【摘要】：背景目前我國65歲以上老年人已達(dá)1.5億之多,占總?cè)丝诘?0.8%,隨著我國老齡化社會時代的到來,由椎間盤退變引發(fā)的頸腰痛疾病也在不斷增多,嚴(yán)重困擾著老年人的身體健康及生活質(zhì)量,同時對于中青年人群來講,腰椎退變性疾病也是導(dǎo)致勞動能力喪失的主要原因之一。根據(jù)最新流行病學(xué)調(diào)查顯示,80%的人一生中至少經(jīng)歷一次腰腿痛疾病的困擾,給個人帶來巨大身心痛苦的同時,也給家庭和社會帶來嚴(yán)重的經(jīng)濟(jì)負(fù)重,因此,尋求一種從根本上有效治療頸肩腰腿痛疾病的方法是醫(yī)療衛(wèi)生領(lǐng)域急需解決的重要問題之一。椎間盤退行性變(Interverbral disc degeneration IDD)發(fā)病機(jī)制復(fù)雜,遺傳、肥胖、吸煙、職業(yè)、年齡、外傷都可以誘導(dǎo)疾病的發(fā)生。IDD是多種因素作用于髓核、纖維環(huán)和終板的結(jié)果,是一系列脊柱退變性疾病的發(fā)生前提和基礎(chǔ)。腰椎間盤退變的病理生理學(xué)方面主要表現(xiàn)為椎間盤內(nèi)髓核細(xì)胞減少,細(xì)胞功能降低,椎間盤細(xì)胞外基質(zhì)的合成減少、降解加速,代謝失去平衡。臨床上主要表現(xiàn)為椎管狹窄、椎間盤突出、椎間盤源性頸腰腿痛等病癥,是一種常見病、多發(fā)病。目前臨床上治療腰椎間盤退變性疾病的方法包括以藥物及推拿、按摩為主的保守治療和以椎間盤髓核摘除、脊柱融合固定及人工椎間盤置換為主的手術(shù)治療。保守治療只是對癥治療,效果不確切,不能延緩椎間盤退變的進(jìn)程;手術(shù)治療方面,髓核摘除會造成椎間盤高度丟失以及相關(guān)的生物力學(xué)、解剖結(jié)構(gòu)變化,可能會進(jìn)一步加重椎間盤退變及腰椎不穩(wěn),甚至影響臨近椎間盤受力的改變,脊柱融合內(nèi)固定僅僅解決病變節(jié)段的壓迫及不穩(wěn)定,不僅不能保持椎間盤功能,甚至可能因為脊柱正常生物力學(xué)平衡的改變,應(yīng)力集中于臨近節(jié)段而發(fā)生鄰近節(jié)段病。因此,尋求一種新的治療方法是目前的當(dāng)務(wù)之急,生物學(xué)治療為椎間盤再生提供一種新的思路和方法。椎間盤退變的主要病理生理學(xué)改變是椎間盤細(xì)胞減少,細(xì)胞功能降低,細(xì)胞外基質(zhì)代謝失衡,因此理想的治療方式應(yīng)能夠維持椎間盤組織內(nèi)正常細(xì)胞的數(shù)量,調(diào)控椎間盤細(xì)胞功能,促進(jìn)細(xì)胞外基質(zhì)的分泌,減緩細(xì)胞外基質(zhì)降解。種子細(xì)胞、生長因子和支架材料是修復(fù)椎間盤退變?nèi)笾鲗?dǎo)因素。骨髓間充質(zhì)干細(xì)胞(bone marrow derived mesenchymal stem cells,BMSCs),來源于骨髓間質(zhì),具有多向分化潛能,在不同的培養(yǎng)基、生長因子等因素的誘導(dǎo)下可分化為骨細(xì)胞、軟骨細(xì)胞、成纖維細(xì)胞、心肌細(xì)胞、脂肪細(xì)胞等。同時BMSCs具備取材方便、體外生長迅速的特點,且來源充足、易于分離培養(yǎng)、增殖能力強(qiáng),無論是在體外還是在體內(nèi)抑或在自體還是在同種異體移植過程中均具有較低免疫原性,是椎間盤組織工程中最理想種子細(xì)胞之一。但是在體內(nèi)BMSCs具體分化成哪一類細(xì)胞,主要決定于細(xì)胞所處的局部環(huán)境,在髓核這種低氧的微環(huán)境及在轉(zhuǎn)化生長因子-β1誘導(dǎo)下,BMSCs可以向類髓核細(xì)胞分化。轉(zhuǎn)化生長因子-β1(Transforming Growth Factor-β1,TGF-β1)是TGF-β家族最主要成員,含量最高,活性也是最強(qiáng),在BMSCs向軟骨細(xì)胞分化過程中,TGF-β1通過配體結(jié)合的方式與Ⅰ、Ⅱ型受體結(jié)合,導(dǎo)致Ⅰ型受體磷酸化,進(jìn)而激活Smad蛋白,再進(jìn)入細(xì)胞核,調(diào)節(jié)目的基因的轉(zhuǎn)錄,通過改變蛋白酶底物的活性,TGF-β1對靶細(xì)胞起到刺激作用,TGF-β1還具有促進(jìn)軟骨細(xì)胞增殖,維持軟骨細(xì)胞表型的作用,但在早期軟骨細(xì)胞培養(yǎng)中就發(fā)現(xiàn),TGF-β1對軟骨細(xì)胞的增殖分化具有雙向調(diào)節(jié)作用,是否為抑制或促進(jìn)與TGF-β1的濃度高度關(guān)聯(lián)。但是TGF-β1作為外源性生長因子,半衰期短,易被蛋白水解酶水解,局部應(yīng)用還未促使誘導(dǎo)完成及形成一定數(shù)量的細(xì)胞外基質(zhì),生物分子卻已降解,如何使生長因子持續(xù)高效發(fā)揮作用是骨組織工程學(xué)研究亟待解決的關(guān)鍵問題。明膠無毒性,在體內(nèi)可降解,具有良好的生物學(xué)特性,是生產(chǎn)微球的首選材料,目前是制藥緩釋系統(tǒng)研究的重點。明膠微球的性質(zhì)、制備方法已比較成熟,在藥物緩釋載體領(lǐng)域已廣泛應(yīng)用,明膠微球直徑10μm左右,散在分布,可以作為椎間盤組織工程的支架材料,完全降解約需2個月左右,且大分子肽類物質(zhì)自微球內(nèi)部向外部擴(kuò)散速度較慢,這些因素決定了TGF-β1明膠微球具有良好的緩釋性能,而且具有可注射性的優(yōu)點。TGF-β1搭載明膠微球緩釋體長時間誘導(dǎo)BMSCs類髓核細(xì)胞分化有效抑制了椎間盤退變,在椎間盤組織工程研究中有廣闊的應(yīng)用前景。本研究中:我們體外構(gòu)建椎間盤髓核組織工程材料,并將其移植到成功造模的兔退變椎間盤中,TGF-β1復(fù)合明膠微球長時間誘導(dǎo)BMSCs向類髓核細(xì)胞分化、促進(jìn)髓核細(xì)胞增殖并合成蛋白多糖及II型膠原,兩者聯(lián)合應(yīng)用可有效延緩椎間盤退變。第一部分:兔骨髓間充質(zhì)干細(xì)胞(BMSCs)分離、純化、體外培養(yǎng)及鑒定目的:通過密度梯度離心法體將BMSCs從胎兔骨髓中分離純化、培養(yǎng)并對其進(jìn)行鑒定,為探討其聯(lián)合明膠微球復(fù)合TGF-β1修復(fù)椎間盤退變提供實驗基礎(chǔ)。方法:兔髂骨穿刺,抽取骨髓液10ml,密度梯度離心法分離BMSCs原代細(xì)胞,體外培養(yǎng)、擴(kuò)增、傳代,倒置顯微鏡下觀察原代及P3、P5代細(xì)胞形態(tài),進(jìn)行P3代BMSCs生長曲線分析,流式細(xì)胞術(shù)對P3代BMSCs表面抗原CD105、CD90、CD31、CD14進(jìn)行鑒定,BMSCs誘導(dǎo)分化為成骨細(xì)胞、成脂細(xì)胞,茜素紅染色成骨細(xì)胞中鈣結(jié)節(jié),油紅O染色成脂細(xì)胞內(nèi)的脂滴。