基于转录组测序鉴定自噬在小鼠精子发生过程中的动态变化

目的基于NCBI数据库中的小鼠精子发生3个时期(精原细胞、粗线期精母细胞、圆形精子)的转录组测序(RNA-Seq)数据,系统地揭示自噬通路及自噬相关基因在精子发生过程中的动态变化规律。方法从GEO数据库中下载精子发生3个时期的RNA-Seq数据(GSE100964),对相邻阶段的差异表达基因分别进行代谢通路富集分析。从Autophagy Database里面取出783个自噬相关基因,对其中在精子发生3个时期的平均表达值大于1的636个自噬相关基因的表达量进行热图展示。结果发现精原细胞和粗线期精母细胞之间的差异表达基因可以显著富集到自噬通路和自噬相关的通路,而粗线期精母细胞和圆形精子之间的差异表达基因不能富集到自噬相关通路。此外,还发现636个自噬相关基因在精子发生过程中有3种主要表达模式。结论自噬在精子发生减数分裂起始前后的RNA水平发生了显著的变化,且自噬相关基因在精子发生过程中呈阶段特异表达。     

维生素D缺乏模型小鼠子宫转录组的测序分析

背景:近来发现维生素D受体广泛分布在卵巢、子宫等女性生殖系统,与自然流产及妊娠期糖尿病等不良妊娠结局密切相关,但其具体机制尚不清楚。目的:通过对维生素D缺乏小鼠子宫组织进行全转录组分析,寻找潜在差异表达miRNA及相关调控网络。方法:取雌性C57BL/6J小鼠18只随机分为2组,维生素D缺乏组给予维生素D缺乏饲料喂养,正常对照组给予维生素D充足饲料喂养,每周记录小鼠体质量。8周后检测小鼠血清中25,(OH)D₃和激素水平,对小鼠子宫组织进行苏木精-伊红染色并收集两组子宫组织进行转录组测序,进行基因本体、京都基因与基因组百科全书生物信息学分析。结果与结论:(1)两组小鼠间体质量无明显差异;与正常对照组相比,维生素D缺乏组小鼠血清25,(OH)D₃、雌二醇水平显著降低,睾酮水平显著升高;(2)子宫组织苏木精-伊红染色结果显示,维生素D缺乏组子宫内膜皱褶减少;(3)转录组测序结果共筛选出差异表达的miRNA25个,其中上调9个(如miR-541-5p和miR-205-5p),下调16个(如miR-378d和miR-708-5p);(4)同时维生素D...     

基于10×Genomics技术对蓖麻毒素中毒后中性粒细胞的转录组分析

目的 探究中性粒细胞在蓖麻毒素(Ricin Toxin,RT)致毒过程中的作用,寻找解毒的有效策略.方法 采用10x Genomics单细胞转录组测序技术对中毒小鼠外周血单个核细胞(PBMCs)进行转录组测序及分析,并通过流式细胞术测定目的细胞亚群.结果 经降维聚类、差异基因、拟时序分析结果显示CD177-CD121b...     

基于单细胞转录组测序分析骨关节炎软骨细胞分化的分子机制

目的 深度分析骨关节炎（osteoarthritis, OA）软骨细胞分化的单细胞转录组数据,探究其关键性靶基因及分子机制,为临床靶向治疗骨关节炎提供分子理论依据。方法 从GEO数据库下载数据集（GSE104782）,包含10个骨关节炎样本及1 464个软骨单细胞测序结果,运用R语言分析软骨细胞分化中关键Marker基因,并采用质控和数据过滤、PCA、TSNE、细胞轨迹、GO、KEGG信号通路及蛋白互助网络分析揭示软骨细胞分化机制。结果该数据集共包含基因17 380种、20个主成分、9种细胞类型、1 886个Marker基因及6种分化途径,其中软骨祖细胞为软骨最早分化细胞。GO分析涉及软骨发育、结缔组织发育等生物过程;涉及含胶原蛋白细胞外基质、内质网内腔等细胞组分;涉及肝素结合、糖胺聚糖结合等分子功能。KEGG通路分析涉及蛋白质消化吸收、ECM受体相互作用、人乳头瘤病毒感染、补体与凝血级联反应、PI3K-Akt信号通路等信号通路。运用Cytoscape 3.7.2软件获得5个关键Marker基因（COL1A1、COL2A1、VEGFA、COL1A2、DCN）。结论 运用单细胞转录组测序...     

