17β—雌二醇抑制内皮素诱导的血管平滑肌细胞增殖作用

目的和方法：利用组织块贴壁法进行大鼠VSMC培养,胰蛋白酶分散细胞法传代。实验采用第4-6代细胞。采用氚-胸腺嘧啶核苷（[^3H]-TdR)掺入和细胞计数来作为VSMC增殖的指标,以RT-PCR的方法检测ETAR的表达,观察17β-雌二醇（E2）对内皮素-I（endothelin-l,ET-1)介导的血管平滑肌细胞（VSMC）增殖反应以及对内皮素A型受体（ETAR）表达的影响。结果：ETAR特异性拮抗剂BQ123能完全阻断ET-1介导的VSMC增殖反应;E2可明显抑制ET-1促进VSMC增殖的作用,RT-PCR结果显示E2能抑制ETAR的表达,12h时抑制作用最为明显;E2受体阻断剂Tamoxifen亦能部分抑制ET-1对VSMC的增殖及ETAR的mRNA的表达。结论：ET-1促进VSMC增殖作用主要通过ETAR介导的,雌激素可通过抑制ETARmRNA表达来发挥对ET-1促进VSMC增殖的抑制作用。     

诱导型一氧化氮合酶在17β-雌二醇诱导的血管平滑肌细胞周期阻滞中的作用

利用大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC)作为模型,观察17β-雌二醇(17β-estradiol,E2)对VSMC诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性和蛋白表达的影响,并探讨其在内皮素-1(endothlin 1,ET-1)刺激的VSMC周期循环中的作用。检测指标包括同位素法测定iNOS的活性,免疫印迹法(western blot)检测iNOS蛋白表达,流式细胞仪检测细胞周期,观察一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N^G-硝基左旋精氨酸甲酯(N^G-nitro—L—arginine methylester,L—NAME)对E2抑制VSMC细胞周期的影响。结果显示,E2明显增加iNOS的活性和蛋白表达,在30min和12h时能诱导VSMC的iNOS活性明显增加,而60min和24h时VSMC的iNOS活性与对照组无显著差异,不呈明显浓度依赖性,雌激素受体(estrogen receptor,ER)拮抗剂Tamoxifen和L—NAME能明显抑制E2诱导的VSMC iNOS活性增加;E2增加VSMC的iNOS蛋白表达的作用在3h时起效,12h达高峰,以后逐渐下降,呈浓度依赖性,Tamoxifen能明显抑制马诱导的VSMC iNOS蛋白表达;E2明显抑制ET-1诱导的S期细胞百分比和G2 S／G1增加,使VSMC在G1期发生细胞周期阻滞,这些作用可被预先给予L—NAME所明显减轻。上述结果提示,E2使ET—l刺激的VSMC细胞周期循环在G1期发生阻滞与增加VSMC iNOS活性有关,该作用至少部分通过ER介导。     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

Caveolin-1蛋白在171β-雌二醇抑制内皮素-1诱导血管平滑肌细胞增殖中的作用

本工作旨在研究Caveolin-1在17β-雌二醇（17β-estradiol,E2)抑制内皮素-1（endothelin-1,ET-1)诱导血管平滑肌细胞（vascular smooth muscle cells,VSMCs前后ET-1对DNA合成和caveolin-1蛋白表达的影响。结果显示,ET-1可以刺激VSMCs增殖。E2作用24h后,可明显抑制ET-1的上述作用。免疫荧光证实,在VSMCs上有cavellin-1分布,ET-1刺激VSMCs增殖的过程中,VSMCs上caveolin-1蛋白荧光强度下,而事称给予E2可逆转这种下降。Western bolt证实,ET-1可抑制caveolin-1蛋白的表达而E2则可增加caveolin-1蛋白的表达。以上结果表明E2抑制ET-1诱导的VSMCs增殖可能与其增加caveolin-1蛋白表达有关。     

AKT 途径在雌激素调控子宫内膜癌KLE 细胞中乙二醛酶Ⅰ 表达的作用

目的:探讨雌激素是否通过AKT途径调控子宫内膜癌KLE细胞中乙二醛酶Ⅰ(GlyoxalaseⅠ,GloⅠ)的表达。方法:采用RT-PCR和Western blotting检测雌激素(17-β雌二醇)处理或AKT途径抑制剂(LY294002)处理子宫内膜癌KLE细胞后GloⅠ的mRNA或蛋白的表达情况。实验分4组:Con组(对照组);LY294002组(LY294002处理组);E2组(17-β雌二醇处理组);E2+LY294002组(LY294002预处理1小时后再17-β雌二醇处理组)。结果:1、经不同浓度的17-β雌二醇(Con、10-11、10-10、10-9M)作用于KLE细胞24小时后,GloⅠmRNA的相对表达量分别为1,1.58±0.04,1.82±0.03,1.81±0.04,以10-10 M的浓度时最为显著(P<0.05)。以10-10 M的17-β雌二醇作用于KLE细胞48小时后,GloⅠ蛋白的相对表达量为1.79±0.02,高于Con组。2、以LY294002抑制AKT途径后,KLE细胞中GloⅠmRNA的相对表达量为0.69±0.03,蛋白的相对表达量为0.16±0.02,均低于Con组。3、E2+LY294002组GloⅠmRNA和蛋白的相对表达量分别为1.02±0.04、1.01±0.03,均低于E2组中GloⅠmRNA和蛋白的相对表达量,E2组分别为1.34±0.03、1.79±0.02。LY294002组GloⅠmRNA和蛋白的相对表达量分别为0.69±0.03,0.16±0.02,均低于Con组GloⅠmRNA和蛋白的相对表达量。结论:雌激素可上调子宫内膜癌KLE细胞中GloⅠmRNA和蛋白的表达。抑制AKT途径可下调子宫内膜癌KLE细胞中GloⅠmRNA和蛋白的表达。AKT途径在雌激素调控子宫内膜癌KLE细胞中GloⅠ的表达无明显作用。     

