UVA对花背蟾蜍微核及核异常的诱导

为了研究紫外线A对花背蟾蜍的毒性作用,选择微核及核异常为毒理监测指标,对花背蟾蜍红细胞进行观察.结果表明,紫外线A对花背蟾蜍成熟红细胞和非成熟红细胞核异常均有诱导作用并随照射强度的增加而不断增加.在150 min的紫外线A短时间照射内,紫外线对核异常能够产生一定的抑制作用.同时在一定范围内紫外线A能够刺激造血器官释放新的红细胞至外周血中以补充损伤的成熟红细胞.紫外线诱导的核异形主要是核畸形和核膨大.     

转线粒体小鼠的研究进展

线粒体转移技术的发展和成熟使得我们业已成功的建立了转线粒体小鼠动物模型。目前常用的方法主要有:一是直接应用显微注射技术将活的线粒体转入小鼠胚胎;二是通过脱核胞质体与胚胎干细胞融合,再将胚胎干细胞显微注入小鼠囊胚,从而形成嵌和鼠;三是将脱核胞质体与小鼠胚胎直接融合而产生的转线粒体小鼠。随着越来越多线粒体相关疾病的发现,各种不同线粒体疾病的转线粒体小鼠的开发具有十分重要的应用价值和广阔的研究前景。     

家兔胚胎细胞核移植的研究

本研究以兔为实验材料,对细胞核过程中显微操作、电融合、电活化以及移核胚的培养等基本问题进行了研究。对兔进行PMSG－hCG超数排卵,收集成熟母细胞和16－细胞胚;后者经胰蛋白酶消化,去除胶膜和透明带,在不含Ca62 、Mg^2 的分离中分成单个卵裂球;然后,分别对两者做CB预处理;首次尝试采用WQilladsen法,去除卵母细胞核、并将单个卵裂球注入透明带,同时、与McGrath-Solter法进行比较;通过电融合使供体核进入去核的卵母细胞内;将所得移核胚在体外或在中间内体内培养并观察。结果表明：一、Willadsen法与McGrath-Solter法比较,核移植操作的成功率及以后的电融合率均无明显差异（Tab.1)。相对于后者,Willadsen法更简便、易于掌握并提高去核率。二、hCG超排注射后13－15h,观察卵母细胞发现：其中,67．8％保留有第一极体。此时的卵子若去除1／3胞质量,去核率可以达到58．3％。若推迟去核时间,第一极体退化,失去去核标志。三、比较不同电脉冲条件,发现强度为0．63kv/cm,持续160μs的一次电脉冲可获较高移核胚的融合率（70．8％）（Tab.2）;并可使61．1％的成熟卵母细胞活化。四、比较移核胚在体外和在中间有体内两种培养条件,前者只有34．6％能发育到6－8细胞期,而后者有23．0％能发育到桑椹胚或囊胚（Tab.3)。说明：需进一步优化家兔胚体外培养条件。     

hESCs/hiPSCs体外诱导产生红细胞的研究进展及其临床应用的展望

人类胚胎干细胞和多功能诱导性干细胞的诞生,标志着干细胞研究已经跨入了全新的应用时代。干细胞研究领域的一个重要方向是特定谱系成熟细胞的定向诱导分化。在诸多的血细胞中,成熟红细胞因为无核而携带着最小量的遗传物质,可能作为最早的干细胞治疗产品而应用于输血替代治疗。最近,干细胞向造血细胞（包括红细胞）的研究正方兴未艾。但由于方法学上的偏差,诱导产生的红细胞的成熟度各有所不同。该文在结合了作者实验室的工作经验的基础上,对目前人类多潜能干细胞向红细胞特定诱导分化的方法做了综合的描述,并提出了该研究领域亟需解决的重大科学问题。     

