新疆维、哈两民族粪样中奇异菌簇和产气柯林斯菌属水平与2型糖尿病的相关性研究

目的检测新疆维吾尔族、哈萨克族糖耐量正常人群及2型糖尿病患者粪便中奇异菌簇和产气柯林斯菌属的水平,分析奇异菌簇和产气柯林斯菌属与2型糖尿病的相关性。方法收集新疆维吾尔族、哈萨克族糖耐量正常人群及2型糖尿病患者血样和粪便样本,采用16S r DNA实时荧光定量PCR技术检测粪便样本中奇异菌簇和产气柯林斯菌属的水平;运用Pearson分析目标菌属水平与4组人群的空腹血糖(FBG)、年龄、身高、体重、体重指数(BMI)、腰围、胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白胆固醇(LDL-C)的相关性。结果 (1)与哈萨克族糖耐量正常组比较,该民族T2DM组中奇异菌簇、产气柯林斯菌属水平均降低(P=0.012,P=0.024),两民族糖耐量正常组之间奇异菌簇、产气柯林斯菌属水平差异有统计学意义(P=0.025,P=0.007);(2)奇异菌簇水平与HDL-C(r=0.281,P=0.031)呈正相关,与体重(r=-0.514,P=0.000)、BMI(r=-0.400,P=0.002)、TC(r=-0.503,P=0.000)、LDL-C(r=-0.581,P=0.000)水平呈负相关;产气柯林斯菌属水平与HDL-C(r=0.550,P=0.000)水平呈正相关,与体重(r=-0.449,P=0.000)、BMI(r=-0.432,P=0.001)水平呈负相关。结论肠道中奇异菌簇和产气柯林斯菌属,可能与与新疆维吾尔族、哈萨克族2型糖尿病患者的脂代谢紊乱密切相关。     

新疆维吾尔族2型糖尿病人群肠道中Faecalibacterium prausnitzii的定量研究

目的定量检测新疆维吾尔族2型糖尿病(T_2DM)和糖耐量正常(NGT)人群肠道中的普拉梭菌(Faecalibacterium prausnitzii)水平。方法提取上述人群粪便细菌总DNA后,采用16SrDNA基因实时荧光定量PCR对普拉梭菌水平进行定量检测;运用Pearson分析普拉梭菌水平与研究对象的空腹血糖(FBG)、甘油三酯(TG)、总胆固醇(TC)、体重和体重指数(BMI)的相关性。结果 (1)16S rDNA基因实时定量PCR结果显示:与新疆维吾尔族NGT组相比,普拉梭菌水平在T_2DM中较低(t=2.590,P=0.014),差异有统计学意义。(2)新疆维吾尔族上述人群肠道中普拉梭菌水平与FBG呈负相关(r=-0.434,P=0.012),体重呈负相关(r=-0.359,P=0.044),TG呈负相关(r=-0.410,P=0.034)。结论 Faecalibacterium prausnitzii水平在肠道中降低可能与2型糖尿病的发病有关,其机制需进一步研究探讨。     

新疆维吾尔族、哈萨克族血浆LBP，sCD14的含量测定及其与2型糖尿病的相关性研究

目的通过分析新疆维吾尔族、哈萨克族正常糖耐量人群和2型糖尿病患者血浆中脂多糖结合蛋白（LBP）、可溶性白细胞分化抗原14（sCDl4）及LBP／sCDl4水平的变化,探讨LBP、sCDl4、LBP／sCDl4与新疆维吾尔族、哈萨克族2型糖尿病（T2DM）的相关性。方法采用酶联免疫吸附法（Elisa）检测LBP、sCDl4在血浆中的浓度。两组问均数比较采用t检验,运用Pearson分析方法对血浆LBP、sCDl4、LBP／sCDl4与新疆维吾尔族、哈萨克族2型糖尿病和糖耐量正常人群的空腹血糖（FBG）、年龄、身高、体重、体重指数（BMI）、腰围、胆固醇（Tc）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL—C）、低密度脂蛋白胆固醇（LDL—C）做相关性分析。结果与哈萨克族糖耐量正常组相比较,该民族T2DM组中LBP和LBP／sCDl4的表达量升高且差异均有统计学意义（P=0．012,P=0．003）;与哈萨克族糖耐量正常组相比,维吾尔族糖耐量正常组血浆LBP和LBP／sCDl4的表达量升高且差异均有统计学意义（P=0．005,P=0．006）。血浆LBP的含量与相对应的FBG、体重、BMI、TC、TG、HDL—C存在一定的相关性及LBP／sCDl4与FBG、TG显著正相关性。结论与哈萨克族糖耐量正常组相比较,LBP和LBP／sCDl4的表达量在哈萨克族2型糖尿病组和维吾尔族糖耐量正常组均显著升高。     

