骨髓间充质干细胞复合TGF-β1明胶微球治疗椎间盘退变的实验研究

发布时间：2018-09-13 15:31

【摘要】：背景目前我国65岁以上老年人已达1.5亿之多,占总人口的10.8%,随着我国老龄化社会时代的到来,由椎间盘退变引发的颈腰痛疾病也在不断增多,严重困扰着老年人的身体健康及生活质量,同时对于中青年人群来讲,腰椎退变性疾病也是导致劳动能力丧失的主要原因之一。根据最新流行病学调查显示,80%的人一生中至少经历一次腰腿痛疾病的困扰,给个人带来巨大身心痛苦的同时,也给家庭和社会带来严重的经济负重,因此,寻求一种从根本上有效治疗颈肩腰腿痛疾病的方法是医疗卫生领域急需解决的重要问题之一。椎间盘退行性变(Interverbral disc degeneration IDD)发病机制复杂,遗传、肥胖、吸烟、职业、年龄、外伤都可以诱导疾病的发生。IDD是多种因素作用于髓核、纤维环和终板的结果,是一系列脊柱退变性疾病的发生前提和基础。腰椎间盘退变的病理生理学方面主要表现为椎间盘内髓核细胞减少,细胞功能降低,椎间盘细胞外基质的合成减少、降解加速,代谢失去平衡。临床上主要表现为椎管狭窄、椎间盘突出、椎间盘源性颈腰腿痛等病症,是一种常见病、多发病。目前临床上治疗腰椎间盘退变性疾病的方法包括以药物及推拿、按摩为主的保守治疗和以椎间盘髓核摘除、脊柱融合固定及人工椎间盘置换为主的手术治疗。保守治疗只是对症治疗,效果不确切,不能延缓椎间盘退变的进程;手术治疗方面,髓核摘除会造成椎间盘高度丢失以及相关的生物力学、解剖结构变化,可能会进一步加重椎间盘退变及腰椎不稳,甚至影响临近椎间盘受力的改变,脊柱融合内固定仅仅解决病变节段的压迫及不稳定,不仅不能保持椎间盘功能,甚至可能因为脊柱正常生物力学平衡的改变,应力集中于临近节段而发生邻近节段病。因此,寻求一种新的治疗方法是目前的当务之急,生物学治疗为椎间盘再生提供一种新的思路和方法。椎间盘退变的主要病理生理学改变是椎间盘细胞减少,细胞功能降低,细胞外基质代谢失衡,因此理想的治疗方式应能够维持椎间盘组织内正常细胞的数量,调控椎间盘细胞功能,促进细胞外基质的分泌,减缓细胞外基质降解。种子细胞、生长因子和支架材料是修复椎间盘退变三大主导因素。骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs),来源于骨髓间质,具有多向分化潜能,在不同的培养基、生长因子等因素的诱导下可分化为骨细胞、软骨细胞、成纤维细胞、心肌细胞、脂肪细胞等。同时BMSCs具备取材方便、体外生长迅速的特点,且来源充足、易于分离培养、增殖能力强,无论是在体外还是在体内抑或在自体还是在同种异体移植过程中均具有较低免疫原性,是椎间盘组织工程中最理想种子细胞之一。但是在体内BMSCs具体分化成哪一类细胞,主要决定于细胞所处的局部环境,在髓核这种低氧的微环境及在转化生长因子-β1诱导下,BMSCs可以向类髓核细胞分化。转化生长因子-β1(Transforming Growth Factor-β1,TGF-β1)是TGF-β家族最主要成员,含量最高,活性也是最强,在BMSCs向软骨细胞分化过程中,TGF-β1通过配体结合的方式与Ⅰ、Ⅱ型受体结合,导致Ⅰ型受体磷酸化,进而激活Smad蛋白,再进入细胞核,调节目的基因的转录,通过改变蛋白酶底物的活性,TGF-β1对靶细胞起到刺激作用,TGF-β1还具有促进软骨细胞增殖,维持软骨细胞表型的作用,但在早期软骨细胞培养中就发现,TGF-β1对软骨细胞的增殖分化具有双向调节作用,是否为抑制或促进与TGF-β1的浓度高度关联。但是TGF-β1作为外源性生长因子,半衰期短,易被蛋白水解酶水解,局部应用还未促使诱导完成及形成一定数量的细胞外基质,生物分子却已降解,如何使生长因子持续高效发挥作用是骨组织工程学研究亟待解决的关键问题。明胶无毒性,在体内可降解,具有良好的生物学特性,是生产微球的首选材料,目前是制药缓释系统研究的重点。