人软骨终板干细胞与髓核细胞体外共培养实验研究

发布时间：2018-06-09 16:46

本文选题：椎间盘退变 + 软骨终板干细胞　；参考：《第三军医大学》2015年硕士论文

【摘要】：研究背景与目的:在骨科这一学科的门诊及临床实践工作中,腰椎间盘突出症是一种最为常见的严重困扰人们健康、影响广大劳动人民正常工作与生活的慢性腰椎退行性疾患。该疾患具有发病率高、慢性及长期反复发作的特点,伴随着当今生活节奏的加快及我国老龄化人口比例的不断攀升,其患者人数也呈逐渐上升趋势。由于该病较高的人群发生率及其慢性病程给病患带来的巨大痛苦,国内外的很多学者从病因、发病机制、诊断治疗及远期预后等诸多方面对其进行了有益的探索。对于椎间盘的解剖结构,它是由位于上下两端的椎间盘软骨终板、中央区域的髓核、环状包绕外周的的纤维环等三部分组成的一个封闭的血供较差的软骨样组织。作为承担连接相邻椎体的重要部分,其功能的稳定性和结构的完整性对于缓冲机体外部生物负荷及维持机体内部力学稳定性起着至关重要的作用。从椎间盘的病理生理与解剖学角度来看,在椎间盘纤维环、髓核、软骨终板、椎体、黄韧带、小关节的退变的诸多学说中,由髓核细胞退变所导致的腰椎退行性疾病是目前被很多学者所接受的导致该疾患的重要病因。髓核是位于椎间盘中央偏右的呈胶冻状的组织,它由纤维母细胞和软骨样细胞这两种细胞及大量的小分子弹性粘糖蛋白、水分等细胞外基质所构成。儿童时期的椎间盘纤维环与中心区域的髓核界限清楚,伴随着年龄的增长,髓核内的水分与弹性粘糖蛋白含量会减低、髓核与纤维环内层的分界会变的模糊不清,髓核组织会变得发白而坚硬。而椎间盘内含水量的比例和多寡将直接影响椎间盘的弹性状态及其盘内的压力水平,最终会导致椎间盘的生物力学性质发生改变。在轻微的外界应力(例如打喷嚏、弯腰)或长期腰部不均衡外力等因素作用下,很容易导致腰椎间盘突出,从而导致腰部及下肢等一系列相应的临床症状的出现。关于腰椎间盘突出症的治疗方法有很多种,归纳起来主要包括传统的保守治疗、手术治疗和以组织工程等为代表的三大类治疗策略。保守治疗适用于腰部或下肢疼痛及麻木感不显著的患者,主要的治疗手段有卧床休息、物理牵引、按摩以及服用非甾体类抗炎药物塞来昔布等。但如果经正规的保守治疗三个月或半年后无明显效果,那么手术则成为目前临床主要的治疗手段。手术疗法包括传统的腰椎间盘髓核切除术及近年新兴的微创手术方式两大类,通过手术,将退变突出的髓核组织摘除,实现解除或缓解突出物对神经根或马尾神经压迫的目的。关于微创与标准手术方法的优劣,目前还一直存在诸多争议。上世纪末,随着组织工程技术等新兴学科的兴起,利用细胞移植等技术手段从根本上延缓髓核细胞退变或直接体外构建人工椎间盘等策略让科研工作者重新看到了解决这一临床难题的曙光。椎间盘细胞移植治疗是一种新兴的治疗手段,通过细胞移植,可以使椎间盘内的细胞总量得到增加,从而实现椎间盘内蛋白聚糖及胶原蛋白含量的升高的目的,这被认为是能够延缓椎间盘退变的一种有效的治疗方法。对于细胞移植的种子细胞来源,自体的未退变髓核细胞由于无排异反应等优点而被公认为是最为理想的种子细胞来源之一,但由于自身髓核细胞数目较少且是一种有创的操作方式而限制了其应用。而来自术中的取自椎间盘退变性疾病患者的髓核细胞又存在细胞活力不足等一些弊端。因此,寻找到一种合适而正确的种子细胞就成为亟需解决的问题。研究方法:本实验的的软骨终板干细胞(cartilageendplatestemcells,cescs)和髓核细胞(nucleuspulposuscells,npcs)取自15例因退变性腰椎疾患而行腰椎椎间盘切除椎弓根螺钉内固定的患者。通过低熔点琼脂糖悬浮培养法获得软骨终板干细胞的克隆,经免疫荧光技术和流式细胞技术进行干细胞表面标记检测,利用扩增出的第三代软骨终板干细胞与第一代髓核细胞进行研究,实验共分为三组:单独cescs组、单组退变npcs培养组、cescs与退变npcs共培养组。在实验开始后的第3、5、7天,利用荧光实时定量pcr(real-timepcr,rt-pcr)检测各个组中Ⅱ型胶原蛋白、sox-9和聚集蛋白聚糖(aggrecan,agc)的mrna表达变化情况;在共培养的第七天,采用蛋白免疫印迹技术(western-blot)检测各组细胞中蛋白聚糖、ii型胶原蛋白、sox-9蛋白的表达变化,利用流式细胞仪检测共培养对髓核细胞凋亡率的影响;利用cck-8检测共培养对髓核细胞增殖能力的影响。结果:从所取标本中进行软骨终板干细胞及退变髓核细胞的分离、鉴定及培养,经流式细胞技术分析,经低熔点琼脂糖悬浮培养法筛选出的为软骨终板干细胞,rt-pcr检测显示,在共培养后,单独培养的cescs在各个时间点几乎均未检测到collⅡ,sox-9及agc的表达;共培养组中的cescs在共培养的第五天开始出现collⅡ,sox-9及Agc表达,与单独培养的CESCs相应基因表达量相比具有统计学意义(p0.05);同样地,共培养组中的NPCs的CollⅡ,SOX-9及Agc等基因的表达量高于NPCs单独培养组(p0.05);其表达量随着共培养时间延长逐渐升高;在共培养的第七天,利用Western-blot技术检测出的各组细胞中蛋白聚糖、II型胶原蛋白、Sox-9蛋白的表达变化与RT-PCR检测结果一致。通过共培养,使髓核细胞的凋亡率降低,增殖能力增强。结论:CESCs与NPCs这两种细胞共培养时的相互作用能促使CESCs表达CollⅡ,SOX-9及Agc等髓核细胞特异性标记物;通过共培养时细胞间的这种相互作用,CESCs有助于NPCs增强自身特异性相关分子的表达。此外,共培养在降低退变髓核细胞的凋亡率同时增强了其增殖能力。CESCs与NPCs这两种细胞共培养时的相互作用表明,对于腰椎间盘突出症细胞移植治疗方法来说,CESCs将会是一个新的种子细胞来源。
[Abstract]:Background and purpose: in the outpatient and clinical practice of this discipline in the Department of orthopedics, lumbar disc herniation is one of the most common chronic lumbar degenerative diseases that seriously perplex people's health and affect the normal work and life of the working people. The disease has the characteristics of high incidence, chronic and long-term recurrent attacks. As the pace of life is quickening and the proportion of aging population is rising in China, the number of patients is increasing gradually. Because of the high incidence of the population and the great pain caused by the chronic course of disease, many scholars at home and abroad have carried out the cause, the pathogenesis, the diagnosis and the long-term prognosis and so on. The anatomical structure of the intervertebral disc is a closed blood donor cartilage like tissue consisting of three parts of the intervertebral disc, the nucleus of the intervertebral disc at the upper and lower ends, the nucleus of the central region, and the fibrous ring around the periphery of the circumference. The integrity of the structure plays a vital role in cushioning the external biological load and maintaining mechanical stability within the body. From the pathophysiological and anatomical aspects of the intervertebral disc, the lumbar disc, nucleus, cartilage endplate, vertebral, ligamentum, and small joints are degenerated by the degeneration of nucleus pulposus cells. Vertebrodegenerative disease is an important cause of the disease, which is now accepted by many scholars. The nucleus pulposus is a frozen tissue located on the right of the intervertebral disc. It consists of two kinds of cells, such as fibroblast and chondroid cell, and a large number of small molecular elastic viscoplastic proteins, water and other extracellular matrix. There is a clear boundary between the nucleus and the nucleus of the central region. With the age increasing, the content of water and elastic protein in the nucleus of the medulla will decrease, the boundary between the nucleus and the inner layer of the fibrous ring becomes blurred, and the nucleus of the medulla will become white and hard. And the proportion and amount of the water content in the intervertebral disc will directly affect the elastic state of the intervertebral disc. And the pressure level in the disc will eventually lead to a change in the biomechanical properties of the intervertebral disc. A slight external stress (such as sneezing, bending down), or a long unbalanced external force of the waist, can easily lead to the protrusion of the lumbar intervertebral disc, which leads to a series of corresponding clinical symptoms such as the lumbar and lower limbs. There are many kinds of treatment methods for intervertebral disc herniation, which include three major types of treatment strategies, such as traditional conservative treatment, surgical treatment and tissue engineering. Conservative treatment is suitable for patients with low waist or lower extremity pain and insignificant numbness. The main treatment hands are bed rest, physical traction, massage, and use. The non steroidal anti-inflammatory drug, celecoxib, etc., but if there is no obvious effect after three months or half a year after regular conservative treatment, then the operation is the main clinical treatment. The surgical treatment includes the traditional lumbar intervertebral disc nucleus excision and the newly emerging minimally invasive surgical recipe in recent years two major categories, through surgery, the degeneration of the pulp protruding the pulp. Nuclear tissue is removed to relieve or alleviate the oppression of the nerve root or the cauda equina. There are still many disputes about the advantages and disadvantages of minimally invasive and standard surgical methods. At the end of last century, with the rise of new disciplines such as tissue engineering technology, cell transplantation was used to retard the degeneration of nucleus pulposus cells. The strategy of constructing artificial intervertebral discs directly in vitro allows researchers to re see the dawn of this clinical problem. Intervertebral disc cell transplantation is a new treatment. Cell transplantation can increase the total amount of cells in the intervertebral disc so that the content of protein and collagen in the intervertebral disc is realized. It is considered to be an effective treatment for degrading intervertebral disc degeneration. For the source of cell transplantation, autologous non degenerative nucleus pulposus is recognized as one of the most ideal source of seed cells because of the advantages of no rejection, but the number of cells in which the nucleus is less