早期人胚胎cDNA文库构建及目的基因筛选

收集受精后３、４和５周龄药物流产胚胎,用改良一步法提取总ＲＮＡ,ｏｌｉｇｏ（ｄＴ）纤维素柱纯化ｍＲＮＡ,逆转录合成一链ｃＤＮＡ,完成二链ｃＤＮＡ的合成后,经碱变性电泳检测,合成ｃＤＮＡ的大小为０．４～９．０ｋｂ之间,且主要集中在１．０～２．０ｋｂ。除去多余的接头,收集大于４００ｂｐ的ｃＤＮＡ片段,与载体ｐＳＰＯＲＴ１和和γＺｉｐＬｏｘ连接,分别得到３、４、５周龄人胚胎质粒文加和噬菌体文库。另外,采     

15-Deoxy-Δ(12,14)-prostaglandin J(2) attenuates the biological activities of monocyte/macrophage cell lines

Monocytes/macrophages link the innate and adaptive immune systems, and in inflammatory disorders their activation leads to tissue damage. 15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has garnered much interest because it possesses anti-inflammatory properties in a number of experimental models. However, whether it regulates monocytes/macrophage pathophysiology is still unknown. This study was designed to examine the effects of 15d-PGJ(2) on the phagocytosis, proliferation and inflammatory cytokines generation in mouse monocyte/macrophage cell line RAW264.7 and J774A.1 cells upon lipopolysaccharide challenge. Our results showed that 15d-PGJ(2) inhibited the phagocytic activity and cell proliferation in a dose-dependent manner, and suppressed proinflammatory cytokines expression, such as tumor necrosis factor-α, transforming growth factor-β1, interleukin-6, and monocyte chemotactic protein-1. These effects were independent of PPARγ, because PPARγ agonist (troglitazone or ciglitazone) and PPARγ antagonist (GW9662) did not affect these activities mentioned above in cells. Treatment of 15d-PGJ(2) also did not modulate expression and distribution of PPARγ. However, these effects of 15d-PGJ(2) were abrogated by antioxidant N-acetylcysteine. Moreover, treatment of 15d-PGJ(2) induced a significant increase in reactive oxygen species production in RAW264.7 and J774A.1 cells. In conclusion, 15d-PGJ(2) attenuates the biological activities of mouse monocyte/macrophage cell line cells involving oxidative stress, independently of PPARγ. These data further underline the anti-inflammation potential of 15d-PGJ(2).     

临床诊断与治疗的新工具——可变淋巴细胞受体

单克隆抗体(Monoclonal antibody, mAb)在癌症以及自身免疫等疾病的诊断与治疗中得到广泛应用, 并且取得了重大进展。当今应用于临床的单克隆抗体是在免疫球蛋白的基础上进行改造研发而得。然而近期发现的无颌类脊椎动物的特异性抗原受体-可变淋巴细胞受体(Variable lymphocyte receptor, VLR), 为抗体类试剂或药物的研发提供了新的视角。与免疫球蛋白(Immunoglobulins, Ig)相比, VLR与抗原结合的特异性、亲和力及稳定性都优于Ig类抗体, 并且抗原特异性单克隆VLR的制备技术日趋成熟。因此, VLR在临床诊断和治疗中具有更高的应用价值, 并可能成为新一代的抗体药物。文章就VLR的基本特征、制备方法及其应用前景进行综述, 为实现VLR在临床诊断与治疗等领域中的应用提供有益参考。     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

孢子丝菌复合体分子分型研究进展

孢子丝菌复合体属于双相真菌,全球分布,可引起人类及动物的慢性深部感染。不同地域的菌株在致病力、传播途径及药物敏感性等方面均存在差异。孢子丝菌病作为一种人兽共患病,其发病率逐年上升,出现多次暴发流行。分子分型不仅是明确感染源和传播途径、预防和控制疾病流行的有力手段,同时有助于了解孢子丝菌基因型与表型的相关性,在研究其致病机制以及临床诊治过程中都具有十分重要的意义。本文对孢子丝菌的分子分型方法的研究进行综述。     

角蛋白酶基因gm2886在密旋链霉菌ACT12中的表达及鉴定

通过生物信息学分析,在本实验室分离得到的1株羽毛高效降解菌微白黄链霉菌Fea-10基因组中发现基因gm2886(GenBank Accession Number:KY368946)可能编码一新的角蛋白酶,通过在该基因5'端和3'端分别连接红霉素抗性基因启动子(PermE)和组氨酸标签编码序列并构建在大肠杆菌-链霉菌穿梭质粒pSET152上,接合转入密旋链霉菌Streptomyces pactum ACT12,从而实现了异源表达,蛋白纯化后对其酶学性质进行了研究。实验结果表明,带有组氨酸标签编码序列的gm2886在密旋链霉菌ACT12中可以表达分泌得到1个大小约为36 kDa的蛋白。多种底物检测表明异源表达得到的重组蛋白GM2886-His6具有蛋白酶活性,可以降解水不溶性的天青角蛋白和羽毛粉;其最适温度和pH分别为50℃和pH 10.0。PMSF可抑制GM2886-His6的酶活,而EDTA不能,说明该酶为丝氨酸蛋白酶。本研究为从分子水平上解析羽毛高效降解菌Fea-10的活性机理,从而进一步开发其应用潜力提供了基础,同时可为该类蛋白酶的研究提供借鉴。     

