人源羊水干细胞的培养、鉴定及影响因素分析

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2 c, n  j( c- {5 m4 `5 L! F王晗 窦忠英 王华岩西北农林科技大学动物医学院 陕西省农业分子生物学重点实验室 陕西省干细胞工程技术研究中心,杨凌712100摘要：取人羊水标本进行羊水干细胞原代，分离培养，并探讨人羊水标本性状等因素对原代分离培养的影响。结果表明，分离的羊水干细胞表达Oct-4等胚胎干细胞和CD29等成体干细胞的特异基因，细胞生长迅速，增殖能力较强，并可分化为Nestin阳性的神经细胞和α-actin阳性的心肌细胞。同时，在羊水干细胞原代培养时，羊水标本性状在一定程度上影响细胞的贴壁数量及贴壁时间。羊水标本细胞的贴壁时间孕中期（4．7±0．6）d与孕晚期（6±0．5）d有明显差异（P0〈0．05），孕晚期贴壁时间较长。羊水中血液污染程度对贴壁时间有明显影响，重度污染组（10．8±0．3）d与无污染组（6±0．5）d、轻度污染组（6．3±0．6）d贴壁时间差异均显著（P〈0．05）。羊水体积对原代细胞的贴壁数量和细胞贴壁时间有一定影响，羊水体积增大贴壁时间延长，但差异不显著，而贴壁细胞数量随羊水体积增加而增加，各组之间差异显著（P〈0．05）。实验系统地检测了羊水干细胞原代培养的影响因素，为羊水干细胞研究提供了可以借鉴的资料，同时分离得到的羊水干细胞，具有分化为神经细胞和心肌细胞的潜能，有望作为种子细胞进行临床应用。
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KeyWord：human; amniotic fluid stem cells; cell attachment numbers; cell attachment time( S: m, X+ L% D

# {- A, t1 Z' }, x关键词：人 羊水干细胞 贴壁细胞数量 贴壁时间
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分类号：S188[著者标引]文献标识码：A文章编号：1006-1304（2008）05-0804-06
/ J4 m. l) R* }0 ^# U5 _4 PGrowth and Identification of Human Amniotic Fluid Stem Cells and Analysis of Their Influencing Factors WANG Han, DOU Zhong-ying, WANG Hua-yan College of Veterinary Medicine, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Shaanxi Stem Cell Engineering and Technology Research Center, Northwest Agriculture and Forestry University, Yangling 712100, China2 B% J# m  Y) {- ]) G' j
Abstract：Amniotic fluid stem （AFS） cells derived from human amniotic fluid were cultured in vitro and the factors of affecting the primary culture of human AFS cells were investigated. The isolated AFS cells, which express embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, grew faster and had high proliferation ability. Moreover, AFS cells could differenti- ate into neurons and cardiomyocytes which expressed Nestin and α-actin, respectively. There were several factors, such as the date of gestation, the level of blood contamination and the volume of amniotic fluid, could significantly affect the attachment time and numbers of AFS cells in the primary culture. The results showed that cell attachment time of the 2nd trimester of gestation （4.7±0.6） d was significantly different from that of the 3rd trimester of gestation （6 ± 0.5） d （P 〈0.05）, suggesting that cells collected from the fluid of 3rd trimester of gestation needed longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of brown-color fluid group （10.8 ± 0.3） d was significantly different with the blood-cell-free fluid group （6± 0.5） d and blood-cell group （6.3±0.6） d （P〈0.05）. The volume ofamniotic fluid influenced on cell attachment numbers and time to some extent. With the increase of amniotic fluid volume, cell attachment numbers significantly increased （P 〈0.05）, and cell attachment time extended but no significant difference （P 〉0.05）. The present studies systematically examined the factors, effection on primary culture of human AFS cells and provided some useful data for AFS cell research. Additionally, the isolated AFS cells maintain the capabilities of differentiation into other cell types and are able to become seed cells for the clinical application.
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- j# F$ _" l$ K+ xKeyWord：human; amniotic fluid stem cells; cell attachment numbers; cell attachment time' o6 G1 R! r& {1 O/ B3 X
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