复合递送基因修饰干细胞与药物的新型凝胶埋植体系的构建及其对脊髓损伤的治疗研究

发布时间：2018-01-11 03:16

本文关键词：复合递送基因修饰干细胞与药物的新型凝胶埋植体系的构建及其对脊髓损伤的治疗研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：干细胞具有多向分化潜能和丰富的细胞功能,对干细胞的基因修饰和基于干细胞的神经组织修复策略长期以来受到广泛关注。其中,对于中枢神经系统疾病,如脊髓损伤(SCI),其发生后呈现复杂的动态病程和病理状况,加之中枢神经组织自愈力低,对损伤后神经功能的恢复不但需要进行神经细胞的补充,还必须克服细胞外基质缺失以及组织微环境炎症反应等多方面的不利因素。如何建立能够在SCI发生后实现有效的神经功能恢复的多功能复合疗法,已经成为科学研究及临床治疗中的热点和亟待解决的难点问题。本文研究针对严重SCI后的神经组织修复这一难题,结合基因传递、干细胞疗法及局部缓控释递药手段,构建新型的多功能复合埋植体系。研究建立脑源性神经营养因子(BDNF)基因修饰的骨髓间充质干细胞(MSC),结合临床治疗脊髓损伤的抗炎药物甲基泼尼松龙(甲泼尼龙,MP),进行脊髓组织的联合埋植治疗。首先,实验通过对MSC基因转染体系的优化和系统研究,制备BDNF基因修饰的MSC,另一方面,构建甲泼尼龙明胶微球(MPGM)缓释制剂以克服MP全身用药的系统毒副作用和脊髓组织利用率低的问题。同时,为实现干细胞与药物制剂的脊髓局部埋植,本研究基于天然细胞外基质成分透明质酸(HA)的交联反应和黏附肽的修饰建立三维凝胶埋植支架载体,利用该载体包载基因修饰的MSC和MPGM进行SCI后的神经组织修复,通过体内外考察,对这一复合埋植体系的治疗效果进行评价,并对复合埋植体系内各组分在神经恢复中的功能及各组分的联合作用方式进行了探讨。为建立高效低毒的MSC基因转染体系,本文对新型脂质载体Screenfect A(SF)在MSC中的基因转染进行了优化和系统研究。基于SF所优化的MSC基因转染体系可以实现较高的基因转染效率,并同时保持MSC良好的细胞活性。进一步考察发现,SF在质粒的细胞摄取和表达阳性率中的表现与LipofectamineTM 2000(Lipo)相当,但SF的转染显示了更高的细胞平均表达水平和MSC增殖活性,且血清对SF转染MSC的效率及细胞活性的影响皆较小。相比常用脂质载体Lipo,基于SF的基因传递明显减少质粒用量,在对基因转染效率及MSC细胞活性的兼顾中显示明显的优势。进一步对SF所携载质粒的胞内转运行为的考察发现,质粒可于转染0.5h之内开始入胞,顺利进行溶酶体逃逸,进而于4h之内逐渐进入MSC细胞核。为了补充受损组织中缺失的细胞外基质,避免所埋植MSC的流失和细胞死亡,并为受损组织提供支撑和桥接,本研究基于HA构建三维凝胶支架埋植载体。研究通过对HA分子量和支架制备处方的优化筛选,获得具有三维多孔网状结构和一定力学强度的凝胶支架。为了提高支架的细胞相容性,促进MSC在支架内的黏附生长,研究引入源于层粘连蛋白链的多肽PPFLMLLKGSTR对凝胶支架进行表面修饰。体外实验结果显示,黏附肽的修饰显著提高了 HA凝胶支架对MSC的细胞相容性,MSC可于肽修饰的支架中黏附生长并长期存活。实验进一步对埋植体系的体内脊髓组织相容性及在体内对MSC的支持作用进行考察,将携载MSC的支架埋植入脊髓组织,肽修饰的支架本身即显示了良好的组织桥接功能,同时可显著提高MSC在组织中的存活。通过对支架-细胞复合埋植及二者单独埋植的研究分析发现,支架载体与干细胞在脊髓修复过程中具有明显的协同作用:支架载体或MSC单独埋植时作用微弱,而二者复合埋植后显著提高了脊髓组织完整性,对大鼠运动功能恢复和组织神经丝重建发挥了显著的促进作用。进一步对组织内继发性损伤初期情况的考察初步揭示了复合埋植体系发挥显著修复作用的可能原因,即干细胞与支架载体的埋植对炎症细胞浸润和胶质疤痕形成具有协同抑制作用。基于对支架载体体内外相容性和与MSC之间协同作用的探讨,本研究进一步利用所构建的支架载体携载BDNF基因修饰的MSC进行SCI后的神经组织修复治疗。体外实验结果显示,支架载体可支持基因修饰的MSC的长期存活及对所修饰基因的表达。体内埋植后,对MSC的BDNF基因修饰及支架的携载,都显著增强了 MSC细胞促进脊髓修复的作用,埋植治疗可明显抑制胶质疤痕形成,同时重建组织中的神经丝,在桥接脊髓神经组织的基础上,明显改善脊髓组织内的细胞形态。对于与支架共同埋植的MSC,BDNF的基因修饰可促进MSC存活以及对神经组织的修复作用,但对于缺少支架携载的MSC,其BDNF基因修饰对细胞功能的增强效果则极其有限,进一步证明支架载体对MSC脊髓埋植的重要作用。为将抗炎药物MP进行局部埋植以克服其全身用药的毒副作用和利用率低的问题,本研究制备甲泼尼龙明胶微球缓释制剂。分别对甲泼尼龙琥珀酸钠明胶微球(MPSS-GM)、琥珀酸甲泼尼龙明胶微球(MPH-GM)和甲泼尼龙明胶微球(MPGM)三种微球进行药物释放性质的研究,考察支架的肽修饰体系对微球最终包载药量和释药性质的影响,结果发现,经过支架肽修饰体系的处理后,MPSS-GM只保留极少量药物,MPH-GM保留部分药物且释放较快速,而MPGM保留了最多的药物且释药时间最长。实验建立MP的高效液相分析方法学,对MPGM释药性质的进一步研究显示其可实现400小时以上的持续释药。为了实现MPGM在脊髓组织中的埋植,实验进一步利用肽修饰支架包载MPGM。体外考察发现,包载MPGM的肽修饰支架仍具有药物缓释功能和良好的MSC细胞相容性。利用肽修饰的支架携载MPGM和基因修饰MSC进行联合埋植,体内试验结果显示,MP和BDNF-MSC在脊髓组织中的联合埋植可有效调节组织中炎症反应,埋植7天时对促炎因子的下调作用和抑炎因子的上调作用最为明显,14天和28天时,联合埋植仍显示明显的抑炎作用,并显著促进了脊髓组织再生。对包载空白GM的支架携载BDNF-MSC进行埋植的研究,显示其也具有一定的炎症缓解作用,与前期对支架和MSC埋植的结果一致,也揭示了复合埋植体系中微球和MP的作用。本课题为了实现基因修饰的干细胞和甲泼尼龙药物在脊髓组织局部的联合递送,对MSC的非病毒基因转染体系和甲泼尼龙的缓释制剂进行研究,并构建一种对干细胞和神经组织相容性良好的肽修饰的HA埋植支架载体。研究通过对携载埋植后的脊髓修复情况的研究,逐步揭示了干细胞、BNDF基因修饰、甲泼尼龙缓释给药和埋植支架载体在神经组织修复中的功能,本研究为共载干细胞与药物的埋植体系的构建提供了理论与实验依据。
[Abstract]:Stem cells have the potential of multi-directional differentiation and abundant cell function, gene modification on stem cells and stem cells based on neural tissue repair strategy has long attracted widespread attention. Among them, the central nervous system diseases, such as spinal cord injury (SCI), the present course and pathological status of the complex, and the healing power the central nervous tissue is low, supplement to the restoration of neural function after injury is not only the need for nerve cells, must also overcome the disadvantage of extracellular matrix and lack of tissue microenvironment inflammation and other aspects. How to establish a multi function composite therapy can effectively realize the recovery of nerve function after SCI, has become a hot topic of scientific research and in the clinical treatment and the problems to be solved. For repair of severe nerve tissue after SCI this problem in this paper, combined with the gene transfer, stem cell Local therapy and controlled-release drug delivery methods, construction of multifunctional composite implant system. A novel study on establishment of brain-derived neurotrophic factor (BDNF) gene modified bone