【摘要】：背景:椎間盤退行性病變(intervertebral disc degeneration,IDD)是一系列脊柱疾患發(fā)生的前提和病理基礎(chǔ),在臨床上主要表現(xiàn)為頸腰痛、椎管狹窄、椎間盤突出、椎體不穩(wěn)等病癥。研究表明,椎間盤退行性病變所引起頸腰痛在所有到醫(yī)院就診的患者中占第2位,僅次于呼吸道感染感染性疾病。在椎間盤退變過程中,椎間盤組織的組分、結(jié)構(gòu)及功能都發(fā)生了改變,其中包括髓核組織中蛋白聚糖成分和水分的逐漸丟失,纖維環(huán)結(jié)構(gòu)排列的紊亂,以及軟骨終板的鈣化及血管化,鄰近椎體骨贅的形成等。然而椎間盤退變的具體病理生理機制目前還不清楚。椎間盤是脊柱運動節(jié)段中最為關(guān)鍵的結(jié)構(gòu),并且是較早發(fā)生退行性改變的組織。缺氧、異常應(yīng)力的作用和酸性環(huán)境都與椎間盤退變密切相關(guān),其中應(yīng)力扮演了非常重要的角色。大量的研究證實,異常力學(xué)因素的持續(xù)作用是導(dǎo)致椎間盤退變的主要原因之一,然而在此過程中所隱藏的具體病理生理機制目前還不清楚。在之前的研究中,我們發(fā)現(xiàn)人退變軟骨終板中存在類似于間充質(zhì)干細胞樣的細胞(Cartilage endplate derived stem cells,CESCs)并成功分離出來。此外我們的團隊在后期的研究中還發(fā)現(xiàn):CESCs具有較強的克隆形成能力以及多向分化潛能。近年來,應(yīng)力在椎間盤退變過程中的作用及相關(guān)機制被廣泛關(guān)注和研究。本研究旨在前期的研究基礎(chǔ)上,以軟骨終板干細胞為研究對象,使用周期性拉伸應(yīng)力裝置FX-4000對軟骨終板干細胞進行應(yīng)力加載,初步探討周期性拉伸應(yīng)力對軟骨終板干細胞分化的影響。目的:臨床上獲得退變椎間盤的軟骨終板標(biāo)本,對所獲得的軟骨終板進行消化,原代細胞貼壁培養(yǎng)、擴增后,采用我們既往的方法,應(yīng)用瓊脂糖懸浮培養(yǎng)系統(tǒng)從第一代細胞中篩選出軟骨終板干細胞。通過觀察應(yīng)力對軟骨終板干細胞成骨分化的影響,并初步探討其在椎間盤退變過程中的意義,以期為椎間盤退行性疾病的預(yù)防和治療提供新的思路和理論依據(jù)。臨床上獲得退變椎間盤的軟骨終板標(biāo)本(在腰椎融合手術(shù)中分離),于分離后2小時之內(nèi)帶入超凈工作臺。對獲取的軟骨終板組織在解剖顯微鏡下,使用眼科手術(shù)器械對獲取組織標(biāo)本進行再次清理,清理結(jié)束后,磷酸鹽緩沖液(phosphate buffer,PBS)沖洗后。將洗凈的組織剪切成體積約為1mm×1mm×1mm大小的組織塊,將剪碎組織塊轉(zhuǎn)移入25cm2培養(yǎng)瓶中,加入約5倍體積含0.15%Ⅱ型膠原酶無血清DMEM/F12培養(yǎng)基,靜置于37℃,5%CO2培養(yǎng)箱過夜消化。消化完成后,用70μm的細胞濾網(wǎng)過濾消化液,將濾液轉(zhuǎn)移至無菌的離心管中,1000r/min離心5min。離心結(jié)束后,取出離心管,倒掉上清液。收集細胞沉淀,加入含有棄上清,加入含有10%FBS、1%雙抗的BMEM/F12的完全培養(yǎng)基重懸細胞,置于細胞培養(yǎng)箱中培養(yǎng)。每3天更換一次細胞培養(yǎng)液,倒置相差顯微鏡下觀察軟骨終板細胞的生長情況。當(dāng)原代培養(yǎng)的細胞達90%融合后,利用瓊脂懸浮培養(yǎng)系統(tǒng)篩選出CESCs進行進一步培養(yǎng)。然后對篩選出來的細胞行三系誘導(dǎo)分化鑒定和流式細胞術(shù)表型分析。選取生長良好的第三代CESCs,接種于Bio Flex 6孔培養(yǎng)板中。待細胞貼壁生長至80-90%融合后,換成FBS體積分數(shù)為1%的DMEM/F12培養(yǎng)液繼續(xù)培養(yǎng)12小時,使各組細胞同步化。在無誘導(dǎo)因子的作用下,將培養(yǎng)板置于Flexcell-4000TM應(yīng)力加載系統(tǒng)中,施加1h、6h、12h、24h,頻率為1Hz、拉伸率為10%的牽拉刺激,同時做靜態(tài)對照實驗。牽拉完成后,收集細胞,應(yīng)用Western blot檢測BMP-2的表達情況,使用q PCR測定部分成骨、成軟骨基因的表達變化結(jié)果:1.將獲得的軟骨終板細胞進行篩選后,應(yīng)用流式細胞術(shù)及三系誘導(dǎo)分化鑒定提示篩選出來的細胞具有干細胞特性。2.將篩選出來的CESCs進行周期性應(yīng)力加載后,發(fā)現(xiàn)與成骨有關(guān)的基因(BMP-2、ALP、Runx2)的表達量較對照組明顯增加,差異具有統(tǒng)計學(xué)意義。而與成軟骨有關(guān)基因(SOX9)表達量隨拉伸時間的延長,表達量卻逐漸降低。結(jié)論:1.通過流式細胞術(shù)和三系誘導(dǎo)分化實驗證實,我們經(jīng)過篩選所得的細胞具有干細胞的特性,這和我們之前的實驗結(jié)果相一致。2.周期性拉伸應(yīng)力可以促進軟骨終板干細胞中的BMP-2的表達,同時上調(diào)成骨相關(guān)基因的表達,促使軟骨終板干細胞向成骨細胞分化。
[Abstract]:Background: intervertebral disc degeneration (IDD) is the precondition and pathological basis of a series of spinal disorders. It is mainly characterized by cervical lumbago, spinal stenosis, disc herniation, and vertebral instability. The study shows that the cervical lumbago is caused by lumbar disc degeneration in all hospital patients. In the process of intervertebral disc degeneration, the components, structures and functions of the intervertebral disc were changed, including the gradual loss of the proteoglycan composition and moisture in the nucleus pulposus, the disorder of the structure of the fibrous ring, the calcification and vascularization of the cartilage endplate, and the adjacent vertebral body. The formation of osteophyte and so on. However, the specific pathophysiological mechanism of intervertebral disc degeneration is not yet clear. Intervertebral disc is the most critical structure in the spinal segment, and it is an early degenerative tissue. Anoxia, abnormal stress and acidic environment are closely related to intervertebral disc degeneration, and stress plays a very important role. A large number of studies have confirmed that the persistent effect of abnormal mechanical factors is one of the main causes of disc degeneration. However, the specific pathophysiological mechanism hidden in this process is not yet clear. In previous studies, we found that human degeneration cartilage endplates are stored in cells like mesenchymal stem cells (Cartilag E endplate derived stem cells, CESCs) has been successfully separated. In addition, our team also found that CESCs has strong clone formation ability and multidirectional differentiation potential. In recent years, the role and mechanism of stress in the process of disc degeneration have been widely concerned and studied. This study aims at the previous research basis. At the same time, the cartilage endplate stem cells were used as the research object. The periodic tensile stress device (FX-4000) was used to load the cartilage endplate stem cells. The effect of periodic tensile stress on the differentiation of cartilage endplate stem cells was preliminarily investigated. We used our previous methods to screen the cartilage endplate stem cells from the first generation cells by using the agarose suspension culture system. By observing the effect of stress on the osteogenesis of the cartilage endplate stem cells, the significance of it in the process of intervertebral disc degeneration was preliminarily discussed in order to degenerate the intervertebral disc. The prevention and treatment of the disease provides new ideas and theoretical basis. The cartilage endplate specimens of the degenerative intervertebral disc (separated from the lumbar spinal fusion surgery) are carried out within 2 hours after the separation. The obtained cartilage endplate tissue is obtained again under an anatomical microscope with an ophthalmic surgical instrument for obtaining the tissue specimen again. After cleaning, after the cleaning, the phosphate buffer solution (phosphate buffer, PBS) was washed. The tissue was cut into a tissue block of about 1mm * 1mm * 1mm size. The cut tissue block was transferred into the 25cm2 culture bottle and added about 5 times the volume of 0.15% type collagenase without serum-free DMEM/F12 medium, at 37, and the 5%CO2 incubator was digested overnight. After the digestion was completed, the digestive juice was filtered with 70 m cell filter net, and the filtrate was transferred into a sterile centrifuge tube. After centrifugal 5min. centrifugation, the centrifuge tube was removed, and the supernatant was removed. The cell precipitation was collected, and the complete medium suspension cell containing BMEM /F12 containing 10%FBS and 1% double resistance was added to the cell culture box. Culture. Change the cell culture medium every 3 days and observe the growth of cartilage endplate cells under the inverted phase microscope. When the primary cultured cells were 90% fusion, CESCs was screened by agar suspension culture system. Then the selected cells were identified by three line differentiation and flow cytometry. The third generation CESCs, which was well growing, was inoculated in the Bio Flex 6 Hole culture plate. After the cell wall growth to 80-90% fusion, the DMEM/F12 culture medium with FBS volume fraction of 1% continued to be cultured for 12 hours to synchronize the cells. Under the action of no inducible factor, the culture plate was placed in the Flexcell-4000TM stress loading system, and 1H was applied. 6h, 12h, 24h, a stretch stimulation with a frequency of 1Hz and a stretch rate of 10%, and a static control experiment. After the traction was completed, the cells were collected, the expression of BMP-2 was detected by Western blot, the partial osteogenesis of the cartilage was measured with Q PCR, and the expression of the chondrogenic gene was changed: 1. after screening the obtained cartilage endplate cells, flow cytometry and three were used. Induced differentiation identification suggested that the selected cells had stem cell characteristics.2. to carry out periodic stress loading of the screened CESCs, and found that the expression of BMP-2, ALP, Runx2, which was related to osteogenesis, was significantly increased compared with the control group, and the difference was statistically significant. The expression of cartilage related gene (SOX9) was associated with the stretching time. The expression level was gradually reduced. Conclusion: 1. through flow cytometry and three lines induced differentiation experiments, we found that the cells we screened have the characteristics of stem cells. This is consistent with our previous experimental results that.2. periodic tensile stress can promote the expression of BMP-2 in cartilage endplate stem cells, while up regulation of osteogenic correlation. Gene expression promotes the differentiation of cartilage endplate stem cells into osteoblasts.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R681.53

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