and is a kind of one. A invasive operation restricts its application, and the nucleus cells derived from the patients with degenerative disease of the intervertebral disc have some disadvantages, such as the insufficiency of cell vitality. Therefore, to find a suitable and correct seed cell is a problem to be solved urgently. Dplatestemcells, cescs) and nucleus pulposus cells (nucleuspulposuscells, NPCs) were obtained from 15 patients with lumbar disc resection of lumbar disc pedicle screw fixation due to degenerative lumbar disease. The cloning of cartilage endplate stem cells was obtained by low melting point agarose suspension culture, and the surface of stem cell surface was carried out by immunofluorescence technique and flow cytometry. The amplified third generation cartilage end plate stem cells and the first generation nucleus pulposus cells were studied. The experiment was divided into three groups: single cescs group, single group of degenerative NPCs culture group, cescs and degenerative NPCs co culture group. On day 3,5,7 after the experiment, fluorescence real-time quantitative PCR (real-timepcr, RT-PCR) was used to detect type II in each group. The mRNA expression of collagen, Sox-9 and aggregation proteoglycan (aggrecan, AGC) was changed. In the seventh days of co culture, protein immunoblotting (Western-blot) was used to detect the changes in the expression of proteoglycan, II type collagen and Sox-9 protein in each cell, and the effect of co culture on the apoptosis rate of nucleus pulposus cells was detected by flow cytometry. The effects of co culture on the proliferation of nucleus pulposus cells were detected by CCK-8. Results: the isolation, identification and culture of cartilage endplate stem cells and degenerative nucleus pulposus cells from the specimens were analyzed by flow cytometry, and the cartilage endplate stem cells were screened by the low melting point agarose suspension culture method. The RT-PCR detection showed that after co culture, the cells were screened by the low melting point agarose suspension culture. The expression of Coll II, Sox-9 and AGC was not detected at all time points in the single cultured cescs, and the cescs in the co culture group began to appear coll II, Sox-9 and Agc in the fifth days of co culture, and was statistically significant compared with the corresponding gene expression of the isolated CESCs (P0.05); similarly, NPCs Coll II in co culture group was found. The expression of 9 and Agc was higher than that of NPCs alone (P0.05), and its expression increased gradually with the time of co culture. In the seventh day of co culture, the expression of proteoglycan, II type collagen and Sox-9 protein in all the cells detected by Western-blot was in accordance with the results of RT-PCR detection. The nucleus pulposus was produced by co culture. The apoptosis rate of the cells is reduced and the proliferation ability is enhanced. Conclusion: the interaction between the two cells of CESCs and NPCs can induce CESCs to express the specific markers of nucleus pulposus cells such as Coll II, SOX-9 and Agc, and CESCs helps NPCs to enhance the expression of its own specific related molecules by CO culture. The interaction between.CESCs and NPCs, which reduces the apoptosis rate of degenerative nucleus pulposus cells and increases the proliferation of.CESCs and NPCs, indicates that CESCs will be a new seed cell source for the treatment of herniated intervertebral disc herniation.
【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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