长时间力学载荷抑制体外培养成骨细胞的增殖和分化  

Effects of Long-term Mechanical Loading on the Proliferation and Differentiation of Osteoblast Cultured in Vitro

作　　者：郭勇[1,2] 王亮[2] 汪洋[1] 赵玉敏[1] 张新昌[2] 张西正[2] 贝朝涌[3] 

机构地区：[1]桂林医学院生物技术学院基础医学院,广西桂林541004 [2]军事医学科学院卫生装备研究所 [3]桂林医学院附属医院骨科

出　　处：《中国运动医学杂志》2015年第7期675-680,共6页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金项目(No.11372351;No.11432016;No.81160223);广西高校科研基金(No.KY2015LX241)资助

摘　　要：目的：观测长时间生理强度力学载荷对体外培养小鼠成骨前体细胞MC3T3-E1增殖和成骨分化的影响,探讨长时间生理载荷影响骨组织的细胞生物学机理。方法：采用四点弯曲加载装置,向成骨前体细胞MC3T3-E1施加2000微应变（με）、0.5 Hz的周期性张应变,持续时间为0-24 h。然后采用MTT法检测细胞增殖活性;Annexin V-FITC/PI流式细胞术检测细胞凋亡;比色法检测碱性磷酸酶（ALP）和乳酸脱氢酶（LDH）活性;RT-PCR检测ALP、骨钙蛋白（OCN）、I型胶原（Col I）的m RNA表达;免疫印迹法检测OCN和Col I蛋白,以及骨形成蛋白表达。结果：短时间的力学载荷（12 h以内）对成骨前体细胞的增殖无影响,24 h的力学载荷抑制成骨细胞增殖活性,但效果不显著;0-24 h的力学载荷均对成骨细胞的凋亡率无明显影响。短时间力学载荷可提高ALP活性和ALP m RNA表达,12 h后开始下降;短时间的力学载荷可提高OCN和Col I的m RNA和蛋白水平,但载荷作用24 h后,OCN和Col I的蛋白表达开始降低。0-8 h的载荷促进骨形成蛋白2和4的表达,12 h载荷后,蛋白表达降低。另外,长时间载荷可提高细胞培养上清的LDH活性。结论：长时间的生理力学载荷对细胞增殖的抑制作用不显著,对细胞凋亡也没有明显影响,但可显著促进细胞坏死,并抑制细胞成骨分化。Objective To observe the effects of long-term physiological mechanical loading on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells in vitro in mice. Methods MC3T3-E1 cells were exposed to mechanical loading of 2000 microstrain（με）at 0.5 Hz for 0 to 24 hours using four-point bending loading device,and then the proliferative activity of the cells was assayed with MTT reagent,the apoptosis was detected with Annexin V-FITC/PI binding assay,the activity of alkaline phosphatase（ALP）and lactate dehydrogenase（LDH） was determined by colorimetric methods,the m RNA levels of ALP,osteocalcin（OCN）,collagen type I（Col I） by RT-PCR,and the protein levels of OCN,Col I,the bone morphogenetic proteins（BMPs） by immunoblotting. Results The cells proliferation changed insignificantly under short-term mechanical loading（within 12 hours） and tended to be inhibited under long-term mechanical loading（24hours）. The cells apoptosis rate changed insignificantly under 0 to 24 hours of mechanical loading. The activity and m RNA expression level of ALP elevated first and then decreased after 12 hours under short-term mechanical loading. The m RNA and protein expressions of OCN and Col I elevated first,and then increased under short-term mechanical loading,but began to decrease after 24 hours of mechanical loading. The protein expressions of both BMP-2 and BMP-4 elevated under 0 to 8 hours of mechanical loading,and then decreased after 12 hours of mechanical loading. Additionally,LDH activity in supernatant of cell culture medium elevated under the long-term loading. Conclusion Long-time physiological mechanical loading caused insignificant effect on osteoblast proliferation and cell apoptosis,but promoted the cell necrosis and inhibited osteoblastic differentiation.

关 键 词：力学载荷 成骨细胞 增殖 分化 凋亡 坏死 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]