基于转录组测序技术研究高原低氧对小鼠脾脏组织的影响北大核心CSCD

目的:利用转录组学测序技术探究免疫系统在高原低氧胁迫适应过程中相关基因的表达及响应的分子机制。方法:本研究在高、低海拔环境分别饲养C57BL/6小鼠30 d,取脾脏组织利用RNA-Seq进行转录组测序,将得到的差异基因(DEGs)进行GO和KEGG富集分析,并通过荧光定量PCR验证测序数据的准确性。结果:与平原常氧组相比,共富集到4218个DEGs(P<0.05)。其中,ANXA1、S100A8、S100A9和HSPB1等基因显著富集;GO结果表明DEGs主要富集于B细胞激活、免疫球蛋白复合体和抗原结合分类,且JAK-STAT及NOD样受体信号通路为显著富集通路。结论:免疫系统响应高原低氧胁迫的转录调控分子可能致使机体内部免疫调节和炎症反应失衡,为相关高原病的病因学探究提供了新的理论依据。     

蚊虫转录组学研究进展?

转录组学作为最早发展起来且应用最为广泛的二代测序技术,为蚊虫基因的功能分析及寻找防治蚊虫的潜在靶标提供了有效手段。目前,转录组学已在蚊虫的吸血分子基础、唾液腺蛋白的分类构成、滞育的发生机制、消化代谢及免疫机理等方面取得突破性进展,有助于深化对蚊虫生化和生理代谢途径的认识,改善蚊虫防治效果提供理论依据。     

应用转录组和外显子组测序分析不同年龄组的胶质母细胞瘤中基因表达与突变的差异

目的 探讨通过转录组和外显子组测序分析不同年龄组的胶质母细胞瘤的差异。 方法 多形性胶质母细胞瘤（GBM）数据下载自TCGA项目。将样本分为2组,不高于60岁的样本为中低年龄组,共78例;高于60岁的样本为高年龄组,共76例。使用R语言DESeq2软件包对RNA-seq原始基因水平的reads数目数据进行差异表达分析。使用Cytoscape插件ClueGO进行不同年龄组别差异表达基因功能富集分析。 结果 GBM致病基因在两组之间大部分基因表达趋势一致,但存在差异基因。在中低龄组中差异基因主要和神经前体细胞增殖相关,而高年龄组偏重于代谢方面。在两组中高突变的基因有很多重叠。样本突变率不同的基因主要为中低年龄组特有,且具有高样本突变率的基因。 结论 中低年龄组和高龄组GBM患者在基因表达和基因突变上存在明显差异。     

模拟失重后骨髓来源巨噬细胞转录组高通量测序及分析北大核心CSCD

目的探索模拟失重对小鼠骨髓来源巨噬细胞基因表达的影响。方法采用尾悬吊法构建模拟失重小鼠模型,对照组不做尾吊处理。模拟失重28 d后提取小鼠骨髓来源巨噬细胞并将其分别诱导为M0型和M1型巨噬细胞。随后,提取细胞总RNA进行转录组测序及分析,并通过实时定量RT-PCR验证重要基因的差异表达。结果转录组测序结果显示,模拟失重组与对照组之间的M0型巨噬细胞共有807个基因差异表达;模拟失重组与对照组之间的M1型巨噬细胞共有898个基因差异表达。GO分析表明,M0型巨噬细胞差异表达基因主要集中于免疫应答、趋化等生物学过程中;M1型巨噬细胞差异基因主要富集于炎症反应、细胞迁移的正调控和单核细胞趋化等生物学过程中。KEGG通路分析发现M0型巨噬细胞的差异基因主要涉及趋化因子、细胞因子-细胞因子受体相互作用等信号通路;M1型巨噬细胞的差异基因主要涉及造血细胞谱系、流体剪切应力等信号通路。进一步分析发现,尾吊组与对照组在M0或M1型巨噬细胞中的差异基因均在细胞因子-细胞因子受体相互作用信号通路中富集,其中尾吊组的M0与M1型巨噬细胞中调节单核细胞/巨噬细胞迁移和浸润的关键趋化因子Ccl2均显著高表达,并进一步通过RT-PCR实验得到验证。结论模拟失重可显著影响小鼠巨噬细胞中以Ccl2为代表的与炎症发生和加重密切相关的基因表达水平,这些改变的基因富集于多个生物学过程,可能对巨噬细胞的黏附、迁移等功能产生影响。     