結(jié)果:使用密度梯度離心法BMSCs提取成功,倒置相差顯微鏡下觀察,BMSCs在24 h后已有部分細(xì)胞開始貼壁生長,原代BMSCs經(jīng)選擇性消化法傳代至第三代,細(xì)胞純度約達(dá)到90%以上,且第三代BMSCs和第五代BMSCs倒置相差顯微鏡下觀察細(xì)胞狀態(tài)良好;CCK-8細(xì)胞增殖活性檢測顯示:P3代BMSCs細(xì)胞增殖活性良好,細(xì)胞于第2天開始進(jìn)入對數(shù)生長期,一直維持到5-6天,于第6天后進(jìn)入平臺期;細(xì)胞陽性標(biāo)志為CD90和CD105,表達(dá)率分別為93.67%和92.03%;陰性標(biāo)志為CD14和CD31,表達(dá)率分別為0.11%和1.08%;P3代BMSCs經(jīng)特殊成骨、成脂誘導(dǎo)劑誘導(dǎo)培養(yǎng),均可定向分化為成骨細(xì)胞、成脂細(xì)胞,茜素紅染色發(fā)現(xiàn)鈣結(jié)節(jié),油紅O染色示細(xì)胞內(nèi)的脂滴。結(jié)論:在胎兔骨髓液中通過密度梯度離心法順利提取BMSCs,然后通過選擇性消化法對細(xì)胞進(jìn)行純化,可獲得高增殖活性、高純度的BMSCs。通過對細(xì)胞表面分子和多向分化潛能進(jìn)行鑒定,證實所提取細(xì)胞為BMSCs。為后續(xù)BMSCs聯(lián)合已復(fù)合TGF-β1的明膠微球治療椎間盤退變提供實驗基礎(chǔ)。第二部分:TGF-β1明膠微球的制作、體外釋放及對BMSCs增殖活性的研究目的:探討TGF-β1明膠微球制作工藝及體外釋放及對BMSCs增殖活性的影響。方法:乳化交聯(lián)法制備空白明膠微球,光鏡及電鏡觀察明膠微球形態(tài)特征及粒徑分析,振搖-離心法將TGF-β1載到空白微球中,ELISA法檢測TGF-β1的體外釋放,計算微球載藥量及包封率,CCK-8法檢測對BMSCs增殖活性的影響。結(jié)果:經(jīng)乳化交聯(lián)法制作明膠微球,普通顯微鏡下觀察交聯(lián)后、凍干前的微球,可見粒徑較均勻,成球性良好,無粘連,掃描電鏡下觀察可見微球表面光滑,粒徑分布約為1-20μm,平均粒徑10.55±1.25μm;每mg明膠微球加入60ng TGF-β1包封率為98%以上,載藥質(zhì)量為58.76±0.026ng,載藥量為5.9%,體外釋放14天,累積釋放TGF-β1為96%以上;吸附TGF-β1的明膠微球隨著時間緩慢釋放TGF-β1,對BMSCs增殖其促進(jìn)作用。結(jié)論:通過乳化法成功制作出明膠微球,明膠微球電鏡下觀察,微球形態(tài)圓整,表面光滑,粒徑均勻;其吸附TGF-β1緩釋可達(dá)14天;且與BMSCs共培養(yǎng),可顯著增強(qiáng)BMSCs增殖活性。第三部分:椎間盤退變動物模型的制作及鑒定目的:探討髓核抽吸法建立腰椎間盤退變動物模型及通過影像學(xué)、分子生物學(xué)進(jìn)行模型鑒定的可行性。方法:側(cè)臥位經(jīng)肌間隙入路,18G針頭的注射器(10ml)依次刺破L3/4、L4/5、L5/6椎間盤,深度約5mm抽吸時間約20秒,建立椎間盤退變動物模型;術(shù)后1w、2w、3w、4w磁共振檢查,測量L3/4、L4/5、L5/6椎間盤磁共振指數(shù),免疫組織化學(xué)法檢測椎間盤髓核中蛋白聚糖變化,HE染色觀察髓核細(xì)胞排列及形態(tài)的變化。結(jié)果:經(jīng)肌間隙入路進(jìn)行兔L3/4、L4/5、L5/6椎間盤髓核抽吸,平均抽出髓核質(zhì)量10mg,無意外死亡;術(shù)后1周到術(shù)后4周,從術(shù)后第2周開始,磁共振指數(shù)持續(xù)降低,差異有顯著統(tǒng)計學(xué)意義,術(shù)后第2周開始,椎間盤退變模型內(nèi)的蛋白聚糖表達(dá)即開始出現(xiàn)差異,且隨著時間的延長,差異越明顯;髓核抽吸組,隨著時間的延長,髓核與纖維環(huán)分局模糊,髓核細(xì)胞減少,分布不均勻,髓核外基質(zhì)結(jié)構(gòu)紊亂。結(jié)論:經(jīng)肌間隙髓核抽吸法成功構(gòu)建椎間盤退變動物模型,此種方法具有操作簡便,手術(shù)時間短,重復(fù)性好,方便開展的優(yōu)點。通過此種方法構(gòu)建的腰椎間盤退變模型術(shù)后實驗動物的感染率低,成功率高。而且通過影像學(xué)、組織形態(tài)學(xué)、分子生物學(xué)對已構(gòu)建模型進(jìn)行評估,均符合前期椎間盤退變征象,可以作為椎間盤退變研究的動物模型。第四部分:BSMCs復(fù)合TGF-β1明膠微球治療椎間盤退變的實驗研究目的:探討B(tài)SMCs復(fù)合TGF-β1明膠微球治療椎間盤退變的可行性,為以后椎間盤退變治療的研究提供一定的借鑒。方法:體外通過骨髓間充質(zhì)干細(xì)胞(BMSCs)聯(lián)合吸附TGF-β1的明膠微球構(gòu)建的椎間盤組織工程材料移植至兔椎間盤制作4周后動物模型中。將60只新西蘭大白兔隨機(jī)分成5組,每組12只動物:A組空白對照組(Control);B組生理鹽水組(NS);C組BMSCs組;D組BMSCs+未吸附TGF-β1的明膠微球注射組(BMSCs+gelatin);E組BMSCs+吸附TGF-β1的明膠微球注射組(BMSCs+gelatin/TGF-β1)。在移植后3w、6w、12w分別通過MRI、RT-q PCR、HE染色和免疫組化(IHC)對椎間盤的修復(fù)程度進(jìn)行評估。結(jié)果:經(jīng)MRI檢測顯示,除A組外,各組椎間盤內(nèi)信號均不同程度降低,B組降低最明顯,E組信號降低最緩慢;分別在移植術(shù)后3w、6w、12w使用MRI指數(shù)對各組進(jìn)行比較發(fā)現(xiàn):3w時,只有A組與B組相比較具有顯著性差異;6w時,除A組外各組椎間盤MRI指數(shù)均不同程度降低,B、C、D組三組與A組相比,差異具有統(tǒng)計學(xué)意義,但A組和E組相比,無顯著性差異;12w時,除A組外各組MRI指數(shù)繼續(xù)降低,但降低趨勢較3w-6w時變緩;分別在3w、6w、12w時取各組椎間盤髓核提取細(xì)胞總RNA,通過RT-q PCR檢測各組蛋白聚糖、II型膠原m RNA表達(dá),經(jīng)統(tǒng)計學(xué)分析顯示,E組蛋白聚糖、II型膠原m RNA表達(dá)量最高,且差異具有統(tǒng)計學(xué)意義。各組蛋白聚糖、II型膠原蛋白表達(dá)顯示A組與E組均無顯著性差異;12w光鏡下觀察HE染色顯示,A組椎間盤病理切片椎間盤髓核完整,髓核與纖維環(huán)分界清晰,纖維環(huán)結(jié)構(gòu)接近正常,髓核細(xì)胞呈圓形空泡狀,分布均勻,B組,髓核與纖維環(huán)分局模糊,髓核細(xì)胞明顯減少,大小不等,分布不均勻,髓核外基質(zhì)結(jié)構(gòu)紊亂,C組髓核細(xì)胞減少,纖維環(huán)排列稍清晰。D組髓核細(xì)胞較C組少,椎間盤核外基質(zhì)排列紊亂,E組纖維環(huán)結(jié)構(gòu)接近正常,髓核細(xì)胞分布略均勻,髓核外基質(zhì)結(jié)構(gòu)規(guī)則。結(jié)論:將構(gòu)建的椎間盤組織工程材料移植到已退變的椎間盤中,椎間盤退變顯著變緩。通過MRI可以觀察到移植的組織工程材料可以顯著延緩椎間盤內(nèi)含水量的丟失;RT-q PCR和免疫組化檢測細(xì)胞外基質(zhì)(蛋白聚糖、II型膠原)顯著較其他干預(yù)組表達(dá)高;通過病理學(xué)檢查,可以觀察到椎間盤組織工程材料能夠維持椎間盤的結(jié)構(gòu),延緩了椎間盤進(jìn)一步退變,促進(jìn)退變椎間盤修復(fù)。
