昆明种系小鼠胚胎干细胞的分离培养及特性鉴定

目的 提高昆明小鼠胚胎干细胞（ES细胞）建株的成功率。方法 收集小鼠3．5d的囊胚培养,用小鼠胚胎成纤维细胞作为饲养层,形成的ES细胞样集落,经两次亚克隆分离培养ES细胞;进行相差显微镜观察、碱性磷酸酶染色和体内外分化能力鉴定。结果 获得两个稳定ES细胞集落,传至第11代。ES细胞呈集落样生长,碱性磷酸酶染色强阳性,体外分化的细胞沿拟胚体外向性生长,形态多样,在体分化可形成来源于3个胚层的组织。结论 两次亚克地获得的细胞具有ES细胞主要的生物学性状,有助于提高昆明小鼠ES细胞建株的能力。     

体外受精与卵细胞内单精显微注射后受精和胚胎质量的比较

目的 评估 ICSI与 IVF后的卵子受精和胚胎发育,了解不明原因不孕的病人在同一治疗周期中,通过 ICSI受精的胚胎是否较 IVF更好。方法 将 30例不明原因不孕的病人同一周期的卵子随机分为两组,分别行 ICSI和 IVF授精,比较各组间卵子的受精和胚胎质量。结果 不明原因不孕中 IVF授精后发生受精障碍 3例,占 10％ (3/30),而 ICSI授精后无受精障碍发生; ICSI授精组与 IVF授精组比较,卵子受精率无明显差异,而 a类胚胎率差异显著 (P     

不同细胞外基质在大鼠胚胎心脏中表达规律的比较

目的比较不同细胞外基质(ECM)及其共同基质受体在大鼠胚胎心脏中的表达规律,探讨ECM在胚胎期心脏发生、发育过程中的作用。方法将胎龄天数(ED)为11～19d的胚鼠用酶免疫组织化学染色检测ECM中的FN、LMN、Vn、CoⅣ及其受体β1在胚胎心脏中的表达。结果①ECM及其受体在胚胎各期心脏的空间性分布各不相同,FN及LMN有明显的空间分布互补关系,β1的空间分布主要与FN相似,但在空间隔及心外膜中又与LMN相似;②FN、LMN及β1在不同心脏部位的表达高峰及持续时间各不相同,β1的表达高峰要早于FN及LMN。结论ECM(主要是FN与LMN)及其受体β1在胚胎心脏的发生发育中起着一定的介导作用,其作用的时期、部位及作用机制各不相同。     

试论英法百年战争与英国资本主义萌芽的成长

长达百年的英法百年战争给英法两国带来了巨大影响。在政治和经济利益的驱动下 ,英王室调整了商业政策、扶持本国商人 ,大力发展生产 ,积极拓展海外市场 ,建立海上霸权。战争在一定程度上刺激、推动了正在成长中的资本主义生产关系萌芽。     

An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells

1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [³H]-adenine pre-labelling methodology.
2. Adenosine analogues showed the following rank order of potency (pD₂ value): 5′-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) ⩾ adenosine (4.19)=N⁶-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A_2A-selective agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 μM.
3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76±8% NECA response), 2-chloroadenosine (70±6%) and APNEA (40±3%) were significantly reduced.
4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA₂ value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93)=8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13).
5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A_2B adenosine receptor.
6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A_2B subtype.

     

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human -globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 g/egg) alone, mitomycin C (10 g/egg) alone, carboplatin (10 g/egg) alone, and cisplatin (10 g/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.     

Signs of the principle body axes prior to primitive streak formation in the rabbit embryo

An early common element during anterior-posterior axis formation amongst amniotes is the primitive streak, running longitudinally in the two-layered embryonic disc. In mammals the primordium of this transient structure is the first definite morphological sign of the anterior-posterior axis, while in avian embryos the axis is visible and apparently defined earlier. Here we scrutinize suggestions that in mammals also there are earlier signs of axis formation by using correlative low and high-resolution light microscopy on tissues from rabbit embryos at 6.3 and 6.5 days post-conception, i.e. immediately before and after primitive streak formation. A series of semithin sections were cut from resin-embedded embryonic discs that had been photographed previously at low power. In embryos at 6.5-days post-conception the primitive streak is as long as up to half the diameter of the embryonic disc, extending anteriorly from a thickening, here called the posterior node, at the posterior margin, which contains the first mesoderm cells ingressing from the epiblast. On both sides of the primitive streak there is a triangular area that appears light in surface views of fixed embryos and correlates with stretches of low-columnar simple epithelium in an otherwise high-columnar pseudostratified epiblast. Within the anterior margin, which has a sharper contour than the rest of the circumference of the embryonic disc, there is a narrow, crescent-shaped dark zone caused by increased cellular height and number in both epiblast and hypoblast. These characteristics of the anterior margin are also found at 6.3 days post-conception, at which stage there is no sign of a primitive streak or a posterior node. The posterior margin, in contrast, is ill-defined in these earlier embryos, or there is a light crescent within the posterior margin, which has the same histological characteristics as the bilateral posterior triangular areas of primitive streak stages. Because the anterior differentiation occurs prior to primitive streak formation and is a sign of both the anterior-posterior and the transverse axes of the embryonic disc, and because some of its histological characteristics are found in primate and human embryos, we propose to name this structure the anterior marginal crescent and to add it to the list of transient structures that gradually establish the principal body axes in mammals. The anterior manifestation of body axes in mammals is thus essentially different from axis development in the avian embryo, where differentiation of these axes is first manifest at the posterior margin.Supported by the Deutsche Forschungsgemeinschaft (Vi 151/1-1); part of the results were presented at the First Joint Meeting of the Anatomical Society of Great Britain and Ireland and of the Anatomische Gesellschaft, Southampton, December 1994, and will be published in abstract form (J Anat, in press)     

