流体静压力及雌激素共同作用对骨髓间充质干细胞成骨及成软骨分化的影响

Combined effects of hydrostatic pressure and estrogen on osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells

目的观察流体静压力联合雌激素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、细胞骨架及成骨、成软骨向分化的影响,检测这两种刺激共同作用是否具有生物叠加效应。方法采用全骨髓贴壁分离法分离培养BMSCs,流式细胞仪进行BMSCs表面标志物分子鉴定。细胞分为对照组(C组)、1 nmol/L17β-雌二醇作用组(E组)、1 nmol/L雌激素受体拮抗剂TAM作用组(T组)、90 kPa压力加载1 h组(P组)、17β-雌二醇预处理12 h后再加压1 h组(P+E组)以及TAM预处理12 h后再加压1 h组(P+T组)。流式细胞仪检测各组细胞的细胞周期变化;FITC-鬼笔环肽染色后激光共聚焦显微镜下观察F-actin细胞骨架表达与重组情况;各组细胞经成骨诱导7 d和14 d后茜素红染色观察钙结节形成量、Real-time PCR检测成骨细胞标志基因Col I、ON、OPN及BSP的mRNA表达;成软骨诱导14 d和28 d后甲苯胺蓝染色检测蛋白多糖表达、Real-time PCR检测成软骨细胞标志基因Sox9、Aggrecan及ColⅡ的mRNA表达。采用SPSS 16.0对数据进行单因素方差分析,后续Dunnett t检验进行各时间点上实验组与空白对照组数据之间及各实验组之间的组间比较。结果流体静压力(90 kPa,1 h)及1 nmol/L 17β-雌二醇作用下细胞增殖能力以及细胞骨架的活性均增强,但并不具有生物整合效应。成骨诱导14 d后,钙结节形成。Real-time PCR结果显示,17β-雌二醇作用组中成骨标志基因Col I、ON、OPN及BSP的表达水平显著增加。两种刺激在成骨诱导分化7 d时均可提高标志基因的表达,具有生物协同效应,但成骨诱导14 d时两者则表现出相互拮抗的作用。成软骨诱导28 d后,甲苯胺蓝染色阳性。成软骨诱导过程中,单纯压力刺激组成软骨标志基因Sox9、Aggrecan及ColⅡ的表达显著升高,雌激素预调则削弱压力对间充质干细胞的促成软骨分化作用。结论流体静压力及雌激素仅在骨髓间充质干细胞成骨分化早期表现生物整合效应,而在增殖、细胞骨架活性方面则无此效应。在成骨分化晚期及成软骨分化中两种刺激因素表现出拮抗作用,雌激素可促成骨分化,而压力则表现为促成软骨分化的作用。

Objective To investigate the effect of hydrostatic pressure and estrogen on the proliferation, F-actin cytoskeleton, osteogenic and chondrogenic differentiation of rat bone marrow mesenchymal stem cells （BM- SCs）, and to test whether combined stimulation can exert the fortified stimulating effort on BMSCs. Methods BMSCs were separated by using the whole bone marrow culture method and purified by differential adherence method. BMSCs surface markers were detected by flow cytometer. BMSCs were randomly assigned to six groups.blank control group （Group C）, t nmol/L 1713-Estradiol treatment group （Group E）, 1 nmol/L tamoxifen treatment group （Group T）, 90 kPa pressure treatment group for ] h （Group P） ; 17β-Estradiol pretreatment for 12 h and 90 kPa pressure group for 1 h （Group P ＋ E） ; and tamoxifen pretreatmet for 12 h and 90 kPa pressure group for t h （ Group P ＋ T）. Cell cycle was measured by flow cytometry. Fluorescent staining under laser scan- ning confocal microscope observation was observed for F-actin cytoskeleton expression and re-assembly. Affer osteogenic differentiation for ？ d and ]4 d, calcified nodules were detected with alizarin red staining. Further, the osteogenic markers including Col I, ON, OPN and BSP were analyzed by real-time PCR. Following chondrogen- esis of BMSCs for ]4 d and 28 d, proteoglycan contents were detected with toluidine blue staining, and chondro- genic markers including Soxg, Aggrecan and Col II were evaluated by real-time PCR. ANOVAs followed by the Dunnett t tests were adopted for comparisons among subgroups. All the experimental data were analyzed by SPSS 16.0 software. Results Both hydrostatic pressure （90 kPa, 1 h） and 1 nmol/L 17β-estradiol could in- crease the proliferation of BMSCs and F-actin activation, but no bio-cooperation effects appeared. Calcified nod- ules were observed after ]4 d osteogenic induction. Real-time PCR showed the estrogen enhanced osteogenetic gene （Col I, ON, OPN and BSP） expression in 7 d and 14 d. Combined effects of pressure and estrogen showed synergistic improving effects on early osteogenetic differentiation, but oppositional effects on advanced osteogenetic differentiation. Toluidine blue staining was positive after 28 d chondrogenic induction. With the hy- drostatic pressure loading regime, the mRNA expression of chondrogenic genes （ Soxg, Aggrecan and Col 11 ） was increased significantly, but with oppositional effects from estrogen on advanced chondrogenic differentiation. Conclusions The superposition effects of mechanical stimulation and estrogen acting only enhanced the differen- tiation of BMSCs in the early osteogenetic differentiation, but no effect was found in the proliferation and F-actin activation. Hydrostatic pressure and estrogen show antagonistic action in advanced osteogenetic differentiation and chondrogenic differentiation. Estrogen promotes osteogenetic differentiation, while hydrostatic pressure can enhance chondrogenic differentiation of BMSCs.