二维回转培养对MLO-Y4骨样细胞PKD2表达定位及胞内钙信号的影响

目的：PKD2（polycystin2,多囊肾病蛋白2）能够在细胞膜上形成无选择性的阳离子通道,在肾上皮细胞中PKD2与初级纤毛共定位,通过改变胞内的钙信号过程参与细胞对力学刺激的响应。本实验通过二维回转培养来模拟失重效应,旨在探讨二维回转培养对MLO-Y4骨样细胞PKD2表达定位,及胞内钙信号的影响。初步了解PKD2在小鼠骨样细胞MLO-Y4响应力学刺激过程中起的作用。方法：采用二维回转培养骨样细胞MLO-Y4,用RT-PCR和western blotting检测PKD2的表达,用荧光共聚焦显微镜检测细胞中PKD2与初级纤毛的定位及细胞内钙离子含量。结果：与对照组相比,在二维回转培养后,骨样细胞MLO-Y4的PKD2表达在mRNA和蛋白水平都有明显的下降,PKD2、PKD1（polycystin1,多囊肾病蛋白1）和乙酰化的α-tubulin共定位,同时二维回转培养降低了细胞内钙离子含量。结论：在二维回转培养下,PKD2可能通过调节自身表达来改变细胞膜上PKD通道的数目和开放情况来影响细胞内钙离子含量,参与骨细胞对细胞外应力的感受过程,其详细机制还有待进一步实验研究。这将对探讨骨细胞响应力学刺激的具体机制提供重要的理论依据。     

原纤毛－多囊蛋白复合物在肾和骨组织中的类似作用

纤毛－多囊蛋白复合物的功能或者结构异常,是导致常染色体显性多囊肾病的主要原因.该复合物除了被认为在正常的肾上皮细胞上起着机械和化学感受器的作用,可能在骨细胞中也有类似的作用.本文总结了多囊蛋白和纤毛的结构、分布特点以及在肾发育过程中所发挥的作用;着重综述了纤毛 多囊蛋白复合物在肾上皮细胞上作为机械和化学感受器,通过影响细胞内一系列的信号途径,调控细胞的基因转录和蛋白合成的最新研究进展,包括与细胞内钙离子变化有关的钙调神经磷酸酶－NFAT途径和PI3K－Atk途径,调控细胞周期的JAK－STAT途径,及维持正常肾结构的Wnt/β连环蛋白信号途径等;还将通过比较在肾上皮细胞上纤毛 多囊蛋白复合物所激活的信号传导途径和在骨细胞中传导机械刺激的信号转导途径的类同,提示在骨细胞中,纤毛 多囊蛋白复合物可能起着在肾上皮细胞上类似的机械感受器作用,为系统性阐明多囊肾病的发病机制,以及揭示失重或负重状态下骨细胞机械感受的分子机制提供了一个新思路.     

PKHD1基因的分子特征和生物功能研究进展

PKHD1(polycystic kidney and hepatic disease 1)基因定位于人染色体6p12.2.该基因在基因组内约占500 kb,其中大约含86个外显子.目前所知,其最长ORF至少由67个外显子组成,编码一个由4 074个氨基酸组成的单次跨膜受体样蛋白,被称为纤囊素(fibrocystin/polycystin,FPC).PKHD1是人类常染色体隐性遗传多囊肾病(autosomal recessive polycystic kidneydjsease,ARPICD)的致病基因.在小鼠中,FPC于胚胎期9.5天可在发育中的神经管、气管、原肠管等含管道的器官中被探测到.在人类,FPC在胎肾即开始表达于输尿管芽,并持续表达于输尿管芽分支演变为集合管的整个过程.在成体肾,FPC也主要表达于肾内集合管上皮细胞,其亚细胞定位于上皮细胞的管腔面的顶端,主要分布在细胞纤毛和基体附近.FPC的主要生物功能目前仍未完全明了,新近的研究表明,FPC可能作为一个膜受体样蛋白,将细胞外的信号通过结合与调节TRPP2(PKD2)钙离子介导的通道传递到细胞内,调控体内各管道上皮细胞分化、增殖、极化和移行,从而促成各种牛理管道的形成.     

