红花花青素合成酶基因的克隆及表达分析

根据红花转录物测序结果中得到的中间序列,采用R11-PCR和RACE方法从红花花瓣中克隆到1个4嬲基因的全长cDNA,该基因全长序列1226bp,具有完整的开放阅读框（ORF）,共1050bp,编码349个氨基酸。生物信息学软件分析显示,该基因编码的蛋白理论分子量约为82．27kDa,等电点为5．09,序列里含有典型的加尾信号序列AATAA和Poly（A）。保守结构域预测表明,该基因编码的蛋白具有典型的ANS蛋白功能结构域,其保守结构域中含有铁离子及2．0-酮戊二酸结合位点。结合其他物种的臌因构建系统树表日月,红花ANS蛋基因与其他物种氨基酸具有一定的同源性,其中与芍药的亲缘关系最近。应用实时荧光定量PCR分析表明,ANS基因在红花的初花期和盛花期的表达量最高。     

重组腺病毒介导HGF修饰ADSCs对大鼠肝损伤模型的治疗作用

目的：建立重组腺病毒介导肝细胞生长因子HGF促ADSCs 定向分化肝细胞的方法,并对其参与肝损伤修复能力进行验证,为作为治疗肝损伤细胞来源提供参考。方法：采用消化培养的方法,分离SD 大鼠腹股沟脂肪组织ADSCs 细胞,连续传代3 次对其进行纯化培养,利用形态学鉴定、流式细胞术检测ADSCs 表面标志物方法对其间充质干细胞样特征进行鉴定,加入成脂肪细胞诱导液观察其分化成脂肪细胞的能力;构建腺病毒表达HGF载体Adeno-HGF-EGFP,并转染ADSCs 细胞,利用免疫细胞化学染色方法检测肝细胞标志分子表达水平;最后建立大鼠肝损伤动物模型,观察Adeno-HGF-EGFP 转染的ADSCs 细胞参与肝损伤修复能力情况。结果：分离的ADSCs 细胞形态较为一致,绝大多数呈梭形,排列不规则。流式细胞术结果显示,该细胞表达CD29、CD90、CD106 等间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34、CD45,同时,分离的ADSCs 细胞具有诱导分化成脂肪细胞能力;Adeno-HGF-EGFP 转染ADSCs后,AFP、ALB、CK18 等肝细胞特异性分子表达水平升高;经尾静脉注射ADSCs 细胞后,肝损伤大鼠的AST、ALT、TBIL 等分子表达水平恢复正常。结论：建立了重组腺病毒介导肝细胞生长因子HGF促ADSCs定向分化肝细胞的方法,并且表达HGF的ADSCs 细胞具有修复大鼠肝损伤模型能力,这为通过细胞治疗肝损伤提供了新的细胞来源。     

糖尿病足治疗新进展

糖尿病足溃烂是糖尿病的常见慢性并发症,以高发生率、高致残率、经济负担重,治疗时间长及高死亡率严重影响病人的生活质量甚至威胁生命,我国糖尿病协会于2010年11月14公布我国糖尿病的患病者已达9240万,除此之外糖尿病前期患者已达1.482亿。而在糖尿病患者中糖尿病足的发病率达到1.7%-11.9%;糖尿病患者中有15%的患者一生中有患糖尿病足的倾向,因此对于糖尿病足的治疗显得尤为重要,本文从糖尿病足的病因、机制,微环境变化着手,重点阐述糖尿病足传统的中西医疗法及目前的手术、介入及干细胞治疗方法的近况,旨在探讨各种方法的优点和不足及可开拓的空间,为进一步治疗糖尿病足提供一定的理论依据,为糖尿病足新方法的研究提供一些新的思维方式。     

