周期性拉伸应力调节人椎间盘软骨终板干细胞成骨分化的研究

发布时间：2018-07-23 11:09

【摘要】：背景:椎间盘退行性病变(intervertebral disc degeneration,IDD)是一系列脊柱疾患发生的前提和病理基础,在临床上主要表现为颈腰痛、椎管狭窄、椎间盘突出、椎体不稳等病症。研究表明,椎间盘退行性病变所引起颈腰痛在所有到医院就诊的患者中占第2位,仅次于呼吸道感染感染性疾病。在椎间盘退变过程中,椎间盘组织的组分、结构及功能都发生了改变,其中包括髓核组织中蛋白聚糖成分和水分的逐渐丢失,纤维环结构排列的紊乱,以及软骨终板的钙化及血管化,邻近椎体骨赘的形成等。然而椎间盘退变的具体病理生理机制目前还不清楚。椎间盘是脊柱运动节段中最为关键的结构,并且是较早发生退行性改变的组织。缺氧、异常应力的作用和酸性环境都与椎间盘退变密切相关,其中应力扮演了非常重要的角色。大量的研究证实,异常力学因素的持续作用是导致椎间盘退变的主要原因之一,然而在此过程中所隐藏的具体病理生理机制目前还不清楚。在之前的研究中,我们发现人退变软骨终板中存在类似于间充质干细胞样的细胞(Cartilage endplate derived stem cells,CESCs)并成功分离出来。此外我们的团队在后期的研究中还发现:CESCs具有较强的克隆形成能力以及多向分化潜能。近年来,应力在椎间盘退变过程中的作用及相关机制被广泛关注和研究。本研究旨在前期的研究基础上,以软骨终板干细胞为研究对象,使用周期性拉伸应力装置FX-4000对软骨终板干细胞进行应力加载,初步探讨周期性拉伸应力对软骨终板干细胞分化的影响。目的:临床上获得退变椎间盘的软骨终板标本,对所获得的软骨终板进行消化,原代细胞贴壁培养、扩增后,采用我们既往的方法,应用琼脂糖悬浮培养系统从第一代细胞中筛选出软骨终板干细胞。通过观察应力对软骨终板干细胞成骨分化的影响,并初步探讨其在椎间盘退变过程中的意义,以期为椎间盘退行性疾病的预防和治疗提供新的思路和理论依据。临床上获得退变椎间盘的软骨终板标本(在腰椎融合手术中分离),于分离后2小时之内带入超净工作台。对获取的软骨终板组织在解剖显微镜下,使用眼科手术器械对获取组织标本进行再次清理,清理结束后,磷酸盐缓冲液(phosphate buffer,PBS)冲洗后。将洗净的组织剪切成体积约为1mm×1mm×1mm大小的组织块,将剪碎组织块转移入25cm2培养瓶中,加入约5倍体积含0.15%Ⅱ型胶原酶无血清DMEM/F12培养基,静置于37℃,5%CO2培养箱过夜消化。消化完成后,用70μm的细胞滤网过滤消化液,将滤液转移至无菌的离心管中,1000r/min离心5min。离心结束后,取出离心管,倒掉上清液。收集细胞沉淀,加入含有弃上清,加入含有10%FBS、1%双抗的BMEM/F12的完全培养基重悬细胞,置于细胞培养箱中培养。每3天更换一次细胞培养液,倒置相差显微镜下观察软骨终板细胞的生长情况。当原代培养的细胞达90%融合后,利用琼脂悬浮培养系统筛选出CESCs进行进一步培养。然后对筛选出来的细胞行三系诱导分化鉴定和流式细胞术表型分析。选取生长良好的第三代CESCs,接种于Bio Flex 6孔培养板中。待细胞贴壁生长至80-90%融合后,换成FBS体积分数为1%的DMEM/F12培养液继续培养12小时,使各组细胞同步化。在无诱导因子的作用下,将培养板置于Flexcell-4000TM应力加载系统中,施加1h、6h、12h、24h,频率为1Hz、拉伸率为10%的牵拉刺激,同时做静态对照实验。牵拉完成后,收集细胞,应用Western blot检测BMP-2的表达情况,使用q PCR测定部分成骨、成软骨基因的表达变化结果:1.将获得的软骨终板细胞进行筛选后,应用流式细胞术及三系诱导分化鉴定提示筛选出来的细胞具有干细胞特性。2.将筛选出来的CESCs进行周期性应力加载后,发现与成骨有关的基因(BMP-2、ALP、Runx2)的表达量较对照组明显增加,差异具有统计学意义。而与成软骨有关基因(SOX9)表达量随拉伸时间的延长,表达量却逐渐降低。结论:1.通过流式细胞术和三系诱导分化实验证实,我们经过筛选所得的细胞具有干细胞的特性,这和我们之前的实验结果相一致。2.周期性拉伸应力可以促进软骨终板干细胞中的BMP-2的表达,同时上调成骨相关基因的表达,促使软骨终板干细胞向成骨细胞分化。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.53

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