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标题: 发育毒性的体外干细胞试验方法的改进：在胚胎干细胞试验中使用分子终点 [打印本页]

作者: mominglily 时间: 2011-3-12 21:07 标题: 发育毒性的体外干细胞试验方法的改进：在胚胎干细胞试验中使用分子终点

题名：Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test
发育毒性的体外干细胞试验方法的改进：在胚胎干细胞试验中使用分子终点, ]: g8 S$ P! O1 c! \4 ?1 ]
作者：Andrea Seiler∗, Anke Visan, Roland Buesen, Elke Genschow, Horst Spielmann
摘要：胚胎干细胞试验（EST）试验利用鼠胚胎干细胞体外分化的潜能来测定体外胚胎毒性。EST代表了一种根据化合物的致畸潜力划分化合物毒性等级的可靠的，经科学验证的体外体系，该体系是以胚胎体（EB）集落中跳动的心肌细胞的形态学分析和未分化的鼠胚胎干细胞及分化的3T3成纤维母细胞的细胞毒性作用为基础的。为了鉴定除了显微评价跳动区域之外更多客观的分化终点并将EST试验应用于高通量筛选系统中，我们通过建立分化的分子终点改进及扩展了EST试验方法。
采用细胞内流式细胞技术研究在受试化合物的影响下肌球重链蛋白（MHC）和α-辅肌动蛋白基因的定量表达情况。此外，定量FACS（荧光激活细胞分选技术）分析作为常规的终点对于化学物质的分级表现出了相同的灵敏性，然而却显著缩短了检测周期。在7天内，观测到了肌节标记基因的最大表达。我们的发现表明肌节的结构蛋白--α-辅肌动蛋白和重链肌球蛋白（MHC），看来是由体外数据预测体内发育毒性的最有前景的候选标志物。因此，改进后的EST试验有希望作为关于使用干细胞技术及基因表达分析领域的技术进步对发育毒性风险评估的新的预测筛选方法。
[ Reproductive Toxicology 18 (2004) 231–240 ]9 H! E9 W4 @1 B

作者: tpwang 时间: 2011-3-12 22:23

回复 mominglily 的帖子2 N, e  `: [/ [) y6 m% q

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test1 {' x+ D, e. `, A" Y' {- A
发育毒性的体外干细胞试验方法的改进：在胚胎干细胞试验中使用分子终点 . P! W7 J! X# B5 Y1 C% k+ J2 Z2 X/ {& _
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endpoint这里是指检验的（终极）指标的意思。同样的用法还可以用在实验中，比如“该实验的endpoint（指标）是……”。因此，原题的翻译大致应该是……在胚胎干细胞检验中采用分子水平的指标。/ v& k2 L; V9 W2 _2 c/ [! v4 z

为了鉴定除了显微评价跳动区域之外更多客观的分化终点并将EST试验应用于高通量筛选系统中，我们通过建立分化的分子终点改进及扩展了EST试验方法。( J. u- ?7 C! y7 {) ]1 o
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“分化终点”=以分化为指标
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此外，定量FACS（荧光激活细胞分选技术）分析作为常规的终点对于化学物质的分级表现出了相同的灵敏性，; c( n8 d; l( g* I% |( g

“常规的终点”（conventional endpoint）=常规指标。1 S& w' u1 z& f! D4 G2 B

原文中有一个关于各种检验方法及其指标（Endpoints）示意图，可以参考。
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Abstract+ j# ^/ C4 b: z, I" T( V5 Q' e
The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of “beating areas” and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular& y  L+ W$ Z5 k, D- b8 L; h
endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and -actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and -actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, -actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.* D% J$ ?& O6 [- k5 s
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一点意见，仅供参考。

作者: mominglily 时间: 2011-3-12 23:39

回复 tpwang 的帖子
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嗯，您的建议很好，受教了，谢谢！

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