氯沙坦对机械通气大鼠肺组织TNF-α和MPO表达的影响

目的通过观察AngⅡ受体拮抗剂——氯沙坦对呼吸机所致肺组织TNF-α水平和髓过氧化物酶（MPO）活性的影响,探讨氯沙坦对呼吸机所致肺损伤的保护作用。方法 24只健康雄性Wister大鼠随机分为对照组、呼吸机所致肺损伤组（VILI）和氯沙坦干预组（LAP）,分别采用ELISA法测定大鼠肺组织中血管紧张素II（AngⅡ）含量,采用免疫组织化学染色法测定大鼠肺组织TNF-α蛋白表达水平,采用化学比色法测定肺组织匀浆中MPO活性。结果 VILI组大鼠肺组织AngⅡ含量、TNF-α蛋白表达水平以及肺组织匀浆中MPO活性均明显高于对照组（均P〈0.01）。氯沙坦干预组大鼠肺组织AngⅡ含量与VILI组比较无明显差异（P〉0.05）,但肺组织TNF-α蛋白表达水平、肺组织匀浆中MPO活性和肺W/D比值均较VILI组明显降低（均P〈0.01）,同时肺组织病理损伤程度也较VILI组明显减轻。结论 AngⅡ通过调控肺组织中TNF-α的表达在VILI发病中起重要作用,氯沙坦可阻断AngⅡ与AT1受体的结合,下调肺组织TNF-α表达,降低MPO的活性,对VILI具有一定防治作用。     

小地老虎谷胱甘肽-S-转移酶AiGSTs1的分子特性及其对杀虫剂胁迫的响应

[目的]鉴定小地老虎Agrotis ipsilon sigma家族谷胱甘肽-S-转移酶(GST)基因,明确其编码蛋白质的催化活性,阐明该基因在小地老虎不同发育期、不同组织及在毒死蜱和高效氯氟氰菊酯胁迫下的表达模式,为深入探究小地老虎GST在杀虫剂解毒代谢中的功能提供理论基础.[方法]利用同源检索方法从小地老虎转录组中鉴定GST基因,利用原核表达系统表达重组蛋白并使用试剂盒检测其活性,使用实时荧光定量PCR技术分析基因的表达模式.[结果]从小地老虎转录组中鉴定了一个编码sigma家族GST的cDNA序列,命名为AiGSTs1.其编码的AiGSTs1蛋白含有谷胱甘肽结合位点和底物结合位点,具有GSTs的典型特征.在大肠杆菌中成功表达了重组AiGSTs1蛋白,测得此蛋白不仅具有谷胱甘肽转移酶活性,还具有过氧化物酶活性.毒死蜱和高效氯氟氰菊酯均可抑制重组AiGSTs1蛋白的活性.AiGSTs1在供试的小地老虎不同发育期和组织中均有表达,但在蛹期和幼虫脂肪体中表达量最高.使用LD5o剂量毒死蜱和高效氯氟氰菊酯处理小地老虎幼虫后,AiGSTs1的表达水平显著上升.[结论]本研究明确了小地老虎AiGSTs1的序列、所编码蛋白的活性和表达模式.毒死蜱和高效氯氟氰菊酯能够诱导AiGSTs1表达水平上调,表明该基因可能在这2种杀虫剂的解毒代谢中起重要作用.     

