中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (32): 5967-5972.doi: 10.3969/j.issn.2095-4344.2012.32.015

• 胚胎干细胞 embryonic stem cells • 上一篇    下一篇

小鼠胚胎干细胞向成骨样时段性细胞分化的形态学特点

史筱璐1，李化静1，马 艳2，毕晓娟2，谢凤莲3，王鲁香4，刘立中1   

  1. 新疆医科大学第一附属医院，
    1耳鼻咽喉科，
    2医学研究中心，新疆维吾尔自治区乌鲁木齐市 830054；
    3新疆医科大学，新疆维吾尔自治区乌鲁木齐市 830054；
    4新疆大学，新疆维吾尔自治区乌鲁木齐市 830000
  • 收稿日期:2012-01-30 修回日期:2012-04-11 出版日期:2012-08-05 发布日期:2012-08-05
  • 通讯作者: 刘立中，新疆医科大学第一附属医院干部保健科，新疆维吾尔自治区乌鲁木齐市 830054 liulizhongno.1@163.com
  • 作者简介:史筱璐★，女，1984年生，新疆维吾尔自治区乌鲁木齐市人，汉族，2011年新疆医科大学毕业，硕士，主要从事耳科学的研究。 lauren84103@sina.com

Time-dependent morphological characteristics of mouse embryonic stem cells differentiating into osteoblasts

Shi Xiao-lu1, Li Hua-jing1, Ma Yan2, Bi Xiao-juan2, Xie Feng-lian3, Wang Lu-xiang4，Liu Li-zhong1   

  1. 1Otorhinolaryngologic Department,
    2Medical Research Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uyghur Autonomous Region, China;
    3Xinjiang Medical University, Urumqi 830054, Xinjiang Uyghur Autonomous Region, China;
    4Xinjiang University, Urumqi 830000, Xinjiang Uyghur Autonomous Region, China
  • Received:2012-01-30 Revised:2012-04-11 Online:2012-08-05 Published:2012-08-05
  • Contact: Liu Li-zhong, Otorhinolaryngologic Department, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uyghur Autonomous Region, China liulizhongno.1@163.com
  • About author:Shi Xiao-yu★, Master, Otorhinolaryngologic Department, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uyghur Autonomous Region, China lauren84103@sina.com

PDF

349

被引次数

1

摘要：

背景：纳米羟基磷灰石具有良好的生物活性和相容性，在体外可与细胞短时间内形成紧密结合，是骨组织工程可植入性的材料。
目的：观察小鼠胚胎干细胞分化为成骨样时段性细胞的形态学特点。
方法：取C57小鼠胚胎干细胞进行培养，将P3胚胎干细胞制备拟胚体。通过碱性磷酸酶、免疫组织化学OCT-4、SOX2、NANOG对原代胚胎干细胞进行鉴定；通过茜素红S、Ⅰ型胶原、冯库萨染色对成骨诱导后的细胞(L-维生素C、β-磷酸甘油、地塞米松)进行检测；扫描电镜观察与纳米羟基磷灰石材料复合培养时细胞的形态及增殖情况。
结果与结论：拟胚体贴壁后7 d，各组碱性磷酸酶表达即发生变化，14 d后碱性磷酸酶表达逐渐增加，光镜下可见轮廓清晰大小不等的绿色结节。茜素红S染色镜下可见橙红色、边界清晰和直径大小不等的结节。冯库萨染色14 d可见细胞团内有黑色沉淀，21 d后黑色沉淀面积增大。胚胎干细胞与纳米羟基磷灰石材料体外复合培养1，3，5，7，10 d，细胞大部分呈细胞团样生长。提示在维生素C、β-磷酸甘油、地塞米松共同作用下，有效促进了胚胎干细胞成骨细胞的产生，胚胎干细胞与支架材料复合培养结合程度高，可用来构建组织工程骨。

关键词: 胚胎干细胞, 成骨细胞, 时段性, 诱导分化, 纳米羟基磷灰石, 干细胞

Abstract:

BACKGROUND: Nano-hydroxyapatite has good biocompatibility and bioactivity. It can tightly connect with in vitro tissues within a short time. Therefore, nano-hydroxyapatite has been widely used for bone tissue engineering.
OBJECTIVE: To investigate the time-dependent morphological characteristics of mouse embryonic stem cells differentiating into osteoblasts.
METHODS: P3 embryonic stem cells from C57 mouse were prepared into embryoid bodies. Primary embryonic stem cells were identified by alkaline phosphatase staining and immunohistochemical analysis (oct 4, sox2, nanog). After osteogenic induction by L-vitamin C, β-phosphoglycerol, and dexamethasone, cells were identified by alizarin red S, collagen Ⅰ, and von Kossa staining. The morphology and proliferation of cells when cultured with nano-hydroxyapatite were observed using scanning electron microscope.
RESULTS AND CONCLUSION: The expression of alkaline phosphatase showed no obvious change in each group within 7 days after adherence of embryoid bodies, but increased gradually after 14 days. Green nodules of varying size with clear border could be seen under optical microscope. After Alizarin red S staining, orange red nodules of varying size with clear border could be observed. Black precipitates appeared in the cell mass after von Kossa staining for 14 days, and amplified after 21 days. After co-culture with nano-hydroxyapatite for 1, 3, 5, 7, 10 days, the majority of embryonic stem cells grew in cell mass. These findings suggest that co-application of vitamin C, β-phosphoglycerol and dexamethasone effectively promote embryonic stem cells to differentiate into osteoblasts; embryonic stem cells composited onto scaffold material can be used in bone tissue engineering.

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