天然雌核发育银鲫卵子控制异源精核发育的受精学机制

作者对两性融合生殖鱼和雌核发育银鲫脱膜卵受精的精核发育进行了观察,并采用鱼类卵子无细胞系对以上两类卵质提取物体外诱导经Ｔｒｉｔｏｎ—Ｘ１００处理的精子及其发育进行了初步研究,结果表明在两性融合生殖型脱膜鱼卵中精核通过解凝最终形成原核,而在雌核发育的银鲫脱膜卵子中部分精核体积虽有一定程度的增加,但始终没有观察到原核的发育;在体外诱导实验中,经Ｔｒｉｔｏｎ—Ｘ１００处理的精子在两类卵质提取物中充分发育,都出现了类似体内原核的状态。该现象提示在银鲫卵质中存在有促使精核形成原核的因子,但在正常受精状态下,由于银鲫卵质促使精核核膜解体的功能的异常,使覆盖精子头部的核膜不能象在两性融合生殖受精卵子中进行崩解,精核进一步的原核发育受到抑制。另外,建立体外诱导系统的重要意义,在于它为研究雌核发育调控的分子学机制提供了一条有效途径。     

克隆猪技术及其在转基因猪研究中的应用

哺乳动物核移植技术是一种可以获得基因组遗传信息完全相同的后代的生物技术。猪体细胞核移植技术包括以下几个环节：卵母细胞的体外成熟、供体细胞的分离和处理、体细胞的核转移、重构胚胎的人工激活、胚胎体外培养和胚胎移植。由于该技术在最近几年的迅速发展,很多实验室已通过该技术成功获得了克隆猪后代。核移植克隆猪技术的出现为生产转基因猪提供了一种有效的方法,并且是目前生产基因打靶猪的惟一方法。至今利用克隆猪技术已经成功获得了一系列的转基因猪和基因敲除猪。以核移植技术产生基因修饰猪目前正处于从基础研究走向应用的过渡阶段。尽管猪体细胞核移植克隆的效率（出生克隆猪数占所用卵数的比例）还不高,但是由于通过该技术能够对猪基因组进行特定的修饰,确保生产的克隆动物100％为转基因动物,从而大大提高了转基因猪的制作效率,可以预料猪核移植技术将会对医药业和农业产生重大的影响。     

痒觉信号通路与相关受体研究进展

痒是一种引起机体产生抓挠欲望的不愉快感觉．与疼痛一样,痒觉也是机体的一种自我防护反应,可以促使机体及时避免有害刺激,但持续较长时间的慢性痒往往是一种病理性特征,严重影响人的生活质量．目前已发现两条痒觉传导通路,组胺依赖的信号通路和组胺非依赖的信号通路．组胺、阿片类物质、大麻素、一些肽类物质及氯喹均可刺激相关受体诱导痒觉产生．痒觉产生的机制,有多种学说,比较有影响的是选择性学说和特异性学说．本文综述了近年来痒觉信号通路及其相关受体的研究进展．     

成熟促进因子对克隆重构胚核重编程的调控

成熟促进因子(maturation promoting factor,MPF)由催化亚单位P34cdc2和调节亚单位cyclin组成,对细胞周期的调控起着重要作用。目前,在核移植研究中发现：供体核在MPF的作用下发生核膜破裂(nuclear envelop breakdown．NEBD)和早熟染色体凝集(premature chromosome condensation,PCC),促进了核、质蛋白质因子的交换,有利于核重编程的进行。PCC还会对供体核的倍性及形态产生影响。     

浅谈剧烈运动后的肌肉酸痛感

对剧烈运动后的肌肉酸痛是人人关心的问题,本文对这个问题进行了分析。肌肉酸痛可分为急性肌肉酸痛和慢性肌肉酸痛。前者产生的原因一般用“乳酸堆积”理论解释,后者产生的原因,目前有肌肉损伤学说和肌肉痉挛学说。本文并介绍了消除肌肉酸痛的五种简单方法。     

