荷载干细胞的TGF-β3/地塞米松/PLGA微球用于退变椎间盘的研究
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摘要
背景
椎间盘(The intervertebral disc, IVD)由层状纤维环(annulus fibrosus, AF)包裹的髓核(nucleus pulposus, NP)构成。髓核中有大量的椎间盘相关基质蛋白,是由负电荷蛋白聚糖(proteoglycans, PG)和Ⅱ型胶原形成的网状结构。椎间盘在许多情况下都会发生退变,临床上极为常见,给国家医疗花费增加了沉重的负担。现有文献证明,代谢因素如基质金属蛋白酶(matrix metalloproteinases, MMPs),细胞凋亡,炎症相关细胞因子都会加速椎间盘的退变。目前临床上针对椎间盘都以对症治疗为出发点,以减轻痛苦和修补功能为目标,并没有对椎间盘病变进行根治或是对其在生物力学功能上进行修复。对于椎间盘的修复,针对退变早期,新型的生物学治疗方案是对椎间盘的基质进行再生以及正常功能的全面恢复。体外研究显示,TGF-β1, BMP-7, GDF-5和TGF-β3等生长因子会刺激椎间盘细胞的增殖及提高细胞外基质的合成能力。虽然一些研究提示GDF-5和BMP-7会椎间盘的退变产生抑制作用,但Walsh等的研究中,退变椎间盘的环状纤维只有对大剂量TGF-β1的体内注射产生即时效应,这些生长因子在体内短暂的半衰期成为阻碍生长因子治疗效果的瓶颈。微创治疗的微粒组装系统不但可以作为细胞和蛋白质缓释载体,而且在可以用于各种微创组织工程研究。明确的生物组织相容性和安全性,使得PLGA微球作为组织相容复合物被广泛应用于各种各样的生物组织工程研究。但是,培养在PLGA微球上的椎间盘细胞和间充质干细胞(mesenchymal stem cells, MSCs),在常规培养基条件下常常从软骨样细胞表型的干细胞分化为成纤维样细胞。因此,一些试验都在尝试着在椎间盘内髓核组织分化中,将特殊的生长因子和药物包装到PLGA微球上。在材料表面修饰的过程中,纳米颗粒在空间上可以被有序加工,用其制作的各式各样新型材料已被用于光学,电学,磁学设备,此外,生物支架材料的表面修饰可以增加细胞表面的相互作用增强细胞功能。值得一提的是,不同电荷的纳米微粒的层层组装(layer-by-layer assembly, LBL)已经用于新型纳米复合材料的制作,而且纳米多层结构材料也已经用于细胞培养和细胞移植。基于上述研究进展,本课题主要从以下五个方面进行了研究：(1)SD ADSCs的分离培养及鉴定；SD GFP/ADSCs和SD TGF-β3/ADSCs过表达细胞系的构建和鉴定；(2)椎间盘微环境对不同年龄来源ADSCs的活力、增殖和基质合成的影响；(3) DEX/TGF-β3/PLGA纳米微球的制备及对SD ADSCs活性、增殖及分化能力的研究；(4) DEX/TGF-β3/PLGA纳米微球/SD ADSCs复合物移植修复SD大鼠退变椎间盘的研究。
第一部分SD ADSCs的分离培养及鉴定；SD GFP/ADSCs和SD TGF-β3/ADSCs过表达细胞系的构建和鉴定
目的分离培养出SD大鼠ADSCs,然后通过三系分化和流式细胞术鉴定其干细胞特征。构建携带TGF-β3和GFP基因的慢病毒载体,转染到.ADSCs,进而药物筛选获得GFP/ADSCs和TGF-β3/ADSCs细胞系,同样鉴定其干细胞特征,为椎间盘退变性疾病的细胞学及基因治疗提供实验依据。
方法和结果我们首先采用机械剪碎组织,酶消化和贴壁法分离培养出SD大鼠ADSCs,通过研究干细胞形态,生长曲线,表面抗原标记表达谱及三系分化能力,对分离的ADSCs进行鉴定。然后利用Gateway技术构建携带TGF-β3和GFP基因的慢病毒载体,转染到ADSCs,进而药物筛选获得稳定表达TGF-β3和GFP基因的细胞系GFP/ADSCs和TGF-p3/ADSCS。最后,本文按照干细胞形态,表面抗原标记表达谱及三系分化能力,对细胞系GFP/ADSCs和TGF-β3/ADSCs进行鉴定；同时利用RT-PCR和Western Blot检测TGF-β3基因在三种细胞系ADSCs, GFP/ADSCs和TGF-β3/ADSCs中的表达情况。体外成功建立SD大鼠ADSCs培养扩增的方法,并成功诱导出成骨、成脂和成软骨细胞。ADSCs造血干细胞表型标志表达为阴性,而间充质干细胞表型标志表达为阳性。基因转染48h后观察到较强的绿色荧光表达,Lenti-GFP-TGF-β3及Lenti-GFP的转染率均达到90%以上。与GFP/ADSCs和ADSCs细胞相比,RT-PCR、Western blot检测到目的基因TGF-β3在TGF-β3/ADSCs中出现过表达(P<0.05)。此外,GFP/ADSCs和TGF-β3/ADSCs细胞系仍然具有干细胞特征。
结论分离培养出的SD大鼠ADSCs体外培养扩增容易,具有多向分化潜能和间充质干细胞特征。成功构建了含有TGF-β3基因慢病毒载体,转染ADSCs后,建立了GFP/ADSCs和TGF-β3/ADSCs细胞系。
第二部分椎间盘微环境对不同年龄来源ADSCs的活力、增殖和基质合成的影响
目的人脂肪间充质干细胞(ADSCs)可能是椎间盘再生细胞的理想种子细胞,但恶劣微环境是否会改变ADSCs生物活性和代谢活力,最终削弱它们的修复潜力。因此,本章主要研究人ADSCs在正常和退变椎间盘微环境条件下的活力,增殖能力及主要基质蛋白的表达情况。
方法和结果分别从儿童(年龄在8-12岁,n=6)和成人(年龄33-42岁,n=6)男性捐助者脂肪中培养ADSCs,然后把它们在标准培养基和模拟椎间盘微环境(低糖,酸性,高渗透压和混合环境)的条件下培养2周。Annexin V-FITC (AV-PI)流式细胞术方法检测细胞生物活性,而细胞增殖率测定采用MTT法检测。实时定量聚合酶链反应(RT-PCR)和Western blot分析检测蛋白多糖,胶原蛋白-Ⅰ和胶原蛋白-Ⅱ基因表达的情况。模拟椎间盘微环境的低糖培养条件不但可以保证ADSCs的活力和以较低的正常水平增殖,而且可以提高ADSCs合成蛋白聚糖的能力,所以我们推测椎间盘内的低糖条件可能对ADSCs移植治疗椎间盘退变是一个有利因素。相反,模拟椎间盘微环境的高渗透压和低pH值微环境,损害ADSCs活力,降低ADSCs增殖速度和基质合成能力。此外,供体年龄差异对ADSCs的在椎间盘微环境中活力和增殖的影响甚微。结论对体外培养的ADSCs存活和增殖而言,低糖培养条件是其有利因素,而高渗透压和酸性环境是其有害因素。这些研究结果有利于促进ADSCs用于下腰痛的转化医学研究。