marrow mesenchymal stem cells (MSC) combined with anti-inflammatory drugs, Kakip Nixonn Ron clinical treatment of spinal cord injury (methyl prednisolone, MP), combined with implant treatment the spinal cord tissue. First, through research and system optimization of MSC gene transfection system, preparation of BDNF gene modified MSC, on the other hand, methylprednisolone gelatin microspheres (MPGM) sustained-release preparation construction to overcome the problem of low system toxicity and spinal cord tissue by MP systemic administration. At the same time, in order to achieve dry spinal cord cells and local implantation of pharmaceutical preparations, this research is based on the natural extracellular matrix of hyaluronic acid (HA) modified by cross-linking reaction and adhesion peptides to establish three-dimensional gel implant scaffolds, Zaiji package by using the carrier By the modified MSC and MPGM SCI after the repair of nerve tissue, through in vivo study, to evaluate the therapeutic effect of compound implant system, and the composite implant system components in the combined effects of each nerve function and recovery of the points are discussed. For the establishment of high efficiency and low toxicity of MSC gene transfection this new type of lipid carrier system, Screenfect A (SF) gene transfection in MSC were studied. MSC gene transfection system optimization and SF optimization system can achieve higher gene transfection efficiency based on the activity of MSC cells and maintain good. Further study found that SF and LipofectamineTM positive expression in cells and the rate of uptake of plasmid in 2000 (Lipo), but SF transfection showed higher mean cell expression and MSC proliferation, and the efficiency of transfection of MSC and serum on SF cell activity Effect are small. Compared with common lipid carrier Lipo, SF gene transfer significantly reduced the amount of the plasmid based shows obvious advantages in both the efficiency of gene transfection and MSC cell activity in the study of SF. Further carrying the plasmid intracellular transport behavior that can begin to enter the cell transfected with plasmid 0.5h, smooth for lysosomal escape, and then gradually into the MSC within 4H in the nucleus. In order to supplement the extracellular matrix in the damaged tissue loss, avoid implanting MSC loss and cell death, and provide support for bridging and damaged tissue, this study based on the HA 3D gel scaffold implant carrier. By optimizing the research on the molecular weight of HA and support system the preparation prescription screening, obtain a gel scaffold with three-dimensional porous structure and good mechanical strength. In order to improve the compatibility of the scaffold cells, promote MSC adhesion in stent grow, research The introduction of PPFLMLLKGSTR polypeptide from laminin chain for surface modification of the scaffold. In vitro experiment results show that the modified adhesion peptide significantly increased cell HA gel scaffold on the compatibility of MSC and MSC to support peptide modified adhesion growth and long-term survival. Further experiments on implant system in spinal cord tissue and inspect the support effect in vivo of MSC, with MSC stent implanted spinal cord tissue scaffold peptide modified itself shows a good bridging function, and can significantly improve the MSC in the organization's survival. Through the study of stent implants and two single cells composite implantation analysis found that the carrier and the stem cells have obvious synergistic effect on spinal cord repair process: support vector or MSC alone when implanted mild effect, and the two composite after implantation significantly improves the spinal cord Tissue integrity, played a significant role in promoting the recovery of motor function of rats and nerve tissue reconstruction. Further investigation on silk tissue secondary injury early situation reveals the possible causes of composite implant system play a