PKHD1基因的分子特征和生物功能研究进展

PKHD1 (polycystic kidney and hepatic disease 1) 基因定位于人染色体6p12.2．该基因在基因组内约占500 kb,其中大约含86个外显子．目前所知,其最长ORF至少由67个外显子组成,编码一个由4 074个氨基酸组成的单次跨膜受体样蛋白,被称为纤囊素(fibrocystin/polycystin,FPC)．PKHD1是人类常染色体隐性遗传多囊肾病(autosomal recessive polycystic kidney disease,ARPKD)的致病基因．在小鼠中,FPC于胚胎期9.5天可在发育中的神经管、气管、原肠管等含管道的器官中被探测到．在人类,FPC在胎肾即开始表达于输尿管芽,并持续表达于输尿管芽分支演变为集合管的整个过程．在成体肾,FPC也主要表达于肾内集合管上皮细胞,其亚细胞定位于上皮细胞的管腔面的顶端,主要分布在细胞纤毛和基体附近．FPC的主要生物功能目前仍未完全明了,新近的研究表明,FPC可能作为一个膜受体样蛋白,将细胞外的信号通过结合与调节TRPP2 (PKD2) 钙离子介导的通道传递到细胞内,调控体内各管道上皮细胞分化、增殖、极化和移行,从而促成各种生理管道的形成．     

三维回转骨细胞条件培养基对成骨细胞功能的调节作用

骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P0.05,**P0.01,***P0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.     

多囊蛋白2离子通道功能及在常染色体显性多囊肾发病中的作用机制

多囊蛋白2 (polycystin-2,PC2,或称TRPP2,PKD2)是一种瞬时受体电位通道(transient receptor potential channel,TRP),在维持细胞正常的Ca²⁺信号传导中起着关键作用,也是最常见的单基因常染色体显性遗传多囊肾病(transient receptor potential channel,ADPKD)的潜在病因之一。PC2可自身组装为同源四聚体离子通道或与其他蛋白质形成异源受体-离子通道复合物,参与调节机械感觉、细胞极性、细胞增殖和凋亡等多种生理功能,导致囊性细胞从正常的吸收、静止状态转变为病理性分泌、增殖状态。本文阐述了PC2蛋白相关结构域以及通道特性在维持细胞内Ca²⁺信号传导中的关键作用,并总结了PC2在细胞膜、纤毛、内质网以及线粒体等特定亚细胞定位形成多囊蛋白复合物,参与多种细胞分化、增殖、存活和凋亡相关信号通路,为确定特异性的有效的ADPKD干预治疗途径和靶点药物提供新的思考方向。     

多囊蛋白2对低频脉冲电磁场促进成骨细胞分化成熟的影响北大核心CSCD

已知低频脉冲电磁场(pulsed electromagnetic fields,PEMFs)可以促进体外培养大鼠颅骨成骨细胞(rat calvarial osteoblasts,ROBs)的分化成熟,但ROBs感知PEMFs物理信号并启动成骨性分化的机制至今不明。本研究探究了0.6 mT 50 Hz PEMFs促进ROBs成骨性分化与位于ROBs表面的初级纤毛上的多囊蛋白2(polycystin2,PC2)的关系。首先用免疫荧光染色法研究了PC2是否位于ROBs初级纤毛内,然后通过Western blotting检测了经PEMFs处理不同时间后,ROBs内PC2蛋白表达的变化情况。接着用PC2阻断剂盐酸阿米洛利(amiloride HCl,AMI)预处理ROBs,检测碱性磷酸酶(alkline phosphatase,ALP)活性和PC2蛋白表达受PEMFs处理的影响情况,以及与骨形成相关的Runx-2、Bmp-2、Col-1、Osx的蛋白及基因表达的变化情况。再用RNA干扰法抑制ROBs内PC2的表达后,检测与骨形成相关基因表达情况。结果发现,PC2被定位于ROBs初级纤毛上,PEMFs处理增加了PC2蛋白表达量。PC2被AMI阻断后,PEMFs不再能提高PC2蛋白表达水平及ALP活性,PEMFs对成骨相关蛋白和基因表达的促进作用也被抵消。使用RNA干扰法抑制PC2的表达后,PEMFs也不再能提高骨形成相关基因的表达。结果表明:存在于成骨细胞初级纤毛表面的PC2在感知并传递PEMFs发出的物理信号中扮演着不可或缺的角色,PEMFs促进ROBs成骨性分化依赖于PC2的存在。本研究为阐明低频脉冲电磁场促进骨形成及治疗骨质疏松的机制研究奠定了基础。     

