摘要
目的采用微载体旋转立体培养法对成人退变髓核细胞进行扩增,研究细胞凋亡情况,探讨髓核细胞凋亡在椎间盘退变中的作用。方法手术切除的退变髓核组织行原代培养,传代后随机进行单层培养和微载体旋转立体培养,采用Annexin-V-PI染色,流式细胞仪定量检测早期细胞凋亡,应用免疫组化方法检测凋亡抑制因子Bcl-2的表达,DeadEndTM比色法检测细胞晚期凋亡情况。结果流式细胞仪定量检测可见细胞在稳定生长期时,微载体旋转立体培养法较单层培养法髓核细胞的凋亡率低(P<0.05),在对数生长期两种培养方法无明显差别(P>0.05)。Bcl-2指标及DeathEndTM比色法检测显示在两个时期内,微载体旋转立体培养法培养的细胞凋亡率较低(P<0.05)。结论微载体旋转立体培养法可较好地降低髓核细胞凋亡率,是一种较有前途的髓核细胞培养方法,为组织工程化椎间盘髓核种子细胞的研究奠定了基础。
Objective To investigate the effects of nucleus pulposus cells apoptosis on degenerated intervertebral discs by using cell amplification with microcarrier system and studying cell apoptosis. Methods After primary culture of the degenerative nucleus pulposus tissues by operation, nucleus pulposus cells were randomly separated into two groups. Of the two groups, one was cultured with monolayer cluture, another with microcarrier system. In order to detect apoptosis of different stages, Annexin-V-PI staining was used for early stage apoptosis, DeadEndTM staining for late stage apopto- sis, immunohistochemical staining for Bcl-2 protein. Results Annexin-V-PI staining demonstrated that apoptosis rate of nucleus pulposus cultured with microcarrier system was lower in stable phase （ P 〈 0.05）. However, there was no signifi- cant difference between the two different culture methods in logarithmic growth phase （P 〉 0.05 ）. The data of Dead- EndTM staining and immunohistochemical staining showed that apoptosis rate of nucleus pulposus cultured with microcan＇ier system was lower in both phases （P 〈 0.05）. Conclusion Microcarrier system, which can efficiently reduce the apop- tosis rate of nucleus pulposus cells, is a promising culture method applied in tissue engineering of intervertebral discs.
出处
《山东大学学报（医学版）》
CAS
北大核心
2012年第12期98-102,共5页
Journal of Shandong University:Health Sciences
关键词
微载体
细胞凋亡
细胞培养
髓核
退变
Microcarrier
Apoptosis
Cell culture
Neucleus pulposus
Degenerative
