Proteomic Analysis of Baboon Cerebral Artery Reveals Potential Pathways of Damage by Prenatal Alcohol Exposure,

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Highlights

• •We studied mid-pregnancy alcohol exposure and baboon fetal cerebral artery.
• •238 proteins differed between control and alcohol-exposed fetuses near-term.
• •Proteins of metabolic pathways represented one of the major targets of alcohol.
• •Alcohol effect on the development of fetal brain vessels is persistent.

     

不同可行走式分娩镇痛方法对分娩结局及胎儿血氧饱和度的影响

目的:探讨不同可行走式分娩镇痛方法对产妇分娩结局及胎儿血氧饱和度(FSaO_2)的影响。方法:选取2017年1月至2018年2月期间于成都市第二人民医院妇产科住院分娩的123例初产孕妇作为研究对象,分为罗哌卡因结合氢吗啡酮可行走式分娩镇痛组(A组)45例、罗哌卡因结合舒芬太尼可行走式分娩镇痛组(B组)45例以及常规分娩组(C组)33例。比较三组产妇的剖宫产率、产后出血量、胎儿FSaO_2,并对比三组胎儿窒息程度。结果:三组产妇年龄、孕周、胎儿体重、剖宫产率以及产后2 h、24 h出血量比较无统计学差异(P0.05),A组胎儿轻度窒息率高于B、C组,A组胎儿正常率低于B、C组(P0.05),B、C组胎儿的轻度窒息率、正常率比较无统计学差异(P0.05),A组的第一产程、第二产程胎儿FSaO_2低于B、C组(P0.05),B、C两组第一产程、第二产程胎儿FSaO_2比较无统计学差异(P0.05)。结论:罗哌卡因结合舒芬太尼的可行走式分娩镇痛与常规分娩均不影响产妇的分娩结局和胎儿FSaO_2,相较罗哌卡因结合氢吗啡酮在分娩镇痛中具有可行性及安全性。     

Generation of human cortical neurons from a new immortal fetal neural stem cell line

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.     

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

BackgroundSialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.MethodsNeuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.ResultsClostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.ConclusionsImmobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.General significanceImmobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.     

Caspase-9抑制剂对SD大鼠椎间盘软骨终板细胞凋亡的影响

目的探讨caspase-9抑制剂对低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡影响的研究。方法取3月龄SD大鼠椎间盘软骨终板,序贯消化法获取细胞原代培养,以1%FBS培养48 h为诱导凋亡条件。实验分为1%FBS凋亡组、caspase-9抑制剂组（Z-LEHD-FMK）及DMSO对照组,分别处理细胞48 h,后经流式细胞仪检测细胞凋亡率、Western blot检测procaspase-9,active caspase-9及active caspase-3的表达。结果流式细胞仪检测显示,caspases-9抑制剂组细胞凋亡率（26.3±2.56）%与1%FBS组（40.8±0.84）%及DMSO组（40.2±1.56）%相比凋亡率较低,有显著统计学差异（P〈0.05）;Western blot检测caspases-9抑制剂组active caspase-9及active caspase-3较1%FBS凋亡组及DMSO对照组表达均明显减少,有显著统计学意义（P〈0.05）。结论 Caspase-9抑制剂能明显抑制低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡,有望成为治疗椎间盘退变的新型药物。     

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

Fetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by HTS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures.     

Cholesterol acceptor capacity is preserved by different mechanisms in preterm and term fetuses

Fetal serum cholesterol and lipoprotein concentrations differ between preterm and term born neonates. An imbalance of the flow of cholesterol from the sites of synthesis or efflux from cells of peripheral organs to the liver, the reverse cholesterol transport (RCT), is linked to atherosclerosis and cardiovascular disease (CVD). Preterm delivery is a risk factor for the development of CVD. Thus, we hypothesized that RCT is affected by a diminished cholesterol acceptor capacity in preterm as compared to term fetuses. Cholesterol efflux assays were performed in RAW264.7, HepG2, and HUVEC cell lines. In the presence and absence of ABC transporter overexpression by TO-901317, umbilical cord sera of preterm and term born neonates (n = 28 in both groups) were added. Lipid components including high density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein A1, and apolipoprotein E were measured and related to fractional cholesterol efflux values. We found overall, fractional cholesterol efflux to remain constant between the study groups, and over gestational ages at delivery, respectively. However, correlation analysis revealed cholesterol efflux values to be predominantly related to HDL concentration at term, while in preterm neonates, cholesterol efflux was mainly associated with LDL. In conclusion cholesterol acceptor capacity during fetal development is kept in a steady state with different mechanisms and lipid fractions involved at distinct stages during the second half of fetal development. However, RCT mechanisms in preterm neonates seem not to be involved in the development of CVD later in life suggesting rather changes in the lipoprotein pattern causative.     

大鼠门静脉分支残端置管模型构建及细胞移植途径的评价

目的：大鼠肝部分切除模型被广泛的应用于肝脏疾病的研究,随着干细胞治疗肝损伤及护肝药物研究的发展,对大鼠肝损伤模型也提出了很多新的要求。本实验拟在大鼠肝部分切除术的基础上改进以建立大鼠肝断面门静脉分支残端的静脉置管模型,并进行细胞移植实验,对比分析新模型的优劣。方法：60只F344大鼠分为三组。A、B组行行85％肝切除术;C组行85％肝切除术＋肝断面门静脉分支残端置管术。术中B组经门静脉注入4×105个表达GFP（greenfluorescenceprotein,GFP）的胎肝干细胞（fetalliverstern／progenitorcells,FLSPCs）。c组经留置导管注射入同等量的FLSPCs,A组注射同等剂量的培养液。72小时取血清,测定肝功能ALT、AST,统计死亡率;取肝脏组织切片观察其修复情况。统计学采用方差分析和LSD—t检验。结果：B、C组F344大鼠72小时肝功指标（ALT、AST）均明显优于A组;B组、C组肝脏组织学的病理损伤的恢复分别较A组快。B、C组间肝功指标无统计学意义。结论：经门静脉分支残端置管途径移植FLSPCs效果等同于经门静脉穿刺途径,且该模型具有可反复、可选时、减少创伤等优点。