[Abstract]:Background At present, the number of elderly people over 65 years old in China has reached 150 million, accounting for 10.8% of the total population. With the advent of the age of aging society, cervical and lumbar pain caused by intervertebral disc degeneration is also increasing, seriously plaguing the health and quality of life of the elderly. At the same time, for young and middle-aged people, lumbar degenerative disease is also. According to the latest epidemiological survey, 80% of the people suffer from low back and leg pain at least once in their lifetime, which brings great physical and mental pain to individuals, but also brings serious economic burden to families and society. Therefore, we should seek a radical and effective treatment for cervical, shoulder, waist and leg pain. Interverbral disc degeneration (IDD) is a complex pathogenesis, inherited, obese, smoking, occupation, age, trauma can induce the occurrence of disease. IDD is the result of a variety of factors acting on the nucleus pulposus, annulus fibrosus and endplate, is a series of spinal degeneration. Pathophysiology of lumbar intervertebral disc degeneration mainly manifests as reduction of nucleus pulposus cells, decrease of cell function, reduction of extracellular matrix synthesis, accelerated degradation and metabolic imbalance. At present, the clinical treatment of lumbar intervertebral disc degeneration disease includes drug and massage, massage-based conservative treatment and surgical treatment based on discectomy, spinal fusion fixation and artificial disc replacement. Intervertebral disc degeneration process; surgical treatment, nucleus pulposus removal will cause disc height loss and related biomechanical, anatomical structure changes, may further aggravate disc degeneration and lumbar instability, and even affect the stress changes of adjacent intervertebral disc, spinal fusion and internal fixation only solve the compression and instability of the lesion segment, not. Therefore, it is urgent to find a new method of treatment. Biological treatment provides a new idea and method for intervertebral disc regeneration. Physiological changes are the reduction of intervertebral disc cells, the decrease of cell function and the imbalance of extracellular matrix metabolism. Therefore, the ideal treatment should be able to maintain the number of normal cells in the intervertebral disc tissue, regulate the function of intervertebral disc cells, promote the secretion of extracellular matrix, and slow down the degradation of extracellular matrix. Bone marrow derived mesenchymal stem cells (BMSCs), derived from bone marrow mesenchymal stem cells (BMSCs), have multiple differentiation