摘要
间充质干细胞的分化受多种因素的影响,其中力学是重要因素之一.为了探讨力学信号在间充质干细胞分化中的传导机制,利用力学加载装置在成骨细胞诱导体系条件下,对小鼠骨髓间充质干细胞系(D1细胞)加载不同拉伸应变,运用RT-PCR方法、Flou-3-AMCa2+染色技术、激光共聚焦显微镜技术及5种信号阻断剂,SB203580(p38MAPK特异抑制剂)、PD98059(MEK-1/2MAPK特异抑制剂)、LY294002(PI3Ks特异抑制剂)、细胞松弛素B(微丝结构阻断剂)、EGTA(Ca2+螯合剂),探讨力学信号的基本传导途径.结果显示:3%的拉伸应变能明显提高细胞内Ca2+水平;细胞微丝结构破坏后,延迟了3%拉伸应变对细胞内Ca2+水平增加的影响;细胞外Ca2+螯合后,拉伸应变不能促进细胞内Ca2+水平的升高,该结果提示,拉伸应变对细胞内Ca2+水平的影响主要通过细胞外Ca2+的内流实现.5种信号阻断剂能完全阻断干细胞向成骨细胞分化过程中关键基因骨钙蛋白(osteocalcin,OCN)和OSX mRNA的表达.p38MAPK途径、MEK-1/2MAPK途径被阻断后,拉伸应变激活了OCN和Osterix(OSX,与成骨细胞分化相关的关键基因)mRNA的表达;PI3Ks途径阻断后,拉伸应变部分激活了OCN和OSX mRNA的表达;细胞微丝破坏及胞外Ca2+螯合后,拉伸应变不能促进OCN和OSXmRNA的表达.上述结果表明:力学信号通过Ca2+信号、细胞微丝结构以及PI3Ks信号途径引起细胞的应答反应和生物学效应.
In order to study the effect of mechanical strain on mesenchymal stem cells （MSCs） differentiation into osteogenic, the cyclic substrate deformation instructment was applicated to MSCs line （D1 cell） in osteogenic media for different strain and times. The cells were cyclically stretched for periods of 1, 2, 3, 5 and 10 min and treated with cytochalasin B（CB） and EGTA. Ca^2＋, which were loaded with （15 Ixl） Fluo-3 AM, were detected by eonfocal laser scanning microscope（CLSM）. The signaling inhibitors, such as SB203580 （p38MAPK specific inhibitor）, PD98059 （MEK-1/2MAPK specific inhibitor）, LY294002 （PI3Ks specific inhibitor）, cytochalasin B （microfilament specific inhibitor）, EGTA （Ca^2＋ specific inhibitor）, were used to investigate mechanical signaling pathway by （3% 0.5Hz） Cyclic strain. The results indicated that 3% strain stimulus could induce increasement of [Ca^2＋] i- dependent fluorescence at 2 min. Its fluorescence intensity were 143.68,which were significantly different compared with 1,3,5,10min groups and 1% and 10% strain groups （P 〈 0.01）. The increasement of Ca^2＋ levels were delayed upto 10 min, when the cells were treated with CB. EGTA （5 mmol/L） could inhibited strain induction increasment of Ca^2＋ levels. Strain （3%, 0.5 Hz） could induce Ca^2＋ spark event in D1 cells at 2 min, however the cells were treated with CB Ca^2＋ spark event were delayed until 5min. The expression of OCN and OSX mRNA were completely inhibited by five specific inhabitors in unstrained condition. Inhibition of ERK1/2 and p38 pathway promoted the expression of OSX and OCN mRNA by strain.The expression of OSX and OCN mRNA to inhibit PIrKs pathway were partly activated by strain. Application of strain could not activate OSX and OCN mRNA expression when extracellular Ca^2＋ greatly inhibited with EGTA （5 mmol/L） and microfilament were damaged with CB.These results demonstrate that mechanical signals regulate MSCs function, suggested Ca^2＋ signaling, PI3Ks pathway and microfilament play an important role in transduction mechanical signal in MSCs differentiation.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2007年第7期718-723,共6页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30470438)
军事医学科学院创新基金项目(CX-04028).~~