新疆哈萨克族2型糖尿病患者粪便中韦荣球菌的分离鉴定

目的从新疆哈萨克族2型糖尿病患者粪便中分离培养并鉴定韦荣球菌。方法收集新鲜哈萨克族2型糖尿病患者的粪便样本,用韦荣球菌专属培养基进行分离培养,微量生化反应管进行初步鉴定;提取所得菌株的DNA,使用韦荣球菌属引物对此DNA进行特异性扩增,结合全自动细菌鉴定仪对菌株进行鉴定。结果 (1)所得到的菌株经微量生化反应管初步鉴定为韦荣球菌;(2)使用韦荣球菌属引物对此菌株DNA进行特异性扩增获得预期产物;(3)经全自动细菌鉴定仪最终确定所得菌株为韦荣球菌。结论从新疆哈萨克族2型糖尿病患者粪便中分离培养得到韦荣球菌。     

新疆维吾尔族、哈萨克族2型糖尿患者群肠道菌群中直肠真杆菌与多形拟杆菌的定量研究

目的分析新疆维吾尔族、哈萨克族正常糖耐量人群和2型糖尿患者群肠道菌群中直肠真杆菌与多形拟杆菌的量变差异,探讨肠道菌群与2型糖尿病（T2DM）的相关性。方法采用16S rDNA实时荧光定量RT-PCR技术相对定量法。结果分别比较4组人群肠道菌群中直肠真杆菌与多形拟杆菌数量的对数值,与维吾尔族正常组比较,该民族T2DM组中直肠真杆菌的量差异有统计学意义（P=0.0125）,与哈萨克族正常组比较,该民族T2DM组中直肠真杆菌的量差异有统计学意义（P=0.0261）,两民族正常组、T2DM组之间直肠真杆菌的量差异均未见统计学意义;与维吾尔族正常组比较,该民族T2DM组中多形拟杆菌的量差异有统计学意义（P=0.0003）,哈萨克族T2DM组与正常组中多形拟杆菌的量差异有统计学意义（P=0.0055）,两民族正常组之间多形拟杆菌的量差异有统计学意义（P=0.0154）,两民族T2DM组之间多形拟杆菌的量差异未见统计学意义。结论直肠真杆菌与多形拟杆菌数量的变化,与新疆维吾尔族、哈萨克族2型糖尿病的发生（可能）相关,需要进一步深入探讨。     

新疆哈萨克族正常血压人群和高血压人群肠道菌群中拟杆菌属、梭菌属结构特征分析

目的分析新疆哈萨克族正常血压人群和高血压人群肠道菌群中拟杆菌属、梭菌属的结构特征,探讨两人群肠道两菌属的差异。方法使用16S DNA-PCR-DGGE技术比较哈萨克族正常血压人群和高血压人群肠道菌群中两菌属结构的差异,将差异条带克隆、测序,与GenBank数据库提供的序列进行比对,确定细菌种类。结果哈萨克族高血压人群肠道梭菌属的16S DNA V3区DGGE图谱中出现一条优势带,其在2组人群中出现的频率经分析后差异具有统计学意义(P0.05)。基因序列在GenBank数据库上用Blast程序进行比对,确定为Uncul-tured bacterium clone nbw1009b01c1。结论这种细菌可能会影响哈萨克族高血压的发生和发展。     

维吾尔族新发2型糖尿病患者肠道 乳杆菌属和多形拟杆菌的定量研究

目的定量研究维吾尔族新发2型糖尿病(T2DM)患者和糖耐量正常(NGT)人群肠道中乳杆菌属(Lactobacillus genus)和多形拟杆菌(Bacteroides thetaiotaomicron)的相对水平。方法严格按照纳入标准、排除标准收集维吾尔族新发T2DM患者96例,NGT 98例。提取所有研究对象的粪便细菌总DNA后,采用16SrDNA基因实时荧光定量PCR(Real-time PCR)对乳杆菌属和多形拟杆菌的水平进行定量检测;运用Pearson相关性分析乳杆菌属与研究对象的BMI、腰围、臀围、收缩压(SBP)、舒张压(DBP)、空腹血糖(FBG)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)和低密度脂蛋白(LDL-C)的相关性。结果 16SrDNA基因Real-time PCR结果显示:(1)与新疆维吾尔族NGT组相比,乳杆菌属水平在新发T2DM中较低,差异有统计学意义(t=8.557,P=0.000)。但是多形拟杆菌在两组差异无统计学意义(t=0.524,P=0.601);(2)新疆维吾尔族上述人群肠道中乳杆菌属水平与FBG呈负相关(r=-0.334,P=0.000),腰围呈负相关(r=-0.170,P=0.018),TC呈负相关(r=-0.178,P=0.013),TG呈负相关(r=-0.157,P=0.030),收缩压呈负相关(r=-0.255,P=0.000)。结论 Lactobacillus genus水平在肠道中降低可能与2型糖尿病的发病,血糖和血脂代谢有关,其机制需进一步研究探讨。     

