小鼠胚胎实验对新建人类体外受精实验室质量控制的评价

目的 利用小鼠体外受精技术对我院新建人类体外受精实验室进行质量控制.方法 手术获取小鼠配子,经体外受精或卵胞浆内单精子显微注射后,形成胚胎体外培养5d,观察受精率、优胚率、囊胚率.结果 本研究进行11个常规体外受精周期,取卵371枚,受精率82.2％(305枚),优胚率91.3％ (274枚),2-细胞囊胚形成率85.3％(256枚)；10个周期卵胞浆内单精子显微注射,取卵子206枚,受精率84.5％(174枚),优胚率92.9％(157枚),2-细胞囊胚形成率89.9％(152枚).结论 通过鼠胚实验对我科新建IVF实验室进行质量控制,结果符合标准.     

利用胚胎发育潜能评估IVF实验室培养系统的稳定性

体外受精-胚胎培养实验室(IVF实验室)培养系统包括温度、湿度、CO2浓度、以及培养胚胎的各种试剂、耗材,其稳定性直接关系到辅助生殖的结局,因此对IVF实验室的培养系统定期进行定期检测是辅助生殖技术中的重要组成部分[1].目前检测培养系统的方法有小鼠胚胎实验、人精子存活实验和内毒素实验等[2].本研究通过分析不同批号耗材的胚胎发育情况评估耗材的稳定性,并进一步反应IVF实验室培养系统的稳定性.报告如下.     

不同培养液对鼠胚在新建试管婴儿实验室中体外发育的影响

目的 利用三种不同的培养液EBSS(SIGMA系列),G1和G2(Vitrolife系列),Quinn’s1026和Quinn’s1029(SAGE系列)对昆明系小白鼠胚胎进行体外培养,以对新建IVF实验室进行评估。方法 取7周龄的昆明白雌性小鼠,用人绝经期促性腺激素(HMG)10 IU促排卵,48 h后注射人绒毛促性腺激素(HCG)10 IU促卵泡成熟,同时与雄性小鼠1:1合笼,再经过48 h后获取形态正常的2细胞鼠胚。将胚胎分成三组,A组使用EBSS(Earle’s Balanced Salt Solution)培养液,B组使用G1和G2培养液,C组使用Quinn’s1026和Quinn’s1029培养液,三种培养液均添加10%人血清白蛋白。分析对比三组结果。结果 72 h后,鼠胚总体囊胚形成率为71.54%(382/534),其中A组的囊胚形成率为30.56%(22/72),B组为70.49%(129/183),C组为82.80%(231/279),B、C组的囊胚形成率显著高于A组(P<0.01)。结论 通过鼠胚体外培养,对新建试管婴儿实验室进行了较好的质控检测,序贯培养液Vitrolife系列的G1和G2以及SAGE系列的Quinn’s1026和Quinn’s1029在鼠胚囊胚形成率上要高于简单培养液EBSS。     

红花注射液生物活性测定方法的筛选

目的:从多种活血化瘀类药物的药效学模型中筛选红花注射液生物活性测定的方法。方法:用4个不同厂家的红花注射液,根据功能主治相关的5种药效学实验方法(包括大鼠急性心肌缺血实验、小鼠急性脑缺血缺氧实验、小鼠体内血栓实验、小鼠断头存活实验、家兔体外抗血小板聚集实验),筛选1～2种设计合理、操作简便、指标明确、灵敏度高、重现性好的方法作为其生物活性测定方法。结果:小鼠体内血栓和家兔体外抗血小板聚集实验符合要求。结论:小鼠体内血栓实验和家兔体外血小板聚集实验为红花注射液生物活性测定的方法。     

甲磺酸甲酯对小鼠精子染色体畸变的影响

利用(C_(57)×CBA)F_1小鼠精子与NIH小鼠体外受精、经培养制备的受精卵染色体标本,观察了甲磺酸甲酯(MMS)对小鼠精子染色体畸变的影响。MMS体内(25、50、100mg/kg),体外(75、150μg/ml)处理精子后,精子染色体畸变率明显升高,呈剂受量-反应关系。畸变类型以染色体型为主。本研究表明,由MMS诱导的精子DNA损伤可传递至精卵;所用模型是一种监测化学物质对生殖细胞(精、卵子)诱变作用的可靠方法。     