第三部分DEX/TGF-β3/PLGA纳米微球的制备及对SD ADSCs活性、增殖及分化能力的研究
目的这项研究旨在确定利用层层组装方法把肝素／多聚L-赖氨酸／生长因子与包埋地塞米松的PLGA微球上,然后评估该三维纳米PLGA微球作为髓核组织工程支架的可行性。
方法和结果我们的结果证明PLGA微球结构可以在近似零级动力条件下同时释放TGF-β3生长因子和地塞米松。经过乳酸脱氢酶(LDH)检测和CCK-8检测得知,双重缓释的PLGA微球对ADSCs没有细胞毒性,,其还可促进SDADSCs的增殖。体外4周培养后,PLGA微球组的ADSCs细胞的GAG/DNA和Ⅱ型胶原表达显著高于对照组。此外,定量实时PCR检测发现,与对照组相比,PLGA微球组培养的ADSCs有较高水平的Ⅱ型胶原,聚集蛋白聚糖及多能聚糖的表达,但是成骨分化标记物(Ⅰ型胶原)含量较低。
结论综上,DEX/TGF-β3/PLGA微球可应用于髓核组织工程中,不但作为支架促进ADSCs细胞生长并促进向髓核样细胞进化,而且降低局部微环境的炎症反应。
第四部分DEX/TGF-β3/PLGA纳米微球/SDADSCs复合物移植修复SD大鼠退变椎间盘的研究
目的下腰痛常常是由椎间盘髓核退变引起的。组织工程是一个功能强大的治疗模式,其以恢复人脊柱正常的生物力学运动功能为目的。第三章我们报道了一个新的纳米结构的3D PLGA微球,体外研究发现,这种通过层层组装地塞米松和生长因子到3D PLGA微球,可以控释两种生物活性因子,是一种优良的髓核组织工程的细胞载体。本章研究的目的是调查移植3D PLGA微球荷载ADSCs复合物到大鼠椎间盘退变模型,是否可以再生退化的椎间盘。
方法和结果大鼠共分为四组：正常对照组(NC组,无操作),退变对照组(DC组,针刺操作),PLGA微球治疗组(PM组,针刺操作+PLGA微球移植),PLGA微球荷载ADSCs复合物治疗组(PMA组,针刺操作+PLGA微球荷载ADSCs复合物移植)。分别在移植后的第4、8、16和24周,对四组大鼠椎间盘行X先平片检测椎间盘高度的变化,T2加权磁共振成像(MRI)检查,组织学染色和评分,免疫组化和RT-PCR检测相关基因表达情况。结果表明,移植24周后,以NC组为正常参照,PM和PMA组的椎间盘平均高度值分别约为63%和76%,MRI信号强度分别约47%和76%。生物化学,免疫组织化学和基因表达分析表明在PM和PMA组椎间盘有较多的蛋白多糖积累。
结论这些数据表明,在大鼠椎间盘退变模型中,移植3D PLGA微球荷载ADSCs复合物能在一定程度再生退化的椎间盘。PLGA微球在细胞移植治疗椎间盘退变性疾病可能会是一种很有前途的生物支架载体。
Introduction
The intervertebral disc (IVD) is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), and the endplates (EP). Among them, NP contains a large amount of disc-matrix proteins which are negatively charged proteoglycans (PG) within a collagen type Ⅱ network1. Consequently, a swelling pressure is present due to the high concentration of water in the NP2. Notably, IVD has poor self-repair capacity and its degeneration often progresses to an irreversible change. This process usually starts in the NP. In this condition, there is an increase of collagen type Ⅰ and denatured collagen type Ⅱ in extracellular matrix (ECM), whereas proteoglycans are reduced because of the activation of matrix-degrading enzyme which degrades the components of ECM3,4.
Growth factor therapy is one of the promising modalities to regenerate IVD. It has been shown that transforming growth factor β1(TGFβ1), bone morphogenetic protein (BMP)-7, growth differentiation factor-5(GDF-5), basic fibroblast growth factor (bFGF) and transforming growth factor β3(TGFβ3) could stimulate IVD cells to proliferate and produce ECM in vitro5,6. Moreover, TGFβ3and GDF-5could inhibit IVD degeneration in animal models7,8. However, the short half-life of growth factors represents a problem for their application in vivo. For example, Walsh et al. reported that the anular fibrochondrocytes in degenerated IVDs are responsive to TGF-β1only after multiple injections in vivo9.