高尔基体对小鼠卵母细胞体外发育进程的影响

目的初步探讨高尔基体在小鼠卵母细胞体外发育进程中的作用。方法布雷菲德菌素A（Brefeldin A,BFA）处理小鼠未成熟,成熟卵母细胞,利用特异性标记物阻COP标记高尔基体。激光扫描共聚焦显微镜观察BFA处理对高尔基体产生的影响;同时。观察并比较不同处理组小鼠未成熟／成熟卵母细胞的体外成熟率、孤雌激活率、体外受精率及2-细胞率。结果GV期卵母细胞经BFA处理后,高尔基体的形态和分布发生明显改变。其体外成熟率（2．5％）与对照组（70．4％）比较统计学差异显著（P〈0．001）;洗掉BFA后,其体外成熟率（67．2％）与对照组无统计学差异（P〉0．05）。另外,成熟卵母细胞经BFA处理后。其体外受精率及2．细胞率均与对照组差异无统计学意义（P〉0．05）。结论小鼠卵母细胞体外成熟的正常进行需要高尔基体主导的膜运输。而体外受精和受精卵卵裂过程中不需要功能性的高尔基体。     

Preferential radioprotection to DNA of normal tissues by ferulic acid under ex vivo and in vivo conditions in tumor bearing mice

Our previous study showed that ferulic acid (FA) offered good radioprotection under in vitro and in vivo conditions to DNA and enhanced the DNA repair process in the peripheral blood leucocytes of mice in vivo. This study concerns radioprotection of normal versus tumor cells. Administration of FA (50 mg/kg body weight) to mice bearing fibrosarcoma tumor, 1 h prior to/ or immediately after radiation exposure (4 Gy) showed preferential radioprotection to normal cells i.e. peripheral blood leucocytes and bone marrow cells in comparison to tumor cells. This preferential protection under in vivo conditions could be attributed to poor vasculature in the tumor or peculiar characteristics of the tumor cells either to restrict its entry inside the cells or metabolize or inactivate the drug. To resolve these ex vivo study was carried out using bone marrow and tumor cells. It was found that under ex vivo condition also only bone marrow cells were protected by FA. Thus the studies revealed that FA showed preferential protection to normal cells under both in vivo and ex vivo conditions. (Mol Cell Biochem xxx: 1–10, 2005)     

Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells

In vitro erythropoiesis has been studied extensively for its application in the manufacture of transfusable erythrocytes. Unfortunately, culture conditions have not been as effective as in vivo growth conditions, where bone marrow macrophages are known to be a key regulator of erythropoiesis. This study focused on the fact that some erythroblasts are detached from macrophages and only contact other erythroblasts. We hypothesized that additional factors regulate erythroblasts, likely through either physical contact or secreted factors. To further elucidate these critical factors, human erythroblasts derived from cord blood were cultured at high density to mimic marrow conditions. This growth condition resulted in a significantly increased erythroid enucleation rate and viability. We found several novel contact-related signals in erythroblasts: intercellular adhesion molecule-4 (ICAM-4) and deleted in liver cancer-1 (DLC-1). DLC-1, a Rho-GTPase-activating protein, has not previously been reported in erythroid cells, but its interaction with ICAM-4 was demonstrated here. We further confirmed the presence of a secreted form of human ICAM-4 for the first time. When soluble ICAM-4 was added to media, cell viability and enucleation increased with decreased nuclear dysplasia, suggesting that ICAM-4 is a key factor in contact between cells. These results highlight potential new mechanisms for autonomous control of erythropoiesis. The application of these procedures to erythrocyte manufacturing could enhance in vitro erythrocyte production for clinical use.     