小鼠胚泡移植法在研究羟基脲发育毒性中的应用

目的探讨体外染毒后胚泡移植方法评价羟基脲发育毒性的可行性。方法由C57BL/6J雌性小鼠和DBA雄性小鼠交配产生BDF1小鼠,以BDF1雌性小鼠作为供胚鼠进行超排卵,并与BDF1雄性小鼠交配后采集受精卵,再以20μg/ml的羟基脲对着床前胚胎进行体外染毒,将染毒后可胚泡化的胚胎移植于假孕昆明种代母鼠体内继续生长发育,观察出生前胎鼠形态学改变和出生后子鼠的行为改变,并对子鼠大脑进行病理检查。结果(1)在实验剂量下,羟基脲染毒组胎鼠出生前形态学检查未见异常。(2)羟基脲染毒组子鼠出现行为异常:回旋运动、阴性趋地运动、翻正反射和断崖回避阳性日均迟于阴性对照组(P<0.05,P<0.01,P<0.05,P<0.05);悬挂时间短于阴性对照组(P<0.05);10日龄游泳积分低于阴性对照组(P<0.01)。(3)电镜超微结构显示染毒组子鼠大脑中毒性损伤。结论将胚胎体外培养和胚泡移植技术结合起来,排除母体因素的影响探究羟基脲的发育毒性,是一可行的方法。     

不同的培养方法对鼠胚体外发育的影响

目的比较鼠胚在不同的培养体系中的体外发育情况,探讨培养液的体积对体外培养的影响。方法把收集的小鼠2-细胞胚胎分成两组,A组于20μl培养液的微滴中培养,B组与50μl培养液的微滴中培养,观察胚胎发育情况。结果 96h后A、B组的囊胚形成率分别为83.64%和81.63%,差异无统计学意义(P>0.05)。结论适当体积的培养滴可促进胚胎的体外发育。     

玉叶解毒颗粒抗甲1型流感病毒的作用

目的:研究玉叶解毒颗粒体内外抗甲1型流感病毒(Influenza virus)作用。方法:体内试验检测玉叶解毒颗粒对小鼠感染流感病毒所致肺炎的抑制作用和死亡保护;体外实验通过对狗肾细胞(MDCK)的培养,探讨玉叶解毒颗粒在细胞上对感染流感病毒的抑制作用。结果:玉叶解毒颗粒对小鼠肺内的流感病毒有一定的清除作用,能减轻小鼠肺内的炎性病变,在剂量为15g.kg-1时对小鼠有死亡保护作用,体外实验在125g.L-1时能抑制流感病毒。结论:玉叶解毒颗粒在体内外具有明显的抗流感病毒作用。     

精源性双酚A对小鼠体外受精和胚胎发育的影响

目的探讨双酚A(BPA)对小鼠体外受精和胚胎发育的精源性影响作用。方法健康昆明种小鼠52只,雌雄各半,随机分为两组,实验组雄鼠用双酚A[10μg/(kg·d)]胃灌洗45d后进行精子参数分析,同时取出正常喂养的雌鼠卵子进行体外受精,对受精情况、胚胎发育情况进行跟踪观察,对照组雄鼠采用相同体积的玉米油胃灌洗。结果 (1)双酚A灌洗组精子的活力33.15%显著低于正常组58.13%(P0.01);畸形率显著高于正常对照组,分别为9.84%和2.11%(P0.01);(2)双酚A灌洗组的受精率为43.28%,显著低于正常组78.13%(P0.01),囊胚形成率为26.14%,略低于正常对照组30.11%,但差异无统计学意义(P0.05)。结论双酚A可致小鼠精子质量明显下降,受精和胚胎发育能力降低,是影响小鼠生育能力的重要的精源性因素。     

MDCK细胞基质与鸡胚基质单价流感疫苗在小鼠中诱导的免疫反应的比较研究

目的:比较鸡胚基质和细胞基质的H1N1单价流感疫苗在小鼠体内诱导的免疫反应的差异.方法:分别采用鸡胚基质和MDCK细胞基质H1N1流感疫苗注射C57BL/6小鼠,通过体外中和实验,胞内细胞因子染色及多细胞因子检测比较2种基质生产的单价流感疫苗诱导的体液及细胞免疫反应的差异.结果:体液免疫方面,细胞基质疫苗组血清滴度与鸡...     

