【作者】 胡胜利；

【导师】 冯华；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 脊髓损伤(spinal cord injury,SCI)在全球呈高发生率、高致残率、高耗费、发病年轻化等特点。脊髓损伤后由于广泛的神经元死亡、大量的轴突变性、弥漫性的脱髓鞘造成患者劳动能力丧失、生活不能自理以及各种并发症,其后果是终身性和毁灭性的,不仅给病人造成极大的痛苦,也给家庭和社会带来沉重的负担。一个多世纪以来,医学界先后采用了手术、药物、物理、基因以及细胞治疗等多种方法来治疗脊髓损伤,但都不能有效地解决患者不同程度的瘫痪这一难题。因此,寻找有效而安全的脊髓损伤后的治疗方法仍然是困扰医学界的一个难题且具有非常重要的科学意义、经济意义和社会意义。外源性干细胞移植是近十几年来脊髓损伤治疗的研究热点,近些年来在胎儿附属物如脐带中发现有丰富的间充质干细胞,具有低免疫原性、高增殖能力以及来源更方便等独特的优越性,体外研究显示能够向骨、软骨、心肌、血管内皮以及神经系细胞等方向分化。因此,本研究利用人源性脐带间充质干细胞移植治疗大鼠脊髓损伤,探讨其疗效和机制。第一部分人脐带间充质干细胞的分离、培养与鉴定目的:探讨人源性脐带间充质干细胞的培养方法并研究其生物学特性。方法:1.获取健康、足月、剖腹产胎儿脐带,剥离脐带wharton’s jelly胶,充分剪碎,组织块贴壁培养法获得脐带间充质干细胞,体外传代、纯化、扩增,倒置显微镜下观察细胞形态。2.流式细胞仪检测细胞表面标志物CD73、CD90、CD105、CD14、CD34、CD45、HLA-DR。结果:1.人脐带wharton’s jelly胶组织块培养5-7 d后组织块周围即可见新生细胞,为长梭形或多角形,培养至16-20 d细胞明显增多,达90%以上融合,类似成纤维样细胞,放射状或漩涡状分布。传代后细胞增殖迅速,生长3-4d细胞即呈80%-90%以上融合。2.流式细胞仪检测人脐带间充质干细胞高表达CD73、CD90、CD105,不表达CD14、CD34、CD45、HLA-DR。结论:1.应用组织块贴壁培养法能够从脐带wharton’s jelly胶中培养出大量增殖能力较强的成纤维样细胞。2.流式细胞仪检测显示脐带来源间充质干细胞和骨髓、脐血、胎盘等其它组织来源的间充质干细胞具有相似的表面标志。3.脐带能为间充质干细胞移植提供充足的干细胞来源。第二部分人脐带间充质干细胞磁标记后生物学特性及磁共振信号研究目的:探讨人脐带间充质干细胞超顺磁性氧化铁(superparamagnetic iron oxide, SPIO)纳米颗粒标记及磁共振示踪的可行性。方法:1.细胞的SPIO标记共分为5个浓度组,分别为对照(0μg)、5.6μg、11.2μg、22.4μg和44.8μg Fe/ml,其中每个浓度有四个孵育条件,即12h-pll,24h-pll,12h+pll和24h+pll。2.普鲁士蓝染色计数SPIO标记细胞,计算标记阳性率,MTT法检测SPIO标记细胞生长和增殖活性。3.体外磁共振GRE T2*WI和SE T2WI成像检测标记细胞的磁共振信号。4.标记细胞大鼠脊髓内移植后,磁共振TSE T2WI成像追踪体内移植磁标记细胞。结果:1.细胞标记阳性率随着孵育浓度和时间的延长而升高,在22.4μg Fe/ml浓度、24h-pll条件下,细胞的标记率达到94.1%,提高浓度到44.8μg Fe/ml、24h-pll,阳性率不再升高;在5.6μg、11.2μg、22.4μgFe/ml三个浓度下(孵育12h),增加pll可以显著提高标记率;在24h+pll条件下,细胞生长受到明显影响,大部分细胞坏死脱落。2.低于22.4μgFe/ml浓度标记,细胞生长和增殖活性不受到明显影响,浓度达到44.8μgFe/ml(孵育24h),二者均显著减弱。3. 22.4μg Fe/ml SPIO标记24h后,体外磁共振检查示GRE T2*WI和SE T2WI上均呈低信号,且随着细胞数目的增加,信号不断降低,与未标记细胞组具有统计学差异,且信号强度与细胞数目呈直线相关。4.细胞移植3d后MRI检查发现标记细胞注射点呈明显的低信号,而未标记细胞注射点信号稍有减低。14d后标记细胞注射点仍然可以追踪到标记细胞低信号。脊髓标本普鲁士蓝和核固红染色,可见标记细胞注射点有大量SPIO阳性细胞,而未标记细胞注射点则见到少量阳性细胞。结论:1.超顺磁性氧化铁纳米颗粒能够有效标记人源性脐带间充质干细胞,且不影响细胞的生长和增殖活性。2.磁标记细胞体外磁共振GRE T2*WI和SE T2WI成像可以产生特征性低信号转变,其信号强度与细胞数量成直线相关。3.磁共振TSE T2WI成像可以追踪体内移植磁标记细胞,持续时间达2w以上。第三部分人脐带间充质干细胞移植治疗脊髓损伤疗效及机制研究目的:研究人脐带间充质干细胞移植治疗脊髓损伤的疗效并初步探讨其机制。方法:1. 36只SD健康成年雌性大鼠随机分为:假手术组12只,大鼠只行T9-T11椎板切开术,不行脊髓打击伤;对照组12只,行T10段脊髓打击伤,伤后第1d脊髓内注射DMEM/F12;实验组,行T10段脊髓打击伤,伤后第1d脊髓内注射第5代人源性脐带间充质干细胞。2.于伤后1d、1w、3w、5w、7w、8w进行行为学BBB运动功能评分;细胞免疫荧光染色观察GDNF、BDNF和NT-3的表达;于伤后3w采用ELISA检测大鼠脊髓标本GDNF、BDNF和NT-3的含量。3.于伤后1m、2m采用免疫荧光染色观察脐带间充质干细胞在宿主脊髓内的迁移和分化。4.于伤后2m采用免疫组织化学染色观察GAP-43、NF-200、GFAP在脊髓内的表达。结果:1.假手术组运动功能于1w后基本正常,对照组和实验组随着时间的延长,运动功能逐渐恢复,在伤后3w内恢复明显。第5w后实验组的BBB评分与对照组相比明显升高。2.细胞免疫荧光染色结果显示BDNF、nestin无表达,GDNF、NT-3弱表达。ELISA检测可见实验组GDNF、NT-3含量比对照组明显增多,而BDNF两组间无明显差别。3.移植后2m,人脐带间充质干细胞在宿主脊髓内存活并且呈纵向迁移,距离达到5mm,损伤区可见大量hNu染色阳性细胞汇集。移植后第1m、2m未见到脐带间充质干细胞向神经元、少突胶质细胞和星形胶质细胞方向分化。4.移植后2m,GFAP免疫组化染色结果显示对照组脊髓损伤区周围灰质和白质GFAP表达明显比实验组和假手术组增强,对照组脊髓损伤程度比实验组重,GFAP形成致密的胶质瘢痕;NF-200免疫组化染色结果显示对照组脊髓损伤区周围NF-200阳性神经纤维长度明显比实验组缩短;GAP-43免疫组化染色结果显示实验组脊髓损伤区周围GAP-43阳性细胞比对照组明显增多,并且有较多典型的再生轴突生长锥样结构,对照组未见到典型的生长锥。结论:1.脐带间充质干细胞移植后能够在宿主脊髓内存活,并且沿着脊髓纵轴迁移。但是不能见到其向神经元、少突胶质细胞和星形胶质细胞方向分化。2.脐带间充质干细胞移植后能够分泌GDNF和NT-3促进大鼠脊髓损伤后后肢运动功能评分增加,从而改善行为学功能。3.脐带间充质干细胞移植后能够抑制大鼠脊髓损伤后胶质瘢痕的形成,促进神经纤维再生。更多还原

