大丽轮枝菌粗毒素在棉花根冠细胞生物学测定方法上的研究

在棉花根冠细胞生物测定过程中,棉花黄萎病病菌通过提纯复壮而达到较强的侵染能力,并制备出较多的黄萎病菌粗毒素。介绍了粗毒素的简易制备、根冠细胞培养与制备过程以及染色过程中染色剂的选择、染色时间的长短。实验与传统方法相比有几点改进:总结出细胞振荡时间在1~2 min之内;之后黑暗条件下静置培养2~6 h;选用pH为6.5的0.01%中性红染色3~5 min,之后加入一滴0.2%伊文思兰染色,伊文思兰的染色时间应严格掌握在3min以内等,为根冠细胞测定方法的推广提供了一定的理论支持。     

两种菊酯类农药对鲤血细胞的影响

采用对鲤肌肉注射不同浓度的高效反式氯氰菊酯和功夫菊酯,研究了两种菊酯类农药对鲤血细胞形态、红细胞渗透脆性的影响。结果显示:随两种农药攻毒浓度增大、时间延长,鲤血细胞异形率增大。低浓度(6、60μg/kg)中毒鲤成熟红细胞大小不一,核稍有畸形,淋巴细胞、单核细胞增多,核浆界限不清,有空泡,甚至伴有核溶解的白细胞;而高浓度(600、6 000μg/kg)中毒鲤血细胞中除伴有低浓度中毒症状,还出现了染色质模糊,部分白细胞核浆界限不清,甚至溶解,退化更明显。红细胞渗透脆性亦随攻毒浓度增大、时间延长而增高,且开始溶血点的变化幅度较完全溶血点大,表明最小抵抗值更易受环境因子影响。     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

支持细胞中促卵泡素信号通路研究进展

支持细胞在精子生成过程中起重要作用,支持细胞的数量决定睾丸大小、生精细胞数量以及最终生成精子数量。促卵泡素作为保证雄性生育能力的重要激素之一,通过与支持细胞上的促卵泡素受体结合,诱导5种信号途径,导致支持细胞中各类转录因子被激活,引起基因表达发生变化,从而保证生精细胞顺利发育成精子。文章主要围绕支持细胞中促卵泡素信号途径,及其对细胞发育过程中相关基因表达的影响等方面进行了综述。     

瑞香狼毒化学成分抑制PC-12细胞神经突起生长活性研究

对瑞香狼毒(Stellera chamaejasme L.)的化学成分进行研究,采用硅胶色谱、凝胶色谱、高效液相色谱等多种色谱手段对瑞香狼毒的乙酸乙酯提取物进行分离纯化,利用NMR、MS波谱分析鉴定3个木质素、1个二萜原酸酯、3个双黄酮、2个黄酮。枇杷素(5)、异落叶松树脂醇(7)和(-)-落叶松树脂醇(8)为首次从该植物中分离得到。PC-12细胞活性试验表明,双黄酮类化合物(狼毒色原酮(2),新狼毒素B(3),异新狼毒素A(4))对NGF依赖的PC-12细胞突起生长有较强的抑制活性(P0.05)。     

