牵张应力对大鼠骨髓基质干细胞化学诱导成脂分化作用的影响  

Mechanical strain inhibits adipogenic differentiation of bone mesenchymal stem cells during cytochemical induction in Sprague Dawley rats

作　　者：梁亮[1] 许长鹏[1] 黎润光[1] 余斌[1] 

机构地区：[1]南方医科大学南方医院创伤骨科,广州510515

出　　处：《中华创伤骨科杂志》2014年第4期334-339,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（81101366、81101366[匹配课题LT201212]、81272052）;广东省自然科学基金（S2011010003855）;广东省产学研结合项目（20118090400016）

摘　　要：目的探讨周期性牵张应力对Sprague Dawley（SD）大鼠骨髓基质干细胞（BMSCs）成脂分化的影响。方法选取4～5周龄，体质量为80—100g的sD大鼠6只，雌雄不限。体外培养sD大鼠BMSCs至第3代，采用流式细胞仪检测CD29、CD34、CD44、CD45，并利用Flexcell-5000XT牵张应力系统加载力学信号（应力拉伸幅度为5％，6h／d，10次／min，持续3d或5d，正弦波形），采用显微镜观察BMSCs的形态。将实验对象BMSCs根据培养基不同（普通培养基和促成脂分化培养基）、是否加载牵张应力及牵张应力持续时间（3、5d）共分为8组，受力组为实验组，未受力组为对照组。油红O染色法观察各组细胞成脂分化水平。荧光定量聚合酶链式反应（Q．PCR）法检测BMSCs成脂分化指标：过氧化物酶体增殖物激活受体（PPAR．叮）、脂联素、CAAT增强子结合蛋白a（c／EBPct）的mRNA表达水平。结果镜下观察原代BMSCs生长良好、形态排列正常。流式细胞检测结果显示第3代BMSCs的CD29、CD34、CD44、CD45的表型阳性率分别为93．6％、5．3％、91．4％和4．6％。BMSCs受成脂诱导剂诱导3、5d，细胞内出现脂滴并为油红O所染色，PPARγ-2、脂联素和C／EBPα的mRNA表达均明显增高，而在诱导过程中。加载应力信号后，未见明显脂滴出现，应力刺激3d和5d，细胞内PPARγ-2、脂联素、C／EBPct的基因表达分别下降64．80％、84。45％、67．12％和70．11％、68．12％、80．39％，3种基因的表达明显受到抑制。结论适当的牵张应力可抑制BMSCs向脂肪方向分化的能力。Objective To study the effect of mechanical strain on the adipogenic differentiation of bone mesenchymal stem cells （BMSCs） in Sprague Dawley （SD） rats. Methods We isolated and cultured primary BMSCs from 6 SD rats （random gender） weighing from 80 to 100 grams and aged from 4 to 5 weeks old. After the levels of CD29, CD34, CD44 and CD45 in the third passage of BMSCs were determined by flow cytometry, mechanical stretch signals （5% strain, sine wave type, 10 cycles per minute for 6 hours daily, and lasting for 3 or 5 days） were delivered to cultures using the Flexcell-5000X tension system. After microscopic observation was performed, the BMSCs were divided into 8 groups according to their medium, mechanical strain and/or duration of mechanical strain. Experimental groups received mechanical strain while control ones no mechanical strain. The adipogenie level was observed in every group by oil red O staining; the mRNA expression levels of PPARγ-2, adiponectin and C/EBPα were measured by RT-PCR. Results Microscopic observation showed that the primary MSCs were morphologically normal; flow cytometry showed that the phenotypic positive rates of CD29, CD34, CD44 and CD45 in the third generation of BMSCs were 93.6%, 5.3%, 91.4% and 4.6%. After adipogenic differentiation induction for 3 and 5 days, the lipid was appreciable in BMSCs and the levels of intracellular PPARγ-2, adiponectin and C/EBPα mRNA were significantly higher. However, their expression was inhibited significantly and the cells stained by oil red O were hardly shown after the BMSCs were stimulated by mechanical strain. The expression levels of PPARγ-2, adiponectin and C/EBPα mRNA were reduced respectively by 64.80%, 84.45% and 67. 12% after mechanical strain stimulation for 3 days and reduced by 70. 11%, 68. 12% and 80. 39% after mechanical strain stimulation for 5 days. Conclusion The data suggest that proper mechanical strain may inhibit the adipogenic differentiation of BMSCs in rats.

关 键 词：应力 物理 间质干细胞 成脂分化 过氧化物酶体增殖物激活受体 脂联素 

分 类 号：R318[医药卫生—生物医学工程]