低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

内皮细胞生长状态对血管平滑肌细胞增生迁移的影响

实验通过建立细胞共培养体系,探讨内皮细胞生长状态对血管平滑肌细胞增生迁移的影响及机制。检测指标包括~3H-TdR掺入、细胞周期、细胞迁移计数和α-SM-actin mRNA表达。结果显示,融合生长内皮使平滑肌细胞~3H-TdR掺入量明显降低,增加平滑肌细胞停留在G_0/G_1期的比例,上调平滑肌细胞α-SM-actin mRNA表达;而对数生长内皮细胞使平滑肌细胞~3H-TdR掺入量明显升高,促进平滑肌细胞由 G_0/G_1期进入G_2/M和S期,下调平滑肌细胞α-SM-actin mRNA表达。对照组平滑肌细胞在基础状态下存在少量迁移,对数增殖内皮细胞组平滑肌迁移数比对照组增高约4倍(P<0.01),而融合生长内皮细胞组平滑肌迁移数仅为对照组的0.5倍(P<0.05)。结果提示内皮细胞生长状态不同,对平滑肌细胞生物学特性的影响也不同,增殖期内皮明显促进平滑肌细胞增生迁移、下调平滑肌细胞α-SM-actin mRNA表达。     

DDPH对低氧内皮细胞条件培养液诱导肺动脉平滑肌细胞增殖及α-SM-actin表达的影响

目的探讨1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙氨基)丙烷盐酸盐(DDPH)抑制低氧内皮细胞条件培养液(HECCM)诱导肺动脉平滑肌细胞增殖及对α-SM-actin表达的影响.方法利用低氧内皮细胞条件培养液建立猪肺动脉平滑肌细胞(PASMC)的增殖模型;以四甲基偶氮唑盐(MTT)比色法、α平滑肌肌动蛋白(α-SM-actin)为指标,采用免疫细胞化学染色法观察低氧内皮细胞条件培养液对肺动脉平滑肌细胞增殖的影响以及DDPH对低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后的逆转效应.结果低氧内皮细胞条件培养液显著促进肺动脉平滑肌细胞增殖,低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后,肺动脉平滑肌细胞的表型发生转化,由收缩表型转化为合成表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量下降;DDPH能显著抑制低氧内皮细胞条件培养液对肺动脉平滑肌细胞的增殖作用,并使肺动脉平滑肌细胞的表型发生逆转,即由合成表型逆转为具有执行正常收缩功能的收缩表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量回升.结论提示DDPH能显著抑制低氧内皮细胞条件培养液促肺动脉平滑肌细胞的增殖作用,其作用机制可能是通过肺动脉平滑肌细胞的表型发生逆转来实现的.     

ERK1/2对低氧大鼠肺动脉平滑肌细胞Kv1.5通道表达的影响

目的:探讨ERK1/2对低氧大鼠肺动脉平滑肌细胞(PASMCs)电压门控性钾离子通道(Kv1.5)表达的影响及其机制。方法:原代培养SD大鼠PASMCs,选3~6代PASMCs随机分组:1正常组(N);2低氧组(H);3DMSO组(HD);4U0126组(HU):10μmol/L U0126;5茴香霉素组(HA):10μmol/L茴香霉素。每组3皿细胞,正常组于常氧培养箱(5%CO2,37℃),其余各组均加入0.05%二甲基亚砜(DMSO)于低氧培养箱(5%CO2,2%O2,37℃),均培养60 h。采用RT-PCR和Western blot法测定PASMCs Kv1.5 mRNA和蛋白表达。结果:与N组相比,H、HD、HA组Kv1.5 mRNA和蛋白表达均明显降低(P0.05);较之H和HD组,HU组Kv1.5 mRNA和蛋白表达明显上升((P0.05),与HU组比较,HA组Kv1.5 mRNA和蛋白表达均明显降低(P0.05)。结论:低氧降低Kv1.5 mRNA和蛋白表达,U0126能够对抗低氧引起的Kv1.5通道的低表达,茴香霉素对低氧条件下Kv1.5通道表达无明显影响,但其表达仍显著低于常氧组,提示低氧可能通过干预ERK1/2信号通路抑制Kv1.5通道表达引起低氧性肺动脉高压。     