萝卜硫素诱导RASD1的表达促进宫颈癌HELA细胞凋亡及周期阻滞的机制研究

目的:探讨萝卜硫素靶向RASD1,对宫颈癌细胞(HELA)凋亡及周期的影响机制。方法:不同浓度的萝卜硫素(SFN)作用于HELA细胞48h,采用CCK-8法检测SFN对HELA细胞增殖的影响;采用RT-PCR法检测SFN对RASD1基因mRNA表达水平的影响;采用Western印迹法检测SFN对RASD1蛋白表达水平的影响;采用流式细胞技术检测加药以及过表达RASD1对HELA细胞凋亡及周期的影响,采用Western blot检测HELA中cleaved-caspase3和cleaved-parp相关凋亡蛋白以及p21和cdc2周期相关蛋白的影响。结果:SFN抑制宫颈癌细胞增殖,且加药组中RASD1的mRNA和蛋白表达水平明显高于对照组,差异有统计学意义(P0.05)。流式细胞凋亡分析结果提示:加SFN或是过表达RASD1细胞组凋亡率明显高于对照组,Western blot结果表明加SFN或过表达RASD1后细胞cleaved-caspase3以及cleaved-parp蛋白水平升高,差异具有统计学意义(P0.05);ModFit LT软件分析结果表明加SFN或过表达RASD1细胞S期显著低于对照组,G2/M期细胞增多,同时Western blot结果示p21蛋白的表达水平增高,而cdc2蛋白表达降低,其差异有统计学意义(P0.05)。结论:在宫颈癌HELA细胞中RASD1作为SFN的作用靶点,诱导细胞凋亡和细胞周期阻滞。     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

结肠癌组织RASSF1A基因转录表达和启动子区甲基化研究

目的:研究Ras相关区域家族1A基因(ras association domain family 1A,RASSF1A)启动子区甲基化对结肠癌组织中该基因转录和表达的影响.方法:应用甲基化特异性PCR(Methylation-special PCR,MSP)、RT-PCR和Western blot方法检测30例结肠癌组织和癌旁组织中的RASSF1A基因启动子区甲基化状态、mRNA和蛋白表达水平.结果:①RASSF1A基因启动子区在结肠癌纽织和正常组织中的甲基化频率分别为57%(17/30)和20%(6/30),甲基化频率在两组具有统计学差异(p＜0.01),,结肠癌组织中RASSF1A基因启动子区甲基化频率显著高于癌旁正常组织(x2=8.531,p＜0.01);②结肠癌组织中RASSF1A基因mRNA和蛋白袁达均显著低于癌旁组织(癌组织和癌旁正常组织中mRNA相对表达量分别为0.2836±0.0493和0.5092±0.0433,P＜0.001;以上组织中蛋白相对表达量分别为0.3124±0.0472和0.5320±0.0440,P＜0.01);③在结肠癌组织中,甲基化组RASSF1A基因mRNA和蛋白表达明显低于非甲基化组(甲基化组和非甲基化组mRNA相对表达量分别为0.0686±0.0174和0.5511±0.0486,P＜0.0001;以上组中蛋白相对表达量分别为0.1219±0.0326和0.5614±0.0380,P＜0.0001).结论:结肠癌组织中RASSF1A基因启动子区甲基化明显增高,与该基因蛋白表达减少显著相关,这可能是导致结肠癌中RASSF1A抑癌基因失活的主要因为.     

FBXO31基因对宫颈癌细胞增殖的影响

为了探讨FBXO31在宫颈癌中的表达情况及其对宫颈癌细胞增殖的影响及其可能机制,本研究采用实时定量PCR法检测FBXO31在宫颈癌组织中的表达水平;MTT法检测Hela细胞增殖能力;流式细胞术检测Hela细胞周期分布;Western blotting检测Hela细胞FBXO31、β-catenin、CyclinD1和c-Myc蛋白的表达水平。研究结果表明,FBXO31在宫颈癌组织中表达明显下调(p0.05)。FBXO31过表达能够明显抑制宫颈癌Hela细胞增殖能力。与空载质粒组比较,过表达FBXO31组的G1期细胞数显著增加,S期细胞数明显降低(p0.05)。本研究还发现FBXO31过表达能明显下调β-catenin蛋白、cyclin D1和c-Myc蛋白水平(p0.05)。本研究结论表明,FBXO31基因在宫颈癌中低表达;过表达FBXO31基因可通过抑制Wnt/β-catenin通路从而抑制宫颈癌细胞增殖。     

MAPK信号途径在一氧化氮抑制大鼠心肌肥大中的作用

实验观察了一氧化氮（NO）前体L－精氨酸对肾性高血压大鼠心肌组织eNOS蛋白表达及亚硝酸盐／硝酸盐含量、MKP－1蛋白表达及MAPK活性的影响,以及与心肌肥厚的关系,采用两肾一夹Goldblatt肾性高血压模型,随机分为5组：L－精氨酸高、中、低剂量组,分别于术后第5周给予L－精氨酸50、150及450mg/kg;L－NAME组,腹腔注射L－NAME 10mg/kg,同时给予L－精氨酸150mg/kg;高血压对照组,正常饮水,以及另设的一假手术对照组。用药8周后,用插管法测量大鼠动脉血压、左心室重与体重比值,用胶内原位磷酸化法测MFAPK活性、免疫印迹法检测心肌组织eNOS及MKP－1蛋白表达、酶还原法测定心肌组织亚硝酸盐／硝酸盐－硝酸盐含量。结果表明：（1）L－精氨酸可明显抑制肾动脉狭窄术后的血压升高、左心室重与体重比增加,增加心肌组织eNOS、MKP-1蛋白表达及亚硝酸盐－硝酸盐含量,降低心肌组织MAPK活性,其中以150mg/kg组作用最为明显;（2）NOS抑制剂L－NAME可明显抑制－精氨酸的以上作用,肾性高血压大鼠心肌组织eNOS蛋白表达下降。NO生成减少及MKP－1蛋白表达下降以及MAPK活性增强可能与高血压及心肌厚形成有关,L－精氨酸通过促进心肌组织eNOS蛋白表达、增加NO产生和MKP－1表达、减弱MAPK活性而发挥抗高血压及心肌肥厚的作用。     

针对Mcl-1的shRNA重组质粒体外增强肝癌细胞对化疗敏感性的实验研究

目的构建针对髓细胞白血病基因-1(Mcl-1)的shRNA的重组质粒,探讨该重组质粒对肝癌细胞化疗敏感性的影响。方法设计3对针对Mcl-1基因不同位点的shRNA片段的真核表达载体psiRNA-hH1neo-Mcl,用阳离子脂质体法将重组质粒转染至人肝癌细胞株HepG2中,采用RT-PCR和Western blot方法检测转染后Mcl-1mRNA及蛋白表达情况,筛选Mcl-1基因表达沉默效应最好的重组质粒。用MTT和流式细胞仪分别检测单独使用丝裂霉素(Mitomycin,MMC)、单独使用重组质粒以及联合使用重组质粒与MMC对HepG2细胞的增殖和凋亡的影响。结果成功构建含不同shRNA片段的重组质粒,依次命名为pMclsi-1、pMclsi-2、pMclsi-3。经测序证实,插入的DNA片段的序列与设计序列完全一致。重组质粒转染HepG2细胞后,Mcl-1基因的mRNA水平及蛋白水平明显下调,其中以pMclsi-1下调效应最强。MTT检测显示,pMclsi-1联合化疗药物MMC对HepG2细胞增殖的抑制活性显著高于单用MMC组或单用pMclsi-1组,流式细胞仪检测显示,pMclsi-1联合化疗药物MMC促进HepG2细胞凋亡的活性显著高于单用MMC组或pMclsi-1组。结论针对Mcl-1基因的siRNA可特异阻断Mcl-1基因的表达,从而明显增强肝癌细胞对化疗药物MMC的敏感性。     