人和动物胃肠道微生物丁酸合成途径相关酶基因的研究进展

人和动物胃肠道中都栖息了大量的产丁酸微生物。研究表明,参与丁酸合成中心途径的酶基因成簇存在,其相关基因的排列形式多样并具有属种特异性。参与丁酸合成最后一步的(丁酰辅酶A/乙酸)辅酶A(CoA)转移酶在胃肠道产丁酸微生物中广泛存在,并在丁酸合成中发挥重要作用。本文结合我们的研究工作,综述了国内外丁酸合成相关酶基因和基因簇的最新研究进展。     

干预措施对噪声污染大鼠脑组织基因表达及去甲肾上腺素水平的影响

为了探索干预措施对噪声污染大鼠脑组织基因表达水平的影响, 将50只SPF级Wistar大鼠随机分为空白对照组、噪声污染组(分为30、60、80 dB三个组)、干预组(利血平+80 dB), 每组10只动物。每天刺激1次, 每次刺激30 min, 连续刺激15 d。第16天解剖出脑组织用酶联免疫吸附法(ELISA)检测基因表达水平。结果发现, 噪声污染组大脑前额叶皮质(PFC)和海马(Hipp)组织中去甲肾上腺素(noradrenaline,NA)水平比对照组分别升高了22.87%、50.35%、94.65%和 12.00%、31.76%、61.83%; 干预组NA水平比对照组分别降低了33.66%和52.06%; 去甲肾上腺素转运蛋白(noradrenaline transporter, NAt)水平比对照组分别升高了22.87%、50.35%、94.65%和12.00%、31.76%、61.83%, 干预组NAt水平比对照组分别降低了33.66%和52.06%; 脑源性神经营养因子(brain derived neurotrophic factor, BDNF)水平比对照组分别升高了24.87%、39.27%、67.41%和44.97%、80.81%、95.84%, 干预组BDNF水平比对照组分别升高了16.36%和14.34%, 升高程度明显低于噪声污染组; 酪氨酸激酶受体B(Tyrosine kinase B, TrkB)水平比对照组分别升高了32.64%、59.95%、82.64%和31.02%、57.31%、80.23%, 干预组TrkB水平比对照组分别升高了4.75%和10.52%, 升高程度明显低于噪声污染组。结果显示, 噪声污染使动物体内去甲肾上腺素等水平升高, 去甲肾上腺素是噪声污染引起组织器官损伤的主要因素, 脑源性神经营养因子和酪氨酸激酶受体B防止神经元受损死亡, 改善神经元的病理状态, 利血平使去甲肾上腺素耗竭, 保护组织器官免受噪声污染的损伤。     

FBXW7 suppresses epithelial‐mesenchymal transition and chemo‐resistance of non‐small‐cell lung cancer cells by targeting snai1 for ubiquitin‐dependent degradation

Objectives

FBXW7 acts as a tumour suppressor by targeting at various oncoproteins for ubiquitin‐mediated degradation. However, the clinical significance and the involving regulatory mechanisms of FBXW7 manipulation of NSCLC regeneration and therapy response are not clear.

Materials and Methods

Immunohistochemical staining and qRT‐PCR were applied to detect FBXW7 and Snai1 expression in 100 samples of NSCLC and matched tumour‐adjacent tissues. FBXW7 manipulation of cancer biological functions were studied by using MTT assay, immunoblotting, flow cytometry, transwells, wound healing assay, and sphere‐formation assays. Immunofluorescence and co‐immunoprecipitation were used to analyse the possible interaction between Snai1 and FBXW7.

Results

We detected the decreased FBXW7 expression in majority of the NSCLC tissues, and lower FBXW7 level was correlated with advanced TNM stage. Furthermore, those patients with decreased FBXW7 expression tend to have both poorer 5‐year survival outcomes, and shorter disease‐free survival, comparing to those with higher FBXW7 levels. Functionally, we found that FBXW7 enforcement suppressed NSCLC progression by inducing cell growth arrest, increasing chemo‐sensitivity and inhibiting Epithelial‐mesenchymal Transition (EMT) progress. Results further showed that FBXW7 could interact with Snai1 directly to degrade its expression through ubiquitylating alternation in NSCLC, which could be partially abrogated by restoring Snai1 expression.

Conclusions

FBXW7 conduction of tumour suppression was partly through degrading Snai1 directly for ubiquitylating regulation in NSCLC

     

miRNA-20a促进卵巢癌细胞OVCAR3的转移

目的检测miRNA．20a对卵巢癌细胞系OVCAR3转移能力的影响。方法通过实时定量RT-PCR验证反义寡核苷酸与小干扰RNA封闭与过表达的效果,然后利用MTF、软琼脂集落形成和transwell侵袭实验检测封闭和过表达miRNA．20a对OVCAR3细胞增殖及转移能力的影响。结果封闭内源性miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显降低。过表达miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显升高。结论miRNA-20a可能参与了卵巢癌细胞OVCAR3的转移。