Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

肠杆菌科细菌对β-内酰胺类抗生素耐药机制的研究现状

肠杆菌科细菌是革兰阴性杆菌中最常见的一类细菌,在一定条件下可引起医院和社区感染。临床首选β-内酰胺类抗生素来抗感染,但由于抗生素的不合理使用导致肠杆菌科细菌产生相应的灭活酶及水解酶或菌株细胞结构改变,从而破坏β-内酰胺环使其对抗生素作用的敏感性下降甚至耐药,给临床抗感染治疗带来了极大的挑战;为更好地指导临床用药,拟就肠杆菌科细菌的耐药表型、对β-内酰胺类抗生素的耐药机制做一综述。     

流感病毒H7N9病毒样颗粒疫苗的研发

<正>最近,中国出现了由禽源性流感病毒A(H7N9)引起的严重性传染病,为此全球付出努力,来实现候选疫苗的快速研发。目前,非流感病毒A(H7N9)H7亚型流感候选疫苗在面对新近出现的流感病毒A(H7N9)时,其免疫原性低,保护效果差。一种流感病毒A(H7N9)重组候选疫苗,是将A/Anhui/1/2013株的血凝素(HA)、神经氨酸苷酶(NA)和A/Indonesia/05/2005(H5N1)株的基质蛋白1(M1)克     

基于CRISPR-Cas系统的基因组定点修饰新技术

CRISPR(clustered regulatory interspersed short palindromic repeat)序列源于原核生物的一种获得性免疫系统,协同Cas(CRISPR-associated)蛋白家族参与抵抗噬菌体或其它病毒的二次感染,广泛存在于细菌(60%)和古菌(90%)中.病菌和宿主的共同进化导致了CRISPR-Cas系统具有多样性,可分为3大类(Ⅰ-Ⅲ),又分为10亚类.在Ⅱ型CRISPR-Cas系统基础上建立了RNA介导的CRISPR-Cas系统来修饰(删除、添加、激活、抑制)靶细胞中特定的基因序列,现已在人类细胞、小鼠、斑马鱼、酵母、细菌、果蝇、线虫、拟南芥中得以应用.本文主要介绍了Ⅱ型CRISPR-Cas系统的结构特点、作用机理及作为新型基因组定点修饰技术的研究进展,分析该技术优势,并展望CRISPRCas系统的应用前景.     

肝细胞癌中转录因子E2F7对Wnt/β-catenin信号通路活性的影响

探讨肝细胞癌(HCC)中非典型性E2F家族成员E2F7在肝癌细胞生长、分化中的作用及可能涉及的分子机制.本研究运用实时荧光定量PCR检测38例原发性肝细胞癌及对应的癌旁组织中E2F7基因mRNA的表达情况;分别通过基因过表达和RNA干扰技术上调或下调E2F7基因表达,并运用实时荧光定量PCR和Western印迹检测肝癌细胞株MHCC-H中β-catenin及其靶基因cmyc的表达情况;双荧光素酶报告基因系统检测E2F7对Wnt/β-catenin信号通路活性的影响;核浆分离实验检测过表达E2F7基因对β-catenin入核的影响;免疫共沉淀实验检测异位表达E2F7与内源β-catenin的相互作用.结果显示,肝细胞癌组织中E2F7基因的表达量显著高于相应的癌旁组织(P0.001);转录因子E2F7可与β-catenin相互作用并促进β-catenin进入细胞核.转录因子E2F7可以促进Wnt/β-catenin信号通路的活性.     

益肾填精中药拮抗环境内分泌干扰物引致青春前期大鼠性腺发育不良的作用机制

邻苯二甲酸二(2-乙基己基)酯(di-2-ethylhexylphthalate,DEHP)及氯氰菊酯(cypermethrin,CYP)是我国广泛存在的两种环境内分泌干扰物(environmental endocrine disruptors,EEDs),具有显著的抗雄激素活性及生殖毒性,可致雄性性腺发育不良.祖国传统医学认为,性腺发育不良属肾精亏虚、肾气不足,临床采用益肾填精中药治疗取得显著疗效,但其具体机制尚不清楚.本实验主要研究益肾填精中药拮抗EEDs——DEHP及CYP引致青春前期大鼠性腺发育不良的作用机制.实验中染毒组分别饲喂500 mg/kg DEHP,80 mg/kg CYP及500 mg/kg DEHP+80 mg/kg CYP,治疗组采用40 mg/kg益肾填精中药与相应染毒物质同时饲喂.研究结果显示,DEHP、CYP单独及联合染毒组的青春前期大鼠睾丸重量、睾丸系数及血清睾酮水平均显著下调;睾丸氧化应激指标MDA含量、GSH-Px活性明显上升;病理组织及超微结构显示睾丸形态萎缩;睾丸支持细胞功能相关的基因与蛋白表达均出现不同程度的下调.益肾填精中药治疗干预后,睾丸重量、睾丸系数及血清睾酮水平均显著增加并接近对照组水平;睾丸形态明显改善,细胞数量增加;睾丸氧化应激水平下降;实时荧光定量PCR及Western印迹显示睾丸支持细胞功能相关的基因与蛋白的表达水平显著上调.本研究证实,益肾填精中药对DEHP及CYP的抗雄激素活性及生殖毒性有显著拮抗作用,可明显拮抗染毒物质诱导的氧化应激作用,促进睾酮分泌,并改善睾丸支持细胞功能,这可能是益肾填精中药有效拮抗EEDs抗雄激素活性及其生殖毒性的主要作用机制之一.     

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-alpha treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4+ T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-gamma. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohn's disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-alpha treatment had no effect on the proliferation of CD4+ T lymphocytes. In contrast, the number of IFN-gamma-releasing CD4+ T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-gamma release to a similar extent. In vitro addition of TNF antagonists to CD4+ T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF+ CD4+ T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4+ T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4+ T lymphocytes rapidly releasing IFN-gamma upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4+ T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation.