【作者】 李香琴；

【导师】 刘天庆；

【作者基本信息】 大连理工大学 ， 化学工程， 2012， 博士

【摘要】 中枢神经系统(central nervous system, CNS)疾病与损伤,如帕金森病(Parkinson’s disease, PD)、脊髓损伤等比较难以自我修复,神经干细胞(neural stem cells, NSCs)移植被认为是具有潜在应用价值的治疗手段,但NSCs来源困难。骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)以其多分化潜能、可源于自体、免疫源性低、易扩增、能分泌多种细胞因子并参与细胞微环境的构建等优势,为CNS疾病与损伤的治疗提供了一个新的思路。本文旨在研究BMSCs扩增后通过与NSCs之间的相互作用促进PD体外实验细胞模型的修复,进一步为BMSCs移植治疗与修复CNS疾病与损伤提供相应的实验依据。本文首先采用胶原凝胶、中空纤维膜(hollow fiber membranes, HFMs)和自行研发设计的气升式环流生物反应器(air-lift loop bioreactor, ALB),建立一种可使细胞免受剪切力损伤的三维动态扩增体系,将BMSCs在体外先行扩增。在扩增的过程中,通过取样检测吸光度及计细胞数,绘制细胞生长曲线,获得细胞扩增倍数；检测细胞代谢参数,确定细胞的新陈代谢状况；扩增7天后,检测细胞在支架内的活性,观察细胞在支架上的生长形态,获得细胞进一步增殖、分化、保持自身功能的能力；流式细胞仪分析细胞表面特异性蛋白表达,检测细胞多向分化潜能,鉴定所扩增的细胞。结果表明,初始密度为5×105cells·mL-1的BMSCs在此系统中扩增时代谢旺盛,一周后,动态条件下扩增约17倍,静态条件下扩增约13倍；扩增的细胞仍保持较高的活性,活率约为92%,且在支架内仍保持长梭形的贴壁生长形态,仍表达CD29和CD44,不表达CD34和CD45,并仍具有较强的成骨、成软骨及成脂肪的多向分化能力；动态条件下扩增的细胞呈现出更加充分伸展的形态与较强的多向分化潜能。将以上扩增的BMSCs与生长于支架内的NSCs进行短期静态共培养,考察BMSCs与三维条件下NSCs之间的相互作用。实验中采用优化的海藻酸钙胶珠制备工艺,包囊适宜密度的NSCs,待微囊化的NSCs长至一定大小的神经球时,与BMSCs进行共培养。共培养过程中观察神经球结构及BMSCs形态的变化；共培养结束后计算NSCs及BMSCs的增殖倍数,对增殖条件下共培养的NSCs表型和多向分化潜能以及BMSCs向NSCs或神经细胞分化进行免疫荧光染色鉴定；对分化条件下共培养的NSCs向不同神经细胞分化的能力进行流式细胞仪检测,对BMSCs向NSCs或神经细胞分化进行免疫荧光染色鉴定。结果表明,1.5%(wt%)的海藻酸钠溶液与3.5%(wt%)的CaCl2溶液凝胶化反应10min,以0.8x105cells·mL-1为初始密度包囊NSCs,制备直径约2mm的海藻酸钙胶珠,与BMSCs进行共培养是比较适宜的。BMSCs可使生长于支架内的NSCs迁出细胞球,对NSCs的增殖没有明显影响；但能够明显影响NSCs的分化,使其向少突胶质细胞分化的能力增加3倍,向星形胶质细胞分化的能力减弱1倍,而向神经元细胞分化的能力没有明显变化；NSCs可促进BMSCs向NSCs及星形胶质细胞、少突胶质细胞、神经元细胞分化,且在分化共培养时这种分化比例增加,分别由增殖共培养时的约8%,11%,6%及7%增加至约18%,25%,15%及11%。根据上述BMSCs与NSCs之间的相互作用,本文进一步利用去血清诱导PC12细胞凋亡,建立PD体外实验细胞模型,将诱导凋亡的PC12细胞与NSCs及BMSCs以不同方式进行共培养,考察BMSCs在NSCs修复这一细胞凋亡模型中的作用。实验采用海藻酸钙胶珠或transwell小室作为共培养隔离手段,通过观察PC12细胞的形态,检测PC12细胞的存活率、细胞周期和培养基中胶质细胞源性神经营养因子(glial cell line derived neurotrophic factor, GDNF)的含量,确定BMSCs在NSCs保护去血清诱导PC12细胞凋亡中的作用。结果表明,NSCs通过分泌神经营养因子GDNF抑制了去血清诱导的PC12细胞的凋亡,使受损细胞的凋亡率由模型组的44.0%降低至24.3%,存活细胞处于G1期的百分数由模型组的79.9%降至53.1%,处于S期和G2期的百分数分别由模型组的15.3%和4.9%增加至35.5%和9.8%,与正常培养的PC12细胞所处周期没有较大差异,G1期细胞向S期和G2期过渡基本未受阻断,起到了神经保护作用；引入BMSCs后,两种干细胞通过相互促进向神经细胞方向分化,使培养体系中细胞分泌GDNF的量比单独NSCs作用时平均增加约33.4%,细胞分泌GDNF的能力明显增强,提高了对去血清诱导凋亡的PC12细胞的保护作用,使受损细胞的凋亡率由单独NSCs作用时的24.3%继续降低至15.1%,且存活细胞处于G1期、S期和G2期的百分数分别为53.3%,36.7%和10.3%,仍与正常培养的PC12细胞所处的周期没有较大差异。更多还原