选择性环氧合酶-2抑制剂联合奥曲肽诱导人胃癌细胞的凋亡

目的探讨选择性环氧合酶-2抑制剂NS-398与奥曲肽联合应用对人胃癌细胞株BGC-823生长、凋亡的影响。方法体外培养BGC-823细胞,分别用NS-398(100μmol/L)与奥曲肽(1μmol/L)单独及联合处理不同时间后,倒置显微镜观察细胞形态学变化;观察生长曲线的变化;流式细胞仪检测细胞凋亡率;实时定量(Real-time)PCR检测COX-2mRNA的表达;Western blot法检测Caspase-3蛋白表达。结果倒置显微镜下,对照组BGC-823细胞生长良好,药物处理后,细胞变小、变圆,悬浮,联合组细胞形态学改变显著强于单纯用药组;药物作用后,细胞生长受抑制,出现负增长,联合组作用明显强于单纯用药组;流式细胞仪检测表明联合用药组诱导BGC-823细胞的凋亡率明显高于单一用药组和对照组(P0.01);各处理组均使BGC-823细胞COX-2mRNA表达下调(P0.05);药物处理后细胞Caspase-3蛋白表达明显增加。结论 NS-398、奥曲肽联合可协同抑制BGC-823细胞生长、增殖,其机制可能与下调COX-2mRNA表达、诱导肿瘤细胞凋亡相关。     

秸秆、氮肥和食细菌线虫交互作用对土壤活性碳氮和温室气体排放的影响

采用3因素2水平交互设计室内恒温培养试验,通过调控秸秆施用、氮肥用量及食细菌线虫,探讨三者对土壤微生物生物量碳氮(C_mic和N_mic)、可溶性碳氮(DOC、DON)、矿质氮(NH₄⁺-N和NO₃^--N)及温室气体排放(CO₂、N₂O和CH₄)的交互影响.结果表明： 施用秸秆显著增加了食细菌线虫数量、C_mic和N_mic,而随着氮肥用量增加,C_mic和N_mic降低,食细菌线虫对C_mic和N_mic的影响则依赖于秸秆和氮肥用量.秸秆、氮肥和食细菌线虫对可溶性碳氮和矿质氮表现出强烈的交互作用,其中秸秆和氮肥均增加了DOC、NH₄⁺ -N和NO₃^--N;食细菌线虫对DOC的抑制作用和对矿质氮的促进作用达到显著水平.秸秆处理对CO₂、N₂O的促进及对CH₄的抑制均达到显著水平,而线虫和氮肥的影响则更多表现出交互作用.在培养第56天,有秸秆时,低量氮肥下食细菌线虫显著促进了CO₂的排放,而高量氮肥下则表现出对CO₂和N₂O显著的抑制作用.总之,土壤生态功能的发挥不可忽视土壤动物的作用.     

Regulatory Phenotype,PD-1 and TLR3 Expression in T Cells and Monocytes from HCV Patients Undergoing Antiviral Therapy: A Randomized Clinical Trial

Background & Aims

The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment.

Methods

Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4⁺ T-cells or CD8⁺ T-cells and toll-like receptor (TLR) 3 expressing CD14⁺ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls.

Results

Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14⁺ monocytes at baseline had a high rate of cEVR (P<0.05).

Conclusions

Low peripheral TLR3 expressing CD14⁺ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells.

Trial Registration

Chinese Clinical Trial Registry ChiCTR10001090.     

Role of Moesin in Renal Fibrosis

Background

Renal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.

Method

Human proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.

Result

HK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.

Conclusion

We find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin.     

石墨烯对植物的生理毒性效应研究进展

石墨烯是当前研究最热的碳纳米材料,具有独特的理化特性,在各领域广泛应用。随着石墨烯生产和使用量的不断增大,其不可避免地进入到环境中,给生态环境和人类健康带来风险。阐明石墨烯的潜在毒性效应及其作用机制对于客观评价石墨烯的生态环境健康风险具有重要意义。迄今为止,已有诸多研究报道了石墨烯的植物生理毒理效应。研究表明,石墨烯对植物的生理学响应及其生理变化过程存在影响,涉及萌芽、幼苗生长、氧化应激、光合特性、植物激素和代谢过程等,且多呈浓度效应。今后亟需构建一套被广泛认可的植物石墨烯毒性评价体系,为石墨烯的安全生产和使用提供指导。