明胶微球的性质、制备方法已比较成熟,在药物缓释载体领域已广泛应用,明胶微球直径10μm左右,散在分布,可以作为椎间盘组织工程的支架材料,完全降解约需2个月左右,且大分子肽类物质自微球内部向外部扩散速度较慢,这些因素决定了TGF-β1明胶微球具有良好的缓释性能,而且具有可注射性的优点。TGF-β1搭载明胶微球缓释体长时间诱导BMSCs类髓核细胞分化有效抑制了椎间盘退变,在椎间盘组织工程研究中有广阔的应用前景。本研究中:我们体外构建椎间盘髓核组织工程材料,并将其移植到成功造模的兔退变椎间盘中,TGF-β1复合明胶微球长时间诱导BMSCs向类髓核细胞分化、促进髓核细胞增殖并合成蛋白多糖及II型胶原,两者联合应用可有效延缓椎间盘退变。第一部分:兔骨髓间充质干细胞(BMSCs)分离、纯化、体外培养及鉴定目的:通过密度梯度离心法体将BMSCs从胎兔骨髓中分离纯化、培养并对其进行鉴定,为探讨其联合明胶微球复合TGF-β1修复椎间盘退变提供实验基础。方法:兔髂骨穿刺,抽取骨髓液10ml,密度梯度离心法分离BMSCs原代细胞,体外培养、扩增、传代,倒置显微镜下观察原代及P3、P5代细胞形态,进行P3代BMSCs生长曲线分析,流式细胞术对P3代BMSCs表面抗原CD105、CD90、CD31、CD14进行鉴定,BMSCs诱导分化为成骨细胞、成脂细胞,茜素红染色成骨细胞中钙结节,油红O染色成脂细胞内的脂滴。结果:使用密度梯度离心法BMSCs提取成功,倒置相差显微镜下观察,BMSCs在24 h后已有部分细胞开始贴壁生长,原代BMSCs经选择性消化法传代至第三代,细胞纯度约达到90%以上,且第三代BMSCs和第五代BMSCs倒置相差显微镜下观察细胞状态良好;CCK-8细胞增殖活性检测显示:P3代BMSCs细胞增殖活性良好,细胞于第2天开始进入对数生长期,一直维持到5-6天,于第6天后进入平台期;细胞阳性标志为CD90和CD105,表达率分别为93.67%和92.03%;阴性标志为CD14和CD31,表达率分别为0.11%和1.08%;P3代BMSCs经特殊成骨、成脂诱导剂诱导培养,均可定向分化为成骨细胞、成脂细胞,茜素红染色发现钙结节,油红O染色示细胞内的脂滴。结论:在胎兔骨髓液中通过密度梯度离心法顺利提取BMSCs,然后通过选择性消化法对细胞进行纯化,可获得高增殖活性、高纯度的BMSCs。通过对细胞表面分子和多向分化潜能进行鉴定,证实所提取细胞为BMSCs。为后续BMSCs联合已复合TGF-β1的明胶微球治疗椎间盘退变提供实验基础。第二部分:TGF-β1明胶微球的制作、体外释放及对BMSCs增殖活性的研究目的:探讨TGF-β1明胶微球制作工艺及体外释放及对BMSCs增殖活性的影响。方法:乳化交联法制备空白明胶微球,光镜及电镜观察明胶微球形态特征及粒径分析,振摇-离心法将TGF-β1载到空白微球中,ELISA法检测TGF-β1的体外释放,计算微球载药量及包封率,CCK-8法检测对BMSCs增殖活性的影响。结果:经乳化交联法制作明胶微球,普通显微镜下观察交联后、冻干前的微球,可见粒径较均匀,成球性良好,无粘连,扫描电镜下观察可见微球表面光滑,粒径分布约为1-20μm,平均粒径10.55±1.25μm;每mg明胶微球加入60ng TGF-β1包封率为98%以上,载药质量为58.76±0.026ng,载药量为5.9%,体外释放14天,累积释放TGF-β1为96%以上;吸附TGF-β1的明胶微球随着时间缓慢释放TGF-β1,对BMSCs增殖其促进作用。结论:通过乳化法成功制作出明胶微球,明胶微球电镜下观察,微球形态圆整,表面光滑,粒径均匀;其吸附TGF-β1缓释可达14天;且与BMSCs共培养,可显著增强BMSCs增殖活性。第三部分:椎间盘退变动物模型的制作及鉴定目的:探讨髓核抽吸法建立腰椎间盘退变动物模型及通过影像学、分子生物学进行模型鉴定的可行性。