人丙氨酸氨基转移酶同工酶在人体组织中的分布

文中拟通过采用基因重组技术获得ALT1和ALT2同工酶重组蛋白,分别制备筛选出高特异性、高活性的ALT1和ALT2单克隆抗体 (ALT1单克隆抗体已成功制备并发表),初步探讨ALT1和ALT2同工酶在人体组织中的定位、分布及表达情况。采用RT-PCR方法从人肝癌细胞 (HepG2) 中扩增ALT2基因,将成熟的ALT2基因亚克隆至pET32a-ALT2原核表达载体中,并将其连接产物转化至BL21(DE3) 感受态细胞中经IPTG诱导表达ALT2蛋白,镍柱 (Ni+) 亲和层析法纯化ALT2重组蛋白。ALT2重组蛋白免疫Balb/c小鼠,选取阳性血清小鼠脾细胞和骨髓瘤细胞SP2/0进行细胞融合,间接ELISA法挑选阳性细胞株,有限稀释法进行亚克隆,采用亲和层析柱法纯化ALT2抗体。通过RT-PCR和Western blotting方法检测ALT1和ALT2在人体正常组织中的表达和分布,研究结果显示组织中的ALT同工酶基因在mRNA水平和蛋白水平上的表达几乎一致。ALT1在肝脏、肾脏、骨骼肌高表达,胃肠道平滑肌中等表达;ALT2在脂肪、骨骼肌、心肌中高表达,胃肠道平滑肌低表达。免疫组化研究表明,ALT1在肝细胞、肾髓质小管和肌纤维中高表达,ALT2在脂肪细胞、心肌细胞中高表达,胃肠道组织ALT1和ALT2主要表达于肠壁上部区域黏膜,上述结果显示同工酶ALT1和ALT2在组织中分布广泛,为理解不同病理条件下ALT活性升高的变化机制提供理论依据。     

人源NOVA1蛋白的真核表达及其抗低氧活性鉴定

构建人NOVA1基因的真核表达载体pCMV-Myc-NOVA1,转染PC12细胞后筛选最佳转染条件,进而结合细胞免疫组织化学研究NOVA1蛋白在PC12细胞中的表达分布,并探究其抗低氧活性。根据NCBI数据库NOVA1基因序列设计上下游引物,以pCR4-TOPO-NOVA1载体为模板采用聚合酶链式反应扩增获得NOVA1基因的全长c DNA编码序列,限制性内切酶SalⅠ和XhoⅠ双酶切后插入pCMV-Myc真核表达载体,酶切及直接测序验证后采用脂质体转染法转染入PC12细胞,针对转染比例和转染时间进行优化,进而采用实时定量PCR和Western blotting检测NOVA1蛋白的表达,最后采用细胞免疫组织化学检测NOVA1蛋白在PC12细胞中的表达定位及其抗低氧活性。通过酶切和直接测序验证,成功构建了真核表达载体pCMV-Myc-NOVA1;质粒和Lipo2000最佳转染比例为1:2.5,最佳转染时间为72 h;最佳转染条件下NOVA1基因和蛋白的表达水平显著增加,转染pCMV-Myc-NOVA1质粒后,NOVA1蛋白主要分布于细胞核和细胞质;过表达NOVA1的PC12细胞增殖活性明显增加。本文采用分子克隆的方法成功构建了NOVA1基因的真核表达载体,通过条件优化实现了高效表达并测定过表达NOVA1蛋白具有明显的抗低氧活性,不仅为深入揭示NOVA1蛋白的作用机理提供了重要参考,而且为NOVA1蛋白潜在的药物开发提供了重要技术支撑。     

番木瓜果实扩展蛋白Cp-EXP1基因表达的荧光定量PCR分析

目的：对番木瓜果实扩展蛋白Cp-EXP1的基因进行荧光定量PCR分析，以便对其功能做进一步研究。方法：以不同成熟阶段的番木瓜果实总RNA为模板，设计引物进行荧光定量PCR。结果：Cp-EXP1基因在不同成熟阶段的番木瓜果实中的表达是有差异的，其表达水平受乙烯调控。结论：Cp-EXP1基因的表达与番木瓜的成熟软化密切相关，其表达分析为研究番木瓜的生长发育及其品种改良打下了良好的基础。     