骨质疏松中破骨细胞活性与其RyR表达的关系研究

目的:研究骨质疏松病理状态下破骨细胞活性的变化与其线粒体表面RyR表达之间的关系。方法:构建骨质疏松SD大鼠模型,2周后取长管骨骨髓血提取破骨细胞,筛选出发育成熟细胞核大于3的破骨细胞,高速离心法分离细胞胞质Cytosol和细胞内质网SR,Western-Blot分析骨质疏松状态下OC表达RyR通道蛋白的变化;进一步用FlaxStation 3分析OC细胞[Ca2+]i释放;最后评测骨质疏松环境下OC细胞活性和骨吸收能力变化。结果:与对照组相比,骨质疏松实验组大鼠OC细胞内质网SR上RyR蛋白表达显著减少;,同时破骨细胞[Ca2+]i释放减少导致OC细胞活性骨吸收能力显著增加。结论:破骨细胞内RyR通过调控其细胞内钙释放程度对OC活性产生影响。     

骨样细胞MLO-Y4与成骨样细胞MC3T3-E1生物学特性的比较

分别采用倒置显微镜观察法、细胞计数法、RT-PCR法、磷酸对硝基苯酚法(PNPP法)和ELISA法来比较小鼠骨样细胞MLO-Y4与小鼠成骨样细胞MC3T3-E1的细胞形态、增殖、相关基因的表达和分泌功能的差异。结果显示MC3T3-E1细胞呈长梭形,具有少量短的突触;而MLO-Y4细胞呈星状或树枝状且具有很多长的突触。MC3T3-E1细胞的增殖能力强于MLO-Y4细胞,两者的倍增时间分别是18 h和20 h。MC3T3-E1细胞中原癌基因c-fos和骨桥蛋白基因OPNmRNA的表达明显高于MLO-Y4细胞,而骨钙素基因OCmRNA的表达则是MC3T3-E1细胞远低于MLO-Y4细胞,白细胞分化抗原44基因CD44 mRNA在两种细胞中的表达差异不明显。ALP的分泌在MC3T3-E1细胞中高于MLO-Y4细胞,NO的分泌在两种细胞中没有显著性差异,M-CSF在MLO-Y4细胞中的分泌较高。由此可见骨样细胞MLO-Y4与成骨样细胞MC3T3-E1在形态、ALP和MCSF分泌及c-fos、OPN和OCmRNA表达方面差异明显。     

原子力显微镜技术检测几种骨组织细胞力学性能

为了探究几种骨组织细胞系的力学性能及其与细胞功能的关系,该文采用原子力显微镜压陷法分别检测了前成骨细胞系（2T3和MC3T3-E1）、前骨细胞系（MLO-A5）和骨样细胞系（MLO-Y4）的杨氏模量,利用激光共聚焦显微镜观察了这几种细胞微丝和微管的排布。结果显示,2T3、MC3T3-E1、MLO-A5和MLO-Y4细胞的杨氏模量分别为（7000±2015）Pa、（6600±2024）Pa、（4700±644）Pa和（4500±1622）Pa,与原代骨组织细胞的杨氏模量及变化趋势保持一致,但两种前成骨细胞的杨氏模量要显著高于前骨细胞和骨细胞。细胞荧光染色结果表日月＇前成骨细胞细胞核周围的微丝和微管分布密度要高于前骨细胞和骨细胞,而前骨细胞MLO-A5,尤其是骨细胞MLO-Y4的骨架主要集中于细胞突触和边缘,这可能是导致几种细胞力学性能差异的原因。该研究从生物力学的角度为进一步深入理解骨组织细胞结构与功能的关系提供了实验依据。     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

拟南芥血红蛋白1(AtGLB1)与过氧化氢的相互作用

拟南芥的血红蛋白1(AtGLB1)属于非共生的血红蛋白。在低氧胁迫中对植物细胞中过氧化氢(H2O2)内稳态的维持起了很重要的作用。为了检测AtGLB1与H2O2能否直接相互作用,我们扩增了拟南芥的AtGLB1基因,并将其克隆到原核表达质粒pET32a中,测序鉴定正确后转化大肠杆菌BL21。IPTG诱导目的蛋白表达后,镍离子亲和层析柱(Ni2+-NTA)纯化了靶蛋白。体外表达的氧合的AtGLB1能与H2O2直接相互作用。因此,与H2O2反应可能是AtGLB1清除低氧胁迫下产生的H2O2的一种方式。     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