吸烟和慢性阻塞性肺疾病患者肺血管重建及蛋白激酶C-α表达的研究

目的探讨吸烟和慢性阻塞性肺病(COPD)患者肺血管重建及蛋白激酶C-α(PKC-α)mRNA和蛋白质的表达情况.方法非吸烟非COPD(Controls组)、吸烟非COPD(Smokers组)和吸烟COPD(COPD组)男性肺癌患者各11人,年龄相匹配,取其行肺叶切除术后的外周肺组织.采用免疫组织化学方法检测肺动脉平滑肌细胞增殖细胞核抗原(PCNA)及肺小动脉α-SM-actin染色以显示肺血管重建,逆转录-聚合酶链反应(RT-PCR)对肺小动脉PKC-αmRNA表达进行半定量,免疫荧光及Western blot法检测肺小动脉PKC-α蛋白质表达.结果吸烟非COPD和吸烟COPD患者肺血管重构明显,两组患者肺动脉平滑肌细胞的增殖指数和α-SM-actin表达量均明显高于非吸烟非COPD(P<0.001).吸烟COPD患者肺血管重构更明显,肺动脉平滑肌细胞的增殖指数和α-SM-actin表达量均高于吸烟非COPD(P<0.001,P<0.05).吸烟非COPD患者肺小动脉PKC-α在转录和翻译两个水平的表达均明显高于非吸烟非COPD患者(P<0.001);吸烟COPD患者动脉血氧分压PaO2明显低于吸烟非COPD患者(P<0.05),肺小动脉PKC-αmRNA及蛋白质表达水平均明显高于吸烟非COPD患者(P<0.01).结论长期吸烟导致肺血管重建,既有香烟的直接作用,又有长期吸烟诱导COPD的低氧因素,其机制可能是通过细胞内PKC生物信号传导通道.     

急性低氧和间断低氧习服对HepG2细胞内血管内皮细胞生长因子基因表达的影响

目的:观察急性低氧和间断低氧习服对人HepG2细胞内血管内皮细胞生长因子(VEGF)及转录因子低氧诱导因子-1α(HIF-1α)的mRNA和蛋白含量的影响及其可能的生物学意义.方法:HepG2细胞随机分为常氧对照组,急性低氧组和间断低氧习服组.采用Northern blot和Western blot分别检测不同组别HepG2细胞内VEGF和HIF-1α mRNA表达和蛋白含量的变化.结果:急性低氧诱导HepG2细胞内VEGF和HIF-1α基因的转录,增加两种蛋白在细胞内的含量.间断低氧习服组的细胞内VEGF和HIF-1α的mRNA含量分别为常氧对照组细胞的(108.6±17.7)%和(116.74±19.8)%,与常氧对照组相比无显著差异(P＞0.05);而其蛋白表达的含量分别为对照组细胞的1.4和2.7倍,都明显低于急性低氧组细胞内两种蛋白的含量(P＜0.05).结论:HepG2细胞达到低氧习服状态后,抑制急性低氧对HepG2细胞内VEGF基因表达的促进作用,其中HIF-1α可能起着重要的调节作用.     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。     

间歇低氧对大鼠骨骼肌IGF-1和myostatin基因表达的影响

目的:旨在探讨低氧对骨骼肌胰岛素样生长因子-1(IGF-1)和肌肉生长抑制素(myostatin)表达的影响。方法:SD大鼠分为常氧对照组(C)、低氧暴露组(HO)、复氧1周组(H1)。于氧浓度13.6%的低氧舱内进行间歇性低氧暴露。采用RT-PCR方法测定腓肠肌myostatin mRNA和IGF-1 mRNA的表达。结果:与常氧对照组比,低氧暴露组骨骼肌IGF-1 mRNA表达显著下降,myostatin mRNA表达显著上升;与低氧暴露组比,复氧1周组骨骼肌IGF-1mRNA表达显著上升,myostatin mRNA表达显著下降。结论:低氧暴露后骨骼肌myostatin mRNA和IGF-1mRNA表达发生反向变化,提示二者可能以相反的作用共同参与低氧对肌肉生长的调控。     

