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。     

多形拟杆菌对糖尿病模型小鼠的影响

目的研究多形拟杆菌(BT)干预糖尿病模型小鼠后对血糖、体重和C肽的影响。方法 (1)用四氧嘧啶(200 mg/kg腹腔注射)制备糖尿病模型小鼠,并分成四组:空白组(n=10)、空白给菌组(n=10)、四氧嘧啶糖尿病模型组(n=11)和模型给菌组(n=11),15 d。(2)BHI血琼脂培养基培养ATCC 29148标准菌株,比浊法测定混悬菌液数量。(3)用多形拟杆菌菌液干预空白给菌组和模型给菌组小鼠,观察15 d中四组小鼠的体重、空腹血糖水平的变化;实时荧光定量PCR测定小鼠肠道内多形拟杆菌的数量;运用酶联免疫法(ELISA)测定血清中C肽的水平。结果 (1)给予菌悬液后,空白给菌组与空白组相比,BT在第3天就可以定植并维持到第15天。与模型组相比,模型给菌组在第15天时可以定植;(2)线性回归相关性分析显示,肠道内多形拟杆菌与体重呈负性相关(r=-0.70,P0.05);与空腹血糖(FPG)呈正性相关(r=0.71,P0.05);与C肽呈负性相关(r=-0.62,P0.05);与胰岛素抵抗指数(HOMA-IR)呈正性相关(r=0.55,P0.05);与胰岛素分泌指数(HOMA-IS)呈负性相关(r=-0.43,P0.05)。结论 (1)外源性灌胃给予多形拟杆菌可以在肠道内定植。(2)肠道内多形拟杆菌的数量变化与糖尿病有相关性。     

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（T2DM）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16S rDNA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T2DM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。     

实时荧光定量PCR法研究附子、红参不同比例配伍对大鼠肠道菌群的影响

目的 应用实时荧光定量PCR法对灌胃给予不同配伍比例的附子和红参大鼠粪便中的总菌、乳杆菌和拟杆菌的相对含量进行检测分析。方法 将15只SD大鼠随机分成A、B、C三组,每组5只。A组：附子单独给药7 d,B组：附子、红参配伍比例1∶1给药7 d,C组：附子、红参配伍比例1∶2给药7 d。各组灌胃7 d结束之后,于第8天收集大鼠的粪便,提取粪便中细菌的DNA,并根据细菌的16S rDNA基因序列设计总菌、乳杆菌和拟杆菌的种属特异性引物,进行实时荧光定量PCR反应,分析不同细菌的相对含量。结果 C组大鼠中总菌、乳杆菌和拟杆菌的相对含量要显著高于A组和B组;A组和B组中总菌、乳杆菌和拟杆菌的相对含量没有明显差异。结论 当附子和红参的配伍比例为1∶2时,能在一定程度上改善肠道的微生态环境,促进有益菌的生长。     

多形拟杆菌对肥胖大鼠减肥影响的初步研究及毒性实验

目的研究人体肠道内的多形拟杆菌对肥胖大鼠的减肥作用。方法从人体肠道中提取、分离和鉴定1株多形拟杆菌。建立高能饲料诱发的大鼠肥胖模型,给大鼠灌胃多形拟杆菌菌液,25 d后观察大鼠体重及肠道内多形拟杆菌的数量变化。结果灌胃多形拟杆菌菌液的给药组的大鼠体重与灌胃生理盐水的模型组相比,增长慢（P〈0.05）,差异有显著性,且体内拟杆菌数量多于模型组,差异有显著性（P〈0.05）。结论提示多形拟杆菌菌液对肥胖大鼠有一定的减肥作用。     

