不同共培养条件下兔NPCs诱导BMSCs向类髓核细胞分化的实验研究
发布时间：2018-11-15 09:44
【摘要】：目的建立体外BMSCs、NPCs非接触式共培养体系,探讨不同条件下NPCs诱导BMSCs向类髓核细胞分化的效果及影响因素。方法1.取新西兰大白兔4只,采用改良全骨髓细胞贴壁法获取BMSCs,进行BMSCs体外培养、纯化、扩增；2.通过细胞形态学观察、绘制细胞生长曲线、流式细胞仪检测,观察BMSCs的生物学性状并对其进行鉴定：3.取新西兰大白兔4只,采用改良酶联消化法获取NPCs,体外细胞培养技术对NPCs进行原代、传代培养；4.通过细胞形态学观察、绘制细胞生长曲线、甲苯胺蓝染色、免疫组织化学染色、RT-PCR检测,观察NPCs的生物学性状并对其进行鉴定；5.取培养鉴定的BMSCs、NPCs,根据BMSCs与NPCs不同的细胞比例分为共培养组(2：1)、共培养组(1：1)、共培养组(1：2)、共培养组(1：4)、共培养(1：1)+TGF-β1组,以TGF-β1诱导组作为对照,建立BMSCs、NPCs非接触式共培养体系。各组培养15天后对经诱导的BMSCs进行实时荧光定量PCR检测,通过检测Aggrecan、 Collagen Ⅱ基因的相对表达量,从基因水平观察诱导效果,并对结果进行分析。结果 1.采用改良全骨髓细胞贴壁法能够获取BMSCs,经流式细胞仪检测纯度较高；2.采用改良酶联消化法能够获取NPCs,经基因水平、蛋白水平鉴定,均有Aggrecan、Collagen Ⅱ表达；3.不同条件下BMSCs、NPCs非接触式共培养体系和TGF-β1诱导体系均可诱导BMSCs向类髓核细胞分化,实时荧光定量PCR检测Aggrecan、Collagen Ⅱ基因表达阳性；4.不同细胞比例的共培养体系下,较高NPCs比例的共培养组与较低NPCs比例的共培养组相比,其诱导的类髓核细胞Aggrecan、Collagen Ⅱ基因表达量更高,组间差异有统计学意义；5.共培养条件下,经1：1细胞比例+10ng/ml TGF-β1诱导后类髓核细胞Aggrecan基因表达量较1：4比例共培养组的更高,差异有统计学意义。Collagen Ⅱ基因表达量与1：4比例共培养组接近,差异无统计学意义。6.体外TGF-β1诱导BMSCs向类髓核细胞分化后,类髓核细胞Aggreca、 Collagen Ⅱ基因表达量与细胞比例1：2共培养条件下诱导的类髓核细胞基因表达量相近,差异无统计学意义；结论体外TGF-β1共培养条件下NPCs均可诱导BMSCs向类髓核细胞分化,体系中BMSCs/NPCs的比例、TGF-β1含量对诱导效果均起重要作用。
[Abstract]:Objective to establish a non-contact co-culture system of BMSCs,NPCs in vitro and to investigate the effect of NPCs on the differentiation of BMSCs into nucleus pulposus cells under different conditions. Method 1. BMSCs, was obtained from 4 New Zealand white rabbits by modified whole-bone marrow cell adherence method. BMSCs was cultured, purified and amplified in vitro. 2. The biological characters of BMSCs were observed and identified by cell morphology observation, cell growth curve and flow cytometry. NPCs, cells were obtained from 4 New Zealand white rabbits by modified enzyme-linked digestion (Elisa) and NPCs was subcultured in vitro. 4. Cell growth curve, toluidine blue staining, immunohistochemical staining and RT-PCR detection were used to observe and identify the biological characteristics of NPCs. According to the ratio of BMSCs and NPCs, BMSCs,NPCs, was divided into three groups: co culture group (2:1), co-culture group (1:1), co-culture group (1:2), co-culture group (#timea-) TGF- 尾 1 group. BMSCs,NPCs non-contact co-culture system was established with TGF- 尾 1 induction group as control. After 15 days of culture, the induced BMSCs was detected by real-time fluorescence quantitative PCR. The relative expression of Aggrecan, Collagen 鈪，
本文编号：2332941
[Abstract]:Objective to establish a non-contact co-culture system of BMSCs,NPCs in vitro and to investigate the effect of NPCs on the differentiation of BMSCs into nucleus pulposus cells under different conditions. Method 1. BMSCs, was obtained from 4 New Zealand white rabbits by modified whole-bone marrow cell adherence method. BMSCs was cultured, purified and amplified in vitro. 2. The biological characters of BMSCs were observed and identified by cell morphology observation, cell growth curve and flow cytometry. NPCs, cells were obtained from 4 New Zealand white rabbits by modified enzyme-linked digestion (Elisa) and NPCs was subcultured in vitro. 4. Cell growth curve, toluidine blue staining, immunohistochemical staining and RT-PCR detection were used to observe and identify the biological characteristics of NPCs. According to the ratio of BMSCs and NPCs, BMSCs,NPCs, was divided into three groups: co culture group (2:1), co-culture group (1:1), co-culture group (1:2), co-culture group (#timea-) TGF- 尾 1 group. BMSCs,NPCs non-contact co-culture system was established with TGF- 尾 1 induction group as control. After 15 days of culture, the induced BMSCs was detected by real-time fluorescence quantitative PCR. The relative expression of Aggrecan, Collagen 鈪，
本文编号：2332941
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