17β-雌二醇对谷氨酸诱导的SH-SY5Y细胞增殖和凋亡的影响

目的:探讨17β-雌二醇对谷氨酸诱导的SH-SY5Y细胞损伤的保护作用及可能机制。方法:选取SH-SY5Y细胞传代培养,分为四组:(1)阴性对照组;(2)氧化损伤组:0.1 mmol·L~(-1)谷氨酸作用24 h;(3)17β-雌二醇低、高浓度组:加入(1.0×10~(-4) mmol·L~(-1)和1.0×10~(-3)mmol·L~(-1))17β-雌二醇作用24 h之后,加入谷氨酸作用24 h。采用MTT比色法检测细胞存活率,流式细胞仪检测细胞活性氧(ROS)水平,Hoechst-PI染色观查细胞凋亡,分光光度计检测上清液中Caspase-3及Caspase-9含量。结果:7β-雌二醇能明显抑制谷氨酸诱导的细胞活性的下降,减少谷氨酸所致SH-SY5Y细胞内ROS的生成,降低细胞凋亡率,减少凋亡因子的活性。结论:17β-雌二醇对神经细胞损伤具有保护作用,这可能与其抗氧化作用有关。     

ERK在17β-雌二醇抑制大鼠血管损伤后平滑肌细胞增殖中的作用

本实验旨在研究细胞外信号调节激酶(extmcellular signal-regulated kinase,ERK)在17β-雌二醇(17β-estra-diol,E2)介导的一氧化氮(nitric oxide,NO)抑制血管损伤后平滑肌细胞(vascular smooth musclecell,VSMC)增殖中的作用。在去势雌性大鼠中建立颈总动脉球囊损伤模型,实验分单纯去势组(OVX)、去势给予E2治疗组(E2 OVX)、去势后球囊损伤组(OVA Inj)和去势后球囊损伤给予E2治疗组(E2 OVA Inj)。分别检测各组血管壁的厚度、血浆中NO的浓度、ERK蛋白表达和活性的变化以及eNOS蛋白表达情况。结果显示,与OVX组相比,OVA Inj组血浆NO含量明显下降和血管壁厚度明显增厚,E2可增加血浆中NO含量和抑制球囊损伤后血管壁的增厚;E2可以抑制ERK蛋白表达和活化,诱导eNOS蛋白的表达。血浆中：NO含量与eNOS蛋白的表达呈正相关,与血管壁厚度和ERK蛋白表达呈负相关。以上结果提示,E2可通过增加血管组织eNOS蛋白表达,促进NO生成,抑制ERK蛋白的表达和活性,从而抑制血管损伤后VSMC的增殖。     

Caveolin-1蛋白在17β-雌二醇抑制内皮素-1诱导血管平滑肌细胞增殖中的作用

本工作旨在研究Caveolin-1在17β-雌二醇(17β-estradiol,E2)抑制内皮素-1(endothelin-1,ET-1)诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用.在培养的VSMCs上,使用[3H]Thymidine([3H]TdR)掺入法、荧光免疫组化方法和Western blot观察E2处理VSMCs前后ET-1对DNA合成和caveolin-1蛋白表达的影响.结果显示,ET-1可以刺激VSMCs增殖.E2作用24 h后,可明显抑制ET-1的上述作用.免疫荧光证实,在VSMCs上有caveolin-1分布,ET-1刺激VSMCs增殖的过程中,VSMCs上caveolin-1蛋白荧光强度下降,而事先给予E2可逆转这种下降.Western blot证实,ET-1可抑制caveolin-1蛋白的表达而E2则可增加caveolin-1蛋白的表达.以上结果表明E2抑制ET-l诱导的VSMCs增殖可能与其增加caveolin-1蛋白表达有关.     

