【作者】 李建华；

【导师】 林新平；

【作者基本信息】 浙江大学 ， 口腔正畸学， 2012， 硕士

【摘要】 目的：初步探讨流体静压力和转化生长因子-β3(transforming growth factor-β3, TGF-β3)在诱导骨髓间充质干细胞(BM-MSC)向软骨细胞分化过程中的内在联系,研究BM-MSCs的分化机制,为最终控制MSCs的定向分化打下基础。方法：取健康雄性SD大鼠骨髓,采用贴壁法分离培养大鼠MSCs, MTS法测定细胞生长曲线,流式细胞仪检测细胞表面抗原。取第四代MSCs培养于三维藻酸盐凝珠,采用Hoechst333258/PI荧光染色和Annexin V/PI直标双染流式细胞仪检测细胞活性。用转化生长因子β3(TGF-β3)和流体静压力诱导MSCs向软骨细胞特征方向分化。利用组织化学染色和Real time荧光定量RT—PCR测定其向软骨细胞特征方向分化过程中转录因子SOX9、特异性细胞外基质II型胶原(collageII)和聚集蛋白聚糖(aggrecan)的表达情况。实验分成3个实验组和对照组,分别为：TGF-β3(10ng/ml)；静压力组(100kPa,4h/d)；TGF-β3和静压力联合组和对照组(不添加任何生长因子和静压力)。各组于诱导的第7天,分别进行HE染色,甲苯胺蓝染色及PAS染色(Periodic Acid-Schiff stain);于诱导第1、3和7天分别提取各组细胞总RNA,进行RT-PCR扩增,检测Sox9,COLⅡ和AGG的表达量。结果：本实验成功分离、纯化、培养了大鼠MSCs,原代及传代细胞贴壁生长,细胞生长状态良好,形态为长梭性。流式细胞仪检测第2、3、4代大鼠MSCs细胞表面标记：CD29、CD90阳性(≥80%、≥98%、≥98%),CD11、CD45阴性(≤2.0%、≤0.4%、≤0.2%)。MTS法细胞增殖实验结果显示大鼠MSCs生长性状稳定,第1、3、5代细胞的生长曲线相似,传代后2天为潜伏期,第4-6天进入对数增殖期,第8天后进入平台期。倒置显微镜观察藻酸盐凝珠内的MSCs,细胞分布均匀,形状为圆形或椭圆形,胞体透亮,随着培养时间的增加,局部可见细胞团簇。Hoechst333258/PI荧光染色显示藻酸盐凝珠内大部分细胞呈低蓝色,核呈正常结构,随培养时间的增加未见明显核呈红色荧光的细胞增多。AnnexinV/PI直标双染流式细胞仪检测结果：对照组、压力组和细胞因子组细胞包裹于藻酸盐凝珠培养7天后,存活率较高,分别为79.7%,71.4%和89.4%。组织切片HE染色：TGF-β3,压力,TGF-β3+压力组与对照组的凝珠内细胞分布均匀,形状为圆形或椭圆形,细胞呈皱缩状,细胞周围为空隙,形似软骨陷窝。甲苯胺蓝染色和PAS染色：TGF-β3,压力,TGF-β3+压力组结果阳性,证实骨髓间充质干细胞经诱导后细胞呈现软骨细胞特征。RT-PCR结果表明TGF-β3,压力,TGF-β3+压力组细胞在诱导1、3、7天后均有Sox9、COL Ⅱ和AGG表达,在整个实验期间内,随着培养时间的增加,各实验组的Sox9和COL Ⅱ基因表达量呈现不同程度的增加。第3、7天的Sox9、COL Ⅱ和AGG mRNA表达水平较对照组高,其中TGF-β3+压力组的Sox9基因表达量,压力组和TGF-β3+压力组的COL Ⅱ基因表达量,TGF-β3和TGF-β3+压力组的AGG基因表达量显著增加,具有统计学差异(P≤0.05)。另外,藻酸盐凝珠培养第三天,TGF-β3+压力组的COL Ⅱ和AGG基因的表达水平明显高于单纯TGF-β3组和压力组(P≤0.01),说明压力和细胞因子的联合作用能获得更为显著的效果。结论：藻酸盐凝珠可以作为大鼠MSCs三维立体培养的载体,为其提供粘附生长、向软骨细胞分化、合成和分泌细胞外基质(ECM)的场所。细胞因子TGF-β3能够有效促进大鼠MSCs向软骨细胞分化,表达软骨细胞特异性的基因和蛋白；静压力促进MSCs表达Sox9, COL Ⅱ和早期AGG的表达。静压力和TGF-β3的组合作用较复杂,不是简单的协同或拮抗作用。因此,生物因子TGF-β3和合适的压力刺激可诱导间充质干细胞向软骨细胞分化并维持其细胞表型,是维持关节软骨稳态的重要因素。更多还原

【Abstract】 Objectives:Bioactive factors, such as TGF β3as well as mechanical factors i.e. hydraulicpressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells(BMSCs). Since these factors are intracellularly transduced through different mechanisms, it is hypothesized that TGF β3and hydraulic pressure may act synergistically on chondrogenic differentiation of BMSCs. The purpose of this study was to investigate the effects of Hydrostatic pressure and TGF-β3on chondrogenesis employing rat Bone-Marrow Derived Mesenchymal Stem Cells(BM-MSCs).METHODS:BM-MSCsderived from the tibias and femurs of rats, were grown in α-modified Eagle medium supplemented with10%fetal calf serum, which were characterized through flow cytometry using specific antibodies. BM-MSCs encapsulated in alginate beads, were divided into four groups:control; transforming growth factor (TGF-β3)(with TGF-P3treatment); loading (with stimulation of Hydrostatic pressure loading); and TGF-β31oading (with TGF-β3treatment and loading stimulation) groups. In the loading experiment, specimens were subjected to100kPaby an in-house designed pressure chamber for4hours a day.Experiments were conducted for1,3, and7consecutive days. Cell differentiation was verified through histochemistry, quantitativereverse transcription-polymerase chain reaction (RT-PCR) analysis.RESULT:MSCs of passage2.3,4were positive(≥80%、≥98%、>98%)for CD29, CD90and negative(≤2.0%、≤0.4%、≤0.2%)for CD11and CD45. Staining of the extracellular matrix was positive. TGF-(33, loading, and TGF-β3loading exhibitedsignificantly higher levels of expressions of chondrogenicmarkers (collagen II and AGG) at the third and seventh day.TGF-β3loading exhibited higher collagen type II and AGG than medium with TGF—β3alone or loading at the third day.CONCLUSION:The used concentrations of TGF-β3and parameters for hydraulicpressure were able to induce and modulatechondrogenesis of rat BMSCs in some extend, but the co-effect of TGF-β3and hydraulicpressure is complicated, there are no synergistic effects were observed.更多还原