结直肠腺瘤及结直肠癌患者肠道中梭杆菌属与产丁酸菌的定量研究

【目的】通过观察梭杆菌属(Fusobacterium spp.)和两株产丁酸菌(Eubacterium rectale、Faecalibacterium prausnitzii)在结直肠癌患者及结直肠腺瘤患者粪便样品中的丰度差异,研究梭杆菌属和产丁酸菌数量变化在结直肠腺瘤和结直肠癌发生发展中的作用和意义。【方法】收集结直肠癌患者(n=19)、结直肠腺瘤患者(n=12)及健康人(n=19)3组粪便样品,提取细菌基因组DNA,利用实时荧光定量PCR技术定量检测3组样品中梭杆菌属(Fusobacterium spp.)、直肠真杆菌(Eubacterium rectale)、普拉梭菌(Faecalibacterium prausnitzii)以及总菌的16S rRNA基因的拷贝数,然后利用秩和检验两两比较3组样品中目标菌群的数量和丰度差异。【结果】结直肠癌组的梭杆菌属丰度显著高于结直肠腺瘤组(P=0.013)和健康组(P=0.000),结直肠腺瘤组的梭杆菌属丰度显著高于健康组(P=0.002);结直肠腺瘤组普拉梭菌的丰度显著低于健康组(P=0.033);结直肠腺瘤组的总菌16S rRNA基因拷贝数也显著低于健康组(P=0.002);直肠真杆菌的水平在3组样品间没有显著差异。【结论】与健康人的粪便样品相比,结直肠腺瘤病人的粪便中产丁酸菌普拉梭菌数量下降,而结直肠腺瘤和结直肠癌病人的粪便样品中梭杆菌属数量增加;梭杆菌属和产丁酸菌数量上的变化提示它们可能与结直肠腺瘤和结直肠癌的发生密切相关。     

老年2型糖尿病患者肠道双歧杆菌生长与血糖相关性

目的 探讨老年2型糖尿病患者肠道双歧杆菌生长与血糖的相关性,从肠道菌群角度为2型糖尿病的防治与研究提供新的途径。方法 根据纳入和排除标准,抽取2015年4月‒10月福建某三甲医院糖尿病健康教育俱乐部的2型糖尿病患者90例,收集其粪便和血液标本,检测患者肠道双歧杆菌以及血糖状况,采用Spearman等级相关法分析两者之间的相关性。结果 老年2型糖尿病患者肠道长双歧杆菌、短双歧杆菌、青春双歧杆菌、婴儿双歧杆菌和双歧杆菌总菌的定量结果分别是（0.0001±0.0006）、（4.405±8.475）、（4.752±2.779）、（0.003±0.005）和（8.069±8.206）logN/g。空腹血糖和早餐餐后血糖分别为（8.638±1.913）、（9.763±1.910）mmol/L。相关分析显示：青春双歧杆菌和婴儿双歧杆菌与早餐后2 h血糖呈现负相关（r=-0.213、-0.217,P<0.05）,但相关性不显著;双歧杆菌总菌与空腹血糖和早餐后2 h血糖有负相关关系（r=-0.302、-0.559,P<0.01）。结论 老年2型糖尿病患者肠道内双歧杆菌数量减少,血糖与肠道双歧杆菌的数量呈负相关,尤其是早餐后2 h血糖,其相关机制有待进一步研究。     

肠道菌群与2型糖尿病相关性的研究进展

摘要：糖尿病是一种常见的代谢性疾病,发病率高,易引起严重的微血管、大血管并发症,造成多重器官损伤;缺乏有效的治疗手段,患者生存质量普遍偏低,致残率、致死率走高;发病机制异常复杂,目前仍未能完全阐明。近年来,大量报道显示,肠道菌群作为人体不可分割的部分,参与了宿主的健康维持和疾病发生,与2型糖尿病的发生、发展有着直接的关系。本文就肠道菌群与2型糖尿病的相关性研究进展进行简要综述,为肠道菌群及2型糖尿病的相关研究提供参考。     

实时荧光定量PCR法研究结直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化

目的通过研究结、直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化,揭示肠道相关菌群改变在大肠癌发病中的作用及意义。方法收集术前结、直肠癌患者粪便标本40例及正常对照标本40例,根据细菌的靶基因序列设计特异性引物。提取待测粪便标本细菌DNA,应用SYBR Green I实时荧光定量PCR测定不同细菌的数量。结果正常对照组与实验组粪便中细菌数量分别为拟杆菌属(8.76±0.77;9.85±0.88)、梭杆菌属(7.94±1.25;10.0±1.65)、梭菌属(3.54±0.70;6.56±0.68),拟杆菌属中的脆弱拟杆菌为(2.12±0.48;4.07±1.77)、梭杆菌属中的坏死梭杆菌为(2.31±0.26;7.62±2.68)及梭菌属中的肉毒梭菌为(2.76±1.16;5.43±1.21),实验组数量均明显增多(P0.05)。结论结、直肠癌患者粪便中拟杆菌属、梭杆菌属和梭菌属的数量较正常对照明显增多,提示结、直肠癌的发生发展与肠道菌群有明显关系。     

青春双歧杆菌对2型糖尿病模型大鼠肠道菌群和脂质代谢的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠肠道菌群的变化,和血清中总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL-C）、超氧化物歧化酶（SOD）和丙二醛（MDA）的水平,探讨青春双歧杆菌对2型糖尿病模型大鼠肠道功能和脂质代谢的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取粪便检查正常菌群,取血和脏器检测TC、TG、HDL-C、SOD和MDA含量。结果青春双歧杆菌导致肠道内双歧杆菌、乳杆菌的数量增加,而肠杆菌、肠球菌数量下降;TC、TG和MDA水平下降,而HDL-C和SOD水平升高。结论青春双歧杆菌具有改善2型糖尿病模型大鼠肠道功能和降血脂作用,与二甲双胍联合应用效果更佳。     

腹部脂肪组织APN基因DNA甲基化及mRNA表达与维吾尔族T2DM的相关性

为探讨APN基因启动子区DNA甲基化及mRNA表达与新疆维吾尔族T2DM发生、发展的相关性,文章选择新疆维吾尔族正常个体50例、肥胖个体48例、肥胖伴T2DM个体26例,收集腹部网膜脂肪组织,利用变性高效液相色谱技术检测APN基因启动子区DNA甲基化情况,应用Real-time PCR方法检测APN 基因mRNA表达情况。结果显示,APN基因启动子区DNA甲基化阳性率在正常对照(34%)、肥胖(47.9%)及T2DM组(65.4%)逐渐增加,差异具有统计学意义(P<0.05)。Real-time PCR结果显示,正常对照组APN mRNA相对拷贝数(0.7162)显著高于肥胖(0.4244)及T2DM组(0.4093),差异具有统计学意义(P<0.05)。非T2DM个体相关性分析提示,APN mRNA相对拷贝数与空腹血清葡萄糖(Fasting plasma glucose, FPG)、糖化血红蛋白(Glycosylated hemoglobin, HbA₁c)、甘油三酯(Triglyceride, TG)水平显著负相关(P<0.05)。APN基因启动子区DNA甲基化与其mRNA表达负相关,甲基化阳性组相对拷贝数(0.2700)显著低于阴性组(0.7870),差异具有统计学意义(P<0.01)。以上结果提示,APN基因启动子区DNA甲基化通过抑制其 mRNA表达导致糖脂代谢紊乱,可能参与了新疆维吾尔族肥胖及T2DM的发生、发展过程。     

肠道菌群与2型糖尿病相关性的研究进展

肠道菌群是人体肠道微生态的重要组成部分,以多种途径影响宿主的代谢与生理功能,通过调节肠道菌群结构与多样性,改善慢性疾病的发生发展已成为国内外的研究热点。本研究归纳总结了近年来肠道菌群影响2型糖尿病的主要途径,为阐明肠道菌群与2型糖尿病的相关性以及基于肠道菌群靶点的药物研制和临床治疗提供新的思路。     

Contribution of genetic variant identified in HHEX gene in the overweight Saudi patients confirmed with type 2 diabetes mellitus