Quantitative assessment of a prognostic or predictive biomarker panel

ABSTRACT

Methods for assessing whether a single biomarker is prognostic or predictive in the context of a control and experimental treatment are well known. With a panel of biomarkers, each component biomarker potentially measuring sensitivity to a different drug, it is not obvious how to extend these methods. We consider two situations, which lead to different ways of defining whether a biomarker panel is prognostic or predictive. In one, there are multiple experimental targeted treatments, each with an associated biomarker assay of the relevant target in the panel, along with a control treatment; the extension of the single-biomarker scenario to this situation is straightforward. In the other situation, there are many (nontargeted) treatments and a single assay that can be used to assess the sensitivity of the patient’s tumor to the different treatments. In addition to evaluating previous approaches to this situation, we propose using regression models with varying assumptions to assess such panel biomarkers. Missing biomarker data can be problematic with the regression models, and, after demonstrating that a multiple imputation procedure does not work, we suggest a modified regression model that can accommodate some forms of missing data. We also address the notions of qualitative interactions in the biomarker panel setting.     

The effect of dimethylaminoadamantane on neuronal membranes.

The effect of dimethylaminoadamantane (DMAA), an amantadine derivative with an anti-Parkinson property, on rat sensory nerve fibres was studied with the sucrose gap method. DMAA 10(-4) M in normal Locke solution reduced the spike amplitude without changing the resting potential, increased the membrane resistance and depressed repetitive spike activity elicited by depolarizing currents. From experiments performed with changed concentrations of sodium, potassium, calcium and chloride ions in the suspension medium it appears that the permeability of sodium, potassium and chloride ions is reduced by DMAA. The possible implication of the membrane effects of the drug in its action on dopaminergic transmission in the brain is discussed.     

In vitro and in vivo genotoxic evaluation of Bothrops moojeni snake venom

Abstract

Context: Bothrops moojeni Hoge (Viperidae) venom is a complex mixture of compounds with therapeutic potential that has been included in the research and development of new drugs. Along with the biological activity, the pharmaceutical applicability of this venom depends on its toxicological profile.

Objective: This study evaluates the cytotoxicity and genotoxicity of the Bothrops moojeni venom (BMV).

Material and methods: The in vitro cytotoxicity and genotoxicity of a pooled sample of BMV was assessed by the MTT and Comet assay, respectively. Genotoxicity was also evaluated in vivo through the micronucleus assay.

Results: BMV displayed a 50% cytotoxic concentration (CC₅₀) on Vero cells of 4.09?µg/mL. Vero cells treated with 4?µg/mL for 90?min and 6?h presented significant (p?<?0.05, ANOVA/Newman–Keuls test) higher DNA damage than the negative control in the Comet assay. The lower DNA damage found after 6?h compared with the 90?min treatment suggests a DNA repair effect. Mice intraperitoneally treated with BMV at 10, 30, or 80?µg/animal presented significant genotoxicity (p?<?0.05, ANOVA/Newman–Keuls test) in relation to the negative control after 24?h of treatment. Contrary to the in vitro results, no DNA repair seemed to occur in vivo up to 96?h post-venom inoculation at a dose of 30?µg/animal.

Discussion and conclusion: The results show that BMV presents cyto- and genotoxicity depending on the concentration/dose used. These findings emphasize the importance of toxicological studies, including assessment of genotoxicity, in the biological activity research of BMV and/or in the development of BMV-derived products.     

The human tumor colony-forming chemosensitivity assay: A biological and clinical review

Summary Over forty papers describing correlations between in vitro human tumor sensitivity to a variety of chemotherapeutic agents and the in vivo response of patients to those agents have been published since the publication in 1978 by Salmon and Hamburger of their results of a human tumor colony-forming chemosensitivityassay (CFCA). The true positive rate in over 1600 correlations is 71% and the true negative rate is 94%. The biological elements of the assay, its developmental history, its place in the spectrum of in vitro chemosensitivity assays, and its theoretical and practical limitations are discussed. The scope, design, and limitations of key clinical trials are presented and an analysis of the potential errors of statistical interpretation of the trials as well as the results of the trials is given.     

Genotoxicity studies on HM10760A,recombinant human erythropoietin conjugated to globin fragment

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100?μg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100?μg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600?μg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600?μg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.     