雌激素对成年和老年雌性大鼠血管平滑肌细胞雌激素受体α表达的影响及其机制

目的探讨雌激素对成年和老年雌性大鼠血管平滑肌细胞(VSMC)雌激素受体α(ERα)表达的影响及其机制。方法采用RT-qPCR、Western blot及重硫酸盐修饰后测序(BSP)的方法,检测体外培养的3-4代成年(2-3月龄)和老年(≥20月龄)雌性SD大鼠VSMC在无或有生理剂量(10-10 mol/L、10-8 mol/L)17β-雌二醇(E2)存在下,ERα的表达及其启动子区CpG岛甲基化水平的变化。结果成年雌性大鼠VSMC无论在有或无雌激素存在时均表达ERα,且表达水平随雌激素浓度增加而上升。而老年雌性大鼠VSMC无论是在mRNA水平还是蛋白质水平几乎检测不到ERα表达,即使补充雌激素达最高生理剂量也无法诱导ERα的重新表达,其ERα基因启动区CpG岛呈现高水平甲基化。结论成年大鼠VSMC表达ERα,且生理剂量雌激素对其表达具有诱导作用。而老年大鼠VSMC ERα基因由于CpG岛已发生高度甲基化而抑制,生理剂量雌激素对ERα表达的诱导作用丧失。     

17β雌二醇对大鼠血管平滑肌细胞凋亡及Bax、Bcl-2蛋白表达的影响

目的研究17β雌二醇(E2)对大鼠血管平滑肌细胞(VSMC)凋亡的影响及其可能机制.方法应用流式细胞术检测不同浓度E2 (0-100nmol/L)在有或无血清状态下对传代VSMC凋亡及其相关蛋白Bcl-2和Bax表达的影响;并采用免疫荧光染色和共聚焦激光扫描荧光显微镜术对Bax的分布进行定位.结果在有或无血清条件下,当浓度达到10 nmol/L以上时,E2 引起VSMC Bax蛋白表达上升及Bax/Bcl-2比值的改变,并促进VSMC凋亡.这一效应有随E2剂量和作用时间增高的趋势.结论 E2可通过上调Bax蛋白表达而诱导VSMC凋亡.     

Antibiotics in the treatment of wounds in the victims of the earthquake in the Armenian SSR

Purulent wounds in 88 victims of the Armenian earthquake with the syndrome of long-term squeezing were examined microbiologically and the experience with using antimicrobial drugs for their treatment was analyzed. In all the cases microbial associations were detected, coliform and nonfermenting gram-negative bacilli, enterococci, anaerobes and yeast-like fungi being isolated from 80, 20, 15 and 25 per cent of the patients respectively. 130 bacterial strains were studied in detail. 80 to 100 per cent of the bacterial isolates were resistant to the antibiotics routinely used in surgical practice, i.e. benzylpenicillin, tetracycline, levomycetin, kanamycin, cephalothin and cefazolin and highly sensitive to new antimicrobial drugs belonging to ureidopenicillins (mezlocillin), quinolones (ciprofloxacin) and cephalosporins of the 3rd generation (cefoperazone and ceftazidime). Retrospective estimation of the antibiotic therapy revealed its inadequacy in 55.7 per cent of the patients for the most part because of the isolates resistance. The 19 variants of the antibiotic use prescribed by the physicians mainly included penicillins, aminoglycosides and their combinations (in 67.2 per cent of the cases). The use of the highly efficient drugs of the groups of ureidopenicillins and quinolones was extremely rare which was likely due to lacking of information on the drugs.     

Histogenesis of the taste buds of the vallate papilla in the in the rat in the postnatal stages of development

The developing taste buds of vallate papillae were studied with electron microscope in rats during the first 7 days after birth. Two types of cells--light and dark--are identified in the taste buds of a one day old animal. The apical parts of dark cells are characterized by numerous dark granules. A distinguishing feature of light cells is the presence of synaptic contacts with afferent intragemmal nerves. On the 4th day of development on the top of the apical parts of the cell, a microvillar apparatus is seen to form, which does not yet communicate with the oral cavity. On the 7th day, basal cells appear in the taste buds. Some of these cells are seen mitotically dividing. The differentiated microvillar apparatus now communicates with oral cavity. The structure of the taste buds is getting similar to that in the adults. The structural and functional peculiarities of the developing taste buds are discussed in association with the period of ontogenesis under consideration.     

Changes in the size of the nucleoli of the Purkinje cells in the canine cerebellum in the postresuscitation period

The changes in the size of Purkinje cell (PC) nucleolus in the lateral and medial cerebellum zones were studied in dogs with different degree of neurologic status recovery after clinical death of various etiology and duration. PC always possess one nucleolus in the control and experimental groups. In the case of complete neurologic status recovery of animals the area of PC nucleolus increases in both zones studied, irrespective of the cause of clinical death. In the case of neurologic disorders the increase in PC nucleolus area is clearly expressed only in the medial zone of the cerebellum, being insignificant in the lateral zone. It is suggested that adaptive characteristics of PC are distinct in the two compared zones, which leads to greater PC vulnerability in the lateral zone during deep hypoxia.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

Role of the mediodorsal nucleus of the thalamus in the function of generalization in the cat

In chronic experiments on cats contribution of the thalamic mediodorsal nucleus to the organization of the processes of generalization and abstraction was studied by means of alternation method. Electrolytic ablation of the nucleus retarded the learning and impeded the initial stages of formation of the generalization function. Mechanisms of thalamic dementia are discussed.