本文選題：椎間盤(pán)退變 + 軟骨終板干細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：研究背景與目的:在骨科這一學(xué)科的門(mén)診及臨床實(shí)踐工作中,腰椎間盤(pán)突出癥是一種最為常見(jiàn)的嚴(yán)重困擾人們健康、影響廣大勞動(dòng)人民正常工作與生活的慢性腰椎退行性疾患。該疾患具有發(fā)病率高、慢性及長(zhǎng)期反復(fù)發(fā)作的特點(diǎn),伴隨著當(dāng)今生活節(jié)奏的加快及我國(guó)老齡化人口比例的不斷攀升,其患者人數(shù)也呈逐漸上升趨勢(shì)。由于該病較高的人群發(fā)生率及其慢性病程給病患帶來(lái)的巨大痛苦,國(guó)內(nèi)外的很多學(xué)者從病因、發(fā)病機(jī)制、診斷治療及遠(yuǎn)期預(yù)后等諸多方面對(duì)其進(jìn)行了有益的探索。對(duì)于椎間盤(pán)的解剖結(jié)構(gòu),它是由位于上下兩端的椎間盤(pán)軟骨終板、中央?yún)^(qū)域的髓核、環(huán)狀包繞外周的的纖維環(huán)等三部分組成的一個(gè)封閉的血供較差的軟骨樣組織。作為承擔(dān)連接相鄰椎體的重要部分,其功能的穩(wěn)定性和結(jié)構(gòu)的完整性對(duì)于緩沖機(jī)體外部生物負(fù)荷及維持機(jī)體內(nèi)部力學(xué)穩(wěn)定性起著至關(guān)重要的作用。從椎間盤(pán)的病理生理與解剖學(xué)角度來(lái)看,在椎間盤(pán)纖維環(huán)、髓核、軟骨終板、椎體、黃韌帶、小關(guān)節(jié)的退變的諸多學(xué)說(shuō)中,由髓核細(xì)胞退變所導(dǎo)致的腰椎退行性疾病是目前被很多學(xué)者所接受的導(dǎo)致該疾患的重要病因。髓核是位于椎間盤(pán)中央偏右的呈膠凍狀的組織,它由纖維母細(xì)胞和軟骨樣細(xì)胞這兩種細(xì)胞及大量的小分子彈性粘糖蛋白、水分等細(xì)胞外基質(zhì)所構(gòu)成。兒童時(shí)期的椎間盤(pán)纖維環(huán)與中心區(qū)域的髓核界限清楚,伴隨著年齡的增長(zhǎng),髓核內(nèi)的水分與彈性粘糖蛋白含量會(huì)減低、髓核與纖維環(huán)內(nèi)層的分界會(huì)變的模糊不清,髓核組織會(huì)變得發(fā)白而堅(jiān)硬。而椎間盤(pán)內(nèi)含水量的比例和多寡將直接影響椎間盤(pán)的彈性狀態(tài)及其盤(pán)內(nèi)的壓力水平,最終會(huì)導(dǎo)致椎間盤(pán)的生物力學(xué)性質(zhì)發(fā)生改變。在輕微的外界應(yīng)力(例如打噴嚏、彎腰)或長(zhǎng)期腰部不均衡外力等因素作用下,很容易導(dǎo)致腰椎間盤(pán)突出,從而導(dǎo)致腰部及下肢等一系列相應(yīng)的臨床癥狀的出現(xiàn)。關(guān)于腰椎間盤(pán)突出癥的治療方法有很多種,歸納起來(lái)主要包括傳統(tǒng)的保守治療、手術(shù)治療和以組織工程等為代表的三大類治療策略。保守治療適用于腰部或下肢疼痛及麻木感不顯著的患者,主要的治療手段有臥床休息、物理牽引、按摩以及服用非甾體類抗炎藥物塞來(lái)昔布等。但如果經(jīng)正規(guī)的保守治療三個(gè)月或半年后無(wú)明顯效果,那么手術(shù)則成為目前臨床主要的治療手段。手術(shù)療法包括傳統(tǒng)的腰椎間盤(pán)髓核切除術(shù)及近年新興的微創(chuàng)手術(shù)方式兩大類,通過(guò)手術(shù),將退變突出的髓核組織摘除,實(shí)現(xiàn)解除或緩解突出物對(duì)神經(jīng)根或馬尾神經(jīng)壓迫的目的。關(guān)于微創(chuàng)與標(biāo)準(zhǔn)手術(shù)方法的優(yōu)劣,目前還一直存在諸多爭(zhēng)議。上世紀(jì)末,隨著組織工程技術(shù)等新興學(xué)科的興起,利用細(xì)胞移植等技術(shù)手段從根本上延緩髓核細(xì)胞退變或直接體外構(gòu)建人工椎間盤(pán)等策略讓科研工作者重新看到了解決這一臨床難題的曙光。椎間盤(pán)細(xì)胞移植治療是一種新興的治療手段,通過(guò)細(xì)胞移植,可以使椎間盤(pán)內(nèi)的細(xì)胞總量得到增加,從而實(shí)現(xiàn)椎間盤(pán)內(nèi)蛋白聚糖及膠原蛋白含量的升高的目的,這被認(rèn)為是能夠延緩椎間盤(pán)退變的一種有效的治療方法。