【Abstract】 Spinal cord injury (SCI) results in loss of neurons, degeneration of axons, formation of glial scar, and severe functional impairment. Human umbilical cord mesenchymal stem cells (hUC-MSCs) can be induced to form neural cells in vitro. Therefore, the stem cells may be a valuable source in the repair of spinal cord injury.Part 1 Isolation, culture, and identification of human umbilical cord mesenchymal stem cellsObjectives:To explore the isolation, culture, and expansion method of hUC-MSCs.Methods:1. Human umbilical cords were collected from full-term caesarian section births and processed within 3–6 h. Umbilical arteries and vein were removed, and the remaining tissue was diced into small fragments. The explants were transferred to culture flasks containing DMEM/F12 along with 10% fetal bovine serum. They were left undisturbed for 4–6 days to allow migration of cells from the explants. They were re-fed and passaged as necessary.2. The mesenchymal cells were trypsinized and suspended in DMEM/F12 at a concentration of 3×106/ml. A 100μl sample was incubated for 35 min at 48 oC with 15μl of various mouse anti-human antibodies against following surface markers: CD14, CD34, CD45, CD90, CD73, CD105, HLA-DR. Then, they were washed, centrifuged, and fixed in 1.5 ml of 4% paraformaldehyde. A FACScan machine was used to analyze antibody binding.Results:1. hUC-MSCs were successfully isolated from umbilical cord explants and expanded as primary cultures. The cells demonstrated a fibroblast-like or spindle-shaped morphology in confluent layers in culture. 2. Flow cytometry showed that the cells expressed high levels of matrix markers (CD90) and mesenchymal stem cells (MSCs) markers (CD73, CD105) but did not express hematopoietic lineage markers (CD34, CD45, CD14) and HLA-DR.Conclusions:1. MSCs can be successfully isolated, cultured, and expanded from human umbilical cord Wharton’s jelly using routine technical approaches.2. hUC-MSCs express surface markers similar to MSCs derived from bone marrow, umbilical cord blood, and placenta.3. Wharton’s jelly of umbilical cord is a rich and easily attainable source of MSCs for cell therapy.Part 2 Biological characteristics and MRI of superparamagnetic iron oxide nanoparticles labeled human umbilical cord mesenchymal stem cellsObjectives:The purpose of this study was to evaluate the influence of superparamagnetic iron oxide (SPIO) nanoparticles on hUC-MSCs and the feasibility of tracking for hUC-MSCs by noninvasive magnetic resonance imaging (MRI).Methods:1. The label (Feridex) was added to the cultures at concentrations equivalent to 5.6, 11.2, 22.4, and 44.8μg Fe/ml (diluted with DMEM/F12) and incubated for 12 or 24 h. Control cultures were incubated without SPIO.2. Prussian Blue staining was used to determinate the labeling efficiency (number of Prussian Blue labeled cells/total number of cells in the sample). Cell viability and growth curves were evaluated with tetrazolium dye 3-(4,5-dimethylthiazol-2)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay.3. In vitro MRI Gradient echo T2-weighted (GRE T2*WI) images [repetition time (TR) 3,200 ms; echo time (TE) 354 ms; FOV=172.5×172.5; slice thickness=3 mm] and spin echo T2-weighted (SE T2WI) images (TR 6,000 ms; TE 98 ms; FOV=220×220; slice thickness=4 mm) were applied to examine SPIO-labeled hUC-MSCs.4. In vivo MRI was used to track SPIO-labeled hUC-MSCs transplanted in rat spinal cord. Results:1. A good correlation between SPIO uptake and the iron