胚胎干细胞来源的拟胚体分化早期血管平滑肌标志物的表达时相

目的:研究胚胎血管发育早期SMα-actin、SM22α、myocardin、平滑肌肌球蛋白重链(SMMHC)的表达规律,并初步探讨在此阶段血小板源性生长因子-BB(PDGF-BB)对血管平滑肌细胞(VSMCs)分化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体(EBs),用免疫荧光染色、RT-PCR、Western blot分析SMα-actin、SM22α、myocardin、SMMHC的表达时相;然后分别用0μmol/L(对照组)、10μmol/L、50μmol/L AG1296(血小板源性生长因子受体抑制剂)处理EBs,观察三组SMα-actin、SM22α、myocardin、SMMHC在基因及蛋白水平上的表达变化。结果:胚胎血管发育早期SMα-actin、myocardin、SM22α、SMMHC分别在EBs第0(胚胎干细胞)、8、11、13d开始有表达。AG1296三种浓度处理后SMα-actin、myocardin、SM22α、SMMHC蛋白表达及myocardin、SM22α和SMMHC mRNA表达均无明显差异。结论:EBs发育过程中存在着自发的VSMCs分化,SMα-actin表达最早,依次为myocardin、SM22α、SMMHC;PDGF-BB对EBs分化早期VSMCs标志物表达的调控可能不是必要的。     

线粒体ATP敏感钾通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α表达及细胞增殖的作用

本文旨在探讨线粒体ATP敏感钾（mitochondrial ATP-sensitive K＋,MitoKATP）通道对大鼠肺动脉平滑肌细胞低氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）表达和细胞增殖的影响。原代培养大鼠肺动脉平滑肌细胞,分为常氧对照组、常氧＋diazoxide（MitoKATP通道的选择性开放剂）组、常氧＋5-hydroxydecanoate（5-HD,MitoKATP通道的选择性阻断剂）组、低氧对照组、低氧＋diazoxide组、低氧＋5-HD组,共6组,分别应用罗丹明123荧光技术检测各组大鼠肺动脉平滑肌细胞的线粒体膜电位,免疫组化检测HIF-1α的表达及酶联免疫检测仪检测细胞增殖的变化。结果显示,常氧＋ diazoxide组与常氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0．05）;低氧＋diazoxide组与低氧对照组比较,罗丹明123荧光、HIF-1α表达及细胞增殖明显增强（P〈0.05）：常氧＋5-HD组与常氧对照组比较,罗丹明123荧光、HIF-1α表达、细胞增殖没有明显变化（P〉0．05）;但低氧＋5-HD组与低氧对照组比较,罗丹明123荧光明显减弱、HIF-1α表达及细胞增殖有所减弱（P〈0．05）。结果提示：MitoKATP通道的开放能引起大鼠肺动脉平滑肌细胞线粒体膜去极化,并可以促进HIF-1α的表达及细胞增殖。     

TRANSFORMATION OF ROD-SHAPED ALGAL CELLS INTO SPHERICAL CELLS

Transformation of the rod-shaped algal cells, which before the conversion could be considered as belonging to the genus Stichococcus, into spherical cells was achieved by the action of the tryptic soy broth and some other organic media. Transformation did not involve degradation of the cell wall, and the resulting spheres were not sensitive to osmotic shock. The transformed cells could be propagated as spheres for several months but eventually reverted to the original rod shape. The reversion could be hastened by transferring spherical cells back into inorganic medium and growing the cells autotrophically. For a long time after reversion, the cells maintained characteristics different from those of the original rod-shaped cells which did not go through the conversion and reversion processes. The reverted cells had a higher rate of heterotrophic growth, a lower rate of autotrophic growth, and a lower capacity for being again transformed into spheres under the influence of tryptic soy broth.     

