小鼠外周组织时钟基因启动子区甲基化分析

 摘要：目的 建立一种简单快速经济的检测八个主要时钟基因BMAL1、BMAL2、CLOCK、NPAS2、PER1、PER2、CRY1和CRY2启动子区甲基化状态的方法。同时检测小鼠外周组织肝、心、肾、胸腺、睾丸时钟基因启动子区甲基化状态。方法 用偏重亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。修饰后的DNA为模板,两套不同的引物对：甲基化特异性引物对和非甲基化特异性引物扩增小鼠肝、心、肾、胸腺、睾丸组织时钟基因启动子区。PCR产物进行电泳和测序。结果 扩增产物与预期片段大小相符合。PCR产物经过直接测序得到进一步证实。结论 成功建立了检测小鼠时钟基因启动子区甲基化的方法,为检测小鼠周围组织时钟基因启动子区甲基化提供了新的方法。同时,成年小鼠所有的八个时钟基因均为非甲基化状态。     

PER2对人口腔鳞癌SCC15细胞增殖、凋亡以及生物钟基因表达的影响

目的探讨人口腔鳞癌SCC15细胞中生物钟基因PER2的表达改变对细胞增殖、凋亡以及其他生物钟基因的影响。方法用RNA干扰技术沉默人口腔鳞癌SCC15细胞中PER2基因;流式细胞术检测细胞增殖和凋亡水平;实时荧光定量PCR检测生物钟基因CLOCK、BMAL1、PER1、PER3、DEC1、DEC2、CRY1、CRY2、TIM、CKIε、RORα、NPAS2和REV-ERBαmRNA表达。结果沉默PER2后,SCC15细胞的增殖水平显著增加,凋亡显著下降(P0.05);SCC15细胞中生物钟基因PER3、BMAL1、DEC1、DEC2、CRY2、TIM、RORα和NPAS2 mRNA的表达水平显著降低,PER1和REV-ERBαmRNA的表达显著升高(P0.05)。结论在癌细胞中,生物钟基因PER2对生物钟基因网络中其他生物钟基因具有重要调控作用,PER2表达降低导致细胞增殖增加和细胞凋亡水平下降。     

PCR方法对单细胞SRY基因扩增效率的对比研究

目的探讨单细胞基因扩增时,巢式PCR和引物预扩增(PEP)-巢式PCR两种扩增方法对SRY基因脱扣的影响程度;并应用PEP-巢式PCR方法对单个卵裂球细胞进行SRY基因诊断。方法获取单个正常男女淋巴细胞,随机分为巢式PCR组和PEP-巢式PCR组。同时扩增SRY基因和ZP3基因位点。选用IVF-ET后冻存的4个胚胎,处理后获取单个卵裂球11个,用PEP-巢式PCR扩增,鉴定其性别。结果单个淋巴细胞经巢式PCR和PEP-巢式PCR方法扩增后,其基因扩增成功率分别为92.39%,98.91%;性别诊断正确率分别为86.00%,98.00%;SRY基因的脱扣率分别为16.67%,2.38%。两者有统计学检验均有显著性差异(P<0.05。对单个卵裂球应用PEP-巢式PCR方法进行SRY基因和ZP3基因扩增后,有3个胚胎的8个卵裂球被诊断为男性,而另外1个胚胎的3个卵裂球被诊断为女性。结论1.行单细胞基因扩增时,PCR扩增方法会影响等位基因脱扣的发生率。2.对性连锁遗传病进行植入前遗传学诊断时,采用PEP-巢式PCR方法扩增SRY基因和ZP3基因对单个细胞对进行性别鉴定时,可以有效地降低SRY基因脱扣的发生率,提高性别诊断的特异性和敏感性,能够用于单细胞的性别诊断,可用于性连锁遗传病的植入前遗传学诊断。     

血小板内皮细胞黏附分子的剪接体在胚胎干细胞血管形成过程中的表达

目的探讨血小板内皮细胞黏附分子(PECAM)1的剪接体在胚胎干细胞分化过程中的表达规律。方法体外培养胚胎干细胞,在细胞因子的作用下形成胚胎样小体(EB);然后将EB种植到胶原中,在细胞因子的作用下诱导出芽性血管新生。分别利用免疫组织化学、流式细胞术、逆转录聚合酶链反应等方法检测胚胎干细胞及其分化过程中PECAM1、Oct4及胚胎阶段特异性抗原(SSEA)1的表达。应用克隆分析的方法检测不同的PECAM1剪接体在EB形成和出芽性血管新生过程中的表达分布。     