【Abstract】 The repair of central nervous system (CNS) disorders, such as Parkinson’s disease (PD) and spinal cord injury, is difficult. Neural stem cells (NSCs) transplantation is believed to be effective in the treatment of neural diseases, however, NSCs are limited and difficult to be isolated from brain. Bone marrow mesenchymal stem cells (BMSCs) are a new cell source to CNS diseases because the cells possess the advantages of multi-differentiation potentials, weaker immunogenicity, easy obtaining and expansion, and secreting cytokines in in vivo microenvironment. Here, the expansion of BMSCs and the interaction between BMSCs and NSCs as well as the effect of expanded BMSCs on PD modeled cells in vitro were investigated.BMSCs were expanded in vitro firstly under three dimensional (3D) conditions by the combined use of collagen gel, hollow fiber membranes (HFMs) and air-lift loop bioreactor (ALB). During the expansion period, the cell growth curves and metabolism parameters were measured. And after7days, the cell viability and morphology were assayed microscopically. Moreover, the BMSC characters of expanded cells were identified by specific expressions of CD29, CD44, CD34and CD45and by multilineage differentiation into osteoblasts, chondrocytes and adipocytes. The results showed that the cultured BMSCs metabolized robustly and expanded about17-fold under dynamic condition,13-fold under static condition with the initial density of5×105cells·mL-1; after7days, the cells maintained high viability of about92%and spread with a spindle appearance and long cell protrusions in gel; the expanded cells were CD29and CD44positive, CD34and CD45negative, and they could differentiate into osteoblasts, chondrocytes and adipocytes; the cells expanded under dynamic condition spread more and showed stronger multi-differentiation potentials.The expanded BMSCs were then used to coculture with NSCs growing in scaffolds to investigate the interaction between BMSCs and NSCs. The NSCs encapsulated in calcium alginate beads (Ca-Alg-Bs) with appropriate density firstly grew to neurospheres before being cocultured with BMSCs. During the coculture period, the neurosphere structure and BMSC morphology were observed. And when the coculture ended, the NSC and BMSC expansion folds were measured; the phenotype and multi-differentiation potentials of NSCs and the differentiations of BMSCs into NSCs or neurocytes in the NSC proliferation medium were assayed by immunofluorescent staining; meanwhile the capacity of NSCs to differentiate into neurocytes in the differentiation medium was analyzed with flow cytometry, and the differentiations of BMSCs into NSCs or neurocytes were assayed by immunofluorescent staining. The results showed that2mm-diameter Ca-Alg-Bs prepared with1.5%sodium alginate and3.5%CaCl2solutions gelling for10min were suitable to encapsulate NSCs with cell density of0.8×105cells·mL-1; NSCs in beads could migrate out of neurospheres by contacting with BMSCs indirectly; BMSCs effected NSCs proliferation slightly, while influenced NSCs differentiation obviously so that the oligodendrocyte proportion increased3times and astrocyte proportion decreased1fold, but neuron proportion remained unchanged; part of the BMSCs could be induced into NSCs, astrocytes, oligodendrocytes and neurons by coculture with NSCs, and the proportions of these cells in the proliferation medium were8%,11%,6%and7%, respectively, while in the differentiation medium, the figures increased to18%,25%,15%and11%, respectively.Furthermore, PD cell model in vitro was constructed through PC12cell apoptosis induced by serum deprivation. The apoptotic PC12cells were then cocultured with NSCs alone or with NSCs and BMSCs together by means of Ca-Alg-Bs or transwell inserts. The repair of apoptotic PC12cells after the coculture was investigated and how BMSCs contributed to the repair of PD modeled PC12cells by NSCs was explored. During and at the end of the coculture, PC12cell morphology was observed and PC12cell survival, cell cycle and GDNF concentration in medium were detected. The results showed that NSCs inhibited PC12cell from apoptosis through secreting neurotrophic factor such as GDNF and the PC12cell apoptosis percentage decreased from44.0%to24.3%; the living PC12cells in G1phase reduced from79.9%to53.1%, while the cells in S and G2phases increased from15.3%and4.9%to35.5%and9.8%, respectively; these cell percentages of repaired PC12cells were close to those in normal cultured cell group, which meant that the process from G1phase to S and G2phases was nearly not obstructed. After BMSCs were introduced into the coculture system, GDNF concentration in the medium increased about33.4%comparing to the coculture system with NSCs alone. In the coculture system of BMSCs, NSCs and PC12cells, the ability of the cells to secret GDNF increased and the protection to the apoptotic PC12cells was enhanced additionally; the PC12cell apoptosis percentage further decreased from24.3%to15.1%, and the living cells in G1, S and G2phases were53.3%,36.7%and10.3%, respectively, which remained close to those in normal cultured cell group.更多还原