CELL CONTACT REFORMATION OF DISSOCIATED CELLS FROM EMBRYONIC CELLS OF STRONGYLOCENTROTUS PURPURATUS

Disaggregated single cells from the gastrula of Strongylocentrotus purpuratus were studied as they reaggregated and reformed quasi-normal embryos. In this investigation emphasis was placed on the structural events involved during the reformation of cell contacts vis-a-vis cell migration. The early cell contacts are non-junctional cell appositions, which are characterized by non-parallel apposing membranes. Between post-migratory epithelial cells, there is a shift from non-parallel to parallel apposing membranes. These cell appositions are found between the overlapping lamellapodia along the apical margins of the epithelial cells during blastocoel enlargement. Incipient continuous junctions are formed by the deposition of an electron dense material in the intermembrane space. As the junction develops, electron dense plaques form in the cytoplasm immediately subjacent to the junction and septa form between the apposing membranes.     

RESTING CELLS OF PEDIASTRUM

Davis , Joseph S. (Southern Illinois U., Edwardsville.) Resting cells of Pediastrum. Amer. Jour. Bot. 49(5): 478–481. Illus. 1962.—After approximately 10 weeks in a partially defined mineral medium, cells of vegetative colonies of Pediastrum boryanum lost their chlorophyll and became orange resting cells. The resting cells were colored orange by carotenoid pigment dissolved in fat droplets within the cells. No cell wall thickening occurred during or after resting-cell formation. Starch was abundant in the resting cells. Dried resting cells remained viable for at least 4 years. When placed in the culture medium, 4-year-old dried resting cells became green and reproduced like vegetative cells.     

乳腺癌中CD55高表达细胞的生物学特性

目的探讨乳腺癌细胞株MCF7中CD55高表达细胞的生物学特性。方法应用抗CD55抗体及Hoechst33342染料对乳腺癌细胞株MCF7细胞进行荧光染色,应用FACS-Vantage和FACS Vantage SE分选CD55hi和CD55lo细胞、SP和MP细胞,对比两组细胞在两种染色中的分群所在、对C2-ceramide诱导凋亡的耐受能力、及应用RT-PCR和免疫组化来检测Bcl-2、TMEM23在两组细胞中的表达。结果分选的CD55hi和CD55lo细胞再用Hoechst 33342染色时,分别位于SP和MP区域中。在C2-ceramide存在的情况下,CD55hi细胞比CD55lo细胞对凋亡刺激具有更高的耐受性。在CD55hi和SP细胞中Bcl-2及TMEM23的表达水平分别比在CD55lo和MP细胞中高。结论 CD55高表达细胞具有与癌干细胞相似的生物学特性,CD55可以推测为乳腺癌干细胞的一个表面标记。     

Nogo-P4对神经干细胞分化成神经元样细胞的影响

目的观察Nogo-P4对神经干细胞分化成神经元样细胞是否存在抑制作用。方法体外培养大鼠脊髓来源神经干细胞,分为A、B、C和D组,在神经干细胞分化过程中分别加入0μmol/L、2μmol/L、4μmol/L和6μmol/L的Nogo-P4,免疫荧光标记神经元样细胞,计数神经元样细胞分化比率,使用Image-ProPlus5.0软件测量神经元样细胞神经突长度。结果神经干细胞分化第3d,A-D各组神经元样细胞平均神经突长度分别为97.80±6.97μm、88.25±5.83μm、80.54±6.75μm和79.31±6.57μm;神经元样细胞比率分别为(35.82±6.53)%、(35.31±6.11)%、(38.97±5.79)%和(34.75±5.61)%。A-C组神经突长度随着Nogo-P4浓度的升高而下降,P<0.05;C、D组神经元样细胞神经突长度较A组缩短约17%;各组神经元样细胞分化比率无显著差异。结论Nogo-P4对神经干细胞分化过程中形成的神经元样细胞具有神经突生长抑制作用,对神经元样细胞分化比率无影响。     

