摘要
目的:探讨体外培养成肌细胞差速贴壁机制,研究成肌细胞不同贴壁速度与物理性状和成熟度间的关系。方法:分离SD大鼠后肢肌肉获得成肌细胞,体外培养第3代时采用差速贴壁法传代,分别在贴壁20min、1、2、26h时收获细胞,流式细胞仪按照一定阈值检测细胞的前向角散射(forward scatter,FSC)、侧向角散射(side scatter,SSC)分布及CD34、Pax7、M-cadherin的表达情况。将原代成肌细胞进行六步Percoll密度梯度离心分离,流式细胞仪检测各组细胞CD34、Pax7、M-cadherin的表达。结果:流式细胞仪检测显示,成肌细胞第3代经差速贴壁法传代后快速贴壁细胞在阈值范围内FSC的阳性细胞少,SSC的阳性细胞多,而贴壁较慢的细胞则反之,26h后还未贴壁的细胞在阈值范围内FSC、SSC细胞分布更少;说明早期贴壁细胞小,所含颗粒多,稍晚贴壁细胞面积大,所含颗粒少,26h后还未贴壁是更为幼稚的细胞。CD34、Pax7等阳性细胞主要出现在较晚贴壁的细胞中。流式细胞仪检测Percoll密度梯度离心分离原代成肌细胞的不同亚群,结果显示在15%～25%Percoll分离组分中富集CD34、Pax7阳性细胞。结论:细胞贴壁速度与物理性状和成熟度密切相关,可以采用15%～25%Percoll密度梯度离心法快速分离具有干细胞特征的肌肉形成细胞。
Objective ：To seek into the mechanism of the differential adherent velocity of myoblast cultured in vitro. Methods： Myoblasts of the third passage were transferred according to the different adherent velocities and acquired at 20 rain, 1,2 and 26 h. The forward scatter, side scatter distribution and the expressions of CD34, Pax7 , M-cadherin were evaluated by flow eytometry. Then the primary cultured muscle cells were isolated with muhi-Percoll density gradient centrifugation and examined by flow cytometry. Results：The rapidly adherent cells were smaller with thick granules, while the bigger cells assembled with thin granules took more time to adhere because of the buoyancy. The CD34, Pax7 positive cells were mainly presented in the later adherent fractions. CD34, Pax7 positive cells were mainly enriched in the 15% -25% fraction. Conclusion：The adherent velocity of myogenic cells are closely related with physical characteristics and extent of maturation. 15% -25% Pereoll density centrifugation can be used to quickly acquire stem-like cells from muscle.
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2008年第6期775-779,共5页
Journal of Practical Stomatology
基金
教育部博士学科专项基金项目(编号:20050025003)