對(duì)于細(xì)胞移植的種子細(xì)胞來(lái)源,自體的未退變髓核細(xì)胞由于無(wú)排異反應(yīng)等優(yōu)點(diǎn)而被公認(rèn)為是最為理想的種子細(xì)胞來(lái)源之一,但由于自身髓核細(xì)胞數(shù)目較少且是一種有創(chuàng)的操作方式而限制了其應(yīng)用。而來(lái)自術(shù)中的取自椎間盤(pán)退變性疾病患者的髓核細(xì)胞又存在細(xì)胞活力不足等一些弊端。因此,尋找到一種合適而正確的種子細(xì)胞就成為亟需解決的問(wèn)題。研究方法:本實(shí)驗(yàn)的的軟骨終板干細(xì)胞(cartilageendplatestemcells,cescs)和髓核細(xì)胞(nucleuspulposuscells,npcs)取自15例因退變性腰椎疾患而行腰椎椎間盤(pán)切除椎弓根螺釘內(nèi)固定的患者。通過(guò)低熔點(diǎn)瓊脂糖懸浮培養(yǎng)法獲得軟骨終板干細(xì)胞的克隆,經(jīng)免疫熒光技術(shù)和流式細(xì)胞技術(shù)進(jìn)行干細(xì)胞表面標(biāo)記檢測(cè),利用擴(kuò)增出的第三代軟骨終板干細(xì)胞與第一代髓核細(xì)胞進(jìn)行研究,實(shí)驗(yàn)共分為三組:單獨(dú)cescs組、單組退變npcs培養(yǎng)組、cescs與退變npcs共培養(yǎng)組。在實(shí)驗(yàn)開(kāi)始后的第3、5、7天,利用熒光實(shí)時(shí)定量pcr(real-timepcr,rt-pcr)檢測(cè)各個(gè)組中Ⅱ型膠原蛋白、sox-9和聚集蛋白聚糖(aggrecan,agc)的mrna表達(dá)變化情況;在共培養(yǎng)的第七天,采用蛋白免疫印跡技術(shù)(western-blot)檢測(cè)各組細(xì)胞中蛋白聚糖、ii型膠原蛋白、sox-9蛋白的表達(dá)變化,利用流式細(xì)胞儀檢測(cè)共培養(yǎng)對(duì)髓核細(xì)胞凋亡率的影響;利用cck-8檢測(cè)共培養(yǎng)對(duì)髓核細(xì)胞增殖能力的影響。結(jié)果:從所取標(biāo)本中進(jìn)行軟骨終板干細(xì)胞及退變髓核細(xì)胞的分離、鑒定及培養(yǎng),經(jīng)流式細(xì)胞技術(shù)分析,經(jīng)低熔點(diǎn)瓊脂糖懸浮培養(yǎng)法篩選出的為軟骨終板干細(xì)胞,rt-pcr檢測(cè)顯示,在共培養(yǎng)后,單獨(dú)培養(yǎng)的cescs在各個(gè)時(shí)間點(diǎn)幾乎均未檢測(cè)到collⅡ,sox-9及agc的表達(dá);共培養(yǎng)組中的cescs在共培養(yǎng)的第五天開(kāi)始出現(xiàn)collⅡ,sox-9及Agc表達(dá),與單獨(dú)培養(yǎng)的CESCs相應(yīng)基因表達(dá)量相比具有統(tǒng)計(jì)學(xué)意義(p0.05);同樣地,共培養(yǎng)組中的NPCs的CollⅡ,SOX-9及Agc等基因的表達(dá)量高于NPCs單獨(dú)培養(yǎng)組(p0.05);其表達(dá)量隨著共培養(yǎng)時(shí)間延長(zhǎng)逐漸升高;在共培養(yǎng)的第七天,利用Western-blot技術(shù)檢測(cè)出的各組細(xì)胞中蛋白聚糖、II型膠原蛋白、Sox-9蛋白的表達(dá)變化與RT-PCR檢測(cè)結(jié)果一致。通過(guò)共培養(yǎng),使髓核細(xì)胞的凋亡率降低,增殖能力增強(qiáng)。結(jié)論:CESCs與NPCs這兩種細(xì)胞共培養(yǎng)時(shí)的相互作用能促使CESCs表達(dá)CollⅡ,SOX-9及Agc等髓核細(xì)胞特異性標(biāo)記物;通過(guò)共培養(yǎng)時(shí)細(xì)胞間的這種相互作用,CESCs有助于NPCs增強(qiáng)自身特異性相關(guān)分子的表達(dá)。此外,共培養(yǎng)在降低退變髓核細(xì)胞的凋亡率同時(shí)增強(qiáng)了其增殖能力。CESCs與NPCs這兩種細(xì)胞共培養(yǎng)時(shí)的相互作用表明,對(duì)于腰椎間盤(pán)突出癥細(xì)胞移植治療方法來(lái)說(shuō),CESCs將會(huì)是一個(gè)新的種子細(xì)胞來(lái)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.53

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張傳志,周躍,李長(zhǎng)青;兔髓核細(xì)胞體外最佳培養(yǎng)條件的探索[J];中國(guó)矯形外科雜志;2005年14期

，

本文編號(hào)：2000423