体外双轴拉伸应变诱导大鼠骨髓间充质干细胞成骨特异性标志物的表达  

Effect of Biaxial Tensile Strain on Expression of Osteogenic Specificity Markers of Rat Bone Marrow-derived Mesenchymal Stem Cells in Vitro

作　　者：张梦颖[1] 李晋川[2] 李汇明[1] 武庚[1] 曹成建[1] 吴文超[3] 李良[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物医学工程研究所,成都610041 [2]四川大学建筑与环境学院,成都610065 [3]四川大学华西医院再生医学研究中心心血管疾病研究室,成都610041

出　　处：《生物医学工程学杂志》2016年第3期499-505,519,共8页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(11272225;11572209)

摘　　要：探讨体外双轴拉伸应变对大鼠骨髓间充质干细胞（rBMSCs）向成骨细胞分化的影响。分离4周龄SD大鼠BMSCs,将P3～P4代rBMSCs接种于DMEM-LG完全培养基的双轴拉伸应变细胞培养小室中。当细胞生长至亚融合状态后,给予细胞施加拉伸应变,频率为1 Hz,应变幅度为1%、2%、5%,每个应变幅度均分别加载2h/d、4h/d、6h/d,连续作用3d。以未受拉伸应变的rBMSCs为空白对照组。以未受拉伸应变,含100nmol/L雌二醇（E2）培养的rBMSCs为阳性对照组。通过实时荧光定量PCR和Western blot方法检测各组rBMSCs的碱性磷酸酶（ALP）、Ⅰ型胶原（ColⅠ）、Runt相关转录因子-2（Runx2）、骨钙蛋白（OCN）mRNA和蛋白的表达量。实验结果表明：（1）E2组rBMSCs中Runx2、ColⅠ、ALP、OCN mRNA及蛋白表达量均明显高于空白对照组（P〈0.05）;（2）1%拉伸应变组rBMSCs中ALP、Runx2mRNA和蛋白表达量均明显高于空白对照组（P〈0.05）,但与E2组相比明显降低（P〈0.05）;（3）2%拉伸应变组rBMSCs中ALP、ColⅠ、Runx2、OCN mRNA和蛋白表达量均明显高于空白对照组（P〈0.05）,其中,加载时间4h/d组rBMSCs中ColⅠ、Runx2 mRNA和蛋白表达量明显高于E2组（P〈0.05）;（4）5%拉伸应变组,加载时间2h/d、4h/d组rBMSCs中ALP、ColⅠ、Runx2mRNA和蛋白表达量均明显高于空白对照组（P〈0.05）,与E2组相比,加载时间4h/d组的ColⅠ、Runx2mRNA和蛋白表达量也明显上调（P〈0.05）。本实验结果提示,雌二醇和体外双轴拉伸应变均可以诱导rBMSCs向成骨细胞分化,且拉伸应变幅度2%、加载时间4h/d时,促rBMSCs成骨分化的作用最强。The purpose of this study was to investigate the effect of biaxial tensile strain on the osteogenic differentiation of rat bone marrow mesenchymal stem cells （rBMSCs） in vitro. The rBMSCs were isolated from tibia and femur of 4 weeks-old Sprague-Dawley （SD） rats. The rBMSCs were cultured in DMEM-LG complete culture medium and grew to subconfluence in the cell culture device for loading tensile strain. The biaxial tensile strain was applied to the rBMSCs for periods of 2, 4 and 6 hours every day, respectively, lasting 3 days. The amplitude of biaxial tensile strain applied to the rBMSCs were 1%, 2% and 5% respectively, at a frequency of 1 Hz. Unstrained rBMSCs were used as blank control （control group）. The rBMSCs cultured with DMEM-LG complete culture medium containing 100 nmol/L 13-Estradiol （E2） were used as positive control. The mRNA expression of alkaline phosphatase （ALP）, collagen type Ⅰ（Col Ⅰ, Runt-related transcription factor 2 （Runx2） and osteocalcin （OCN） was examined with real-time quantitative PCR and the protein expression of ALP, Col Ⅰ Runx2 and OCN was detected with Western blot method. The results showed as follws： （1） The mRNA and protein expression of the ALP, Col Ⅰ, Runx2, OCN were significantly higher in rBMSCs of the E2 group than those in the control group （P〈.05）. （2） The mRNA and protein expression level of the ALP, Runx2 were higher markedly in the 1% tensile strain groups than those in the control group （P〈0.05）, but lower than those in the E2 group （P〈0.05）. （3） The mRNA and protein expression level of the ALP, Col Ⅰ, Runx2, OCN were significantly higher in the 2% tensile strain groups than those in the control group （P〈0.05）, and the mRNA and protein expression level of Col I and Runx2 in the group applied with 2M amplitude of tensile strain for 4 h/d was significantly higher than those in E2 group （P〈0.05）. （4） The mRNA and protein expression level of the ALP, Col Ⅰ Runx2 were significantly highe

关 键 词：大鼠骨髓间充质干细胞 拉伸应变 Ⅰ型胶原 Runt相关转录因子-2 骨钙蛋白 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]