两种纳米金标记p53抗体探针的制备、表征和性质研究

目的制备IgG-Au-HRP和IgG-Au-DNA-HRP两种纳米金标记p53抗体探针,并对其进行生物学特征表征和性质研究。方法利用纳米金粒子与蛋白质非共价吸附和与巯基修饰的寡核苷酸以Au-S键共价结合的特点,将辣根过氧化物酶(HRP)直接或通过寡核苷酸链间接标记在纳米金表面,制备两种多功能纳米金生物探针(IgG-Au-HRP和IgG-Au-DNA-HRP探针);应用透射电镜、紫外-可见光光谱等对纳米金探针性能进行表征,对纳米金探针表面HRP酶活性进行分析,并通过酶免疫标记技术(enzyme linked immuncsorbent assay,ELISA)优化探针制备条件和比较两种探针标记效率。结果①两种新型探针具有良好的单分散性,大小均一,粒径约10nm;②p53蛋白检测效果对IgG-Au-HRP和IgG-Au-DNA-HRP探针制备条件优化结果表明:制备IgG-Au-HRP探针时,HRP分子和p53抗体的最佳比例为10:1;制备IgG-Au-DNA-HRP探针时,DNA和p53抗体的最佳比例为2.5:1;③HRP分子定量检测显示:一个IgG-Au-HRP探针可标记HRP数量约11个,IgG-Au-DNA-HRP探针可标记HRP数量为20个;④两种探针结合ELISA法初步检测p53蛋白判定IgG-Au-DNA-HRP探针比IgG-Au-HRP探针检测蛋白灵敏度高。结论两种新型纳米金探针制备简单,可作为一种新型探针应用于微量蛋白的高灵敏检测。     

TaqMan探针实时PCR检测人MTHFR基因C677T多态性方法的建立

目的建立TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法。方法设计一对MTHFR基因C677T多态位点的引物及TaqMan探针,采用TaqMan探针实时PCR扩增SNP分型方法检测唇腭裂患者及其父母共100人的MTHFR基因C677T多态性,与常规PCR-RFLP方法进行一致率比较,并对其特异性、敏感性和重复性以及成本-效益等进行评价,同时对部分实时PCR产物样本进行测序验证。结果运用TaqMan探针实时荧光PCR技术对MTHFR基因C677T多态性检测结果准确,特异性好,与常规PCR-RFLP方法结果具有高度一致性,Kappa=0.922>0.75(P=0.000);检测灵敏度可达2×103拷贝;重复性好、高通量、无污染、安全性好;随机样品TaqMan探针分型结果与测序结果完全一致。结论成功建立了TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法;此方法是常规临床诊断及大规模群体研究的良好平台。     

HIF-1α介导低氧诱导的内皮细胞Brm表达上调的机制研究

目的:本研究拟探讨低氧诱导因子1α(HIF-1α)在低氧诱导的血管内皮细胞Brm表达上调中的作用及机制,为低氧性肺动脉高压的防治提供理论依据。方法:在内皮细胞中过表达或siRNA干扰HIF-1α的表达,并给予1%低氧处理24 h后,运用萤光素酶报告基因检测方法检测Brm启动子的转录活性,运用RT-qPCR检测Brm的mRNA表达水平,运用Western blot检测Brm的蛋白表达水平。运用ChIP技术检测低氧条件下HIF-1α与Brm启动子区域的结合变化情况。结果:过表达HIF-1α可显著上调Brm的启动子活性、mRNA及蛋白表达,而siRNA干扰HIF-1α可显著抑制Brm的启动子活性、mRNA及蛋白表达,ChIP实验揭示低氧可显著增强HIF-1α与Brm启动子的结合。结论:低氧上调内皮细胞中Brm的表达依赖HIF-1α的转录调控作用。     

小鼠Brd2基因探针的制备及其在荧光原位杂交实验中的应用

目的:为观察溴结构域包含蛋白2(bromodomain containing protein 2,Brd2)基因在小鼠中枢神经系统的表达与分布,本研究制备了两条地高辛标记的Brd2 cRNA探针。方法:提取小鼠脑组织总RNA,设计两对Brd2引物,通过逆转录聚合酶链式反应(RT-PCR)方法,扩增得到两个Brd2的DNA片段,并将它们分别克隆到pCRⅡ-TOPO载体中。利用体外转录方法合成地高辛标记的cRNA探针,最后通过荧光原位杂交实验分析所标记探针的特异性及杂交效果。结果:本实验成功构建了两个Brd2/pCRⅡ-TOPO质粒,获得了特异性的地高辛标记的Brd2cRNA探针,在荧光原位杂交实验中应用两条探针得到了较好的杂交信号。结论:本实验制备的地高辛标记的cRNA探针可特异地检测Brd2 mRNA,为进一步观察Brd2 mRNA在中枢神经系统的分布及功能研究提供了工具。     