Several groups have investigated whether authentic NP tissue could be engineered in vitro and in vivo by transplanting allogenic donor NP cells and mesenchymal stem cells (MSCs) seeded in3D scaffolds10-12. Among the scaffolds evaluated, mainly solid and preformed scaffolds comprising porous foams and fibrous meshes have been used. However, these types of scaffolds have the major drawback of requiring a surgical incision to position the scaffolds into desired sites in the body. Recently, a wide variety of biodegradable PLG A spherical particles have been utilized for the sustained delivery of bioactive molecules as an injectable depot formulation13,14. Moreover, PLGA is known to be biocompatible that has a broad range of applications in tissue engineering and clinic application15,16. Therefore, PLGA microspheres may be a suitable material for the preparation of injectable scaffolds for NP tissue engineering.
Microparticulate systems have attracted increasing interest over the past few years as carriers for the delivery of cells and proteins due to minimal invasive procedures in tissue engineering16-18. Growth factors and drugs have been co-incorporated in PLGA microspheres using microparticulate systems for NP tissue differentiation19-21. Especially, anti-inflammatory agents such as dexamethasone can minimize implantation-associated inflammation and promote cell differentiation22,23. It is important to administer both anti-inflammatory corticosteroids and growth factors in a localized environment at low doses, so that both agents can function symbiotically without interference.
This study contains five parts:(1) Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-P3/ADSCs cell lines;(2) Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc;(3) Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration;(4) Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration model.
Part I Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-p3/ADSCs cell lines
Aim:The purpose of this study was to construct a lentiviral vector containing TGF-β3/GFP gene and transfect into adipose derived stem cells in vitro. Then we use drugs to select GFP/ADSCs and TGF-β3/ADSCs cell lines, which are always characterized as stem cells. It will facilitated the following development of gene and cell therapy in treating diseases of intervertebral disc degeneration.