聚合酶链反应在免疫学中的应用(文献综述)

<正> 聚合酶链反应(Polymerase Chain Reac-tion,PCR)是近年发展起来的一项新技术。应用该技术可以在上万个基因或表达产物中挑选出所需要的基因,它在试管中经数小时后即可扩增成上百万个同一基因分子,供进一步分析,所以PCR被看作是一种无细胞的分子克隆技术。它具有快速、灵敏和特异     

单细胞巢式PCR诊断软骨发育不全

目的 探讨单细胞水平诊断软骨发育不全(achondroplasia，ACH)的可靠性，为开展ACH的胚胎植入前遗传学诊断(preimplantation genetic diagnosis，PGD)打下基础。方法 采用巢式PCR扩增单淋巴细胞及单卵裂球的成纤维细胞生长因子受体3基因的高发突变位点G380R区域，用限制酶Bfm I消化PCR产物，10％聚丙烯酰胺凝胶电泳检测。结果 单淋巴细胞的PCR扩增成功率为90．4％，等位基因脱扣发生率为8．2％，诊断准确率为91．8％；单卵裂球的扩增成功率为75．4％。结论 单细胞巢式PCR诊断ACH是比较稳定、可靠的。     

增加5''''RACE法特异性的简单改进

5'RACE（即cDNA末端快速扩增）法是克隆特定mRNA5'末端的一种快速、灵敏的方法。尤其在基因捕获（Gene－trap）中很有价值，利用5'RACE法能快速、可靠地鉴定被捕捉的外显子。但是，原有的RACE法特异性低，PCR产物在琼服糖凝胶电泳时通常出现成片条带现象；而且多数情况下特异的PCR产物不能用溴化乙锭（EB）染色显示扩增结果，尤其对于5'端GC含量高或者表达水平低的mRNAs，更易出现这种情况．因此，为了检测到特异的PCR产物，通常还需要进一步做片段大小选择性扩增或进行Southern杂交分析．我们在寻找鼠胚胎发生过程中控制生…     

巢式聚合酶链反应诊断肺炎衣原体感染的临床应用探讨

目的 应用并评价巢式聚合酶链反应技术诊断肺炎衣原体（Chlamyolia pueumoniae,Cp)感染。方法 采用以衣原体属及Cp种的16SrRNA特异片段为目的基因。对已知鼻咽拭子Cp培养结果的81例病例进行连续2次扩增,检测Cp基因组DNA,并对其应用予以评价。结果 以培养结果为标准。PCR方法的灵敏度为5/17（29.41%）,特异度为52/64（81.25%),阳性预计值为5/17（29.41%),特异度为52/64（81.25%)。阳性予计值为5/17（29.41%),阴性邓丈夫值为52/64（81.25%)。结论 这种巢式聚合酶链反应技术尚不能用于临床诊断Cp感染,有必要以诊断金标准对其应用进行评价。     

用PCR分析方法检测体细胞杂交细胞中的人染色体

本文介绍一种快速高效检测人和啮齿类（大鼠和小鼠）体细胞的杂交细胞中人染色体的新方法。这种方法以聚合酶链反应促进人ＤＮＡ扩增为基础，引物为已定位的基因，或人的ＤＮＡ片段。     

用PCR分析方法检测体细胞杂交细胞中的人染色体

本文介绍一种快速高效检测人和啮齿类（大鼠和小鼠）体细胞的杂交细胞中人染色体的新方法。这种方法以聚合酶链反应促进人ＤＮＡ扩增为基础，引物为已定位的基因，或人的ＤＮＡ片段。     

Promoter methylation analysis of seven clock genes in Parkinson's disease

The expression of clock genes is altered in leukocytes from patients with Parkinson's disease (PD). However, the underlying mechanisms are unknown. To determine whether abnormal CpG methylation contributes to the dysregulated expression of these genes, the methylation status of the promoters of seven major human clock genes, PER1, PER2, CRY1, CRY2, Clock, NPAS2, and BMAL1, was examined using methylation-specific PCR (MSP) and sequencing in 206 PD patients and 181 healthy controls. This analysis revealed that most clock gene promoters were devoid of methylation. Methylation was only detectable in the CRY1 and NPAS2 promoters. Interestingly, the methylation frequency of the NPAS2 promoter was significantly decreased in PD patients. These results suggest that altered promoter methylation may contribute to the abnormal expression of clock genes in PD.     