终止分光光度法快速检测酪氨酸多巴氧化酶活性

目的 提高分光光度法检测酪氨酸多巴氧化酶活性(多巴氧化酶)的灵敏度、特异性和稳定性.方法 用高氯酸对分光光度法进行改进,并与多巴色素法和连续检测法比较.结果 终止分光光度法检测多巴氧化酶的灵敏度为多巴色素法10倍,为连续监测法的2倍,且稳定性强,可分析浊样和非浊样样本,并且每小时样本测定量为另两种的2倍.结论 建立的终止分光光度法检测多巴氧化酶活性敏感、快速和特异,是一种可靠的实验诊断手段.     

HPLC法测定交沙霉素的含量及与微生物检定法的比较

目的:建立HPLC法测定交沙霉素含量的方法并与微生物检定法结果相比较.方法:采用RP-HPLC法,以C18为固定相;流动相:水-乙腈(45:55),用三乙胺调节pH值为8.0;检测波长:232 nl.结果:在0.1～1.4 mg·ml-1的浓度范围内呈良好线性关系.结论:本方法色谱系统简单,可靠.经与微生物检定法结果比较,结果准确可靠,精密度高.且能将交沙霉素与其各杂质组分实现良好分离,测定结果更加准确.     

A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke

In the last few years tobacco companies have been developing several research strategies in order to reduce the risks associated with smoking. These strategies include, for example, the refining of alternative cigarette designs that reduce the amount of hazardous chemicals in the mainstream smoke by introducing modified filters, and/or reducing the amount of biologically significant ingredients in tobacco-burning cigarettes. In the last few decades numerous studies have been published to assess the biological activity of tobacco smoke using in vivo and in vitro test systems. In this scenario a general scientific consensus on how to measure and characterize the risk associated with cigarette smoke is still lacking. Short-term in vitro assays, which are widely accepted by regulatory agencies around the world, are useful tools to evaluate both the biological activity and the progress towards a reduction of tobacco smoke toxicity. These assays could be mainly applied to evaluate cytotoxicity and genotoxicity properties on whole cigarette smoke as well as condensates or fractions of whole smoke. Cytotoxicity induction can be measured as cellular viability and growth rates using different end-points. Otherwise, the target of genotoxicity studies is the DNA molecule. For genotoxicity evaluation, the end-points and cell systems should be chosen from those that are relevant and appropriate as clinical surrogate markers. In this respect, the occurrence of early biological effect markers, such as mutational or clastogenic events (point mutations, frameshifts, micronuclei, SCE, DNA adducts) in bacterial and mammalian cells should be studied in a tiered approach following the guidelines of regulatory agencies. The choice of criteria shall be matter of discussion.     

自动加样快速微孔板法在疑难配血中的应用

目的 在疑难配血中建立一种自动化、快速、准确的供血者标本血型抗原筛查方法.方法 使用自动加样快速微孔板法对188个供血者全血标本进行C、e、M、Fya、Jka抗原检测,同时使用手工试管法进行平行实验;记录并对比上述两种方法筛查总过程使用的时间,筛查出的供者标本数.用微板法和试管法对抗-C、抗-e、抗-M、抗-Fya、抗-Jka试剂进行抗体效价测定.结果 微孔板法、试管法均筛查出相同血型抗原阴性供者,微孔板筛查所需要的总时间较短.效价测定显示微板法灵敏度与手工试管法相同.结论 微孔板法在疑难配血中筛查特定抗原阴性的供者,具有快速、准确的效果.     

MTT法检测rhEGF生物活性

目的鉴于待测的生物活性因子均有适于其自身的最佳测定条件,规范了重组表皮生长因子(rhEGF)的MTT测定法。方法根据MTT测定法原理,在3个因素(每孔细胞数、MTT和胎牛血清浓度)及3个水平上进行了正交实验设计。结果MTT检测法的最佳条件是:Balb/c3T3细胞悬液(5×107cells·L-1)100μl·well-1,MTT(8g·L-1)20μl·well-1,FBS(1·5%)100μl·well-1。结论上述正交实验规范的MTT最佳实验条件,适用于Balb/c3T3细胞活性的检测及其药物抑制性实验。但在测定rhEGF生物活性时,为使对照孔细胞数保持在较低水平以便给生物活性因子留有适当的余地以显示其促细胞增殖活性,建议FBS浓度采用0·5%。