沉默Kif5b赋予MDCK细胞干性
投稿时间:2015-10-14  修订日期:2015-11-03  点此下载全文
引用本文:崔菊,金国祥,雷霆,王在,黄建东,蔡剑平.沉默Kif5b赋予MDCK细胞干性[J].医学研究杂志,2016,45(5):58-62
DOI: 10.11969/j.issn.1673-548X.2016.05.014
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作者单位E-mail
崔菊 100730 北京医院、卫生部北京老年医学研究所、卫生部老年医学重点实验室
香港大学李嘉诚医学院生物医学科学学院 
 
金国祥 香港大学李嘉诚医学院生物医学科学学院  
雷霆 香港大学李嘉诚医学院生物医学科学学院  
王在 香港大学李嘉诚医学院生物医学科学学院
100029 北京, 中日友好医院临床医学研究所 
 
黄建东 香港大学李嘉诚医学院生物医学科学学院
518055 中国科学院深圳先进技术研究院 
jdhuang@hku.hk 
蔡剑平 100730 北京医院、卫生部北京老年医学研究所、卫生部老年医学重点实验室 caijp51@vip.sina.com 
基金项目:国家自然科学基金青年基金资助项目(31400995);北京市自然科学基金资助项目(7154234)
中文摘要:目的 观察沉默Kif5b对上皮细胞MDCK的影响并探讨Kif5b表达水平与细胞分化程度的相关性。方法 设计构建靶向Kif5b基因的短发卡RNA(shRNA)质粒载体,并转染犬肾上皮细胞株MDCK,检测其对Kif5b表达的抑制作用;采用Western blot法检测E-钙黏蛋白(E-cadherin),N-钙黏蛋白(N-cadherin),波形蛋白(vimentin)在各组细胞中的表达差异;通过细胞诱导液培养检测细胞分化为脂肪细胞和成骨细胞的能力。结果 从形态上看,对照组MDCK细胞间紧密相连,呈鹅卵石状分布,而Kif5b沉默的细胞呈成纤维细胞状;与对照组相比,转染Kif5b-shRNA显著抑制Kif5b的表达,同时E-cadherin的表达下降,N-cadherin和vimentin的表达上升,细胞抵抗胰蛋白酶消化的能力降低;沉默Kif5b的MDCK细胞能被分化为脂肪细胞和成骨细胞;此外,Kif5b在MDCK中的表达量显著高于小鼠间充质干细胞(mesenchymal stem cell)。结论 在MDCK细胞中沉默Kif5b诱导MDCK细胞经历上皮细胞-间充质转化(epithelial-mesenchymal transition)过程,去分化为间充质干细胞样细胞,去分化的细胞能够被转分化为特化的脂肪细胞和成骨细胞。Kif5b的表达量与细胞的分化程度呈正相关。
中文关键词:Kinesin-1  MDCK细胞  上皮细胞-间充质转化  间充质干细胞
 
Knockdown of Kif5b Confers Stem Cell Properties to MDCK Cells
Abstract:Objective To investigate the function(s) of Kif5b in MDCK cells and to examine the relevance of Kif5b expression level and cell stemness. Methods Stable Kif5b deficient MDCK cells were generated by using pSilencer 3.1-H1 hygro siRNA expression system. Western blot was used to examine the knockdown efficiency of Kif5b and the expression levels of EMT markers, such as E-cadherin, N-cadherin, vimentin and β-catenin. The strengths between cell contacts were evaluated by trypsin digestion. The ability of the cells to be differentiated to adipocyte and osteoblast was examined by culturing the cells with differentiation medium followed by Oil red O or Alizarin Red S staining. Furthermore, the expression levels of Kif5b in MDCK cells and mouse mesenchymal stem cells (MSC) were compared by Western blot. Results MDCK cells transfected with Kif5b-shRNA had 90% knockdown efficiency. Kif5b deficient MDCK cells had profound changes in cell morphology resembling mesenchymal cells, decreased resistance to trypsin digestion and acquired the EMT phenotype, which was characterized by down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. Besides, these cells gained the ability to be differentiated to adipocyte and osteoblast, resembling mesenchymal stem cells. Furthermore, expression level of Kif5b in MDCK cells was significantly higher than that in MSC. Conclusion Silencing of Kif5b in MDCK cells triggers Epithelial-Mesenchymal transition and confers mesenchymal stem cell properties. The expression level of Kif5b is negatively related to cell stemness.
keywords:Kinesin-1  MDCK cell  Epithelial-mesenchymal transition  Mesenchymal stem cell
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