凋亡相关斑点样蛋白寡聚阻断剂对小鼠脊髓损伤急性期局部基因转录表达影响的转录组分析

背景:炎症小体在脊髓损伤后发挥重要作用,凋亡相关斑点样蛋白是炎症小体的共同接头蛋白。作者前期的研究表明,一种特异性的凋亡相关斑点样蛋白寡聚阻断剂CRID3,可以通过抑制凋亡相关斑点样蛋白寡聚化而抑制炎症小体的活化和相应细胞因子的产生,进而改善损伤脊髓局部微环境,发挥神经保护作用。但是其在转录水平对损伤脊髓的影响尚未见报道。目的:采用转录组测序技术分析CRID3对小鼠脊髓损伤急性期(8 h)局部基因转录表达的影响。方法:共取雌性8周龄C57BL/6小鼠30只,体质量18-20 g,随机分为假手术组和脊髓损伤组,将脊髓损伤后的小鼠分为模型对照组和给药组,给药组术后腹腔注射CRID3(50 mg/kg),对照组只注射等体积的生理盐水。脊髓损伤后8 h,每组各灌注取材3只小鼠,取脊髓冰冻切片进行苏木精-伊红染色,确定损伤模型是否成功。损伤后8 h每组各取3只小鼠,取脊髓提取纯化总RNA,进行文库制备和转录组测序,用DESeq2软件分析3组模型的差异基因表达。同转录组测序,模型对照组和给药组各取6只小鼠脊髓提取纯化总RNA,采用反转录实时定量PCR方法验证转录组测序结果。用GOseq R软件和K...     

基于单细胞转录组测序分析人肋来源软骨细胞的异质性

背景:肋软骨是人体组织中一类重要的软骨来源,常作为软骨自体移植和组织工程构建的种子细胞来源。单细胞测序是分析细胞异质性的强大工具,可针对肋软骨细胞进行细胞异质性的深入研究。目的:通过单细胞转录组测序分析人肋软骨组织细胞分群情况,以及每个细胞亚群参与的生物学过程。方法:临床获取1例31岁小耳重建手术后废弃的肋软骨组织制成原代细胞悬液,经10X Genomics平台进行单细胞分离,使用Gel Bead Kit V3构建单细胞RNA-seq文库,Illumina Novaseq6000测序仪对文库进行测序,并利用主成分分析和T分布随机邻域嵌入进行降维,获得4个亚群细胞,进而获得不同亚群细胞的标记基因,再对每个细胞亚群的标记基因进行GO和KEGG分析,分析这些基因可能参与的生物学过程。结果与结论:测序共获取6 634个细胞,符合质控标准。将肋软骨细胞划分为4个细胞亚群,分别为以COL10A1、S100A2为标记基因的肥大软骨细胞群;以BMP2、COL2A1为标记基因的软骨细胞群;以FOS、JUN为标记基因的增殖性细胞群;以MYLK、CD146为标记基因的干细胞群。将肋软骨细胞进一步划分细胞亚群...     

绿原酸改善db/db小鼠血管内皮功能障碍的作用初探

目的:探讨绿原酸(CGA)对肥胖型2型糖尿病小鼠血管内皮功能障碍的作用并初步分析相关的机制。方法:雄性db/db小鼠12只,随机分对照组和CGA组,每组6只。CGA组以含0.02%CGA的饲料喂养,对照组给予普通饲料喂养,干预时间为12周。每周测空腹血糖、体重和鼠尾血压。实验结束后取血分离血浆,采用ELISA法测血红素氧合酶1(HO-1)、过氧化氢酶(CAT)、醌NAD(P)H脱氢酶1(NQO1)和谷胱甘肽过氧化物酶1(GPx-1)的水平。取胸主动脉以DHE和DAF-2 DA染色观察血管内膜超氧阴离子和一氧化氮(NO)水平,以Wire Myograph System观察胸主动脉张力,Western blot观察血管组织中核因子E2相关因子2(Nrf2)、过氧化物酶体增殖物激活受体α(PPARα)、磷酸化AMP活化蛋白激酶(p-AMPK)、磷酸化内皮型NO合酶(p-eNOS)、P22~(phox )和P47~(phox)的蛋白水平。结果:膳食CGA可显著降低小鼠的空腹血糖和体重,增加血浆中HO-1、CAT、NQO1和GPx-1水平,减少血管内皮超氧阴离子的水平,增加NO水平,改善小鼠血管内皮依赖性舒张功能(P0.01)。Western blot结果发现CGA可显著上调血管组织中PPARα、Nrf2、p-AMPK和p-eNOS的蛋白水平,降低P22~(phox )和P47~(phox)的水平(P0.01)。结论:膳食CGA显著改善db/db小鼠血管内皮依赖性舒张功能。这可能与其上调PPARα、Nrf2和AMPK等抗氧化应激分子,降低氧化应激水平,促进eNOS磷酸化有关,但确切机制尚需进一步研究。     

SPARC在db/db小鼠肾脏组织中高表达

目的 检测db/db鼠肾脏组织中的富含半胱氨酸的酸性分泌蛋白(SPARC)的表达情况.方法 RT-PCR、Western blot及免疫荧光方法检测db/db鼠肾脏组织中的SPARC mRNA及蛋白的表达.结果 SPARC在db/db鼠肾脏组织中呈现高表达.结论 db/db鼠肾脏组织中的SPARC呈现高表达(P＜0.05),可能与糖尿病肾病的发生与发展有关.     