concentration in the culture medium was observed. As the concentration of SPIO and the incubation time increased, the amount of intracellular iron increased.2. The data show that neither survival nor proliferation potential was impaired in dealing with 22.4μg Fe/ml for 24 h. However, treatment with 44.8μg Fe/ml for 24 h impairs cell survival and proliferation significantly.3. GRE T2*WI and SE T2WI images revealed the presence of a hypointense signal. A good linear correlation between the number of labeled cells and intensity of the GRE T2*WI images was observed. Similar result was obtained in the SE T2WI images.4. In vivo MRI 3 days and 14 days after injection showed a large reduction in signal intensity in the region transplanted with SPIO-labeled hUC-MSCs. The images from unlabelled hUC-MSCs showed a smaller reduction in signal intensity. Prussian Blue co-stained with nuclear fast red confirmed the presence of SPIO-labeled cells in the region of transplant site with SPIO-labeled hUC-MSCs.Conclusions:1. hUC-MSCs can be labeled efficiently without cell toxicity using SPIO at optimized low dosages.2. Noninvasive imaging of transplanted hUC-MSCs is feasible. These results may be beneficial to cell-based therapies for clinical implementation.Part 3 Transplantation of human umbilical cord mesenchymal stem cells in rats traumatic spinal cord injuryObjectives:To evaluate the effects of hUC-MSCs transplantation on functional recovery in rats traumatic spinal cord injury.Methods:1. Rats were randomly divided into 3 groups, sham operation group (n=12), control group (n=12), and hUC-MSCs group (PC, n=12). All groups were subjected to spinal cord injury by weight drop device except for the sham group. Control group received DMEM/F12 injections, but hUC-MSCs group undertook cells suspension treatments. 2. Rats from each group were examined for neurological function. Enzyme linked immunosorbent assay (Elisa) was used for evaluation of contents of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3).3. Survival, migration, and differentiation of hUC-MSCs was explored with immunofluorescence.4. The expression of GAP-43, NF-200, and GFAP was also assessed using immunohistochemistry and immunofluorescence.Results:1. Significant improvements in locomotion were observed in the hUC-MSCs groups. There is statistical significance compared with control group.2. Transplanted hUC-MSCs produced large amounts of GDNF and NT-3 in the host spinal cord, which may be beneficial to the repair of injured spinal cord.3. Transplanted hUC-MSCs survived for at least 8 weeks. However, they did not differentiate into neural cells.4. The functional recovery was accompanied by increased length of neurofilament positive fibers and increased numbers of growth cone around the lesion site. There were fewer reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the hUC-MSCs group than in the control group.Conclusions:1. hUC-MSCs can survive and migrate in the host spinal cord after transplantation without differentiating into neural cells.2. hUC-MSCs can inhibit the formation of glial scar and enhance neuroregeneration, which promote the functional recovery after SCI.3. These data suggest that hUC-MSCs may possess therapeutic potential for traumatic spinal cord injury.更多还原