猪骨髓基质细胞的分离培养及向软骨细胞分化的研究
发布时间：2018-10-22 08:29
【摘要】： 骨髓基质干细胞(Bone marrow stromal cells,BMSCs)是一类具有多向分化潜能的组织干细胞,在体内外适当的诱导环境下可以分化成为骨、软骨、脂肪、肌肉、神经、肌腱及韧带等多种细胞和组织。该细胞来源充足,取材方便,增殖能力强,可以在体外大规模扩增而不丢失多向分化潜能,并且异体移植免疫排斥反应小。目前,已成为应用较广泛的重要种子细胞之一。为了获得充足的骨髓基质干细胞来源,我们建立了猪骨髓基质干细胞的分离方法,通过体外培养和生物学鉴定,证实其为骨髓基质干细胞,并具有成骨分化性能。通过体外转导pEGFP-C1-TGF-β1入骨髓基质干细胞,在条件培养基作用下诱导其向软骨细胞分化。以期为骨组织工程和软骨损伤修复的研究提供新的策略,获得了以下结果: 1.无菌采取猪股骨头,采用骨钳钳破骨髓腔,收集骨髓液于无菌离心管,沉淀制成悬液接种于培养皿,于37℃、5 % CO2条件下培养。培养的细胞为单层贴壁生长,呈成纤维样形态。CD44因子免疫组化鉴定为阳性;条件培养基诱导成骨分化,ALP染色阳性,并有矿化结节生成;结合细胞形态学观察,结果表明,分离培养的细胞为骨髓基质干细胞。开始时大多为细长梭形,增殖速度快,细胞传代周期延短,待传至第7、8代时细胞铺展得宽大而扁薄,增殖速度减慢,细胞传代周期延长,细胞内颗粒物质增多。 2.从猪外周血淋巴细胞中抽提总RNA,并用TGF-β1特异性引物扩增获得TGF-β1全基因,并将TGF-β1全基因亚克隆到带有绿色荧光蛋白报告基因的真核表达质粒pEGFP-C1。 3.并用脂质体法转染BMSCs,通过直接荧光观察pEGFP-C1-TGF-β1融合蛋白在细胞中的分布定位,结果在转染后观察到绿色荧光,间接免疫荧光法检测TGF-β1表达均为阳性,转染后细胞失去BMSCs典型的成纤维形态,而呈现出三角、多角形态。Ⅱ型胶原免疫组化染色检测显示,转染TGF-β1基因后的BMSCs开始表达软骨细胞表面特异性标志物Ⅱ型胶原。 综上所述,本研究在成功分离培养猪BMSCs的基础上,将TGF-β1基因转入BMSCs后,表达了软骨细胞表面标志物,说明BMSCs已向软骨细胞分化。
[Abstract]:Bone marrow stromal cell (Bone marrow stromal cells,BMSCs) is a kind of tissue stem cells with multiple differentiation potential, which can differentiate into bone, cartilage, fat, muscle, nerve, tendons and ligaments in vivo and in vitro. The cells were abundant in source, convenient in selecting materials, strong in proliferative ability, and could be expanded in vitro without losing the potential of multidirectional differentiation, and the immune rejection of allograft was small. At present, it has become one of the most important seed cells. In order to obtain sufficient bone marrow mesenchymal stem cells (BMSCs), we established a method for isolation of porcine BMSCs, which was proved to be bone marrow mesenchymal stem cells (BMSCs) by in vitro culture and biological identification. Bone marrow mesenchymal stem cells (BMSCs) were transfected with pEGFP-C1-TGF- 尾 1 in vitro and induced to differentiate into chondrocytes. In order to provide a new strategy for bone tissue engineering and cartilage repair, the following results are obtained: 1. The porcine femoral head was sterilized. Bone forceps were used to break the medullary cavity, and bone marrow fluid was collected in aseptic centrifuge tube. The suspensions were precipitated and inoculated in culture dish at 37 鈩，
本文编号：2286651
[Abstract]:Bone marrow stromal cell (Bone marrow stromal cells,BMSCs) is a kind of tissue stem cells with multiple differentiation potential, which can differentiate into bone, cartilage, fat, muscle, nerve, tendons and ligaments in vivo and in vitro. The cells were abundant in source, convenient in selecting materials, strong in proliferative ability, and could be expanded in vitro without losing the potential of multidirectional differentiation, and the immune rejection of allograft was small. At present, it has become one of the most important seed cells. In order to obtain sufficient bone marrow mesenchymal stem cells (BMSCs), we established a method for isolation of porcine BMSCs, which was proved to be bone marrow mesenchymal stem cells (BMSCs) by in vitro culture and biological identification. Bone marrow mesenchymal stem cells (BMSCs) were transfected with pEGFP-C1-TGF- 尾 1 in vitro and induced to differentiate into chondrocytes. In order to provide a new strategy for bone tissue engineering and cartilage repair, the following results are obtained: 1. The porcine femoral head was sterilized. Bone forceps were used to break the medullary cavity, and bone marrow fluid was collected in aseptic centrifuge tube. The suspensions were precipitated and inoculated in culture dish at 37 鈩，
本文编号：2286651
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