Actions of somatostatin on lipid metabolism in mouse cerebral cortex slices

Cerebral cortex slices from mice were used to investigate the variations of lipid metabolism by somatostatin. Somatostatin decreased [14C]acetate incorporation into all lipid fractions significantly. Likewise, the peptide evoked a decrease of triglyceride lipase activity. The incorporation of [32P]orthophosphate into phospholipids was diminished by somatostatin. These results add more information about the effects of somatostatin in cerebral cortex.     

凋亡细胞在db/db自发性糖尿病小鼠颌下腺的分布

目的:观察凋亡细胞在db/db糖尿病小鼠颌下腺中的分布。方法:选取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的dh/m~(?)小鼠颌下腺,应用TUNEL标记方法染色后进行图像分析.统计凋亡细胞在颌下腺组织中分布的细胞阳性率。结果:随着糖尿病的发展,颌下腺组织出现腺体萎缩及颗粒曲管数目减少,实质细胞排列不整齐,呈簇状堆集,纤维及血管增多。凋亡细胞在对照组及糖尿病组颌下腺中均有分布,糖尿病组凋亡细胞阳性率高于对照组。糖尿病组与对照组凋亡细胞阳性率随月龄增大均呈增加趋势。结论:db/db糖尿病可导致颌下腺组织萎缩及实质细胞形态学改变;凋亡细胞阳性率在糖尿病组随疾病发展而增加显著。这与糖尿病腺体萎缩和功能受损相一致。     

The distribution and density of monocarboxylate transporter 2 in cerebral cortex,hippocampus and cerebellum of wild-type mice

Monocarboxylates cannot cross the blood-brain barrier freely to participate in brain energy metabolism. Specific monocarboxylate transporters (MCTs) are needed to cross cellular membranes. Monocarboxylate transporter 2 (MCT2) is a major monocarboxylate transporter encoded by the SLC16A7 gene. Recent studies reported that neurodegenerative diseases of the CNS, such as Alzheimer's disease (AD) and Parkinson's disease (PD), were related to energy metabolic impairment. MCT2 also plays an important role in energy metabolism in the CNS. To provide experimental evidence for future research on the role of MCT2 in the pathological process of CNS degenerative diseases, the distribution and density of MCT2 in different subregions of wild-type mouse brain was examined using immunohistochemistry, western blot and immunogold post-embedding electron microscopic techniques. The amount of MCT2 was higher in cerebellum than in cortex and hippocampus on western blots, and there was no statistical difference between cortex and hippocampus. Immunohistochemistry assay revealed the highest density of MCT2 in the CA3 of the hippocampus. The granular cell layer of the cerebellum contained more MCT2 than the molecular layer. The MCT2 density on the end feet of astrocytes of molecular layer was lower than in hippocampus, but the postsynaptic densities (PSDs) of asymmetric synapses in the molecular layer exhibited a high density using immunogold post-embedding electron microscopic techniques.     

Mutant C57Bl/KsLepr<Superscript>db/+</Superscript> mice as a genetic model of type 2 diabetes mellitus

The genetic model of diabetes mellitus was studied on mutant C57Bl/KsLepr^db/+ mice. These mice were characterized by high concentrations of glucose and glycosylated hemoglobin in the blood, polyuria, polyphagia, polydipsia, progressive obesity, biphasic morphological changes in insular islets of the pancreas (hyperplasia and atrophy), fatty degeneration of the liver, and hypoplasia of the spleen tissue and lymph nodes. Our results indicate that C57Bl/KsLepr^db/+ mice serve as an adequate model of type 2 diabetes mellitus. This model is suitable for testing of therapeutic methods for type 2 diabetes mellitus. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 664–667, December, 2007     

Mibefradil reduces blood glucose concentration in db/db mice

OBJECTIVE:

Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca²⁺]_i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic β-cell function in type 2 diabetes. T-type Ca²⁺ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown.

METHODS:

We examined the effect of the Ca²⁺ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection.

RESULTS:

Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca²⁺ channels, but not by the specific L-type Ca²⁺ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca²⁺ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals.