尾加压素Ⅱ介导卵圆细胞增殖的机制探讨

<正>目的:探讨尾加压素Ⅱ(UⅡ)对肝脏干细胞即卵圆细胞内活性氧(ROS)的影响及其参与UⅡ促细胞增殖的机制探讨。方法:通过流式细胞术,应用DCFH-DA探针检测UⅡ孵育卵圆细胞WB-F344后,胞内ROS水平变化。通过Western blot方法检测胞内NADPH氧化酶亚基、细胞周期蛋白E、细胞周期蛋白依赖性激酶2及信号通路蛋白ERK表达水平。采用流式细胞术     

应用荧光原位杂交技术诊断未培养羊水细胞染色体异常

目的建立运用荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测未培养羊水细胞染色体异常的方法。方法对24例未培养羊水细胞进行FISH检测,其中用21号染色体荧光探针检测19例,X/Y染色体探针检测3例,13、18号染色体探针各检测1例。结果24例产前诊断者未培养羊水细胞检测出一例21三体。X/Y染色体探针检测时1例为2个绿色的X信号,2例均为1个红色Y信号和1个绿色X信号。检测结果与培养后羊水细胞检测结果一致,并与外周血染色体核型检测结果相符。结论本研究方法快速、简便、准确可靠,适于临床运用推广。     

Gd_2O_3:Eu~(3+)双模态分子影像探针的肝毒性分子机制探究

目的探究Gd_2O_3:Eu~(3+)双模态分子影像探针的肝毒性,并初步阐明其肝毒性的分子机制。方法离体LO2肝细胞与Gd_2O_3:Eu~(3+)分子影像探针共孵育,CCK8法评估其细胞毒性作用,流式细胞仪分析其对细胞凋亡的影响。Balb/c小鼠尾静脉注射该分子影像探针建立动物在体模型,肝组织切片免疫组化检测肝组织中凋亡标志蛋白caspase3的激活情况。RT-q PCR检测凋亡相关信号通路p53、Bal-2和Bal-x L的m RNA表达水平,明确该分子影像探针引发肝毒性的相关分子机制。结果细胞实验表明,Gd_2O_3:Eu~(3+)分子影像探针可引发肝细胞凋亡;动物实验表明,在肝组织内激活P53基因,抑制Bcl-2及Bcl-x L基因表达,同时激活caspase3蛋白,进而引发肝细胞的凋亡。结论从基因水平初步阐明Gd_2O_3:Eu~(3+)分子影像探针的肝毒性分子机制,可能是通过激活肝脏组织内的p53,同时抑制Bcl-2、Bcl-x L基因表达,进而激活caspase3蛋白,而引发肝细胞的凋亡。     

卵巢癌特异性MR分子探针的制备

目的 制备针对卵巢癌的特异性磁共振(magnetic resonance,MR)分子探针,并评价其体外转染SKOV3细胞的可行性.方法 琼脂糖凝胶阻滞实验确定探针中的多聚赖氨酸修饰的超顺磁性氧化铁(SPIO-PLL)及质粒pGenesil1-shRNA的最佳质量比,荧光显微镜观察绿色荧光蛋白在细胞中的表达以检测探针是否进入细胞;透射电子显微镜观察探针中铁在细胞中的部位;CCK-8法检测探针的细胞毒性;MR扫描检测探针转染后细胞的T2*信号强度改变.结果 当SPIO-PLL和质粒的质量比达6∶1时,二者有效结合;探针体外转染细胞后,可观察到绿色荧光蛋白表达,检测到铁颗粒存在于胞质内;在探针浓度低于50 mg/L的范围内,细胞存活率高于80％;探针转染后细胞的T2*信号强度明显降低(P＜0.01).结论 成功制备卵巢癌特异分子探针,在体外成功转染卵巢癌SKOV3细胞,对细胞毒性小,可被MR扫描敏感检测.     

不同检测方法检测4种生殖道病原体的结果差异和应用价值

目的:比较PCR荧光探针法、乳胶法、培养法及ELISA法对生殖道分泌物中淋病奈瑟菌(Neisseria Gonorrhoeae,NG)、解脲脲原体(Ureaplasma urealyticum,Uu)、沙眼衣原体(Chlamydia Trachomatis,CT)及单纯性疱疹病毒2型(Herpes Simplex Virus type 2,HSV-2)的检出率,分析其结果差异和应用价值.方法:选取2021年1月至2022年5月我院性病门诊就诊的100例患者作为研究对象.采用PCR荧光探针法对NG、Uu、CT、HSV-2进行检测,同时采用培养法检测Uu、NG;胶体金法检测CT;ELISA法检测HSV-2,并将PCR荧光探针法与其他3种方法检测结果进行比较分析.结果:在NG、Uu、CT的检测结果中,PCR荧光探针法NG、Uu阳性率与培养法无明显差异(P>0.05),PCR荧光探针法CT结果阳性率明显高于胶体金法(P<0.05).在检测HSV-2的结果中,PCR荧光探针法阳性率明显低于ELISA(P<0.05).结论:与胶体金法、ELISA法相比,PCR荧光探针法可显著提高CT、HSV-2检出率,且与培养法NG、Uu的检出率相近,有助于临床实施早期诊断并及时治疗.     