Methods and results:Firstly, we cultured SD rat ADSCs through cutting adipose into pieces, digesting using enzyme and isolating by adherent method. Separated ADSCs were identified by studying stem cell morphology, growth curve, surface antigen marker expression profiles and three-line differentiation capacity. Gateway technology was used to construct lentiviral vectors carrying the TGF-β3, and/or GFP gene, and which were transfected into ADSCs later. Antibiotics were used to obtain a stable expression of TGF-β3and/or GFP gene in cell lines TGF-β3/ADSCs and/or GFP/ADSCs. Finally, in accordance with the morphology, surface antigen marker expression profiles and three-line differentiation capacity of stem cells, TGF-β3/ADSCs and GFP/ADSCs were identified. The expression of TGF-β3gene was detected by RT-PCR and Western Blot methods in all three cell lines. SD rats ADSCs were successfully established, cultured and expanded in vitro, and they were also successfully induced into osteogenic, adipogenic and cartilage cells. The hematopoietic stem cell phenotypic markers of ADSCs were negative, while the expression of mesenchymal stem cell phenotypic markers was positive. After48h transfection, strong green fluorescence could be seen, and the transfection rate of Lenti-GFP-TGF-β3and Lenti-GFP were above90%. Compared with GFP/ADSCs and ADSCs cells, the expression of TGF-β3gene is overexpression in TGF-β3/ADSCs cells by RT-PCR and Western blot detection (P <0.05). In addition, the characteristics of stem cells could still be identified in GFP/ADSCs and TGF-β3/ADSCs cell lines.
Conclusions:SD rats ADSCs were easily isolated and cultured in vitro, which have multiple differentiation potential abilities and mesenchymal stem cell characteristics. Lentiviral vectors containing the TGF-β3/GFP gene were successfully transfected into ADSCs,then GFP/ADSCs and TGF-β3/ADSCs cell lines were established.
Part Ⅱ Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc
Aim:Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions.
Methods and results:ADMSCs were harvested from young (aged8-12years, n=6) and mature (aged33-42years, n=6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for2weeks. Cell viability was measured by annexin V-FITC and PI (AV-PI) staining and cell proliferation was measured by MTT assay. The expression of aggrecan, collagen-Ⅰ, and collagen-Ⅱ was detected by real-time quantitative polymerase chain reaction and Western blot analysis. IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-Ⅰ. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD.
Conclusions:IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain.
Part Ⅲ Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration
Aim:This study aimed to investigate the feasibility of the nanostructured3D poly(lactide-co-glycolide)(PLGA) constructs, which are loaded with dexamethasone (DEX) and growth factor embeded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, to serve as an effective scaffold for nucleus pulposus (NP) tissue engineering.
Methods and results:Our results demonstrated that the microsphere constructs were capable of simultaneously releasing TGF-β3and DEX with approximately zero order kinetics. The dual bead microspheres showed no cytotoxicity, and promoted the proliferation of the rat adipose-derived mesenchymal stem cells (ADSCs) by lactate dehydrogenase assay and CCK-8assay. After4weeks of cultivation in vitro, the ADSCs-scaffold hybrids contained significantly higher levels of sulfated GAG/DNA and collagen type Ⅱ than the control samples. Moreover, quantitative real time PCR analysis revealed that the expression of disc-matrix proteins including collagen type Ⅱ, aggrecan, and versican in the ADSCs-scaffold hybrids was significantly higher than that in the control group, whereas the expression of osteogenic differentiation marker (collagen type I) was decreased.
Conclusions:Taken together, these data indicate that Dex/TGF-β3/PLGA microspheres could be used as a scaffold to improve the ADSCs growth and differentiating into NP like cells, and reduce the inflammatory response for IVD tissue engineering.
Part Ⅳ Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration model
Aim:Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported a new nanostructured3D poly(lactide-co-glycolide)(PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSCs) seeded PLGA microspheres into the rat intervertebral disc degeneration model could regenerate the degenerated disc.
Methods and results:Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix associated gene expressions were evaluated in normal controls (NC)(without operations), degeneration control (DC) group (with needle puncture, only DMEM injection); PLGA microspheres (PM) treatment group (with needle puncture, PLGA microspheres only injection); and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSCs injection) for a24-week period. The results showed that24weeks post-transplantation, PM and PMA groups regained a disc height value of about63%and76%, MRI signal intensity of about47%and76%, respectively, compared to NC group. Biochemistry, immunohistochemistry and gene expression analysis indicated the restoration of proteoglycan accumulation in the discs of PM and PMA groups.
Conclusions:Taken together, these data suggest that ADSCs-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat intervertebral disc degeneration model. PLGA microsphere may serve as a promising carrier in cell transplantation therapy for degenerative disc disease.