拟南芥H_2O_2突变体的筛选及特性分析

通过化学诱变剂甲基磺酸乙酯(EMS)诱变模式植物拟南芥(Arabidopsis thaliana)获得突变体筛选群体.在5 mmol/L H2O2胁迫下,以叶片温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对H2O2不敏感突变体hpi1(hydrogen peroxide-insensitive1)和敏感突变体hps1(hydrogen peroxide-sensitive1).进一步研究发现,两种突变均为单基因隐性突变,气孔密度同野生型一样,而叶片温度、气孔开度和叶片失水率则有明显的差异.种子萌发实验表明,hpi1对甘露醇(Man)和NaCl不敏感而对ABA敏感,hps1则对3种胁迫都表现出敏感特性.     

过氧化氢和氯化钙对香蕉幼苗抗寒性的影响

用H₂O₂和CaCl₂单独或混合使用的方法喷洒香蕉幼苗,并置于低温培养箱中进行冷胁迫处理,发现它们可提高香蕉幼苗冷胁迫期间叶片POD活性,降低细胞质泄漏,增加可溶性糖含量及减缓叶绿素降解,从而减轻冷伤害程度。H₂O₂和CaCl₂混合处理的效果优于单独处理,二者有协同效应。     

流加H_2O_2对提高供氧及微生物代谢的影响

在大部分的需氧发酵中 ,供氧通常是通过向发酵液通气来实现的。在某一临界细胞浓度时 ,供氧不能满足细胞生长所需 ,成为细胞生长的限制基质 ,进而导致细胞密度和产品浓度较低[1] 。传统方法改善供氧主要是从反应器设计和工艺等方面考虑 ,如增大搅拌速率 ,提高通气速率 ,使用纯氧通气 ,提高罐压等。但由于氧气的溶解度低以及机械和操作上的原因 ,其操作范围有限。近年来出现了一些新的方法来改善发酵过程中的供氧问题 ,如加入氧载体[1～ 3 ] ,流加Ｈ2 Ｏ2[4～ 7] ,与藻类共培养[8～ 10 ] ,以及通过基因克隆转入携带氧的基因等方法。本文将着…     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Ca2+和钙调素对H2O2诱导的玉米幼苗耐热性的调控

外源H2O2预处理提高了玉米幼苗内源H2O2的含量和钙调素(CaM)活性,缓解了高温处理过程中CaM活性的下降,增加了玉米幼苗在高温胁迫下的存活率.H2O2诱导的玉米幼苗耐热性的形成可被外源Ca2 处理所加强,被Ca2 螯合剂EGTA、质膜Ca2 通道阻塞剂La3 、胞内Ca2 通道阻断剂RR(钌红),以及CaM抑制剂CPZ(氯丙嗪)和TFP(三氟拉嗪)所抑制,表明Ca2 和CaM参与了H2O2诱导的玉米幼苗耐热性形成的调控.     

Long noncoding RNA Airn protects podocytes from diabetic nephropathy lesions via binding to Igf2bp2 and facilitating translation of Igf2 and Lamb2

Diabetic nephropathy (DN) is a severe diabetic microvascular complication with high mortality. Long noncoding RNAs (lncRNAs) are characterized as important regulators of various biological processes by emerging researches, whereas the molecular mechanisms by which lncRNAs participate in DN progression need to be further clarified. Herein, we conducted a study on the regulatory role in DN of an lncRNA named antisense of Igf2r non‐protein‐coding RNA (Airn). Airn expression was downregulated in renal tissues of diabetic mice, and was negatively related with DN development. Besides, Airn downregulation was detected in high‐glucose‐stimulated podocytes, resulting in poorer cell viability, a higher tendency to cell apoptosis, and a deficiency of laminin level, while Airn overexpression could significantly alleviate these deleterious effects. Mechanistically, using RNA immunoprecipitation and RNA pull‐down assays, we found that Airn could bind to the RNA‐binding protein Igf2bp2, thus facilitating translation of Igf2 and Lamb2 to maintain normal podocyte viability and glomerular barrier function. Collectively, our results demonstrate the protective role of lncRNA Airn in podocytes against DN, providing a new insight into DN pathogenesis and molecular therapy.