E-钙粘附素/β-连环素复合体对孕鼠神经干细胞迁移和归巢的影响  

The impact of E- cadherin /β- catenin complex to the pregnant mice neural stem cell on migration and homing

作　　者：吴众[1] 崔银江[1] 刘杰[1] 王建广[1] 陈栋[1] 余磊[1] 张磊[1] 李少华[1] 

机构地区：[1]同济大学附属第十人民医院骨科,上海市200072

出　　处：《中国矫形外科杂志》2014年第8期747-752,共6页Orthopedic Journal of China

基　　金：国家自然科学基金面上项目(编号:81271990)

摘　　要：[目的]干细胞的微环境是影响干细跑行为的关键因素之一，微环境中的粘附分子E-钙粘附素是其重要组成部分，在微环境中扮演重要角色，它功能的发挥离不开β-连环素，常作为一种复合体研究他们的功能。本课题采用实时定量PCR技术测量神经干细胞在原代和传代4—6代后E-钙粘附素和β-连环素的表达量是否有所变化，同时在蛋白表达水平借助流式细胞技术测量β-连环素相应的表达变化，进而判断这两种粘附分子在神经干细胞的传代过程中有何影响，为神经干细胞的迁移、植入的研究打下基础。[方法]孕鼠神经干细胞若干，为了说明前后实验细胞均为神经干细胞，本实验在取得细胞之时，立刻取少量神经干细胞行特异性Nestin染色，证明其并未分化。取一部分进行实时定量PCR检测E-钙粘附素和β-连环素的含量，毕竟这是在RNA水平借助实时定量PCR来测量的，为了佐证本结论，在蛋白水平借助流式细胞计数测量β-连环素的含量变化，β-连环素是一种胞浆蛋白，在细胞核内也有少量的表达，且核内核外保持一种动态的平衡，口-连环素处于E-钙粘附素／β-连环素复合体的核心部位，它表达的变化也间接体现了E-钙粘附素／β-连环素复合体的变化。剩余的继续培养，保持传代不分化，传代4—6代，即2周左右，再一次重复前面的操作，统计前后的结果有无差别。[结果]原代及传代培养形成的克隆球进行神经干细胞特异性抗体巢蛋白（Nestin）免疫荧光染色时发绿色荧光，呈阳性反应。应用实时定量PCR技术发现神经干细胞传4—6代后，E-钙粘附素和β-连环素的含量明显增多，且差别具有统计学意义（P〈0．05）；相应的流式细胞技术测量β-eat的含量变化，发现β-连环素的含量也明显增多，且差别同样具有统计学意义（P〈0．01）。[结论]试验中神经干细�[ Objective ] Stem cell niche, a specialized ent in which stem cells reside, plays a key role in regu- lating the behaviors of stem cells. E - cadherin, which often times forms complexes together withβ - catenin, is an important component of stem ceils niehe. This issue uses RT - PCR measurement of neural stem ceils in primary and passaged after 4 - 6 on E - cad and β - eat expression and the change in the level of protein expression by flow cytometry measureement of the corre- sponding β- eat expression to determine the impact of these two adhesion molecules in neural stem cells in the process of pas- sage of neural stem cells and to lay the foundation for the future research for migration and implants. [ Method] Firstly a number of pregnant rats were taken, neural stem cells was extracted for Nestin staining to definite that neural stem cells are undifferenti- ated. Then part of them was taken to do RT - PCR detection of E - cad and β- cat content. To assess whether the mRNA levels increase in neural stem cells after passage will lead to a difference at protein level, we measured the expression levels in neural stem eeUs by flow eytometry. Since E - eadherin and β - catenin proteins form complexes,we used flow eytometry to detect the catenin exnmssion levels which catenin expression levels which would represent the level of E - cadherin/β- catenin complexes.β- catenin was mainly a cyto-plasmic protein which could also be observed in nucleus at low levels. There was a dynamic equihbrium ofβ - catenin between the cytoplasmic pool and nuclear pool. The remaining of the cells continued to be fostered to maintain the passage 4 - 6 on behalf of passage and did not divide, that is, about twoweeks. At last we repeated the previous operation, found out whether there were differences between the results. [ Result I Neuro- spheres formed from embryonic rat spinal cord neural cells express neural stem cell marker nestin and were fixed and immunos- tained for Nestin （green）. Application of RT - PCR found that a

关 键 词：E-钙粘附素 Β-连环素 神经干细胞 脊髓损伤 

分 类 号：R651.2[医药卫生—外科学]