BackgroundThe rs7932837 polymorphism in the Hematopoietically expressed homeobox (HHEX) gene was discovered through genome-wide association studies and is a promising candidate for type 2 diabetes mellitus (T2DM), which is one of the risk factors for obesity and other complications. T2DM has been identified as a heterogeneous and multifactorial disease characterized by insulin resistance and secretion.AimThe aim of this study was to investigate the rs7932837 polymorphism in the HHEX gene in overweight patients diagnosed with T2DM in the Saudi Population.MethodsIn this case-control study, one hundred T2DM cases and 100 controls were selected based on inclusion and exclusion criteria. Genotyping was performed with polymerase chair reaction-restriction fragment length polymorphism analysis and statistical analysis was performed between T2DM cases and controls for clinical characteristics, genotype and allele frequencies and multiple logistic regression analysis.ResultsIn this study, T2DM cases were compared with healthy control subjects. Clinical characteristic analysis revealed the statistical analysis between age, weight, BMI, FBG, HDL-c, TC, TG and family history (p < 0.05). HWE analysis was in the accordance (p < 0.05). The rs7932837 polymorphism in the recessive model showed the positive association (AA + AG vs AA: 2.22 [1.25–3.96] & p = 0.006) and none of the genotypes or alleles were in the statistical association. Multiple logistic regression analysis revealed positive association with age, BMI and FBG (p < 0.05).ConclusionThis study concludes as rs7932837 polymorphism in the HHEX gene showed positive association with recessive model and future studies recommend to carry out with large number of sample size with additional polymorphisms in HHEX gene.     

Plasma Adiponectin Levels in High Risk African‐Americans with Normal Glucose Tolerance,Impaired Glucose Tolerance,and Type 2 Diabetes**

Objective: We studied plasma adiponectin, insulin sensitivity, and insulin secretion before and after oral glucose challenge in normal glucose tolerant, impaired glucose tolerant, and type 2 diabetic first degree relatives of African‐American patients with type 2 diabetes. Research Methods and Procedures: We studied 19 subjects with normal glucose tolerance (NGT), 8 with impaired glucose tolerance (IGT), and 14 with type 2 diabetes. Serum glucose, insulin, C‐peptide, and plasma adiponectin levels were measured before and 2 hours after oral glucose tolerance test. Homeostasis model assessment‐insulin resistance index (HOMA‐IR) and HOMA‐β cell function were calculated in each subject using HOMA. We empirically defined insulin sensitivity as HOMA‐IR < 2.68 and insulin resistance as HOMA‐IR > 2.68. Results: Subjects with IGT and type 2 diabetes were more insulin resistant (as assessed by HOMA‐IR) when compared with NGT subjects. Mean plasma fasting adiponectin levels were significantly lower in the type 2 diabetes group when compared with NGT and IGT groups. Plasma adiponectin levels were 2‐fold greater (11.09 ± 4.98 vs. 6.42 ± 3.3811 μg/mL) in insulin‐sensitive (HOMA‐IR, 1.74 ± 0.65) than in insulin‐resistant (HOMA‐IR, 5.12 ± 2.14) NGT subjects. Mean plasma adiponectin levels were significantly lower in the glucose tolerant, insulin‐resistant subjects than in the insulin sensitive NGT subjects and were comparable with those of the patients with newly diagnosed type 2 diabetes. We found significant inverse relationships of adiponectin with HOMA‐IR (r = ?0.502, p = 0.046) and with HOMA‐β cell function (r = ?0.498, p = 0.042) but not with the percentage body fat (r = ?0.368, p = 0.063), serum glucose, BMI, age, and glycosylated hemoglobin A1C (%A1C). Discussion: In summary, we found that plasma adiponectin levels were significantly lower in insulin‐resistant, non‐diabetic first degree relatives of African‐American patients with type 2 diabetes and in those with newly diagnosed type 2 diabetes. We conclude that a decreased plasma adiponectin and insulin resistance coexist in a genetically prone subset of first degree African‐American relatives before development of IGT and type 2 diabetes.     