significant role in the repair, the stem cells and the scaffold implant has a synergistic inhibitory effect on inflammatory cell infiltration and study on the formation of glial scar. Between the scaffold in vitro and in vivo biocompatibility and synergy with MSC based on this study, further support using the constructed vector carrying BDNF gene modified MSC SCI after nerve tissue repair treatment in vitro. The experimental results show that the support vector can support the modified MSC and long term survival to modify gene expression in vivo. After BDNF gene modification and support of MSC carrier, significantly enhanced MSC cells promote spinal cord repair function, Implant treatment can obviously inhibit the formation of glial scar, and reconstruction of neurofilament in the organization, based on spinal cord bridging, improve spinal cord cell morphology. The common implants and support MSC gene modified BDNF can promote the survival of MSC and nerve tissue repair function, but for lack of support carrying the MSC, the BDNF gene modified enhancement effect on cell function is extremely limited, further demonstrated the important role of MSC in the spinal cord scaffold implantation. The anti-inflammatory drug MP for local implantation to overcome the systemic drug toxicity and low utilization rate, the study on Preparation of methylprednisolone sustained-release gelatin microspheres respectively. Methylprednisolone sodium succinate gelatin microspheres (MPSS-GM), methylprednisolone succinate gelatin microspheres (MPH-GM) and methylprednisolone (MPGM) gelatin microspheres three kinds of microspheres drug release The qualitative research system of the peptide modified stent up influence, drug loading and release properties of the microspheres were found after treatment of stent peptide modified system, MPSS-GM only very small amounts of drugs, drug release and retention of some MPH-GM is fast, while MPGM retained the drug most and drug release time long. The establishment of HPLC analysis of MP method, it can achieve more than 400 hours of sustained release drug release characteristics of further research on the MPGM display. In order to realize the MPGM in spinal cord were further experiments with peptide modified stent loaded MPGM. in vitro study showed that peptide modified stent MPGM package is still with slow-release function and good biocompatibility. The use of MSC peptide modified stents carrying MPGM and MSC gene modified joint implants, in vivo experiments showed that MP and BDNF-MSC in spinal cord implantation can be combined The effective regulation of tissue inflammation, after 7 days of proinflammatory cytokine downregulation and upregulation of anti-inflammatory cytokines is the most obvious, 14 days and 28 days, the joint implants still showed significantly inhibitory effect on inflammation, and promote spinal cord regeneration. Articles on BDNF-MSC loaded on the support package for implantation carrying a blank GM, the display also has a certain role in relieving inflammation, consistent with previous results of stent and MSC implantation, also reveals the microspheres and MP composite implant system. The effect of methylprednisolone cells and drug delivery in the spinal cord tissues combined in order to achieve this subject stem gene modified, sustained-release preparation for MSC non viral gene delivery system and methylprednisolone were studied, and to construct a compatible peptide modified good for stem cells and neural tissue scaffold. HA implantation through the study on the repair of spinal cord after implantation of the carrying situation The status of the research, gradually reveals the stem cells, BNDF gene modified, methylprednisolone sustained-release carrier and implanted in the nerve tissue repair of the function, this study provides a theoretical and experimental basis for the construction of stem cells and drug implant system is loaded.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943

【相似文献】