potential. BMSCs can differentiate into osteocytes, chondrocytes, fibroblasts, cardiomyocytes, adipocytes and so on under the induction of different media, growth factors and other factors. At the same time, BMSCs are one of the most ideal seed cells in intervertebral disc tissue engineering, because they have the characteristics of convenient materials, rapid growth in vitro, abundant sources, easy isolation and culture, strong proliferation ability, low immunogenicity in vitro, in vivo or in the process of autologous or allogeneic transplantation. The differentiation of BMSCs into nucleus pulposus-like cells is mainly determined by the local environment in which the cells are located. BMSCs can differentiate into nucleus pulposus-like cells under the hypoxic microenvironment of nucleus pulposus and the induction of transforming growth factor-beta 1. Transforming growth factor-beta 1 (TGF-beta 1) is the most important member of TGF-beta family with the highest content and activity. In the process of BMSCs differentiating into chondrocytes, TGF-beta 1 binds to type I and type II receptors by ligand binding, resulting in phosphorylation of type I receptors, activation of Smad protein, reentry into the nucleus and regulation of target gene transcription. TGF-beta 1 stimulates target cells by altering the activity of protease substrates, and TGF-beta 1 also promotes them. Chondrocytes proliferate into chondrocytes to maintain the chondrocyte phenotype, but in early chondrocyte culture, it was found that TGF-beta 1 has a bidirectional regulatory effect on the proliferation and differentiation of chondrocytes. Whether TGF-beta 1 can inhibit or promote the proliferation of chondrocytes is highly related to the concentration of TGF-beta 1. Gelatin is non-toxic, biodegradable in vivo and has good biological characteristics. It is the preferred material for producing microspheres. Gelatin microspheres are widely used in the field of drug sustained-release carriers because of their mature properties and preparation methods. Gelatin microspheres with a diameter of about 10 microns and dispersed in distribution can be used as scaffolds for intervertebral disc tissue engineering. It takes about 2 months to completely degrade the macromolecular peptides from the inside of the microspheres. These factors determine that TGF-beta 1 gelatin microspheres have good sustained-release properties and injectability. The long-term induction of BMSCs-like nucleus pulposus cells differentiation by TGF-beta 1 gelatin microspheres has effectively inhibited the degeneration of intervertebral discs and has broad application prospects in tissue engineering. In this study, we constructed tissue engineering material of nucleus pulposus of intervertebral disc in vitro and transplanted it into the degenerative intervertebral disc of rabbits. TGF-beta 1 composite gelatin microspheres induced BMSCs to differentiate into nucleus pulposus-like cells for a long time, promoted the proliferation of nucleus pulposus cells and synthesized proteoglycan and type II