摘要
目的观察骨髓基质干细胞(BMSCs)经Ⅰ型胶原作用后细胞粘附能力增加、细胞骨架肌动蛋白的表达和细胞内钙水平变化情况。方法以密度梯度离心法获得成年兔BMSCs,在含10%小牛血清的RPMI 1640培养基条件下,以一定初始浓度分别接种盖玻片(实验组包被Ⅰ型胶原,对照组不做处理)。培养一定时间后,检测细胞的粘附情况,分别以免疫荧光技术并参照El-Amin方法观察细胞骨架肌动蛋白结构分布、以Fluo-3作为钙指示剂行细胞内游离Ca^(2+)测定。结果观察细胞接种12 h内细胞骨架肌动蛋白结构分布显示：细胞接种6 h时,实验组Ⅰ型细胞占56%,Ⅱ型细胞占44%；而对照组Ⅰ型细胞则占79%,Ⅱ型细胞为21%。接种12 h时,实验组Ⅰ型细胞占11%,大部分为Ⅱ型细胞；而对照组Ⅰ型细胞只占45%。提示材料经Ⅰ型胶原修饰后,细胞多数表现为圆形,铺展良好,细胞骨架肌动蛋白的构建活跃。细胞内游离Ca^(2+)测定显示：在Ⅰ型胶原存在的条件下生长的细胞内荧光明显增强,说明细胞内游离Ca^(2+)浓度较对照组高。结论兔BMSCs经Ⅰ型胶原作用后,细胞粘附能力增强不仅与细胞骨架肌动蛋白的构建活跃有关,还与细胞内游离Ca^(2+)浓度明显提升有关。
Objective To investigate the stimulative effects of Collagen Ⅰ on the increased adhesion of rabbit bone marrow stromal cells （BMSCs）, cytoskeleton actin organization and intracellular free Ca^2＋ concentration. Methods The third generation BMSCs isolated from mature rabbits were cultured at different initial concentrations on cover-slice coated by collagen Ⅰ in RPMI1640 containing 10% fetal calf serum, and cultured on the same kind of cover-slice untreated with collagen Ⅰ as control. The cells adhesive behavior at different times was assessed. Cellular actin organization was described as either type Ⅰ or type Ⅱ cells. In general, type Ⅰcells are round and represent a preliminary stage of actin assembly, while type Ⅱ cells are elongated with organized actin fiber network. At the same time intracellular free Ca^2＋ concentration was measured by using calcium fluorescent probe Fluo-3/AM and laser confocal microscope. Results We found more type Ⅱ ceils in BMSCs cultured with collagen type Ⅰ six hours after culture than in the control group. At 12 hours 89% of the BMSCs were type 11 cells, while only 55% were type Ⅱ ceils in the control group. This indicated active cellular actin organization after being modified by collagen type Ⅰ. We also found that the BMSCs cultured with collagen type Ⅰ increased intracellular free Ca^2 ＋ concentration in monolayer culture. Conclusions Collagen Ⅰ is effective in promoting the cellular adhesion, which suggests that a kind of internal relationship or cross-talk may exist between cellular actin organization, intracellular free Ca^2＋ concentration and cell adhesion. Further study, however, is needed.
出处
《中华创伤骨科杂志》
CAS
CSCD
2006年第6期549-552,共4页
Chinese Journal of Orthopaedic Trauma
基金
国家高技术计划新材料领域"九五"项目(009-0160)
军队医药卫生科研基金重点课题(01Z079)