椎间盘(The intervertebral disc, IVD)由层状纤维环(annulus fibrosus, AF)包裹的髓核(nucleus pulposus, NP)构成。髓核中有大量的椎间盘相关基质蛋白,是由负电荷蛋白聚糖(proteoglycans, PG)和Ⅱ型胶原形成的网状结构。椎间盘在许多情况下都会发生退变,临床上极为常见,给国家医疗花费增加了沉重的负担。现有文献证明,代谢因素如基质金属蛋白酶(matrix metalloproteinases, MMPs),细胞凋亡,炎症相关细胞因子都会加速椎间盘的退变。目前临床上针对椎间盘都以对症治疗为出发点,以减轻痛苦和修补功能为目标,并没有对椎间盘病变进行根治或是对其在生物力学功能上进行修复。对于椎间盘的修复,针对退变早期,新型的生物学治疗方案是对椎间盘的基质进行再生以及正常功能的全面恢复。体外研究显示,TGF-β1, BMP-7, GDF-5和TGF-β3等生长因子会刺激椎间盘细胞的增殖及提高细胞外基质的合成能力。虽然一些研究提示GDF-5和BMP-7会椎间盘的退变产生抑制作用,但Walsh等的研究中,退变椎间盘的环状纤维只有对大剂量TGF-β1的体内注射产生即时效应,这些生长因子在体内短暂的半衰期成为阻碍生长因子治疗效果的瓶颈。微创治疗的微粒组装系统不但可以作为细胞和蛋白质缓释载体,而且在可以用于各种微创组织工程研究。明确的生物组织相容性和安全性,使得PLGA微球作为组织相容复合物被广泛应用于各种各样的生物组织工程研究。但是,培养在PLGA微球上的椎间盘细胞和间充质干细胞(mesenchymal stem cells, MSCs),在常规培养基条件下常常从软骨样细胞表型的干细胞分化为成纤维样细胞。因此,一些试验都在尝试着在椎间盘内髓核组织分化中,将特殊的生长因子和药物包装到PLGA微球上。在材料表面修饰的过程中,纳米颗粒在空间上可以被有序加工,用其制作的各式各样新型材料已被用于光学,电学,磁学设备,此外,生物支架材料的表面修饰可以增加细胞表面的相互作用增强细胞功能。值得一提的是,不同电荷的纳米微粒的层层组装(layer-by-layer assembly, LBL)已经用于新型纳米复合材料的制作,而且纳米多层结构材料也已经用于细胞培养和细胞移植。基于上述研究进展,本课题主要从以下五个方面进行了研究：(1)SD ADSCs的分离培养及鉴定；SD GFP/ADSCs和SD TGF-β3/ADSCs过表达细胞系的构建和鉴定；(2)椎间盘微环境对不同年龄来源ADSCs的活力、增殖和基质合成的影响；(3) DEX/TGF-β3/PLGA纳米微球的制备及对SD ADSCs活性、增殖及分化能力的研究；(4) DEX/TGF-β3/PLGA纳米微球/SD ADSCs复合物移植修复SD大鼠退变椎间盘的研究。
第一部分SD ADSCs的分离培养及鉴定；SD GFP/ADSCs和SD TGF-β3/ADSCs过表达细胞系的构建和鉴定
目的分离培养出SD大鼠ADSCs,然后通过三系分化和流式细胞术鉴定其干细胞特征。构建携带TGF-β3和GFP基因的慢病毒载体,转染到.ADSCs,进而药物筛选获得GFP/ADSCs和TGF-β3/ADSCs细胞系,同样鉴定其干细胞特征,为椎间盘退变性疾病的细胞学及基因治疗提供实验依据。
方法和结果我们首先采用机械剪碎组织,酶消化和贴壁法分离培养出SD大鼠ADSCs,通过研究干细胞形态,生长曲线,表面抗原标记表达谱及三系分化能力,对分离的ADSCs进行鉴定。然后利用Gateway技术构建携带TGF-β3和GFP基因的慢病毒载体,转染到ADSCs,进而药物筛选获得稳定表达TGF-β3和GFP基因的细胞系GFP/ADSCs和TGF-p3/ADSCS。最后,本文按照干细胞形态,表面抗原标记表达谱及三系分化能力,对细胞系GFP/ADSCs和TGF-β3/ADSCs进行鉴定；同时利用RT-PCR和Western Blot检测TGF-β3基因在三种细胞系ADSCs, GFP/ADSCs和TGF-β3/ADSCs中的表达情况。体外成功建立SD大鼠ADSCs培养扩增的方法,并成功诱导出成骨、成脂和成软骨细胞。ADSCs造血干细胞表型标志表达为阴性,而间充质干细胞表型标志表达为阳性。基因转染48h后观察到较强的绿色荧光表达,Lenti-GFP-TGF-β3及Lenti-GFP的转染率均达到90%以上。与GFP/ADSCs和ADSCs细胞相比,RT-PCR、Western blot检测到目的基因TGF-β3在TGF-β3/ADSCs中出现过表达(P<0.05)。此外,GFP/ADSCs和TGF-β3/ADSCs细胞系仍然具有干细胞特征。
结论分离培养出的SD大鼠ADSCs体外培养扩增容易,具有多向分化潜能和间充质干细胞特征。成功构建了含有TGF-β3基因慢病毒载体,转染ADSCs后,建立了GFP/ADSCs和TGF-β3/ADSCs细胞系。
第二部分椎间盘微环境对不同年龄来源ADSCs的活力、增殖和基质合成的影响
目的人脂肪间充质干细胞(ADSCs)可能是椎间盘再生细胞的理想种子细胞,但恶劣微环境是否会改变ADSCs生物活性和代谢活力,最终削弱它们的修复潜力。因此,本章主要研究人ADSCs在正常和退变椎间盘微环境条件下的活力,增殖能力及主要基质蛋白的表达情况。
方法和结果分别从儿童(年龄在8-12岁,n=6)和成人(年龄33-42岁,n=6)男性捐助者脂肪中培养ADSCs,然后把它们在标准培养基和模拟椎间盘微环境(低糖,酸性,高渗透压和混合环境)的条件下培养2周。Annexin V-FITC (AV-PI)流式细胞术方法检测细胞生物活性,而细胞增殖率测定采用MTT法检测。实时定量聚合酶链反应(RT-PCR)和Western blot分析检测蛋白多糖,胶原蛋白-Ⅰ和胶原蛋白-Ⅱ基因表达的情况。模拟椎间盘微环境的低糖培养条件不但可以保证ADSCs的活力和以较低的正常水平增殖,而且可以提高ADSCs合成蛋白聚糖的能力,所以我们推测椎间盘内的低糖条件可能对ADSCs移植治疗椎间盘退变是一个有利因素。相反,模拟椎间盘微环境的高渗透压和低pH值微环境,损害ADSCs活力,降低ADSCs增殖速度和基质合成能力。此外,供体年龄差异对ADSCs的在椎间盘微环境中活力和增殖的影响甚微。结论对体外培养的ADSCs存活和增殖而言,低糖培养条件是其有利因素,而高渗透压和酸性环境是其有害因素。这些研究结果有利于促进ADSCs用于下腰痛的转化医学研究。