大蒜新素对人巨细胞病毒感染的人胚肺成纤维细胞凋亡的影响

目的 研究大蒜新素对人巨细胞病毒(HCMV)诱导感染的人胚肺成纤维细胞(HELF)凋亡的动态变化及凋亡调控基因bcl-2和fas mRNA表达的影响。方法 用流式细胞术测定HELF在高、低感染复数(MOI分别为2．5、0．25)HCMV感染后1、12、24、36、48、72、96h凋亡细胞比率；大、中、小剂量大蒜新素(9、6和3μg／mL)处理正常的和感染的HELF细胞(MOI为2．5)后上述各时间点凋亡细胞比率。用原位杂交法检测大剂量大蒜新素处理感染细胞(MIO为0．25)72h后bcl-2和fas mRNA表达强度变化，并与正常细胞和感染细胞对比分析。结果 正常细胞凋亡比率保持恒定低水平(凋亡率平均2．68％)；低、高感染复数感染细胞随时间延长凋亡细胞逐渐增多，凋亡率峰值分别达8．85％(96h)和25．63％(72h)；各剂量大蒜新素处理的正常细胞凋亡率增加，呈剂量依赖性，峰值分别为4．88％、6．47％和8．35％；但各剂量药物处理感染细胞后却使细胞凋亡比率下降，大剂量药物处理72h时效应最为明显，凋亡细胞比率由25．63％降至16．24％。正常细胞高表达bcl-2，低表达fas基因；感染HCMV后，bcl-2表达显著下调，fas表达明显增强；大蒜新素处理感染细胞后使bcl-2表达强度明显高于感染细胞，fas表达水平明显低于感染细胞，但仍未恢复到正常细胞水平。结论HCMV是HELF凋亡的强诱导剂；病毒可通过下调凋亡抑制基因bcl-2和上调凋亡促进基因fas而发挥致凋亡作用。大蒜新素本身可诱导HELF凋亡，但却能明显抑制HCMV诱导的细胞凋亡，其抑制作用可能与药物干扰HCMV影响凋亡调控基因bcl-2和fas表达有关。     

血管内皮生长因子促进小鼠胚胎干细胞生成CD34+细胞的研究

目的：研究血管内皮生长因子(vascular endothelial growth factor, VEGF)体外促进小鼠胚胎干细胞系ESD3生成CD34+细胞的能力。方法将ESD3形成拟胚体，将拟胚体细胞转入含不同浓度的VEGF和VEGF+干细胞因子（stem cell factor, SCF)的培养基中。实验分6组，分别为VEGF 5 μg/L组、VEGF 10 μg/L 组、VEGF 20 μg/L组、VEGF 5 μg/L＋SCF组、VEGF 10 μg/L＋SCF组、VEGF 20 μg/L＋SCF组，同时设不加因子的自发分化对照组。RTPCR检测CD34 mRNA的表达，流式细胞仪检测CD34+细胞的比例，甲基纤维素半固体培养法检测生成造血集落的能力。结果：经过1周的诱导培养，实验组生成的细胞可以表达CD34 mRNA，CD34＋细胞的比例也升高，并可形成造血祖细胞的集落。从CD34 mRNA的表达水平、诱导生成CD34＋细胞的比例和生成的集落数量看，VEGF 20 μg/L＋SCF组和VEGF 10 μg/L＋SCF组的诱导效率最高。结论：VEGF能够促进ESC生成CD34＋细胞，尤以与SCF合用时，其诱导效率更高。     

液态媒质中鸡胚心脏瞬间心电向量的综合

目的和方法在电导率不同的2种液态媒质中,采集60个5日龄鸡胚心脏2组相反方向上若干离场源不同距离的测试点心电图,分别比较相同媒质中相反方向上相同距离点的心电图形态和R(r)波振幅,判断该单腔心瞬间心电向量综合的可能性.结果无论是高电导率的生理盐水,还是低电导率的蒸馏水,相同媒质相反方向相同距离点上的心电图皆为正立,其R(r)波振幅的差异不显著(P>0.05);心电在高电导率液态媒质中的衰减幅度远大于在低电导率的液态中的衰减幅度.结论单腔心相反方向的心肌除极所形成的瞬间心电向量可能由于心腔内高电导率血液的衰减效应而无法实现跨越心腔的相互综合.任一瞬间的心电活动可能无法用单一的综合心电向量来表示.