Obesity alters the expression profile of clock genes in peripheral blood mononuclear cells

Introduction

The aim of this study was to investigate the association between the variation in expression profile of clock genes and obesity using peripheral blood mononuclear (PMN) cells.

Material and methods

The subjects comprised 10 obese patients and 10 healthy volunteers. Blood was collected at different time-points during the day and levels of blood sugar, IRI, adiponectin and leptin were determined. Peripheral blood mononuclear cells were sampled, and expression levels of brain and muscle Arnt-like protein-1 (BMAL1), Period (PER)1, PER2, Cryptochrome (CRY)1, CRY2, and REV-ERBα mRNA were quantified.

Results

During the day, the expression levels of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells of the obese group were all significantly higher compared to those in the non-obese group. In addition, expression of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells increased between 12:00 and 21:00 in the obese group. In PMN cells of both groups, PER1 gene expression showed a bimodal pattern, with high expression at 9:00 and 18:00.

Conclusions

Differences were observed in the expression profile variation of clock genes between the obese and non-obese groups. This study reveals the differences in clock gene expression profiles between obese and non-obese subjects, with evidence for two distinct chronotypes, and suggests a contribution of these chronotypes to fat accumulation in humans.     

Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of approximately 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.     

Effective isolation and culture of endothelial cells in embryoid body differentiated from human embryonic stem cells

We describe the isolation of endothelial cells from the center region of the attached embryoid body (EB) by a two-step enzyme treatment. The isolated cells from the center and outgrowth region of the EB were characterized separately. As human embryonic stem (hES) cells differentiated in EB, they lost expression of the undifferentiated marker Oct-4, whereas expression levels of endothelial-specific markers were increased. Using RT-PCR, fluorescence-activated cell sorting (FACS) analysis, and immunofluorescence, we have shown that various endothelial cell markers, including platelet/endothelial cell adhesion molecule (PECAM), von Willebrand factor (vWF), Flk-1, and Tie2, were expressed on the attached EB. Compared with the outgrowth region of EB, the center region had a higher population of cells with these endothelial cell markers. Once isolated by the twostep enzyme treatment, cells from the center region continued to differentiate into endothelial cell lineage while expression level of endothelial cell markers in cells from the outgrowth region decreased in subcultures. This study has demonstrated that the isolation of EB by a two-step enzyme treatment is a useful technique to obtain endothelial cell marker-positive cells with high yield. Furthermore, a similar approach can be taken to identify the location and distribution of specific cell types in EB and thereby allow us to isolate and expand specific cell types.     

In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.     

Embryoid body-mediated differentiation of mouse embryonic stem cells along a hepatocyte lineage: insights from gene expression profiles

Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.     

Ultrastructure of human embryonic stem cells and spontaneous and retinoic acid-induced differentiating cells

Ultrastructural and immunohistochemical studies of 4 groups of cells-(human embryonic stem cells (hES), embryoid bodies (EB), and spontaneously and retinoic acid (RA)-induced differentiating cells)-were carried out to investigate their detailed phenotype. Immunohistochemically, the EB cells showed strong immunoreactivity for CD34, CD117, and nestin. Differentiating cells expressed pancytokertin, vimentin, CD31, CD56, GFAP, nestin, and NeuN as well as CD34, and c-Kit. However, synaptophysin and neurofilaments were not present in these same differentiating cells. Transmission electron microscopy showed that hES and EB cells were very similar to germ cells or cells of the inner cell mass. Spontaneously and RA-induced differentiating cells exhibited epithelial, mesenchymal, endodermal, and neuronal phenotypes. The perikarya of the neuronal cells had rich RERs (Nissl substance) and long cytoplasmic processes filled with numerous neural tubules. However, neither synaptic junctions nor synaptic vesicles were developed. In our study, RA treatment with brain-derived growth factor and TGFalpha in neuron differentiation medium induced not only neuronal differentiation but also pluripotential differentiation. Full neuronal differentiation did not occur after 2 weeks in culture, as no synaptic junctions and synaptic vesicles developed.