软粪液和小肠液对寒地獭兔肠道菌群定植的影响

目的研究口服软粪液和小肠液的寒地獭兔肠道内拟杆菌、梭菌属、韦荣球菌和大肠埃希菌数量的变化,揭示软粪液和小肠液对肠道菌群定植的影响。方法 90只仔兔分为I、II和III组,分别口服1mL的软粪液、小肠液和纯净水。无菌刮取32和46日龄仔兔的十二指肠、空肠、回肠和盲肠肠壁内容物,提取内容物细菌基因组DNA。根据拟杆菌、梭菌属、韦荣球菌和大肠埃希菌的16SrDNA V3可变区基因序列设计特异性引物,进行实时荧光定量PCR反应,定量不同细菌。结果口服软粪液(I组)的32和46日龄仔兔其空肠中的拟杆菌浓度显著高于对照组(5.77E-93.58E-10、1.10E-93.39E-10)(t值为8.20、2.16,P0.05),回肠中的梭菌属和韦荣球菌的浓度极显著高于对照组(5.27E-63.58E-7、7.41E-61.43E-6、1.57E-51.33E-6、7.99E-61.80E-6)(t值为3.07、4.24、5.87、6.28,P0.01)。口服小肠液(II组)的32日龄仔兔其十二指肠和回肠中的梭菌属浓度极显著高于对照组(3.71E-74.87E-8、6.88E-63.58E-7)(t值为3.42、2.95,P0.01);46日龄仔兔其回肠中的韦荣球菌浓度显著高于对照组(4.13E-61.80E-6)(t值为2.03,P0.05)。结论软粪液和小肠液对拟杆菌、梭菌属、韦荣球菌的定植有促进作用,对大肠埃希菌的定植影响较小。     

新疆昆仑雪菊水提物对 小鼠肠道菌群的调节作用

目的 研究新疆昆仑雪菊水提物对小鼠肠道菌群的影响。方法 C57BL/6小鼠,雄性,8周龄,40只,分为4个组(每组10只):高、中、低三个剂量组和空白对照组,给药14 d,收集给药前后小鼠粪样,16S rDNA实时荧光定量PCR法测定粪样中肠杆菌、肠球菌、乳杆菌和产气荚膜梭菌水平。结果 与空白对照组比较,中剂量组小鼠肠道粪样中乳杆菌显著增多(t=-2.503,P0.05),低剂量组与高剂量组变化不显著;与空白对照组比较,其他组小鼠肠道中肠杆菌属、肠球菌属和产气荚膜梭菌水平均无显著变化。结论 参照《保健食品检验与评价技术规范—2003版》之"调节肠道菌群作用检验方法",新疆昆仑雪菊水提物对小鼠肠道菌群具有一定的调节作用。     

哈萨克族、维吾尔族正常糖耐量人差异代谢物及肠道菌群的比较CSCD

目的 探讨哈萨克族2型糖尿病(type 2 diabetes mellitus,T2DM)发病率低于维吾尔族的可能原因,比较两个民族正常糖耐量人群肠道菌群、粪便及血浆代谢物的差异。方法 纳入哈萨克族正常糖耐量人(KNGT组)及维吾尔族正常糖耐量人(UNGT组)各8名,利用超高效液相色谱串联四极杆飞行时间质谱(UPLC-QTOF-MS/MS)对其粪便及血浆样本进行非靶向代谢组学检测,通过Illumina测序平台对其粪便样本进行宏基因组测序,对结果进行Wilcoxon检验及LEfSe差异分析。结果 非靶向代谢组学结果显示,KNGT组人群AICAR(5-氨基咪唑-4-甲酰胺核糖核苷)等5种粪便代谢物及1,7-Dimethylxanthine等5种血浆代谢物水平更高;宏基因组数据分析结果显示Dysgonomonadaceae等6种肠道细菌在UNGT组人群丰度较高,Alistipes putredinis等26种肠道细菌在KNGT组人群丰度较高,通过分析碳水化合物活性酶发现,两组人群糖苷水解酶和糖基转移酶的水平最高。结论 KNGT组与UNGT组人群中粪便、血浆代谢物及肠道细菌的差异可能是哈萨克族T2DM发病率较低的原因之一。     

Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of arsine and methylarsines in soil and in culture

Arsenate, arsenite, monomethylarsonate, and dimethylarsinate were added to different soils, and evolution of gaseous arsenical products was determined over 3 weeks. Arsine was produced in all three soils from all substrates, whereas methylarsine and dimethylarsine were produced only from methylarsonate and dimethylarsinate, respectively. At least three times more arsine than dimethylarsine was produced in soil incubated with dimethylarsinate. Resting cell suspensions of Pseudomonas and Alcaligenes produced arsine as the sole product when incubated anaerobically in the presence of arsenate or arsenite. In all instances, no trimethylarsine was observed, nor could any evidence be shown for the methylation of any arsenical substrate in soil or in culture. It was concluded that reduction to arsine, not methylation to trimethylarsine, was the primary mechanism for gaseous loss of arsenicals from soil.     

Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo

Guanine (Gua) modification by nitrating and hydroxylating systems was investigated in DNA. In isolated calf thymus DNA, 8-NO(2)-Gua and 8-oxo-Gua were dose-dependently formed with peroxynitrite, and 8-NO(2)-Gua was released in substantial amounts. Myeloperoxidase (MPO) with H(2)O(2) and NO(2)(-) reacted with calf thymus DNA to form 8-NO(2)-Gua dose dependently without release of 8-NO(2)-Gua. The frequency of strand breaks was higher than the sum of 8-NO(2)-Gua and 8-oxo-Gua, particularly in the MPO-treated DNA, indicating the importance of other types of damage. The activation of human neutrophils and lymphocytes with phorbol ester did not induce 8-NO(2)-Gua and 8-oxo-Gua in their nuclear DNA. However, 8-NO(2)-Gua was found in calf thymus DNA co-incubated with activated neutrophils in the presence of NO(2)(-). No significant formation of 8-NO(2)-Gua was found in liver DNA from mice treated with Escherichia coli lipopolysaccharide. The incubation of peroxynitrite or MPO-H(2)O(2)-NO(2)(-)-treated DNA with formamidopyrimidine glycosylase (Fpg) released 8-oxo-Gua, but not 8-NO(2)-Gua, indicating that 8-NO(2)-Gua is not a substrate for Fpg. Although 8-NO(2)-Gua was generated in isolated DNA by different nitrating systems, other types of damage were formed in abundance, and the lesion could not be found reliably in nuclear DNA, suggesting that the biological importance is limited.     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

Activity of native and dextran-modified hyaluronidase in in vitro and in vivo systems

Modified hyaluronidase derivatives have been obtained. Covalent coupling of the enzyme with aldehyde dextran results in 65-85% protein binding to the carrier, residual catalytic activity accounting for 90-100% of the baseline. Modified hyaluronidase is more thermostable than the native enzyme. The data on intravenous drug distribution in the mouse organs are promising and ensure effective use of modified hyaluronidase for the treatment of pulmonary diseases.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro

⁷⁵Se and ¹⁰⁹Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO₃^2? doses and times up to 24 h after the simultaneous subcutaneous administration of SeO₃^2? and CdCl₂ to adult male rats. The simultaneous injection of CdCl₂ and SeO₃^2? markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 μmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO₃^2? does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Biosynthesis of beta-glucuronides of retinol and of retinoic acid in vivo and in vitro

After the intraportal injection of retinol-6,7-(14)C to rats, the O-ether derivative of retinol, retinyl -glucosiduronate, appears in the bile. Both retinoyl -glucuronide and retinyl -glucosiduronate are also synthesized in vitro when washed rat liver microsomes are incubated with uridine diphosphoglucuronic acid (UDPGA) and either retinoic acid or retinol, respectively. The synthesis of retinoyl -glucuronide was also demonstrated in microsomes of the kidney and in particulate fractions of the intestinal mucosa. The glucuronides were characterized by their UV absorption spectra, by their quenching of UV light or fluorescence under it, by their thin-layer chromatographic behavior in two solvent systems, and by the identification of products released during their hydrolysis by -glucuronidase. With retinoic acid as the substrate, the UDP glucuronyl transferase of rat liver microsomes had a pH optimum of 7.0, a temperature optimum of 38 degrees C, and a marked dependence on the concentrations of both retinoic acid and UDPGA, but was unaffected by a number of possible inhibitors, protective agents, and competitive substrates. The conversion of retinal to retinoic acid and the synthesis of retinoyl -glucuronide from retinoic acid could not be detected in whole homogenates, cell fractions, or outer segments of the bovine retina.