【摘要】：背景：腰椎滑脫是骨科的常見病癥之一，以反復(fù)腰痛為主要臨床表現(xiàn)，嚴(yán)重影響患者的生活質(zhì)量。而腰椎滑脫患者最終大多數(shù)需要手術(shù)矯正，術(shù)中用于脊柱融合的材料存在各種不足，如果能通過人工干預(yù)使椎間盤骨化，則有望達(dá)到脊柱融合的目的。有研究顯示，GDF-5在軟骨發(fā)生和長骨發(fā)育中具有重要的作用，能誘導(dǎo)多種細(xì)胞向成軟骨和成骨細(xì)胞分化，，這表明其可誘導(dǎo)類軟骨細(xì)胞-髓核細(xì)胞向成骨細(xì)胞分化。 目的：本實驗通過建立家兔椎間盤髓核細(xì)胞的體外培養(yǎng)模型，觀察髓核細(xì)胞的細(xì)胞學(xué)特性和生物學(xué)特性，然后在給予rhGDF-5和bFGF進(jìn)行人工干預(yù)后，探討rhGDF-5和bFGF對髓核細(xì)胞表型表達(dá)和分化特性的影響，并為直接誘導(dǎo)椎間盤髓核細(xì)胞成骨行椎間融合提供理論依據(jù)。 方法：1.從健康家兔取出椎間盤髓核，用Ⅱ型膠原酶消化法分離原代椎間盤髓核細(xì)胞后，單層培養(yǎng)并以胰酶消化法傳代，采用差速貼壁法進(jìn)行純化。2.觀察髓核細(xì)胞在細(xì)胞因子rhGDF-5和bFGF誘導(dǎo)下成骨表型表達(dá)和細(xì)胞學(xué)特性的變化。按試驗中的細(xì)胞培養(yǎng)條件分為四個培養(yǎng)組：A組：DMEM/15%FBS培養(yǎng)液；B組：DMEM/15%FBS培養(yǎng)液+rhGDF-5；C組：DMEM/15%FBS培養(yǎng)液+bFGF；D組：DMEM/15%FBS培養(yǎng)液+rhGDF-5+bFGF。3.利用光學(xué)顯微鏡觀察細(xì)胞形態(tài)，MTT法測定生長曲線，免疫組化觀察Ⅰ型膠原和Ⅱ型膠原的表達(dá)，放射免疫技術(shù)測定骨鈣素的表達(dá)，茜素紅染色等。 結(jié)果：初貼壁的髓核細(xì)胞呈短梭形、多角形，融合后呈旋渦狀，傳代后呈長梭形或星形，培養(yǎng)后期呈明顯的趨化性生長、極性排列。大約于第五代時細(xì)胞出現(xiàn)生長滯緩，即出現(xiàn)老化現(xiàn)象。rhGDF-5能夠抑制細(xì)胞的增殖；增加骨鈣素的表達(dá)；促進(jìn)鈣鹽的沉積，形成鈣結(jié)節(jié)。bFGF能夠促進(jìn)細(xì)胞的增殖；對骨鈣素表達(dá)無明顯影響；促進(jìn)Ⅰ型膠原的表達(dá)；促進(jìn)鈣鹽沉積，形成鈣結(jié)節(jié)。當(dāng)聯(lián)合使用rhGDF-5和bFGF時，能夠促進(jìn)細(xì)胞增殖；能夠促進(jìn)Ⅰ型膠原、骨鈣素的表達(dá)和鈣鹽的沉積，其效果要比單獨使用任一細(xì)胞因子都好。四個組的Ⅱ型膠原表達(dá)均為陰性。 結(jié)論：1.本實驗成功建立家兔髓核細(xì)胞體外培養(yǎng)模型。原代培養(yǎng)的髓核細(xì)胞主要呈類軟骨細(xì)胞樣表型,逐漸出現(xiàn)向成纖維細(xì)胞轉(zhuǎn)分化。在細(xì)胞傳至第五代時，出現(xiàn)老化現(xiàn)象。2. rhGDF-5能成功誘導(dǎo)體外髓核細(xì)胞成骨，bFGF能加強rhGDF-5對髓核細(xì)胞的成骨誘導(dǎo)作用。
[Abstract]:Background: lumbar spondylolisthesis is one of the common diseases in orthopedic department. However, most of the lumbar spondylolisthesis patients need surgical correction, and the materials used in spinal fusion have various shortcomings. If the intervertebral disc ossification can be made by artificial intervention, it is expected to achieve the purpose of spinal fusion. Some studies have shown that GDF-5 plays an important role in chondrogenesis and long bone development and can induce the differentiation of many kinds of cells into chondroblasts and osteoblasts, which indicates that GDF-5 can induce the differentiation of chondroblast-nucleus pulposus cells into osteoblasts. Objective: to observe the cytological and biological characteristics of nucleus pulposus cells in vitro by establishing a rabbit model of nucleus pulposus cells cultured in vitro, and then to observe the effects of rhGDF-5 and bFGF. To investigate the effects of rhGDF-5 and bFGF on phenotypic expression and differentiation of nucleus pulposus cells, and to provide theoretical basis for direct induction of intervertebral fusion of nucleus pulposus cells. Method 1: 1. The nucleus pulposus of the intervertebral disc was removed from the healthy rabbits. Primary nucleus pulposus cells were isolated by type 鈪

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