黄瓜膨胀素的重组表达及活性分析

目的:提高纤维素的酶水解效率和开发高效的纤维素酶水解过程。方法:采用RT-PCR方法从黄瓜胚轴细胞中分离了膨胀素S1的cDNA,并使之与毕赤酵母表达质粒pPICZ(A连接,形成重组质粒pPICZ(A-exs1。通过电转化方法,用质粒pPICZ(A-exs1转化巴氏毕赤酵母GS115,得到重组菌株P.pastoris-exs1。在该重组菌株中,膨胀素的基因通过同源重组整合在毕赤酵母的染色体上,并处于毕赤酵母甲醇氧化酶启动子的下游。重组菌株P.pastoris-exs1在甲醇诱导下可合成并分泌膨胀素。结果:培养上清液没有纤维素酶活性,但具有破坏滤纸纤维素结晶结构的能力。培养上清液与里氏木霉纤维素酶等量混合后,可使纤维素酶的滤纸酶活力提高50%。结论:采用巴氏毕赤酵母GS115重组成功表达了黄瓜膨胀素,其表达产物可以促进纤维素酶对滤纸的水解。     

Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat

Expansin protein is a component of the cell wall generally accepted to be the key regulator of cell wall extension during plant growth. Plant hormones regulate expansin gene expression as well as plant growth during drought stress. However, the relationship between expansin and plant hormone is far from clear. Here, we studied the involvement of expansin in plant cell growth mediated by the hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) under osmotic stress which was induced by polyethylene glycol (PEG)-6000. Wheat coleoptiles from a drought-resistant cultivar HF9703 and a drought-sensitive cultivar 921842 were used to evaluate cell growth and expansin activity. Osmotic stress induced the accumulation of ABA. ABA induced expansin activity mainly by enhancing expansin expression, since ABA induced cell wall basification via decreasing plasma membrane H⁺-ATPase activity, which was unfavorable for expansin activity. Although ABA induced expansin activity and cell wall extension, treatment with exogenous ABA and/or fluridone (FLU, an ABA inhibitor) suggested that ABA was involved in the coleoptile growth inhibition during osmotic stress. IAA application to detached coleoptiles also enhanced coleoptile growth and increased expansin activity, but unlike ABA, IAA-induced expansin activity was mainly due to the decrease of cell wall pH by increasing plasma membrane H⁺-ATPase activity. Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

扩展蛋白与植物抗逆性关系研究进展

扩展蛋白是一种细胞壁蛋白,可调节细胞壁的松弛和伸展。目前研究表明,扩展蛋白几乎参与调节植物生长发育的整个进程。扩展蛋白还与植物的多种抗性反应有关,在植物对干旱、高盐以及病虫害等生物胁迫和非生物胁迫响应方面起着重要的调节作用。干旱胁迫下扩展蛋白基因的表达与植物的抗旱性有一定的关系;植物的耐盐性受到扩展蛋白基因表达的影响;淹水促进植物的伸长生长与扩展蛋白的表达密切相关;扩展蛋白调节细胞壁松弛为植物抗病性研究提供了新的思路。     

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.     

蜡梅Chimonanthus praecox (L.) Link COR413蛋白基因(Cpcor413pm1)的分子特性与表达分析

为研究蜡梅冬季开花过程的抗寒分子机理,在构建蜡梅花cDNA文库及EST分析的基础上,通过随机克隆测序,克隆了1个蜡梅COR413蛋白的cDNA基因,命名为Cpcor413pm1.Cpcor413pm1 cDNA长946　bp,推测的编码蛋白CpCOR413PM1包含201个氨基酸.CpCOR413PM1蛋白N端保守性差,没有信号肽,具有5个保守的跨膜区,1个潜在的GPI锚点,具有可能对蛋白结构或活性十分重要的位置保守的7个Pro、1个Cys, 1个被富Gly区一分为二的α螺旋区域,以及Tyr、Thr、Ser磷酸化位点各1个.序列比较分析表明,Cpcor413pm1是首次从蜡梅中克隆到的COR413蛋白基因.RT-PCR分析表明, 该基因在蜡梅的萌动期、蕾期、露瓣期、初开期、盛开期、衰老期均有表达,但在初开期和盛开期表达丰度更高,推测Cpcor413pm1与蜡梅花的抗冻性有密切关系.