CONCLUSIONS:

Collectively, these results suggest that T-type Ca²⁺ channels are potential therapeutic targets for antidiabetic drugs.     

The serotonin innervation of the cerebral cortex in the rat—an immunohistochemical analysis

The serotonergic innervation of the cerebral cortex in the rat has been studied by immunohistochemistry employing an antibody directed against the neurotransmitter, serotonin. The dorsal raphe, median raphe and B9 cell groups contain intensely labelled neuronal perikarya. Bundles of large diameter axons suggestive of fibers of passage are observed in successive sections as they ascend through the midbrain tegmentum, medial forebrain bundle, diagonal band and supracallosal stria en route to the cortex. In addition, a lateral pathway to the cerebral cortex traversing the ansa peduncularis is visualized. All regions of the cerebral cortex appear to be innervated by serotonergic axons which have a distinctive morphology: they are fine (0.1–0.5 μm), varicose, and extremely convoluted. Serotonergic axons of passage are thicker and comparatively straight. Throughout the lateral neocortex, as well as in the anterior cingulate cortex, serotonergic axons form a densely arborizing plexus through all cortical layers. Contrary to earlier reports, based on histofluorescence, describing a sparse innervation of the cortex with most of the fibers found in the molecular layer, the present study reveals that the innervation is relatively uniform across all cortical layers. In most of the cortex the density of serotonin-containing axons exceeds that of noradrenergic fibers. A distinctive and different pattern of serotonin innervation is found in the posterior cingulate cortex (cytoarchitectonic field RSg): the serotonergic axons are restricted largely to lamina I and III. A restricted laminar pattern also characterizes the innervation of the hippocampus; dense axonal plexuses occur in the outer rim of the dentate hilus and in the stratum lacunosum-moleculare. The serotonergic afferents to the cortex appear to have at least two different modes of distribution, a relatively uniform pattern in the anterior cingulate and the lateral neocortex and a restricted, laminar pattern in the posterior cingulate and the hippocampus.The density and extent of the serotonin innervation is such that the raphe neurons may contact every cell in the cortex. The widespread arborization of serotonin axons contrasts with the spatially restricted termination of thalamic afferents. The distribution of serotonin-containing fibers also differs substantially from the terminal patterns of noradrenergic and dopaminergic fibers. The differences in axonal morphology and distribution amongst the monoamine afferents reflect differences in their contributions to cortical circuitry. The present findings indicate that the serotonin-containing neurons may exert a profound and global, but not necessarily uniform, influence upon cortical function.     

川芎嗪对大鼠脑缺血再灌注损伤后大脑皮质Bc1-2表达的影响

目的:研究川芎嗪对大鼠脑缺血再灌注损伤后Bcl-2表达的影响.方法:以线栓法制作大鼠大脑中动脉阻塞的局灶性脑缺血再灌注模型,采用免疫印迹、2, 3, 5-氯化三苯四氮唑(TTC)、 H-E染色和神经行为相结合的方法,观测缺血再灌注侧大脑皮质内Bcl-2的表达、脑梗塞体积、脑组织结构及神经功能的变化.结果:与缺血再灌注组(I 2h/R24h)比较,川芎嗪组的Bcl-2表达明显增高,脑梗塞体明显缩小,脑组织的病理损伤和神经异常行为明显减轻.结论:川芎嗪可通过上调Bcl-2的表达,缩小脑梗塞的体积和减轻脑缺血区组织结构的损伤以及明显改善神经症状,对脑缺血再灌注损伤有保护作用.     

脑缺血对大鼠大脑皮质中钙通道Cav1.3表达的影响

目的:探讨脑缺血后大鼠大脑皮质中钙通道Cav1.3表达变化及其在脑缺血损伤中的作用机制。方法:雄性Wistar大鼠随机分为假手术组、缺血7 d组和缺血14 d组。采用免疫组织化学和免疫印迹分别检测各组大鼠大脑皮质中钙通道Cav1.3的表达,采用TUNEL法观察大鼠大脑皮质内细胞的凋亡。结果:与假手术组比较,缺血7 d和14 d组大鼠大脑皮质中钙通道Cav1.3免疫组织化学显色的平均光密度(OD)值和免疫印迹条带的IOD比值都明显降低,缺血14 d组较7 d组降低更明显,细胞凋亡检测显示大脑皮质中凋亡神经元数量随缺血时间延长而增加。结论:缺血导致神经元凋亡的机制可能通过影响钙通道Cav1.3的表达,从而影响皮质神经元的正常功能,导致神经元死亡。