【作者】 孙伟；

【导师】 赵杰；

【作者基本信息】 上海交通大学 ， 外科学， 2014， 博士

【摘要】 目的：1.探讨T2mapping技术作为一种灵敏、无创的工具定量评估椎间盘退变的潜力和可行性。2.研究腺病毒介导的Sox9基因转染对单层培养骨髓间充质干细胞（BMSCs）分化的作用。3.研究Sox9基因转染的BMSCs在三维壳聚糖水凝胶（C/Gp）中的分化情况。4.通过体内动物实验，探讨Sox9基因转染自体BMSCs-C/Gp复合物对退变椎间盘的修复作用。方法：1.建立兔椎间盘退变模型，通过传统MRI、T2mapping和real-timePCR检测，观察髓核横向弛豫时间（T2）和蛋白聚糖、II型胶原基因表达在退变过程中的纵向变化，研究其相关性。2.分离、培养兔BMSCs，将携有Sox9基因的腺病毒导入BMSCs，利用real-time PCR法检测Sox9基因转染的单层培养BMSCs的相关基因表达。3.制备温敏性壳聚糖水凝胶C/Gp，将BMSCs与C/Gp复合，通过死活细胞染色和MTT法检测BMSCs的存活和增殖情况，观察Sox9基因转染对三维C/Gp培养BMSCs相关基因表达的影响。4.制作兔椎间盘退变模型，从影像学、大体形态学、组织学以及免疫组织化学等方面观察Sox9-BMSCs-C/Gp复合物修复退变椎间盘的作用。结果：1.髓核T2与改良Thompson分级间呈明显负相关。髓核T2值与蛋白聚糖、II型胶原的基因表达在诱导椎间盘退变后的前3周降低最明显。髓核T2与蛋白聚糖、II型胶原基因表达在整个退变进程中呈强相关。2. Sox9转染并未影响BMSCs增殖，转染Sox9基因的BMSCs高表达Sox9蛋白，Sox9、II型胶原和蛋白聚糖的基因表达明显上调。3. C/Gp在室温下呈液态，37℃时呈凝胶状。BMSCs可在C/Gp中存活、增殖。转染Sox9基因的BMSCs在C/Gp中高表达Sox9、II型胶原和蛋白聚糖基因。4. Sox9基因转染的BMSCs可在体内存活12周，Sox9-BMSCs组椎间盘在影像学、大体形态学、组织学及免疫组织化学几方面均表现出更少的退变性变化。结论：1.髓核T2与蛋白聚糖、II型胶原基因表达有较好的相关性。T2mapping可以作为一种灵敏的无创的工具定量评估椎间盘退变。2. Sox9基因转染可以诱导单层培养BMSCs向髓核样细胞分化。3. C/Gp具有较好的细胞相容性，转染Sox9基因的BMSCs在三维C/Gp中可以向髓核样细胞分化。4. Sox9基因转染BMSCs-C/Gp复合物可以有效延缓椎间盘退变。更多还原

【Abstract】 Objective:1. To investigate the potential of T2mapping as a sensitive,noninvasive tool for quantitatively characterizing the process ofintervertebral disc degeneration (IDD) in a rabbit model.2. To observe the effect of Sox9gene transfer on the chondrogenicdifferentiation of monolayer BMSCs.3. To study the effect of Sox9gene transfer on the differentiation ofBMSCs encapsulated in the thermogelling chitosan gels (C/Gp).4. To investigate the efficacy and feasibility of Sox9-transducedBMSCs to repair the degenerated intervertebral disc in a rabbit model.Materials and methods:1. Disc degeneration was induced via a validated annular stab method.After T2-weighted MRI and T2mapping analysis, expression analysis ofaggrecan and type II collagen gene in nucleus pulposus (NP) wasperformed using real-time PCR. The longitudinal changes in NP T2andgene expressions as well as their correlations through the process of IDDwere studied.2. BMSCs were harvested, cultured and transduced by adenovirus-Sox9. Real-time PCR was performed for investigating the geneexpressions of monolayer BMSCs.3. The C/Gp gels was prepared. The MTT and Live/Dead assay wereperformed for the viability and proliferation of the encapsulated BMSCs.Real-time PCR was performed for investigating the gene expressions of BMSCs in three-dimension C/Gp gels.4. In vivo study was performed. The repair effect of Sox9-transducedautologous BMSCs encapsulated in C/Gp on degenerated discs wasevaluated by T2-weighted MRI, T2mapping, macroscopic appearance,histology and immunohistochemistry.Results:1. There was a strong inverse correlation between NP T2andmodified Thompson grades. The decline of L4/5NP T2through24weekswas nonlinear, the most significant decrease was observed in3weekspostoperatively. The tendency was confirmed at gene expression levels. NPT2correlated strongly with aggrecan and type II collagen geneexpressions.2. The MTT assay data showed that proliferation proceeded equally inthe Adv-Sox9and Adv-GFP groups. The expression of Sox9protein washigher in Sox9-transduced BMSCs. Sox9gene transfer significantlyup-regulated the gene expressions of Sox9, type II collagen and aggrecanin BMSCs cultured in monolayer.3. The C/Gp was liquid at room temperature and gel at37°C. TheMTT and live/dead cell staining demonstrated that the C/Gp possessessatisfactory biocompatibility. The Sox9-transduced BMSCs encapsulatedin three-dimension C/Gp showed significantly high gene expression levelsof Sox9, type II collagen and aggrecan.4. Sox9-transduced BMSCs could survive in vivo for12weeks. Ahigher T2-weighted signal intensity and T2value, better preserved NPstructure and greater amount of extracellular matrix were observed in discstreated with Sox9-transduced BMSCs compared with those withouttransduction.Conclusions:1. NP T2correlates well with aggrecan and type II collagen gene expressions. T2mapping could act as a sensitive, noninvasive tool forquantitatively evaluating IDD and characterizing the process of IDD.2. Sox9gene transduction can differentiate monolayer BMSCs into aNP-like phenotype.3. C/Gp gels possess satisfactory biocompatibility. Sox9genetransduction can differentiate BMSCs encapsulated in three-dimensionC/Gp into a NP-like phenotype.4. Sox9gene transfer could significantly enhance the repair effect ofBMSCs on the degenerated discs.更多还原