第三部分DEX/TGF-β3/PLGA纳米微球的制备及对SD ADSCs活性、增殖及分化能力的研究
目的这项研究旨在确定利用层层组装方法把肝素／多聚L-赖氨酸／生长因子与包埋地塞米松的PLGA微球上,然后评估该三维纳米PLGA微球作为髓核组织工程支架的可行性。
方法和结果我们的结果证明PLGA微球结构可以在近似零级动力条件下同时释放TGF-β3生长因子和地塞米松。经过乳酸脱氢酶(LDH)检测和CCK-8检测得知,双重缓释的PLGA微球对ADSCs没有细胞毒性,,其还可促进SDADSCs的增殖。体外4周培养后,PLGA微球组的ADSCs细胞的GAG/DNA和Ⅱ型胶原表达显著高于对照组。此外,定量实时PCR检测发现,与对照组相比,PLGA微球组培养的ADSCs有较高水平的Ⅱ型胶原,聚集蛋白聚糖及多能聚糖的表达,但是成骨分化标记物(Ⅰ型胶原)含量较低。
结论综上,DEX/TGF-β3/PLGA微球可应用于髓核组织工程中,不但作为支架促进ADSCs细胞生长并促进向髓核样细胞进化,而且降低局部微环境的炎症反应。
第四部分DEX/TGF-β3/PLGA纳米微球/SDADSCs复合物移植修复SD大鼠退变椎间盘的研究
目的下腰痛常常是由椎间盘髓核退变引起的。组织工程是一个功能强大的治疗模式,其以恢复人脊柱正常的生物力学运动功能为目的。第三章我们报道了一个新的纳米结构的3D PLGA微球,体外研究发现,这种通过层层组装地塞米松和生长因子到3D PLGA微球,可以控释两种生物活性因子,是一种优良的髓核组织工程的细胞载体。本章研究的目的是调查移植3D PLGA微球荷载ADSCs复合物到大鼠椎间盘退变模型,是否可以再生退化的椎间盘。
方法和结果大鼠共分为四组：正常对照组(NC组,无操作),退变对照组(DC组,针刺操作),PLGA微球治疗组(PM组,针刺操作+PLGA微球移植),PLGA微球荷载ADSCs复合物治疗组(PMA组,针刺操作+PLGA微球荷载ADSCs复合物移植)。分别在移植后的第4、8、16和24周,对四组大鼠椎间盘行X先平片检测椎间盘高度的变化,T2加权磁共振成像(MRI)检查,组织学染色和评分,免疫组化和RT-PCR检测相关基因表达情况。结果表明,移植24周后,以NC组为正常参照,PM和PMA组的椎间盘平均高度值分别约为63%和76%,MRI信号强度分别约47%和76%。生物化学,免疫组织化学和基因表达分析表明在PM和PMA组椎间盘有较多的蛋白多糖积累。
结论这些数据表明,在大鼠椎间盘退变模型中,移植3D PLGA微球荷载ADSCs复合物能在一定程度再生退化的椎间盘。PLGA微球在细胞移植治疗椎间盘退变性疾病可能会是一种很有前途的生物支架载体。
Introduction
The intervertebral disc (IVD) is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), and the endplates (EP). Among them, NP contains a large amount of disc-matrix proteins which are negatively charged proteoglycans (PG) within a collagen type Ⅱ network1. Consequently, a swelling pressure is present due to the high concentration of water in the NP2. Notably, IVD has poor self-repair capacity and its degeneration often progresses to an irreversible change. This process usually starts in the NP. In this condition, there is an increase of collagen type Ⅰ and denatured collagen type Ⅱ in extracellular matrix (ECM), whereas proteoglycans are reduced because of the activation of matrix-degrading enzyme which degrades the components of ECM3,4.
Growth factor therapy is one of the promising modalities to regenerate IVD. It has been shown that transforming growth factor β1(TGFβ1), bone morphogenetic protein (BMP)-7, growth differentiation factor-5(GDF-5), basic fibroblast growth factor (bFGF) and transforming growth factor β3(TGFβ3) could stimulate IVD cells to proliferate and produce ECM in vitro5,6. Moreover, TGFβ3and GDF-5could inhibit IVD degeneration in animal models7,8. However, the short half-life of growth factors represents a problem for their application in vivo. For example, Walsh et al. reported that the anular fibrochondrocytes in degenerated IVDs are responsive to TGF-β1only after multiple injections in vivo9.