collagen. Combined application of the two materials can effectively delay the degeneration of intervertebral disc. Part I: Isolation, Purification, Culture and Identification of Rabbit Bone Marrow Mesenchymal Stem Cells (BMSCs). Objective: BMSCs were isolated and purified from fetal rabbit bone marrow by density gradient centrifugation, cultured and identified, providing experimental basis for disc degeneration repair with gelatin microspheres combined with TGF-beta 1. Methods: Rabbit iliac bone puncture, bone extraction. BMSCs primary cells were isolated by density gradient centrifugation with 10 ml myelin. The morphology of primary and P3, P5 cells was observed under inverted microscope. The growth curve of P3 BMSCs was analyzed. The surface antigens CD105, CD90, CD31 and CD14 of P3 BMSCs were identified by flow cytometry. BMSCs were induced to differentiate into osteoblasts, adipocytes and alizarin. Results: BMSCs were successfully extracted by density gradient centrifugation. Under inverted phase contrast microscope, some cells began to adhere to the wall after 24 hours. Primary BMSCs were subcultured to the third generation by selective digestion. The purity of BMSCs was above 90%, and the third generation was more than 90%. CCK-8 cell proliferation assay showed that the proliferation activity of P3 BMSCs was good, and the cells entered the logarithmic growth phase from the 2nd day until 5-6 days, and entered the plateau phase after the 6th day. The positive markers of CD90 and CD105 were 93.67% respectively. The negative markers were CD14 and CD31, the expression rates were 0.11% and 1.08%, respectively. After special osteogenesis and adipogenic inducer induction culture, all the P3 BMSCs could be directionally differentiated into osteoblasts and adipocytes. Calcium nodules were found by alizarin red staining, and intracellular lipid droplets were detected by oil red O staining. BMSCs with high proliferative activity and high purity can be obtained by extracting BMSCs and purifying them by selective digestion. BMSCs were identified by cell surface molecule and multi-directional differentiation potential. This study provides experimental basis for the subsequent treatment of intervertebral disc degeneration with BMSCs combined with gelatin microspheres compounded with TGF-beta 1. Objective: To study the preparation, in vitro release and proliferation activity of TGF-beta 1 gelatin microspheres. Methods: The blank gelatin microspheres were prepared by emulsification and crosslinking method. The morphological characteristics and particle size of gelatin microspheres were observed by light and electron microscopy. TGF was prepared by shaking-centrifugation method. Results: The gelatin microspheres were prepared by emulsifying and crosslinking method. The microspheres before freeze-drying were observed under ordinary microscope. The size of the microspheres was uniform, the sphericity was good and there was no adhesion. The surface of the microspheres was smooth and the particle size distribution was about 1-20 micron with an