不同分离方法对兔骨髓间充质干细胞成软骨分化影响的研究
发布时间：2018-11-27 13:35
【摘要】：目的 用全骨髓法和密度梯度离心两种方法对兔骨髓间质干细胞(MSCs)进行体外分离、培养及扩增,对比在培养基中只加入TGF-β_1一种诱导物质情况下,两种方法得到的MSCs的成软骨能力及差别,旨在前人研究的基础上,建立一个更简易、实用的体外诱导培养体系,为软骨缺损修复提供更多的实验依据。 实验方法 用3%戊巴比妥钠1mg/kg耳缘静脉麻醉,无菌条件下用骨穿针从两侧股骨大转子处穿入股骨髓腔内,骨穿针接10ml注射器(内含3000U/ml的肝素0.4ml),每侧各抽取骨髓3～4ml,混合后等分为两份,一份(A组全骨髓法)中直接加入DMEM-LG培养液,用全骨髓法培养细胞,另一份(B组密度梯度离心法)加入装有Percoll分离液的无菌离心管中,1000r/min离心10min,吸取液面交界处云雾状单核细胞层细胞,制成单细胞悬液,两组的第3代细胞均用转化生长因子β_1(TGF-β_1)诱导BMSCs向软骨方向分化。倒置显微镜观察BMSCs的生长情况,用免疫组织化学、原位杂交方法检测TGF-β_1诱导的细胞Ⅱ型胶原表达情况。 实验结果 1.相差显微镜观察: 1.1 全骨髓培养组 24h后→部分细胞贴附在瓶壁上,可见一些单核、长梭或多角型细胞
[Abstract]:Objective to isolate, culture and amplify (MSCs) from rabbit bone marrow mesenchymal stem cells by whole bone marrow method and density gradient centrifugation in vitro. Under the condition that only TGF- 尾 _ 1 was added into the medium, the chondrogenic ability and difference of MSCs obtained by the two methods were compared. The purpose of this study was to establish a more simple and practical culture system of MSCs induced in vitro on the basis of previous studies. To provide more experimental evidence for cartilage defect repair. Methods 3% pentobarbital sodium (1mg/kg) was used to anesthetize the auricular margin of rats. Under aseptic condition, the femoral medullary cavity was inserted with bone piercing needle from the bilateral trochanter of femur. 10ml syringe (heparin 0.4ml containing 3000U/ml) was connected with bone puncture needle. Each side of bone marrow was extracted from 3ml of bone marrow. After mixing, the bone marrow was divided into two parts. One part (group A, whole bone marrow method) was directly added into DMEM-LG culture medium, and the cells were cultured by whole bone marrow method. The other (group B density gradient centrifugation) was added to the aseptic centrifuge tube containing the Percoll isolate. The 1000r/min was centrifuged for 10 mins, and the monocyte layer cells in the cloud shape at the junction of the liquid level were absorbed, and the monocytes were made into a single cell suspension. Transforming growth factor 尾 _ 1 (TGF- 尾 _ 1) was used to induce the differentiation of BMSCs into cartilage. The growth of BMSCs was observed by inverted microscope, and the expression of type 鈪，
本文编号：2360966
[Abstract]:Objective to isolate, culture and amplify (MSCs) from rabbit bone marrow mesenchymal stem cells by whole bone marrow method and density gradient centrifugation in vitro. Under the condition that only TGF- 尾 _ 1 was added into the medium, the chondrogenic ability and difference of MSCs obtained by the two methods were compared. The purpose of this study was to establish a more simple and practical culture system of MSCs induced in vitro on the basis of previous studies. To provide more experimental evidence for cartilage defect repair. Methods 3% pentobarbital sodium (1mg/kg) was used to anesthetize the auricular margin of rats. Under aseptic condition, the femoral medullary cavity was inserted with bone piercing needle from the bilateral trochanter of femur. 10ml syringe (heparin 0.4ml containing 3000U/ml) was connected with bone puncture needle. Each side of bone marrow was extracted from 3ml of bone marrow. After mixing, the bone marrow was divided into two parts. One part (group A, whole bone marrow method) was directly added into DMEM-LG culture medium, and the cells were cultured by whole bone marrow method. The other (group B density gradient centrifugation) was added to the aseptic centrifuge tube containing the Percoll isolate. The 1000r/min was centrifuged for 10 mins, and the monocyte layer cells in the cloud shape at the junction of the liquid level were absorbed, and the monocytes were made into a single cell suspension. Transforming growth factor 尾 _ 1 (TGF- 尾 _ 1) was used to induce the differentiation of BMSCs into cartilage. The growth of BMSCs was observed by inverted microscope, and the expression of type 鈪，
本文编号：2360966
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