猪骨髓基质细胞的分离培养及向软骨细胞分化的研究
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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。
Bone marrow mesenchymal stem cells (BMSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate into a variety of cells such as osteocytes, chondrocytes, adipocytes, musle cells, neurons tendon cells and ligament cells. Due to the characteristics of sufficient resource, being conveniently derived and having high reproductive activity,BMSCscan be widely amplified in vitro without losing its multi-directional differentiation potentiality. Moreover, when used in allotransplantation, the cells activated minor immunological rejection. Therefore,BMSCs have become an important kind of widespread used seed cells. In order to obtain sufficient BMSCs from pigs, an effective separation method was established. Through the in vitro culture and biological identification, cells isolated from pigs were verified asBMSCsand had the ability of osteoblastic differentiation. In this study, recombinant plasmid pEGFP-C1-TGF-β1 was transfected intoBMSCsin vitro and cells were cultured in conditioned medium and were induced into chondrocytes, providing a new strategy to the research of bone injury repair and bone tissue engineering. Preliminarily findings were as follows:
1. Femoral head was aseptically cut out from pigs, bone marrow cavity was broken down, and bone marrow fluid was extracted. Cells sedimentum was suspended and inoculated into culture dish, and were cultured at 37℃in a 5 % CO2 incubator. The primary cells adhered to the dish bottom on a monolayer, presented fibroblast-like morphous. The immunochemical identification of CD44 factor was positive, cells were induced by conditioned medium to differentiate into osteogenic cells, ALP dyeing result was positive and mineralized nodules were formed. Combined with morphocytology observation, the cells isolated and cultured were determined toBMSCs. At the beginning, cells showed long fusiform, and had a high proliferation rate, and the passage cycle is shortened. After the 7th or 8th passage, cells extended to become magnanimous, flat and thin, and proliferated slow. And the passage cycle was extended, cell particulate matters increased as well.
2. RNA was extracted from peripheral blood lymphocyte of pigs, and the TGF-β1 gene was amplified, and then was cloned into eukaryotic expression vector pEGFP-C1 which included a green fluorescent report gene.
3.BMSCswere transfected with pEGFP-C1-TGF-β1 by Liposome-mediated method. The distribution and location of pEGFP-C1-TGF-β1 fusion protein was observed with fluorescence inverted microscope. Results showed that green fluorescence was detected in transfected cells, and TGF-β1 expression was positive by indirect immunofluorescence detection. Transfected cells lost the typical fibroblast-like morphous ofBMSCs, while showed triangle and polygon morphous. Immunohistochemistry detection of typeⅡcollagen showed thatBMSCsexpressed typeⅡcollagen, the specific surface marker of chondrocyte.
In conclusion, on the basis of the successful and mature techniques ofBMSCsisolation and cultivation,BMSCswere transfected with TGF-β1 and cells differentiated fromBMSCsexpressed the surface marker of chondrocyte, providing a new strategy to gene therapy of cartilage injury repair.
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。
Bone marrow mesenchymal stem cells (BMSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate into a variety of cells such as osteocytes, chondrocytes, adipocytes, musle cells, neurons tendon cells and ligament cells. Due to the characteristics of sufficient resource, being conveniently derived and having high reproductive activity,BMSCscan be widely amplified in vitro without losing its multi-directional differentiation potentiality. Moreover, when used in allotransplantation, the cells activated minor immunological rejection. Therefore,BMSCs have become an important kind of widespread used seed cells. In order to obtain sufficient BMSCs from pigs, an effective separation method was established. Through the in vitro culture and biological identification, cells isolated from pigs were verified asBMSCsand had the ability of osteoblastic differentiation. In this study, recombinant plasmid pEGFP-C1-TGF-β1 was transfected intoBMSCsin vitro and cells were cultured in conditioned medium and were induced into chondrocytes, providing a new strategy to the research of bone injury repair and bone tissue engineering. Preliminarily findings were as follows:
1. Femoral head was aseptically cut out from pigs, bone marrow cavity was broken down, and bone marrow fluid was extracted. Cells sedimentum was suspended and inoculated into culture dish, and were cultured at 37℃in a 5 % CO2 incubator. The primary cells adhered to the dish bottom on a monolayer, presented fibroblast-like morphous. The immunochemical identification of CD44 factor was positive, cells were induced by conditioned medium to differentiate into osteogenic cells, ALP dyeing result was positive and mineralized nodules were formed. Combined with morphocytology observation, the cells isolated and cultured were determined toBMSCs. At the beginning, cells showed long fusiform, and had a high proliferation rate, and the passage cycle is shortened. After the 7th or 8th passage, cells extended to become magnanimous, flat and thin, and proliferated slow. And the passage cycle was extended, cell particulate matters increased as well.
2. RNA was extracted from peripheral blood lymphocyte of pigs, and the TGF-β1 gene was amplified, and then was cloned into eukaryotic expression vector pEGFP-C1 which included a green fluorescent report gene.
3.BMSCswere transfected with pEGFP-C1-TGF-β1 by Liposome-mediated method. The distribution and location of pEGFP-C1-TGF-β1 fusion protein was observed with fluorescence inverted microscope. Results showed that green fluorescence was detected in transfected cells, and TGF-β1 expression was positive by indirect immunofluorescence detection. Transfected cells lost the typical fibroblast-like morphous ofBMSCs, while showed triangle and polygon morphous. Immunohistochemistry detection of typeⅡcollagen showed thatBMSCsexpressed typeⅡcollagen, the specific surface marker of chondrocyte.
In conclusion, on the basis of the successful and mature techniques ofBMSCsisolation and cultivation,BMSCswere transfected with TGF-β1 and cells differentiated fromBMSCsexpressed the surface marker of chondrocyte, providing a new strategy to gene therapy of cartilage injury repair.
引文
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