【摘要】：目的建立體外BMSCs、NPCs非接觸式共培養(yǎng)體系,探討不同條件下NPCs誘導(dǎo)BMSCs向類髓核細(xì)胞分化的效果及影響因素。方法1.取新西蘭大白兔4只,采用改良全骨髓細(xì)胞貼壁法獲取BMSCs,進(jìn)行BMSCs體外培養(yǎng)、純化、擴(kuò)增；2.通過(guò)細(xì)胞形態(tài)學(xué)觀察、繪制細(xì)胞生長(zhǎng)曲線、流式細(xì)胞儀檢測(cè),觀察BMSCs的生物學(xué)性狀并對(duì)其進(jìn)行鑒定：3.取新西蘭大白兔4只,采用改良酶聯(lián)消化法獲取NPCs,體外細(xì)胞培養(yǎng)技術(shù)對(duì)NPCs進(jìn)行原代、傳代培養(yǎng)；4.通過(guò)細(xì)胞形態(tài)學(xué)觀察、繪制細(xì)胞生長(zhǎng)曲線、甲苯胺藍(lán)染色、免疫組織化學(xué)染色、RT-PCR檢測(cè),觀察NPCs的生物學(xué)性狀并對(duì)其進(jìn)行鑒定；5.取培養(yǎng)鑒定的BMSCs、NPCs,根據(jù)BMSCs與NPCs不同的細(xì)胞比例分為共培養(yǎng)組(2：1)、共培養(yǎng)組(1：1)、共培養(yǎng)組(1：2)、共培養(yǎng)組(1：4)、共培養(yǎng)(1：1)+TGF-β1組,以TGF-β1誘導(dǎo)組作為對(duì)照,建立BMSCs、NPCs非接觸式共培養(yǎng)體系。各組培養(yǎng)15天后對(duì)經(jīng)誘導(dǎo)的BMSCs進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè),通過(guò)檢測(cè)Aggrecan、 Collagen Ⅱ基因的相對(duì)表達(dá)量,從基因水平觀察誘導(dǎo)效果,并對(duì)結(jié)果進(jìn)行分析。結(jié)果 1.采用改良全骨髓細(xì)胞貼壁法能夠獲取BMSCs,經(jīng)流式細(xì)胞儀檢測(cè)純度較高；2.采用改良酶聯(lián)消化法能夠獲取NPCs,經(jīng)基因水平、蛋白水平鑒定,均有Aggrecan、Collagen Ⅱ表達(dá)；3.不同條件下BMSCs、NPCs非接觸式共培養(yǎng)體系和TGF-β1誘導(dǎo)體系均可誘導(dǎo)BMSCs向類髓核細(xì)胞分化,實(shí)時(shí)熒光定量PCR檢測(cè)Aggrecan、Collagen Ⅱ基因表達(dá)陽(yáng)性；4.不同細(xì)胞比例的共培養(yǎng)體系下,較高NPCs比例的共培養(yǎng)組與較低NPCs比例的共培養(yǎng)組相比,其誘導(dǎo)的類髓核細(xì)胞Aggrecan、Collagen Ⅱ基因表達(dá)量更高,組間差異有統(tǒng)計(jì)學(xué)意義；5.共培養(yǎng)條件下,經(jīng)1：1細(xì)胞比例+10ng/ml TGF-β1誘導(dǎo)后類髓核細(xì)胞Aggrecan基因表達(dá)量較1：4比例共培養(yǎng)組的更高,差異有統(tǒng)計(jì)學(xué)意義。Collagen Ⅱ基因表達(dá)量與1：4比例共培養(yǎng)組接近,差異無(wú)統(tǒng)計(jì)學(xué)意義。6.體外TGF-β1誘導(dǎo)BMSCs向類髓核細(xì)胞分化后,類髓核細(xì)胞Aggreca、 Collagen Ⅱ基因表達(dá)量與細(xì)胞比例1：2共培養(yǎng)條件下誘導(dǎo)的類髓核細(xì)胞基因表達(dá)量相近,差異無(wú)統(tǒng)計(jì)學(xué)意義；結(jié)論體外TGF-β1共培養(yǎng)條件下NPCs均可誘導(dǎo)BMSCs向類髓核細(xì)胞分化,體系中BMSCs/NPCs的比例、TGF-β1含量對(duì)誘導(dǎo)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，

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