改良贴壁组织块法与改良Ⅰ型胶原酶消法对成骨细胞增殖效果的比较研究  

Comparison of Effects of Two kinds of Culture Methods on Proliferation of Osteoblast

作　　者：倪滔[1] 张文丽[1] 罗应伟[1] 

机构地区：[1]昆明医科大学口腔医学院,云南昆明650031

出　　处：《昆明医科大学学报》2014年第6期23-28,共6页Journal of Kunming Medical University

基　　金：云南省应用基础研究基金资助项目(2010ZC116);昆明医科大学研究生创新基金资助项目(2013N04)

摘　　要：目的 比较改良贴壁组织块法与改良Ⅰ型胶原酶消法对SD大鼠颞下颌关节髁突软骨下骨成骨细胞的增殖效果.方法 取10只出生1 -3dSD乳鼠颞下颌关节髁突软骨下骨,将骨片平分成2份,分别采用改良贴壁组织块法和改良Ⅰ型胶原酶消法培养颞下颌关节髁突成骨细胞,并标记为Ⅰ组和Ⅱ组,通过细胞形态学观察、HE染色、ALP染色鉴定成骨细胞.取第4代成骨细胞进行细胞计数、MTT生长曲线及流式细胞仪周期分析,比较2种原代培养方法对成骨细胞增殖能力的异同.结果 （1）Ⅰ组和Ⅱ组培养的细胞经鉴定均为成骨细胞; （2） Ⅱ组成骨细胞总量约为Ⅰ组细胞总量的1.5倍; （3） Ⅱ组成骨细胞MTT生长曲线较Ⅰ组细胞提前2d进入对数生长期及达到生长高峰; （4）2组细胞在达到第4代时,细胞周期含量差异无统计学意义（P＞0.05）.结论 改良贴壁组织块法和改良Ⅰ型胶原酶消化法均适用于颞下颌关节软骨下骨成骨细胞的培养,但改良Ⅰ型胶原酶消化法可以更快速有效地获得大量成骨细胞.Objective To compare the effects of improved tissue explants adherent method and improved type-Ⅰ collagen enzyme digestion method on proliferation of condylarsubchondral bone osteoblasts of SD rat＇s Temporal mandibular joint （TMJ） Methods Ten neonatal （1-3 d） SD rats were executed and their TMJ condylar were fetched out.The bone fragments were equally divided into 2 parts and names group Ⅰ and group Ⅱ.Group Ⅰ was cultured by tissue explants adherent method,and Group Ⅱ by type-Ⅰ collagen enzyme digestion method.The cell shape,HE staining,ALP staining were used to identify osteoblast.The cells counting,MTT growth curve and cell cycle analysis of the fourth generation of osteoblast were observed.Results （1） Identification：Group Ⅰ and Group Ⅱ had positive expression,the cells were osteoblasts.（2） Cell counting：Group Ⅱ had about 1.5 times as many cells as comparing with group Ⅰ.（3） MTT growth curve：The cells of Group Ⅱ entered logarithmic phase and summit of growth in two days in advance comparing with group Ⅰ.（4） Cell cycle analysis：There was no significant difference in 2 groups （P＞ 0.05）.Conclusion Improved tissue explants adherent method and Type-Ⅰ collagen enzyme digestion method are suitable for condylar subchondral bone osteoblasts culture of SD rat＇s TMJ,but improved type-Ⅰ collagen enzyme digestion method can get more osteoblastes in short time and can provide more cells for laboratory inverstigation.

关 键 词：改良贴壁组织块法 改良Ⅰ型胶原酶消法 髁突 成骨细胞 增殖 细胞周期 

分 类 号：R331[医药卫生—人体生理学]