牙菌斑形成早期微生物定植规律的定量PCR研究

目的观察牙面彻底清洁后24 h内牙面上定植的变异链球菌、伴放线放线杆菌和总微生物的数量变化。方法 8名健康成人接受全口洁治后,分别于6、12和24 h收集龈上菌斑,提取菌斑内细菌的基因组DNA。设计变异链球菌、伴放线放线杆菌和总菌特异性引物,获得目的基因,克隆于大肠埃希菌DH5α感受态细胞,测序后获得质粒标准品。将样本和梯度稀释的质粒标准品进行SYBR Green I实时荧光定量PCR检测,绘制标准曲线,确定样本中变异链球菌、伴放线放线杆菌和总菌DNA拷贝数。结果牙面彻底清洁6 h后即有大量变异链球菌定植,变异链球菌拷贝数占总菌的0.32%,24 h后增加到0.67%。12 h时定植的变异链球菌拷贝数高于6 h,差异有统计学意义（P=0.031）,24 h后继续增加（P=0.024）。12 h时定植的总菌拷贝数高于6 h,差异有统计学意义（P=0.004）,24 h后继续增加（P=0.042）。牙菌斑中伴放线放线杆菌的拷贝数低于103。结论早期牙菌斑中12 h定植的变异链球菌和总菌数量高于6 h,且24 h内不断增加,仅有少量伴放线放线杆菌定植。     

标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量.     

古DNA实时荧光定量PCR实验中标准品的制备

实时荧光定量PCR技术通过对PCR每一循环扩增产物的实时检测,可对模板的精确拷贝数进行绝对定量,从而用于古DNA实验中提取和扩增条件的比较和优化.本研究采用异硫氰酸胍碱裂解-SiO2吸附的方法,从采自黑龙江省的晚更新世斑鬣狗化石材料中提取得到了斑鬣狗线粒体基因组古DNA.经常规PCR扩增后,将纯化的扩增产物克隆到微生物体内使其大量复制,再用M13通用引物扩增出含少量外源DNA的古DNA目标片段,从而建立了适用于古DNA荧光定量PCR扩增的标准品的制备方法.经检测分析,运用该方法制备的标准品性质稳定,能够准确地指示反应体系中较为精确的古DNA模板拷贝数,从而反映古DNA的提取和扩增效率,用于比较并优化古DNA提取和扩增条件.     

TaqMan荧光定量RT-PCR检测水稻RAc1基因mRNA方法的建立

构建包含RAc1基因cDNA片段的质粒,作为水稻肌动蛋白基因RAc1之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参.从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAc1基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析.纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验.建立了RAc1基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102～107拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系(r=1.000),扩增效率高(E=98.2%).建立了基因RAc1实时定量PCR的质粒标准品.     

TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建立

构建包含RAcl基因cDNA片段的质粒,作为水稻肌动蛋白基因RAcl之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAcl基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAcl基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102—1护拷贝,阈值循环数（Ct）与PCR体系中起始模板量的对数值之间有着良好的线性关系（r=1．000）,扩增效率高（E=98．2％）。建立了基因RAcl实时定量PCR的质粒标准品。     

JEV-DNA实时荧光定量标准品的构建

利用TaqMan荧光定量PCR技术,建立JEV-DNA定量标准品的制备方法。通过处理JEV减毒活疫苗提取病毒RNA,进行RT-PCR扩增目的片段,与T载体连接,转化感受态细胞,T-A克隆,测序鉴定后定量。获得预期的重组质粒,建立的标准曲线有较大的线性范围。此法制备的重组质粒标准品可用于对病毒载量进行测定。     

版纳微型猪近交系TNF-α基因mRNA实时荧光定量PCR检测方法的建立

目的快速、灵敏、可靠的检测与临床多种疾病密切相关的重要基因TNF-α的表达情况,构建TNF-α基因及内参GAPDH基因荧光定量PCR质粒标准品。方法利用版纳微型猪近交系4～6月龄猪建立动物模型,提取皮肤创面总RNA,设计特异引物,进行RT-PCR扩增。纯化目的片段与pMD18-T载体连接,转化宿主菌DH5α,提取重组质粒DNA,并经酶切、PCR和测序鉴定,计算重组质粒原液拷贝数浓度并制备梯度浓度标准品,进行实时荧光定量PCR,生成标准曲线。结果建立的TNF-α基因和GAPDH内参基因mRNA表达实时荧光定量PCR检测方法灵敏度分别可达103和105拷贝,线性范围分别为103～109和105～109拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间存在的线性关系R2分别为0.993和0.999,扩增效率E分别为111.073%和95.948%。结论成功的构建了版纳微型猪近交系TNF-α基因质粒标准品和标准曲线,并用内参基因GAPDH进行校正,此方法可为探讨TNF-α基因在临床多种疾病中所发挥的分子机理奠定基础。     

拟除虫菊酯降解酶基因APs实时荧光定量PCR检测方法的建立

本研究建立了一种能够快速、灵敏地检测来源于铜绿假单胞菌GF31中拟除虫菊酯降解酶基因APs的SYBR GreenⅠ相对实时荧光定量PCR方法。通过克隆构建APs目的基因及16S rRNA内参基因重组质粒,分别制备了质粒标准品SgI-pET30a、PA-pET30a、Pep-pET30a以及16S r RNA-p ET30a;绘制标准曲线,建立了实时荧光定量PCR体系。结果显示,在质粒标准品浓度为0.001~10 ng范围内,实时定量PCR标准曲线的线性关系良好,APs和16S r RNA的标准曲线R~20.99,扩增效率=95%~105%;熔解曲线呈单峰,特异性良好;扩增曲线呈S型,CT值在各梯度间均匀变化、重复性好;将建立的相对实时荧光定量PCR方法应用于检测工程菌及野生菌APs基因的m RNA表达水平,2~(-△△Ct)法计算得工程菌SgI、PA及Pep的表达量分别为野生菌的1 898、1 314和956倍。     

SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

目的 建立SYBR Green Ⅰ荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数.方法 巢式RT-PCR扩增SIV病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒.该质粒经限制性内切酶Not I酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品.结果 应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL.结论 制备的RS外标准品纯度高,SYBR Green Ⅰ荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数.     

荧光定量PCR监测五氯酚对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响

通过特异引物扩增环境中氨氧化细菌16S rDNAV2保守区域,将该片段克隆到T-easy载体上,PCR产物经测序和定量PCR扩增体系鉴定,证实PCR扩增产物为氨氧化细菌16S rDNA保守序列,以含该序列的重组质粒作为定量PCR监测氨氧化细菌数量的DNA标准品。用荧光定量PCR技术比较了五氯酚(PCP)对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响。结果表明,不加PCP的反应器中,好氧颗粒污泥和活性污泥中氨氧化细菌的数量分别为4.28×107±5.44×106cells/(g干污泥)和2.51×109±8.61×108cells/(g干污泥)。随着PCP浓度的增加(0~50mg/L),PCP对氨氧化细菌数量的影响不大(P>0.05),而且,污泥中氨氧化细菌的数量与氨氮的去除率无直接的正相关关系(P>0.05),PCP主要是抑制氨氧化细菌的代谢活性导致污泥氨氮去除效率降低。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2017年中国植物科学若干领域重要研究进展

2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Changes in aquatic vegetation in Rhine floodplain streams in Alsace in relation to disturbance

Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.