上转化纳米颗粒介导的光动力治疗对THP-1巨噬细胞凋亡机制研究

<正>目的:探讨上转化纳米颗粒包覆的二氢卟吩(UCNPs-Ce6)介导的光动力治疗对THP-1巨噬细胞凋亡机制。方法:通过DCFH-DA检测光动力治疗后细胞内活性氧(ROS)的变化,通过钙黄绿素探针检测光动力治疗之后线粒体通透性转换孔(MPTP)开放。通过免疫荧光与Western blotting检测光动力治疗后Bax蛋白与细胞色素C蛋白在胞浆和线粒体的表达。结     

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.     

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.     

Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3′ ssDNA extension of human telomeres. POT1–TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1–TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1–TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1–TPP1, in contrast to other single amino acid mutations. Chimeric human–fish telomerases that contained the human TEN domain were active but not stimulated by POT1–TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A–mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1–POT1–telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

1 IntroductionThe POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Human POT1 had been identified in 2001 year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is…     

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.     

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

Characterization of POT1 tumor predisposition syndrome: Tumor prevalence in a clinically diverse hereditary cancer cohort

PurposeGermline variants in POT1 have been implicated in predisposition to melanoma, sarcoma, and glioma in limited studies. Here, we determine the prevalence of cancer types in individuals with POT1 pathogenic variants (PVs) undergoing multigene panel testing (MGPT) for a broad variety of cancer indications.MethodsWe performed a retrospective review of data provided on clinical documents from individuals with POT1 PVs identified via MGPT over a 5-year period. Tumor prevalence in POT1 PV heterozygotes was compared with MGPT-negative wild-type (WT) controls using χ² test.ResultsPOT1 PVs were identified in 227 individuals. POT1 PV and WT (n = 13,315) cohorts had a similar proportion of reported tumors (69.6% and 69.2%, respectively); however, POT1 PV heterozygotes were more likely to be diagnosed with multiple tumors (18.9% vs 8.7%; P < .001). Compared with POT1 WT, we identified a significant increase in melanoma (odds ratio 7.03; 95% CI 4.7-10.5; P < .001) and sarcoma (odds ratio 6.6; 95% CI 3.1-13.9; P < .001).ConclusionThis analysis of the largest POT1 PV cohort to date validates the inclusion of POT1 in hereditary cancer MGPT and has the potential to impact clinical management recommendations, particularly for patients and families at risk for melanoma and sarcoma.     

基于偏序拓扑图的帕金森病语音障碍分析方法

从形式概念分析角度,提出将偏序拓扑图用于帕金森病语音障碍分析与诊断。首先,在属性拓扑的基础上,结合偏序结构表示,构造偏序拓扑图的形式背景表示方法,并利用偏序拓扑图进行概念本体计算,获得原始形式背景的层次化概念树结构。进而结合决策属性,对概念树进行着色与约简,获得约简概念树。根据约简概念树的偏序关系,可获得分析对象的概念分类结构。将该方法应用于帕金森病语音特征数据集进行概念提取,实验表明不但可以在概念层面分析帕金森病与语音特征的关系,同时可以作为诊断依据进行数据诊断。将该方法应用于多个帕金森病数据集(样本数分别为197、5 875、1 040、220)进行分类精度对比测试,表明基于偏序拓扑图的帕金森病语音障碍分析在不同的帕金森病语音数据集下的平均诊断精度达到76.64%,高于LDA(67.36%)、QDA(70.83%)、kNN(71.83%)、parzen窗(70.24%)、SVM(74.61%)等经典分类器的诊断精度,高出经典分类器SVM 2.72%,表明该方法能有效应用于帕金森病语音障碍分析。     

Relations between psychometric profiles and cardiovascular autonomic regulation in physical education students

This study was designed to investigate physical education (PE) students the link between mood disturbances, caused by psychological or physical stressors associated with studying, and the autonomic nervous system modifications. PE students completed the profile of mood state (POMS) questionnaire at the end of the university year. Heart rate variability (HRV) was then measured during a head-up tilt test (HUT) in those with the highest and lowest total mood disturbance (TMD) scores on three successive POMS. Among the 218 students who completed the POMS (85 female and 137 male), 65 had high TMD scores, suggesting mood disturbances and fatigue. The final sample included 12 subjects in the potentially overtrained (POT) group and 16 subjects in the control (CTL) group. A greater decrease of two indices of the autonomic system (SD1 and RMSSD) was observed during the HUT in the POT than in the CTL group (P < 0.05). The depression (Dep) and vigor (Vig) subscales of POMS were correlated with several HRV indices. More specifically, in the POT group, the Vig score was correlated with autonomous activity in the supine position, and the Dep score with percentages of change of sympatho-vagal activity during the HUT. This suggests that (1) POT students could present a weaker autonomic response to HUT, (2) Dep and Vig subscales of the POMS questionnaire may indicate autonomic dysregulations.