雌激素对鸡胚BMSCs诱导的成骨细胞增殖、凋亡及细胞周期的影响

doi:10.11843/j.issn.0366-6964.2014.02.005

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (2): 197-206.doi: 10.11843/j.issn.0366-6964.2014.02.005

雌激素对鸡胚BMSCs诱导的成骨细胞增殖、凋亡及细胞周期的影响

禹泽中^2#，王志美^1#，江莎¹，侯加法^1*

（1.南京农业大学动物医学院，南京 210095； 2.玉溪农业职业技术学院，玉溪 653100）

收稿日期:2013-09-24 出版日期:2014-02-23 发布日期:2014-02-23
通讯作者: 侯加法，教授，博士生导师，主要从事畜禽骨骼生物学研究，E-mail：jfhou@njau.edu.cn
作者简介:禹泽中（1975-），男，云南弥渡人，副教授，主要从事临床兽医教学和科研工作，E-mail：940906290@qq.com；王志美（1985-），女，山东枣庄人，硕士，主要从事畜禽骨骼生物学研究，E-mail：168shiying168@163.com。#为并列第一作者
基金资助:
国家自然科学基金（31272618；30972234；30671546）；江苏高校优势学科建设工程资助项目（PAPD）

Effect of 17β-E₂ on Proliferation，Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos

YU Ze-zhong^2#，WANG Zhi-mei^1#，JIANG Sha¹，HOU Jia-fa^1*

(1.College of Veterinary Medicine，Nanjing Agricultural University，Nanjing 210095, China;2.Yuxi Agriculture Vocation-Technical College，Yuxi 653100, China)

Received:2013-09-24 Online:2014-02-23 Published:2014-02-23

摘要/Abstract

摘要：

本试验建立鸡胚骨髓间充质干细胞（BMSCs）体外培养及其诱导成骨细胞方法，以探讨雌激素对BMSC诱导的成骨细胞的影响。以全骨髓进行体外贴壁培养、传代，取第3代细胞进行成骨细胞诱导培养，用倒置显微镜观察其形态，MTT法测定细胞增殖率，流式细胞仪检测细胞周期率。通过碱性磷酸酶、Van-Gieson及茜素红染色等方法进行成骨细胞鉴定。将诱导的成骨细胞分为5个试验组，分别添加10^-7、10^-8、10^-9、10^-10和10^-11 mol·L^-1浓度的17β-E₂于成骨细胞诱导培养基，并设置对照组。用MTT法和PNPP法测定不同浓度17β-E₂对诱导成骨细胞增殖和分化的影响，判断其最有效浓度。并用该有效浓度作用于诱导培养的成骨细胞，通过流式细胞术观察该浓度17β-E₂对诱导的成骨细胞凋亡的影响。结果表明，鸡胚全骨髓贴壁培养法可获得BMSCs，经典培养基诱导培养获得的细胞具有成骨细胞典型的生物学特征，其ALP、I型胶原及钙化结节染色均为强阳性。10^-9mol·L^-117β-E₂可促进诱导的成骨细胞增殖、分化，抑制其凋亡。经纯化处理的鸡BMSCs可诱导成骨细胞，且雌激素可提高BMSCs诱导的成骨细胞功能。

Abstract:

To investigate the effect of 17β-E₂on osteoblasts induced by chicken bone marrow mesenchymal stem cells(BMSCs) ，the method of in vitro culture of BMSCs and its inducement of osteoblasts were established in this experiment.Chicken BMSCs were purified with whole marrow adherent culture and subculture in vitro.The third generation of cells of BMSCs were selected for inducing osteoblastic culture.The growth and morphology，the cell proliferation rate and the cell cycle of osteoblasts induced-BMSCs were detected with inverted phase contrast microscope，methyl-thiazol-tetrazolium (MTT) and flow cytometry，respectively.Induced-BMSCs obsteoblasts were identified by alkaline phosphates (ALP)，Van-Gieson staining and Alizarin red staining，respectively.The induced osteoblasts were divided into 5 experimental groups and cultured with osteogenic differentiated medium containing different 17β-E₂ concentrations of 10^-7，10^-8，10^-9，10^-10，10^-11 mol·L^-1 for each group，respectively.Cells cultured without 17β-E₂ as a blank control group.Effects of different concentrations of 17β-E₂on the proliferation and differentiation of induced-BMSCs osteoblasts were measured by MTT and PNPP for finding the most effective concentration.Then this effective concentration of 17β-E₂was used for culturing induced-BMSCs osteoblasts，and the percentages of apoptotic cells were measured by flow cytometry.The results showed that BMSCs could be purified with whole marrow adherent culture.Osteoblasts could be induced from purified BMSCs using classic differentiated medium and were typically characteristic of the osteoblastic biology.The induced-BMSCs osteoblasts’ ALP，collagen typeⅠand calcified nodules’ staining were strongly positive compared with control group.10^-9 mol·L^-1 17β-E₂concentration represented the most action of promoting proliferation and differentiation and inhibiting apoptosis of induced-BMSCs osteoblasts among the other concentration groups.Thus，it is shown that osteoblasts can be induced from purified chicken BMSCs and estrogen can enhance the function of induced-BMSCs osteobalsts.

中图分类号:

S831
S814.1

禹泽中，王志美，江莎，侯加法. 雌激素对鸡胚BMSCs诱导的成骨细胞增殖、凋亡及细胞周期的影响[J]. 畜牧兽医学报, 2014, 45(2): 197-206.

YU Ze-zhong，WANG Zhi-mei，JIANG Sha，HOU Jia-fa. Effect of 17β-E₂ on Proliferation，Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(2): 197-206.

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雌激素对鸡胚BMSCs诱导的成骨细胞增殖、凋亡及细胞周期的影响

Effect of 17β-E₂ on Proliferation，Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos

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雌激素对鸡胚BMSCs诱导的成骨细胞增殖、凋亡及细胞周期的影响

Effect of 17β-E2 on Proliferation，Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos

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Effect of 17β-E₂ on Proliferation，Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos