动态压应力与PTHrP对体外培养大鼠前软骨干细胞生物学特性的影响
发布时间：2018-10-04 21:18
【摘要】：目的平板及微球培养纯化的前软骨干细胞(PSCs),对其进行动态压应力刺激及添加甲状腺激素相关肽(PTHrP)干预,进一步揭示前软骨干细胞的增殖、分化规律及其调控机制,为选择有效干预措施治疗骺板发育异常、修复及重建骺板等提供理论及实验依据。方法取材新生SD大鼠股骨干骺端软骨,免疫磁珠分选纯化,分别进行二维及三维分组培养,随机分组后适宜压应力(90mmg)及添加不同浓度PTHrP干预下连续培养,并设对照组(不干预),细胞计数及cck-8法检测细胞增殖情况,免疫组化法测定三维培养软骨细胞成软骨特异性细胞外基质Ⅱ型胶原(CollagenⅡ),RT-PCR法测定CollagenⅡ、聚集蛋白聚糖(Aggrecan)、PTHrP、X型胶原、TGF-β1等。根据实验结果评价应力及PTHrP对前软骨干细胞生物学性状的影响。结果1免疫磁珠分选培养出生长状态良好的前软骨干细胞,免疫组化、免疫荧光鉴定显示细胞稳定表达相对特异性标志物FGFR-3与PCNA;适宜参数压应力刺激与PTHrP干预组细胞培养至5代以上仍状态较好,而对照组细胞致5代已老化;2细胞计数及cck-8法检测细胞增殖能力有效干预组较对照组增大(P㩳0.05)其中应力刺激与PTHrP干预有协同效应。3 RT-PCR检测结果显示有效刺激实验组CollagenⅡ、aggrecan、Sox9表达较空白对照组升高,X型胶原降低,(P㩳0.05)90mmg压力刺激组PTHrP、TGF-β1、IGF-1表达水平增强。(P㩳0.05)结论压应力刺激与体外PTHrP干预均能影响PSCs的增值和分化。其中90mmg压应力间断刺激组细胞增殖作用明显,终末分化时间延长,可能与压应力导致细胞内PTHrP表达增高有关;小剂量(0.1nmol/L)PTHrP干预对细胞增殖有促进作用且延迟其终末分化,Sox9可能为其靶点;压应力刺激及PTHrP干预对PSCs的增殖和细胞外基质的分泌影响有协同作用。
[Abstract]:Objective to investigate the proliferation, differentiation and regulation mechanism of prechondrocyte stem cells (PSCs) by dynamic compressive stress stimulation and (PTHrP) intervention by plate and microspheres culture. To select effective intervention measures for the treatment of epiphyseal plate abnormal development, repair and reconstruction of epiphyseal plate to provide theoretical and experimental basis. Methods the femoral metaphyseal cartilage of newborn SD rats was collected and purified by immunomagnetic beads. The samples were cultured in two and three dimensional groups respectively. After randomly grouping, it was suitable for compression stress (90mmg) and continuous culture with different concentrations of PTHrP. Cell count and cck-8 method were used to detect the proliferation of chondrocytes in the control group, and Collagen 鈪，
本文编号：2251910
[Abstract]:Objective to investigate the proliferation, differentiation and regulation mechanism of prechondrocyte stem cells (PSCs) by dynamic compressive stress stimulation and (PTHrP) intervention by plate and microspheres culture. To select effective intervention measures for the treatment of epiphyseal plate abnormal development, repair and reconstruction of epiphyseal plate to provide theoretical and experimental basis. Methods the femoral metaphyseal cartilage of newborn SD rats was collected and purified by immunomagnetic beads. The samples were cultured in two and three dimensional groups respectively. After randomly grouping, it was suitable for compression stress (90mmg) and continuous culture with different concentrations of PTHrP. Cell count and cck-8 method were used to detect the proliferation of chondrocytes in the control group, and Collagen 鈪，
本文编号：2251910
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