Several groups have investigated whether authentic NP tissue could be engineered in vitro and in vivo by transplanting allogenic donor NP cells and mesenchymal stem cells (MSCs) seeded in3D scaffolds10-12. Among the scaffolds evaluated, mainly solid and preformed scaffolds comprising porous foams and fibrous meshes have been used. However, these types of scaffolds have the major drawback of requiring a surgical incision to position the scaffolds into desired sites in the body. Recently, a wide variety of biodegradable PLG A spherical particles have been utilized for the sustained delivery of bioactive molecules as an injectable depot formulation13,14. Moreover, PLGA is known to be biocompatible that has a broad range of applications in tissue engineering and clinic application15,16. Therefore, PLGA microspheres may be a suitable material for the preparation of injectable scaffolds for NP tissue engineering.
Microparticulate systems have attracted increasing interest over the past few years as carriers for the delivery of cells and proteins due to minimal invasive procedures in tissue engineering16-18. Growth factors and drugs have been co-incorporated in PLGA microspheres using microparticulate systems for NP tissue differentiation19-21. Especially, anti-inflammatory agents such as dexamethasone can minimize implantation-associated inflammation and promote cell differentiation22,23. It is important to administer both anti-inflammatory corticosteroids and growth factors in a localized environment at low doses, so that both agents can function symbiotically without interference.
This study contains five parts:(1) Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-P3/ADSCs cell lines;(2) Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc;(3) Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration;(4) Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration model.
Part I Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-p3/ADSCs cell lines
Aim:The purpose of this study was to construct a lentiviral vector containing TGF-β3/GFP gene and transfect into adipose derived stem cells in vitro. Then we use drugs to select GFP/ADSCs and TGF-β3/ADSCs cell lines, which are always characterized as stem cells. It will facilitated the following development of gene and cell therapy in treating diseases of intervertebral disc degeneration.
