单细胞测序方法研究进展

近年来,高通量测序技术(Next-generation sequencing,NGS)快速发展,已广泛应用于生命科学各个领域,但传统的混合细胞测序(Bulk cell sequencing)检测的是细胞群体的总平均反应,无法反应每个细胞的真实情况,这会影响研究者对细胞功能认知的准确性。单细胞测序技术(Single cell sequencing,sc-Seq)的出现,从一定程度上解决了传统测序固有的缺陷。单细胞测序是针对单个细胞的RNA或DNA进行测序,能够准确测出单个细胞的基因结构和表达状态,从而分析相同表型细胞的异质性。本文首先介绍单细胞测序的原理、测序类型和测序平台,有助于理解单细胞测序和在进行科研项目时设计合适的项目方案。进一步介绍单细胞转录组测序的分析流程和各种常用的分析工具或软件,并重点阐述单细胞转录组测序分析中的细胞聚类和拟时序分析的原理和研究进展,为进行单细胞转录组测序数据分析提供参考。最后,本文简述了单细胞测序研究热度、单细胞测序的应用、挑战和展望等,有助于更全面地认识单细胞测序。     

关联维数法在皮肤激光散斑测量中的应用

本文采用关联维数方法研究了皮肤厚度对皮肤激光散斑测量的影响。计算了６种模拟皮肤厚度情况下的生物散斑关联维数。结果表明,维数随皮肤厚度的增加而减小,而且重复性很好。这说明,关联维数方法可以应用在皮肤激光散斑的研究中     

脂肪间充质干细胞来源外泌体的功能

外泌体是细胞外膜质纳米囊泡,将蛋白质、核酸(DNA和RNA)转运到靶细胞中,介导局部和系统的细胞间通信,从而改变受体细胞的行为.这些小泡在许多生物功能中发挥重要作用,如脂肪合成、免疫调节、神经再生和肿瘤调节等.脂肪间充质干细胞目前被认为是细胞治疗和再生医学领域中一种功能丰富的工具,可产生和分泌多种外泌体,继承细胞的多种...     

Abnormal Epidermal Keratinization in the repeated epilation mutant mouse

Repeated epilation (Er) is a radiation-induced, autosomal, incomplete dominant mutation in mice which is expressed in heterozygotes but is lethal in the homozygous condition. Many effects of the mutation occur in skin: the epidermis in Er/Er mice is adhesive (oral and nasal orifices fuse, limbs adhere to the body wall), hyperplastic, and fails to undergo terminal differentiation. Skin from fetal +/+, Er/+ and Er/Er mice at ages pre- and postkeratinization examined by light, scanning, and transmission electron microscopy showed marked abnormalities in tissue architecture, differentiation, and cell structure; light and dark basal epidermal cells were separated by wide intercellular spaces, joined by few desmosomes, and contained phagolysomes. The numbers of spinous, granular, and superficial layers were highly variable within any given region and among various regions of the body. In some areas, 2-8 layers of granular cells, containing large or diminutive keratohyalin granules, extended to the epidermal surface; in others, the granular layers were covered by several layers of partially keratinized or nonkeratinized cells. In rare instances, a single or small group of cornified cells was present among the granular layers but was not associated with the epidermal surface. Both the granular and nonkeratinized/partially keratinized upper epidermal layers Er/Er skin gave positive immunofluorescence with antiserum to the histidine-rich, basic protein, filaggrin. Proteins in epidermal extracts from +/+, Er/+ and Er/Er mice were separated and identified by radio- and immunolabeling techniques. The Er/Er extract was missing a 26.5- kdalton protein and had an altered ratio of bands in the keratin region. The 26.5-kdalton band was histidine-rich and cross-reacted with the antiserum to rat filaggrin. Several high molecular weight bands present in both Er/Er and +/+ extracts also reacted with the antiserum. These are presumed to be the precursors of filaggrin and to account for the immunofluorescence om Er/Er epidermis even though the product protein is absent. The morphologic and biochemical data indicated that the genetic defect has a general and profound influence on epidermal differentiation, including alteration of two proteins (filaggrin and keratin) important in normal terminal differentiation, tissue architecture, and cytology. Identification of epidermal abnormalities at early stages of development (prekeratinization) and defective structure of other tissues and gross anatomy suggest that the mutation is responsible for a defect in same regulatory step important in many processes of differentiation and development.     

Isolation and characterization of the major form of polyprenyl-phospho- mannose from Mycobacterium smegmatis

We isolated from the endogenous polyprenyl-phospho-sugar pool of Mycobacterium smegmatis two mannose-containing compounds, i.e., a partially saturated C35-octahydroheptaprenyl-P-mannose and a fully unsaturated C50-decaprenyl-P-mannose. The relative amount of C35- polyprenyl-P-mannose in mycobacterial cells was comparable to that of decaprenyl- P-pentoses and, at least, an order of magnitude higher than that of C50-decaprenyl-P-mannose. The major form of mycobacterial polyprenyl-P-mannose was structurally characterized by combined gas chromatography-mass spectrometry, fast-atom bombardment tandem mass spectrometry and proton-nuclear magnetic resonance spectroscopy as beta- d-mannopyranosyl-monophospho-(C35)octahydroheptapren ol of which all three isoprene units have Z ( cis ) configuration. The differences in the structure and cellular concentrations of the mycobacterial mannosyl- P-polyprenols reflect distinct biochemical pathways of the two compounds and suggest the existence of specific GDP-Man:polyprenyl-P mannosyltransferases (synthetases) able to distinguish between C35- octahydroheptaprenyl- and C50-decaprenyl- phosphates of mycobacteria. Since the 6'-O-mycoloylated form of C35-octahydroheptaprenyl-P-mannose isolated from M. smegmatis is apparently involved in mycolate rather than mannosyl transfer reactions, we speculate that a catabolic pathway responsible for degradation of C35-P-mannose and recycling C35- octahydroheptaprenyl phosphate might exist in mycobacteria.      