average diameter of 10.55 (+ 1.25 micron). The entrapment efficiency of gelatin microspheres with 60 ng of TGF-beta 1 was over 98%, the drug loading was 58.76 (+ 0.026 ng), the drug loading was 5.9%, and the cumulative release of TGF-beta 1 was over 96% after 14 days in vitro release. CONCLUSION: Gelatin microspheres were successfully prepared by emulsification, and the morphology of microspheres was round, the surface was smooth, and the size of microspheres was uniform. The sustained release of TGF-beta 1 was up to 14 days, and the proliferation activity of BMSCs could be significantly enhanced by co-culture with BMSCs. Part III: Production and identification of animal model of intervertebral disc degeneration. AIM: To explore the feasibility of establishing animal model of lumbar intervertebral disc degeneration by nucleus pulposus aspiration and identifying it by imaging and molecular biology. 1w, 2w, 3w, 4W magnetic resonance examination, measurement of L3/4, L4/5, L5/6 intervertebral disc magnetic resonance index, immunohistochemical method to detect proteoglycan changes in the nucleus pulposus, HE staining to observe the changes of nucleus pulposus cells arrangement and morphology. External death; 1 week to 4 weeks after surgery, from the second week after surgery, magnetic resonance index continued to decline, the difference was statistically significant. From the second week after surgery, the expression of proteoglycan in the intervertebral disc degeneration model began to show differences, and with the extension of time, the difference was more obvious; nucleus pulposus aspiration group, with the extension of time, nucleus pulposus and fiber. Conclusion: The animal model of lumbar intervertebral disc degeneration was successfully established by intramuscular nucleus pulposus aspiration, which has the advantages of simple operation, short operation time, good reproducibility and easy to carry out. The established models were evaluated by imaging, histomorphology and molecular biology. All of them conformed to the early signs of intervertebral disc degeneration and could be used as animal models for the study of intervertebral disc degeneration. Methods: The tissue engineering material of intervertebral disc constructed by bone marrow mesenchymal stem cells (BMSCs) combined with gelatin microspheres adsorbing TGF-beta 1 in vitro was transplanted into the rabbit intervertebral disc model after 4 weeks. New Zealand white rabbits were randomly divided into 5 groups: control group A, NS group B, BMSCs group C, BMSCs + gelatin injection group without TGF - beta 1 adsorption (BMSCs + gelatin), BMSCs + gelatin injection group adsorbed TGF - beta 1 adsorption (BMSCs + gelatin / TGF - beta 1) group E, BMSCs + gelatin / TGF - beta 1 injection group 3, 6 and 12 weeks after transplantation, respectively, through MRI, RT - gelatin / TGF - beta 1. Q-PCR, HE staining and immunohistochemical staining (IHC) were used to evaluate the degree of repair of intervertebral discs.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R681.53