米格列奈与瑞格列奈分别联合二甲双胍在新诊断2型糖尿病的临床价值比较

目的：探讨米格列奈和瑞格列奈在治疗新诊断2型糖尿病的临床价值比较。方法：将2010年1月至2012年12月间我院新诊断的120例2型糖尿病患者随机分为A、B两组,其中A组患者60例,采用瑞格列奈联合二甲双胍进行治疗,B组患者60例,采用米格列奈联合二甲双胍进行治疗。对两组患者血糖水平及12周后相应指标进行统计分析。结果：治疗3天后两组患者的空腹血糖及餐后血糖水平明显低于治疗前,B组患者血糖降低幅度大于A组（P〈0．05）;治疗12周后,两组空腹血糖及餐后血糖水平显著减小,B组减小程度高于A组;患者空腹及餐后胰岛素水平有一定的增加,B组增加程度高于A组;B组患者HbAlc的降低程度高于A组;两组同时还可以降低HOMA胰岛素的抵抗指数以及增大胰岛素的敏感指数,其中B组效果好于A组;B组患者出现低血糖的人次少于A组。结论：米格列奈与瑞格列奈分别联合二甲双胍可以短期降低新诊断2型糖尿病患者的血糖水平,在中期治疗后米格列奈联合二甲双胍可以更有效的改善患者糖尿病的各项指标。     

新疆维、哈两民族人群血浆中谷氨酰胺的含量及其与2型糖尿病关系的研究

目的 探讨谷氨酰胺与2型糖尿病的关系.方法 采用高效液相色谱仪(HPLC)来测定新疆维、哈两民族血浆中谷氨酰胺的含量;使用放免法测定空腹血清胰岛素(FINS)含量;采用全自动生化分析仪来测定空腹血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDLC)、低密度脂蛋白(LDLC)的含量.结果 维吾尔族正常人与糖尿病患者之间血浆中谷氨酰胺含量差异有统计学意义(P＜0.01);哈萨克族正常人与糖尿病患者之间血浆中谷氨酰胺含量差异无统计学意义;维哈两民族的正常人血浆中谷氨酰胺含量差异有统计学意义(P＜0.01);维哈两民族的糖尿病患者血浆中谷氨酰胺的含量差异有统计学意义(P＜0.05).维哈两民族人群血浆中的谷氨酰胺含量与空腹血糖(FBG)水平呈负相关(r=-0.4858,P＜0.001),与FINS水平呈正相关(r =0.236,P＜0.05),与胰岛素分泌指数(HOMA-IS)呈正相关(r=0.52,P=0.001),与TG呈负相关(r=-0.4330,P＜0.01),与LDLC呈负相关(r=-0.4077,P＜0.01),与HDLC呈正相关(r=0.2553,P＜0.01).结论 谷氨酰胺可能是2型糖尿病的一个保护性的影响因素.     

Evaluation of method performance for oxidative stress biomarkers in urine and biological variations in urine of patients with type 2 diabetes mellitus and diabetic nephropathy

Background

Oxidative stress biomarkers such as superoxide dismutase (CuZnSOD), catalase (CAT) and malondialdehyde (MDA) play an important role in the pathogenesis or progression of numerous diseases. Data regarding the biological variation and analytical quality specifications (imprecision, bias and total error) for judging the acceptability of method performance for oxidative stress biomarkers in urine are conspicuously lacking in the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals (index of individuality) and assessing the significance of changes in serial results from an individual (reference change value; RCV).

Materials and methods

20 patients with type 2 diabetes mellitus (T2DM), 20 patients with diabetic nephropathy (DN) and 14 healthy individuals as control were involved in this study. Timed first morning urine samples were taken from patients and healthy groups on the zero, 1st, 3rd, 5th, 7th, 15th and 30th days. Index of individuality and reference change value were calculated from within-subject and between-subject variations. Methods of oxidative stress biomarkers in human blood were adopted in human urine and markers were measured as spectrophotometrically. Also, analytical quality specifications for evaluation of the method performance were established for oxidative stress biomarkers in urine.

Results

Within-subject variations of oxidative stress biomarkers were significantly higher in patients with DN and T2DM compared to healthy subjects. MDA showed low individuality, and within-subject variances of MDA were larger than between-subject variances in all groups. However, CAT and CuZnSOD showed strong individuality, but within-subject variances of them were smaller than between-subject variances in all groups. RCVs of all analytes in diabetic patients were relatively higher, because of high within-subject variation, resulting in a higher RCV. Also, the described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 6.0-7.9% and 12.5-23.3%, respectively.

Conclusions

RCVs concept for predicting the clinical status in diabetic patients represents an optimization of laboratory reporting and could be a valuable tool for clinical decision. Furthermore, for oxidative stress biomarkers’ measurements in urine, the desirable imprecision goals based on biological variation are obtainable by current methodologies.