方法:侧卧位经肌间隙入路,18G针头的注射器(10ml)依次刺破L3/4、L4/5、L5/6椎间盘,深度约5mm抽吸时间约20秒,建立椎间盘退变动物模型;术后1w、2w、3w、4w磁共振检查,测量L3/4、L4/5、L5/6椎间盘磁共振指数,免疫组织化学法检测椎间盘髓核中蛋白聚糖变化,HE染色观察髓核细胞排列及形态的变化。结果:经肌间隙入路进行兔L3/4、L4/5、L5/6椎间盘髓核抽吸,平均抽出髓核质量10mg,无意外死亡;术后1周到术后4周,从术后第2周开始,磁共振指数持续降低,差异有显著统计学意义,术后第2周开始,椎间盘退变模型内的蛋白聚糖表达即开始出现差异,且随着时间的延长,差异越明显;髓核抽吸组,随着时间的延长,髓核与纤维环分局模糊,髓核细胞减少,分布不均匀,髓核外基质结构紊乱。结论:经肌间隙髓核抽吸法成功构建椎间盘退变动物模型,此种方法具有操作简便,手术时间短,重复性好,方便开展的优点。通过此种方法构建的腰椎间盘退变模型术后实验动物的感染率低,成功率高。而且通过影像学、组织形态学、分子生物学对已构建模型进行评估,均符合前期椎间盘退变征象,可以作为椎间盘退变研究的动物模型。第四部分:BSMCs复合TGF-β1明胶微球治疗椎间盘退变的实验研究目的:探讨BSMCs复合TGF-β1明胶微球治疗椎间盘退变的可行性,为以后椎间盘退变治疗的研究提供一定的借鉴。方法:体外通过骨髓间充质干细胞(BMSCs)联合吸附TGF-β1的明胶微球构建的椎间盘组织工程材料移植至兔椎间盘制作4周后动物模型中。将60只新西兰大白兔随机分成5组,每组12只动物:A组空白对照组(Control);B组生理盐水组(NS);C组BMSCs组;D组BMSCs+未吸附TGF-β1的明胶微球注射组(BMSCs+gelatin);E组BMSCs+吸附TGF-β1的明胶微球注射组(BMSCs+gelatin/TGF-β1)。在移植后3w、6w、12w分别通过MRI、RT-q PCR、HE染色和免疫组化(IHC)对椎间盘的修复程度进行评估。结果:经MRI检测显示,除A组外,各组椎间盘内信号均不同程度降低,B组降低最明显,E组信号降低最缓慢;分别在移植术后3w、6w、12w使用MRI指数对各组进行比较发现:3w时,只有A组与B组相比较具有显著性差异;6w时,除A组外各组椎间盘MRI指数均不同程度降低,B、C、D组三组与A组相比,差异具有统计学意义,但A组和E组相比,无显著性差异;12w时,除A组外各组MRI指数继续降低,但降低趋势较3w-6w时变缓;分别在3w、6w、12w时取各组椎间盘髓核提取细胞总RNA,通过RT-q PCR检测各组蛋白聚糖、II型胶原m RNA表达,经统计学分析显示,E组蛋白聚糖、II型胶原m RNA表达量最高,且差异具有统计学意义。各组蛋白聚糖、II型胶原蛋白表达显示A组与E组均无显著性差异;12w光镜下观察HE染色显示,A组椎间盘病理切片椎间盘髓核完整,髓核与纤维环分界清晰,纤维环结构接近正常,髓核细胞呈圆形空泡状,分布均匀,B组,髓核与纤维环分局模糊,髓核细胞明显减少,大小不等,分布不均匀,髓核外基质结构紊乱,C组髓核细胞减少,纤维环排列稍清晰。D组髓核细胞较C组少,椎间盘核外基质排列紊乱,E组纤维环结构接近正常,髓核细胞分布略均匀,髓核外基质结构规则。结论:将构建的椎间盘组织工程材料移植到已退变的椎间盘中,椎间盘退变显著变缓。通过MRI可以观察到移植的组织工程材料可以显著延缓椎间盘内含水量的丢失;RT-q PCR和免疫组化检测细胞外基质(蛋白聚糖、II型胶原)显著较其他干预组表达高;通过病理学检查,可以观察到椎间盘组织工程材料能够维持椎间盘的结构,延缓了椎间盘进一步退变,促进退变椎间盘修复。
[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.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R681.53

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