℃。结果秦皮甲素线性范围为47.2⁴72 ng,r=0.999 0,秦皮乙素线性范围为45.6472 ng,r=0.999 0,秦皮乙素线性范围为45.6⁴56 ng,r=0.999 2,秦皮甲素和秦皮乙素平均加样回收率分别为101.09%和100.51%。结论本方法简便、准确、可靠,适用于腹泻康片中秦皮甲素和秦皮乙素的质量控制。     

阿德福韦酯联合复方丹参滴丸对乙肝病毒携带者血小板聚集性的影响

目的：检测阿德福韦酯联合复方丹参滴丸对乙肝病毒携带者血小板聚集性的影响。方法：选取乙型肝炎病毒携带者8例,阿德福韦酯联合复方丹参滴丸治疗12周。在治疗前后分别对血小板聚集性以及HBV—DNA进行检测。结果：受试者在经过阿德福韦酯和复方丹参滴丸的治疗后血小板聚集性有所增强[用药前VS用药后（54．8±6．23）％VS（68．2±7．71）％,P〈O．05]。同时乙肝病毒DNA（HBV—DNA）的拷贝数下降,所有受试者用药前均高于106copies／ml,而用药后均低于104copies／ml。结论：阿德福韦酯联合丹参治疗可增加乙肝病毒携带者的血小板的聚集性。血小板的聚集性的增强可能与HBV复制受抑有关。