Comparative Effect of Quercetin and Quercetin‐3‐O‐β‐d‐Glucoside on Fibrin Polymers,Blood Clots,and in Rodent Models

The present study evaluates the in vitro, in vivo, and ex vivo antithrombotic and anticoagulant effect of two flavonoids: quercetin and quercetin‐3‐O‐β‐d ‐glucoside (isoquercetin). The present results have shown that quercetin and isoquercetin inhibit the enzymatic activity of thrombin and FXa and suppress fibrin clot formation and blood clotting. The prolongation effect of quercetin and isoquercetin against epinephrine and collagen‐induced platelet activation may have been caused by intervention in intracellular signaling pathways including coagulation cascade and aggregation response on platelets and blood. The in vivo and ex vivo anticoagulant efficacy of quercetin and isoquercetin was evaluated in thrombin‐induced acute thromboembolism model and in ICR mice. Our findings showed that in vitro and in vivo inhibitory effects of quercetin were slightly higher than that of quercetin glucoside, whereas in vitro and ex vivo anticoagulant effects of quercetin were weaker than that of quercetin glucoside because of their structural characteristics.     

In Vitro Binding of Trypanosoma congolense to Erythrocytes*

Synopsis. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin. Trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.     

ATP formation from deoxyadenosine in human erythrocytes: Evidence for a hitherto unidentified route involving adenine and S-adenosylhomocysteine hydrolase

A novel route of ATP formation has been identified using erythrocytes from patients deficient in four different enzymes associated with ATP formation. It entails prior adenine production from deoxyadenosine (or adenosine) in a reaction involving S-adenosylhomocysteine hydrolase. The postulated route has been demonstrated in human erythrocytes which, unlike other human cells, cannot form ATP from IMP. It is based on studies by others using purified S-adenosylhomocysteine hydrolase preparationsin vitro. The results provide the first confirmation that this reaction occurs in intact human cellsin vitro and thus most probablyin vivo. This adenine production is normally masked in intact cells by further metabolism to ATP. Clinical significance for such a route is suggested by the fact that some adenosine analogues with potent oncostatic and antiviral properties also release adenine (or analogues)in vitro.     

Dysfunctional monocytes from a patient with disseminatedMycobacterium kansasii infection are activatedin vitro andin vivo by GM-CSF

A 27 year-old woman presented with disseminated infection due toMycobacterium kansasii. Signs and symptoms of disseminated infection persisted despite the administration of multiple antimycobacterial agents to which her organism was sensitive for 15 months. She was seronegative for HIV-1 and functional studies of T and B lymphocytes and granulocytes failed to demonstrate any abnormality. Peripheral blood monocytes proved abnormally permissive to the intracellular growth ofMycobacterium avium andM. kansasii, and expressed normal number of receptors to interferon-gamma, but reduced numbers of receptors to granulocyte monocyte colony stimulating factor and tumor necrosis factor. These defects were partially reversed within vitro exposure of her cells to recombinant GM-CSF. In addition, administration of recombinant human GM-CSFin vivo (250 mg/M² per day) for 10 days armed her circulating monocytes as evidenced by increased production of O₂ ^– in response to phorbol esther and, when infectedex vivo withM. kansasii, enhanced inhibition of intracellular growth compared with pre-therapy monocytes. These defects reappeared with discontinuation of GM-CSF and resolved with its re-administration. While a salutary clinical and microbiologic effect was difficult to assess, administration of GM-CSFin vivo was associated within vitro activation of monocytes and enhanced mycobactericidal activity in this patient with a defect in monocyte function.     

CD3-Positive B Cells: A Storage-Dependent Phenomenon

The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.     

Liver stem cells: a two compartment system

Hepatocytes and biliary epithelia are phenotypically very dissimilar, but share a common ancestry. Hepatocytes regenerate very efficiently, and their division potential indicates that many of them are functional stem cells. When hepatocyte-damaging agents also impair the regenerative ability of surviving hepatocytes, a potential stem cell system of biliary origin is activated to generate new hepatocytes — a reversal of ontogeny. Now both bile duct derived cells and hepatocytes can be isolated from the liver, genetically modified in vitro and returned to their in vivo origins where, after considerable population expansion, they can function as hepatocytes — paving the way for ex vivo gene therapy.