山羊卵泡发育相关基因的筛选及分析

【背景】山羊第一卵泡波中的优势卵泡（dominant follicles, DF）和从属卵泡（subordinate follicles, SF）是整个卵泡发育过程中最为关键的两个阶段。随着卵泡的进一步发育,最终DF可能发育成为成熟卵泡,直到排卵;SF将走向闭锁,其中颗粒细胞的凋亡是导致卵泡发生闭锁的关键因素。然而目前对促进卵泡的优势化或导致其闭锁的分子机理尚不清楚。【目的】通过对山羊第一卵泡波中DF和SF颗粒细胞进行高通量测序,旨在筛选影响卵泡发育的关键基因,为深入探究卵泡发育的调控机制提供理论依据。【方法】选取10只1岁龄健康的贵州白山羊分别注射前列腺素F_2α,使其同期发情,此后每天用B超检测并记录卵泡的生长情况,发情3 d后,统一屠宰并采集第一卵泡波中DF (直径4.5—6 mm)与SF (直径3 —4.5 mm),分别分离其中的颗粒细胞,提取总RNA、构建文库后通过Illumina Hiseq 2500平台进行测序。利用FastQC对测序产出raw reads进行质量评估并经过过滤后,获得品质较高的clean reads;使用Trinity对得到的clean reads进行重新组装,从而获得unigenes;使用CLC Genomics Workbench将unigenes与山羊RefSeq数据库进行比对获得mRNA;使用DESeq2 软件对获得的mRNA进行差异表达分析;分别采用goseq和kobas软件对得到的差异表达基因进行GO分析及KEGG信号通路分析;最终通过qRT-PCR对筛选出的可能影响卵泡发育的关键基因进行验证。【结果】分别对测序得到的raw reads进行过滤后,在DF颗粒细胞中获得43 217 934条clean reads,占raw reads的比例为95.19%;SF颗粒细胞中获得40 766 348条clean reads,占raw reads的比例为95.35%。将得到的unigenes与山羊的RefSeq 数据库进行比对后,共得到33 896条带有注释的转录本,再通过设定FPKM>1, q value<0.05,共在两种卵泡颗粒细胞中获得13 644个基因。设定参数：FPKM≥1,SF-FPKM/DF-FPKM>1,P<0.05,获得695个差异表达mRNA,其中233个在SF颗粒细胞中表达显著上调,462个表达显著下调;对所获得695个差异表达mRNA进行GO功能富集分析,共分为三大类42组：其中生物学过程占47.6%,细胞组分占47.6%,分子功能占4.8%;KEGG信号通路分析,发现20条通路,其中与核糖体通路相关的基因富集最为显著。通过在Genecard中进行功能分析后,筛选6个可能与山羊卵泡发育密切相关的基因,其中PRLR、PTX3、RGN在SF颗粒细胞中表现为上调;DKK3、ALDH1A2、RARRES1则表现为下调。qRT-PCR显示PRLR、RGN、DKK3、ALDH1A2、RARRES1的表达趋势与高通量测序结果一致,且RGN在从属卵泡颗粒细胞中的表达量极显著地高于优势卵泡（P<0.01）;DKK3、ALDH1A2、RARRES1在优势卵泡颗粒细胞中的表达量极显著地高于从属卵泡（P<0.01）。【结论】DKK3、ALDH1A2、RARRES1和RGN在优势卵泡和从属卵泡中表达量存在极显著差异,推测在山羊卵泡发育过程中可能促进卵泡的优势化或导致闭锁,对深入探究卵泡发育的调控机制具有重要意义。     

Sargassum horneri as a Functional Food Ameliorated IgE/BSA-Induced Mast Cell Activation and Passive Cutaneous Anaphylaxis in Mice

Sargassum horneri (S. horneri), an edible brown alga, has been proposed as a functional food with an improvement effect on abnormal skin immune responses. The present study investigates the anti-allergic effect of an ethanol extract from S. horneri (SHE) on immunoglobulin E (IgE)/bovine serum albumin (BSA)-mediated activation in bone marrow-derived cultured-mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) reaction in mice. SHE markedly and dose-dependently suppressed the degranulation of BMCMCs by reducing the β-hexosaminidase and histamine release without cytotoxicity. In addition, SHE significantly decreased the FcεRI expression on the surface of BMCMCs and its IgE binding. Moreover, SHE reduced the mRNA expression and the production of allergic cytokines; interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-13; interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α; and a chemokine, thymus and activation-regulated chemokine (TARC), by suppressing the activation of Src-family kinases and nuclear factor (NF)-κB signaling. In further study, the application of SHE reduced the PCA reaction in an IgE/BSA-induced type I allergic mice model. Taken together, we suggest that SHE has an anti-allergic effect in type I allergic responses.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.