人外周血中LAK细胞的克隆化

本文报道首次采用半固体-液体两步法克隆正常人外周血单个核细胞(PBMNC)中的淋巴因子激活的杀伤细胞(LAK)获得成功。先用含重组人白细胞介素2(rhIL-2)的软琼脂半固体克隆外周血中的T细胞,再将T细胞克隆转移至96孔板中继续在含rhIL-2的液体中培养。~(51)Cr释放的结果表明,约10～30％的克隆对NK敏感的K562细胞和NK不敏感的H7402、Anip-1肿瘤细胞均有细胞毒性,即为LAK细胞克隆。LAK细胞克隆能在体外含rhIL-2培养液中增殖1.5～3.0个月,每个克隆可扩增至10~9～10~(10)个细胞,仍然维持LAK细胞活性。表型分析的结果表明,克隆的LAK细胞CD3( )、CD8( ),属T细胞系统。有增殖能力的LAK前体细胞在PBMNC中的频率约为1～3×10~(-4)。用有限稀释法将LAK细胞克隆进一步亚克隆,98％以上的亚克隆均有LAK活性,表型也和原克隆相同,证实原克隆具有克隆源性。本文的两步法克隆LAK细胞程序可在较短时间内获得大量均一的LAK细胞,极大地有助于LAK细胞的深入研究和广泛应用。     

施万细胞条件培养基对大鼠骨髓间质细胞的诱导分化作用

目的　探讨施万细胞条件培养基对大鼠骨髓间质细胞的诱导分化作用。方法　从大鼠骨髓中分离培养间质细胞并传至第 6代 ,诱导前 2 4h加 1μg·L-1碱性成纤维生长因子 (bFGF)入培养液中以促分裂 ,再以施万细胞条件培养基作诱导剂 ,观察细胞形态的变化 ,并采用免疫组织化学法对诱导后一周的细胞进行Map 2及NSE、GFAP表达的检测。结果　诱导后 48小时间质细胞在形态上表现为神经元样 ,神经元样细胞呈Map 2及NSE阳性 ,而GFAP显阴性。结论　施万细胞的上清液能诱导骨髓间质细胞分化为神经元样细胞。     

SECRETORY CELLS OF LILY PISTILS. II. ELECTRON MICROSCOPE CYTOCHEMISTRY OF CANAL CELLS

The secretory cells which line the canal of Lilium longiflorum pistils possess, on the side facing the canal, an elaborate wall which, with associated structures, Rosen and Thomas (1970) termed the “secretion zone.” We examined the secretion zone in the electron microscope following treatment of excised pistil slices with extraction procedures which remove pectin, hemicellulose, cellulose, lipid, or protein. The outer, fibrillar wall (layer 1) of the secretion zone contains protein, pectin, and cellulose. Internal to layer 1 is a granular-fibrillar wall (layer 2) several microns thick. It consists of outer and inner regions which can be distinguished from each other cytochemically. The granular component is composed of pectin which is not esterified with methyl groups and which may be complexed with protein. The short, randomly dispersed microfibrils of layer 2 were sensitive to procedures which dissolve cellulose. The extraction procedures did not reveal the chemical nature of the “osmiophilic islands” of layer 2. Paramural body membranes appear to be composed of glycoprotein and may function in secretion by serving as sites of pinocytic interchange at the plasmalemma. The origin of stigmatic exudate and the release of canal cell secretion product are discussed.     

FEULGEN HYDROLYSIS OF NORMAL CELLS AND MOUSE ASCITES TUMOR CELLS

The effect of HCl hydrolysis on the dye content (Feulgen reaction) of normal cells and mouse ascites tumor cells was examined by means of cytophotometric measurements. After 11 min of hydrolysis, 16-day-old tumor cells showed a hypotetraploid DNA line with doubling peaks. The DNA values were in the ratios of 1:2:4:8 during all the tested hydrolysis times (3 to 21 min). The size of the nucleus and the DNA concentration did not influence the hydrolysis and the dye content. However, the time of the hydrolysis considerably influenced the dye content of normal and tumor cells. The course of the curves obtained by plotting dye absorption against hydrolysis time showed an inflection of the curve at 9 min' hydrolysis time in tumor cells, whereas the inflection occurred at 8 min in mitotic cells. These inflections were statistically significant. The DNA stem-line¹ for tumor cells shifted during different hydrolysis times when compared to normal cells. The possibility is discussed of two types of DNA which differed in their acid sensitivity and which yielded atypical hydrolysis curves.