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 董躍福,王德春,胡有谷;血管內(nèi)皮生長因子與椎間盤退變[J];中國脊柱脊髓雜志;2004年10期

2 張勇;徐永清;王非;陸華拓;唐輝;游永剛;;椎間盤退變模型及其評估指標(biāo)[J];中國脊柱脊髓雜志;2009年06期

3 席志鵬;周臨東;謝林;;椎間盤退變模型研究進(jìn)展[J];山西中醫(yī)學(xué)院學(xué)報;2010年02期

4 武海軍;銀和平;;椎間盤退變的分子生物學(xué)研究進(jìn)展[J];內(nèi)蒙古醫(yī)學(xué)雜志;2011年07期

5 楊松波;高春華;龐曉東;李端明;楊洪;彭寶淦;;椎間盤退變模型的研究進(jìn)展[J];脊柱外科雜志;2012年05期

6 彭寶淦,賈連順;椎間盤退變機(jī)理的研究進(jìn)展[J];中國矯形外科雜志;2000年04期

7 黃宗強(qiáng),劉尚禮,鄭召民;椎間盤退變的分子生物學(xué)研究進(jìn)展[J];中國矯形外科雜志;2003年01期

8 劉彭華;椎間盤退變的性別差異分析(附100例報告)[J];現(xiàn)代醫(yī)用影像學(xué);2003年04期

9 張銀剛,郭雄;椎間盤退變的發(fā)生機(jī)制及其修復(fù)的生物學(xué)進(jìn)展[J];中華骨科雜志;2004年02期

10 趙勇,王文波;基因治療與椎間盤退變[J];中國矯形外科雜志;2004年17期

相關(guān)會議論文 前10條

1 夏冬冬;林勝磊;王向陽;;兔椎間盤退變模型的研究進(jìn)展[A];2012年浙江省骨科學(xué)術(shù)年會論文集[C];2012年

2 夏冬冬;林勝磊;王向陽;;兔椎間盤退變模型的研究進(jìn)展[A];2013中國工程院科技論壇暨浙江省骨科學(xué)學(xué)術(shù)年會論文摘要集[C];2013年

3 張寧;陳維善;陳其昕;徐侃;李方財;吳瓊?cè)A;;酒精與椎間盤退變[A];2008年浙江省骨科學(xué)學(xué)術(shù)年會論文匯編[C];2008年

4 劉q，

本文編號：2241579