Methods and results:Firstly, we cultured SD rat ADSCs through cutting adipose into pieces, digesting using enzyme and isolating by adherent method. Separated ADSCs were identified by studying stem cell morphology, growth curve, surface antigen marker expression profiles and three-line differentiation capacity. Gateway technology was used to construct lentiviral vectors carrying the TGF-β3, and/or GFP gene, and which were transfected into ADSCs later. Antibiotics were used to obtain a stable expression of TGF-β3and/or GFP gene in cell lines TGF-β3/ADSCs and/or GFP/ADSCs. Finally, in accordance with the morphology, surface antigen marker expression profiles and three-line differentiation capacity of stem cells, TGF-β3/ADSCs and GFP/ADSCs were identified. The expression of TGF-β3gene was detected by RT-PCR and Western Blot methods in all three cell lines. SD rats ADSCs were successfully established, cultured and expanded in vitro, and they were also successfully induced into osteogenic, adipogenic and cartilage cells. The hematopoietic stem cell phenotypic markers of ADSCs were negative, while the expression of mesenchymal stem cell phenotypic markers was positive. After48h transfection, strong green fluorescence could be seen, and the transfection rate of Lenti-GFP-TGF-β3and Lenti-GFP were above90%. Compared with GFP/ADSCs and ADSCs cells, the expression of TGF-β3gene is overexpression in TGF-β3/ADSCs cells by RT-PCR and Western blot detection (P <0.05). In addition, the characteristics of stem cells could still be identified in GFP/ADSCs and TGF-β3/ADSCs cell lines.
Conclusions:SD rats ADSCs were easily isolated and cultured in vitro, which have multiple differentiation potential abilities and mesenchymal stem cell characteristics. Lentiviral vectors containing the TGF-β3/GFP gene were successfully transfected into ADSCs,then GFP/ADSCs and TGF-β3/ADSCs cell lines were established.
Part Ⅱ Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc
Aim:Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions.
Methods and results:ADMSCs were harvested from young (aged8-12years, n=6) and mature (aged33-42years, n=6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for2weeks. Cell viability was measured by annexin V-FITC and PI (AV-PI) staining and cell proliferation was measured by MTT assay. The expression of aggrecan, collagen-Ⅰ, and collagen-Ⅱ was detected by real-time quantitative polymerase chain reaction and Western blot analysis. IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-Ⅰ. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD.
Conclusions:IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain.
Part Ⅲ Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration
Aim:This study aimed to investigate the feasibility of the nanostructured3D poly(lactide-co-glycolide)(PLGA) constructs, which are loaded with dexamethasone (DEX) and growth factor embeded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, to serve as an effective scaffold for nucleus pulposus (NP) tissue engineering.
Methods and results:Our results demonstrated that the microsphere constructs were capable of simultaneously releasing TGF-β3and DEX with approximately zero order kinetics. The dual bead microspheres showed no cytotoxicity, and promoted the proliferation of the rat adipose-derived mesenchymal stem cells (ADSCs) by lactate dehydrogenase assay and CCK-8assay. After4weeks of cultivation in vitro, the ADSCs-scaffold hybrids contained significantly higher levels of sulfated GAG/DNA and collagen type Ⅱ than the control samples. Moreover, quantitative real time PCR analysis revealed that the expression of disc-matrix proteins including collagen type Ⅱ, aggrecan, and versican in the ADSCs-scaffold hybrids was significantly higher than that in the control group, whereas the expression of osteogenic differentiation marker (collagen type I) was decreased.
Conclusions:Taken together, these data indicate that Dex/TGF-β3/PLGA microspheres could be used as a scaffold to improve the ADSCs growth and differentiating into NP like cells, and reduce the inflammatory response for IVD tissue engineering.
Part Ⅳ Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration model
Aim:Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported a new nanostructured3D poly(lactide-co-glycolide)(PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSCs) seeded PLGA microspheres into the rat intervertebral disc degeneration model could regenerate the degenerated disc.
Methods and results:Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix associated gene expressions were evaluated in normal controls (NC)(without operations), degeneration control (DC) group (with needle puncture, only DMEM injection); PLGA microspheres (PM) treatment group (with needle puncture, PLGA microspheres only injection); and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSCs injection) for a24-week period. The results showed that24weeks post-transplantation, PM and PMA groups regained a disc height value of about63%and76%, MRI signal intensity of about47%and76%, respectively, compared to NC group. Biochemistry, immunohistochemistry and gene expression analysis indicated the restoration of proteoglycan accumulation in the discs of PM and PMA groups.
Conclusions:Taken together, these data suggest that ADSCs-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat intervertebral disc degeneration model. PLGA microsphere may serve as a promising carrier in cell transplantation therapy for degenerative disc disease.
引文
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