犬MC1R基因R306ter与毛色性状相关性研究

目的　分析犬MC1R基因中R30 6ter位点多态性与犬毛色表型的相关性 ,为遗传育种 ,培育出更加优良的实验用犬奠定基础。方法　采用PCR SSCP技术 ,对MC1R基因R30 6ter位点进行基因多态性检测 ,分析位点多态性与毛色性状之间的相关性 ,并对该位点进行克隆测序。结果　PCR SSCP分析结果表明 ,R30 6ter位点序列具有多态性 ,表现为C、D二个等位基因和CC、CD及DD三种基因型。对R30 6ter多态性片段克隆测序发现 ,MC1R基因在编码第 30 6位氨基酸的密码子处存在一个由CGA到TGA的终止突变。结论　经统计分析结果表明在杂种犬中MC1R基因多态性与毛色性状不存在显著的相关性 ,这可能是由于外科手术学教学用犬是杂种犬 ,其遗传背景不同所致。由于MC1R基因的R30 6ter位点内存在碱基变异 ,因此在杂种犬中表现出明显的PCR SSCP多态性     

吸氧预处理对大鼠脑缺血再灌注损伤的保护作用

目的探讨吸氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法通过大鼠局灶脑缺血再灌注损伤模型,采用SOD、MDA测定、电镜及神经行为学检查的方法,观察吸氧预处理对大鼠脑缺血再灌注损伤后SOD、MDA、神经行为学评分及脑组织病理变化。结果吸氧预处理组SOD活力高于对照组(P<0.05),MDA含量、神经行为学评分均低于对照组(P<0.05),脑组织超微结构损伤均减轻。结论吸氧预处理对大鼠脑缺血再灌注损伤有保护作用。     

Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris

BP230 is a cytoskeletal linker protein of 2649 amino acids originally identified as the target autoantigen in bullous pemphigoid, a potentially devastating autoimmune skin blistering disorder. To better define its function, we sought to generate recombinant forms of BP230 in both Saccharomyces cerevisiae and Pichia pastoris after cloning its entire cDNA. By immunoblot analysis, full-length BP230 was not found in extracts of P. pastoris, whereas minor amounts of degraded BP230 were detected in extracts of S. cerevisiae. In contrast, both S. cerevisiae and P. pastoris were able to produce the 770-amino acid COOH-terminal domain of BP230. Furthermore, the production level of the recombinant BP230 tail in S. cerevisiae was significantly higher than that observed in P. pastoris and that of endogenous BP230 in cultured human keratinocytes. Finally, 12 of 17 (71%) BP sera recognized the recombinant BP230 protein in yeast extracts. Our results indicate that S. cerevisiae occasionally constitutes a better tool for recombinant protein production than P. pastoris. Although both its large size and poor solubility limit production of BP230, the developed yeast system provides cellular fractions enriched in BP230 recombinant proteins that constitute useful tools for the diagnosis of bullous pemphigoid.     

Anti-α-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-α-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sjögren's syndrome (SjS). We looked (in Nijmegen) for anti-α-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS [6], rheumatoid arthritis [RA] [12], systemic lupus erythematosus [SLE] [6], and scleroderma [6]) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-α-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The ELISAs for α-fodrin showed six (Nijmegen) and four (Hanover) anti-α-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-α-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-α-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-α-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-α-fodrin autoantibodies does not add much to the diagnosis of Sjögren's syndrome.     

医学院校新生适应性问题的研究

目的:在教育理论的指导下,通过问卷调查的形式对一年级学生的生活、学习、心理健康状况等方面的适应性问题进行调研。方法:采用随机抽样的原则,以抽到的班级所有符合条件的大学新生作为调查对象。采用自制的一般人口学特征问卷、高考志愿填报情况调查表、教学软硬件建设满意度调查表、目前学习状况及未来就业去向调查表、症状自评量表对46名大学新生进行调查。结果:调查班级学生中,多数学生来自农村家庭;与"70版、80后"相比,"90后"学生在职业选择方面具有更多的自主选择权;对学校在软硬条件方面亦提出更高的要求;受限于近期个人目标,缺乏长远人生目标,可持续发展能力差。心理问题阳性检出率为24%,排名在前4位的心理健康问题依次是:强迫、抑郁、焦虑、躯体化,男女生表现个有不同。结论:传统教育思维仍试图在极短时间里完成适应性教育工作,致使抽象理论与学生现实需求相脱节。陌生的环境、迥然不同的学习方式、全方位转变的人际关系使得新生心理问题出现叠加性、多重性的特点。相关部门应改变适应教育模式,建立新生适应教育活动的长期反馈机制和评估体系,才能更好的服务于新生教育工作。