本文選題：纖維環(huán)細胞 + 髓核細胞��； 參考：《中國人民解放軍軍醫(yī)進修學院》2008年碩士論文

【摘要】： 研究背景: 特發(fā)性脊柱側(cè)凸(adolescent idiopathic scoliosis,AIS)患者最終大多需要手術(shù)矯正,不僅治療方案復雜,費用高,而且術(shù)中使用的各種融合材料包括自體骨、異體骨、人工骨等都存在著各種不足。椎間盤作為脊柱重要的連接單位,如果能通過人工干預使之骨化,則有望達到脊柱融合的目的。 椎間盤由纖維環(huán)和髓核兩部分構(gòu)成,椎間盤纖維環(huán)和髓核的共同特點是細胞外基質(zhì)豐富而細胞數(shù)量較少,類軟骨細胞存在于髓核及纖維環(huán)內(nèi)層,成纖維細胞主要存在于纖維環(huán)的外層。當具有相應遺傳物質(zhì)基礎(chǔ)的細胞處于有利于分化逆轉(zhuǎn)的環(huán)境中,會出現(xiàn)轉(zhuǎn)分化(transdifferentiation)。由一種分化狀態(tài)變?yōu)榱硪环N分化狀態(tài)。 研究目的: 觀察在實驗室模擬的體外環(huán)境中椎間盤細胞的細胞學特性與表型表達的特點,觀察在給予rh-BMP_2和bFGF進行人工干預后,體外培養(yǎng)的椎間盤細胞的表型表達和分化特性。 實驗方法: 大體標本取自16-25歲青年患者,椎間盤本身無病變,標本離體后迅速送到實驗室,仔細分離出纖維環(huán)和髓核,利用體外細胞培養(yǎng)技術(shù)建立腰椎間盤纖維環(huán)細胞和髓核細胞培養(yǎng)體系。觀察這兩種細胞在細胞因子rh-BMP_2和bFGF誘導下的成骨表型及生物學特性的變化。實驗中按培養(yǎng)條件分為四個培養(yǎng)組:A組:DMEM/FBS培養(yǎng)液;B組:DMEM/FBS培養(yǎng)液+rh-BMP_2;C組:DMEM/FBS培養(yǎng)液+bFGF;D組:DMEM/FBS培養(yǎng)液+rh-BMP_2+bFGF。實驗觀察內(nèi)容包括:細胞形態(tài)、生長曲線、堿性磷酸酶活力、Ⅰ型膠原表達、Ⅱ型膠原表達、骨鈣素表達、茜素紅染色等。 實驗結(jié)果: 隨著培養(yǎng)時間的延長,纖維環(huán)細胞呈明顯的趨化性生長,極性排列,局部有細胞基質(zhì)堆積。rh-BMP_2對纖維環(huán)細胞增殖無明顯影響;能夠提高堿性磷酸酶的活力;促進Ⅰ型膠原的表達;提高骨鈣素的表達;促進鈣鹽的沉積,形成鈣結(jié)節(jié)。bFGF能夠明顯促進細胞增殖;降低了堿性磷酸酶的活力;促進Ⅰ型膠原的表達;促進鈣鹽沉積,形成鈣結(jié)節(jié)。當rh-BMP_2和bFGF聯(lián)合使用時,能夠促進細胞增殖;對堿性磷酸酶活力無明顯影響;能夠強力促進骨鈣素、Ⅰ型膠原的表達和鈣鹽沉積,其效果優(yōu)于單獨使用任何一種細胞因子。 原代髓核細胞呈多角形,傳代后變成星形或長梭形,培養(yǎng)后期呈明顯的趨化性生長,極性排列。rh-BMP_2能夠抑制細胞的增殖;提高骨鈣素的表達;促進鈣鹽沉積,形成鈣結(jié)節(jié)。bFGF能夠促進細胞增殖;促進Ⅰ型膠原的表達;促進鈣鹽沉積,形成鈣結(jié)節(jié)。當rh-BMP_2和bFGF聯(lián)合使用時,能夠促進細胞增殖;促進骨鈣素、Ⅰ型膠原的表達和鈣鹽沉積,戍骨誘導效果最佳。四個培養(yǎng)組的Ⅱ型膠原表達均為陰性。 結(jié)論: rh-BMP_2對纖維環(huán)細胞體外有明確的成骨誘導作用,bFGF能夠增強rh-BMP_2的成骨誘導作用。 rh-BMP_2對髓核細胞體外有明確的成骨誘導作用,bFGF能夠增強rh-BMP_2的成骨誘導作用。
[Abstract]:Background: Most of the patients with idiopathic scoliosis who need surgical correction ultimately need surgical correction. Not only are the treatment schemes complex and costly, but also the various fusion materials used during the operation include autogenous bone, allogeneic bone, artificial bone and so on. Intervertebral disc, as an important junction unit of spine, can achieve spinal fusion if it can be ossified by artificial intervention. The intervertebral disc is composed of fibrous ring and nucleus pulposus. The common characteristic of disc fiber ring and nucleus pulposus is that the extracellular matrix is abundant and the number of cells is small. The chondroid cells exist in the nucleus pulposus and the inner layer of the fibrous ring. Fibroblasts mainly exist in the outer layer of the fibrous ring. When the cells with the corresponding genetic material base are in a favorable environment for differentiation reversal, transdifferentiation and transdifferentiation may occur. From one state of differentiation to another state of differentiation. Objectives of the study: The cytological and phenotypic characteristics of intervertebral disc cells were observed in laboratory simulated environment, and the phenotypic expression and differentiation characteristics of intervertebral disc cells cultured in vitro were observed after the artificial intervention of rh-BMP_2 and bFGF. Experimental methods: The specimens were taken from young patients aged 16-25 years, and there was no lesion in the intervertebral disc itself. The specimens were sent to the laboratory in vitro and carefully separated from the fibrous annulus and nucleus pulposus. The cell culture system of fibroannular cells and nucleus pulposus cells of lumbar intervertebral disc was established by cell culture in vitro. The changes of osteogenic phenotype and biological characteristics of these two kinds of cells induced by cytokines rh-BMP_2 and bFGF were observed. In the experiment, we divided into four groups according to the culture conditions: 1: a group: DMEM / FBS culture medium group B: DMEM / FBS medium rh-BMP2C group: DMEM / FBS medium bFGFGF group rh-BMP_2 bFGF. Cell morphology, growth curve, alkaline phosphatase activity, type 鈪，

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