人脐带间充质干细胞治疗脊髓损伤的实验研究

发布时间：2018-05-12 23:15

本文选题：人脐带间充质干细胞 + 超顺磁性氧化铁纳米颗粒　；参考：《第三军医大学》2010年博士论文

【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。
[Abstract]:Spinal cord injury (SCI) is characterized by high incidence, high disability, high consumption, and younger onset in the world. After spinal cord injury, extensive neuronal death, a large number of axons, and diffuse demyelinating causes the loss of labor, life and various complications. The consequences are life-long and destruction. For more than a century, the medical community has used a variety of methods, such as surgery, medicine, physics, gene and cell therapy, to treat spinal cord injury, but it can not effectively solve the problem of the patient's paralysis to varying degrees. The treatment of effective and safe spinal cord injury remains a difficult problem in the medical field and is of great scientific significance, economic significance and social significance. Exogenous stem cell transplantation is a hot spot in the recent decade for the treatment of spinal cord injury. In recent years, there are abundant mesenchymal stem cells found in fetal appendages such as umbilical cord. Cells have unique advantages such as low immunogenicity, high proliferation ability and more convenient sources. In vitro studies have shown that the cells can differentiate into bone, cartilage, myocardium, vascular endothelial cells and nerve cells. Therefore, human umbilical cord mesenchymal stem cells are used to transplant spinal cord injury in rats and explore its therapeutic effect and mechanism.
Part one: isolation, culture and identification of human umbilical cord mesenchymal stem cells
Objective:
Objective to explore the culture methods of human umbilical cord mesenchymal stem cells and study their biological characteristics.
Method:
1. to obtain health, full-term, fetal umbilical cord by caesarean section, umbilical cord Wharton 's jelly glue, full scissors, tissue block wall culture method to obtain umbilical cord mesenchymal stem cells, external generation, purification, amplification, and inverted microscope observation of cell morphology.
2. cell surface markers CD73, CD90, CD105, CD14, CD34, CD45, HLA-DR. were detected by flow cytometry.
Result:
After the 1. human umbilical cord Wharton 's jelly glue tissue mass was cultured for 5-7 D, the new cells were seen around the tissue block, which was long spindle or polygon, and the culture to 16-20 D cells increased obviously, up to 90% fusion, similar to fibroblast like cells, radiate or whirlpool. The cells proliferated rapidly after the passage, and the growth of 3-4d cells was more than 80%-90% fusion.
2. flow cytometry showed that human umbilical cord mesenchymal stem cells were highly expressed CD73, CD90, CD105, and did not express CD14, CD34, CD45, HLA-DR..
Conclusion:
1. the tissue block adherent culture method can produce a large number of fibroblast like cells with strong proliferative ability from the umbilical cord Wharton 's Jelly gel.
2. flow cytometry showed that mesenchymal stem cells from umbilical cord derived mesenchymal stem cells and bone marrow, umbilical cord blood, placenta and other tissue derived mesenchymal stem cells have similar surface markers.
3. umbilical cord can provide sufficient stem cell source for mesenchymal stem cell transplantation.
The second part of human umbilical cord mesenchymal stem cells after magnetic labeling biological characteristics and magnetic resonance signals
Objective:
Objective to investigate the feasibility of iron oxide (SPIO) nanoparticle labeling and magnetic resonance tracing in human umbilical cord mesenchymal stem cells (superparamagnetic).
Method:
The SPIO markers of 1. cells were divided into 5 concentration groups, the control (0 mu g), 5.6 mu g, 11.2 mu g, 22.4 mu g and 44.8 micron g Fe/ml, with four incubation conditions for each concentration, namely 12h-pll, 24h-pll, 12h+pll and 24h+pll..
2. Prussian blue staining was used to count SPIO labeled cells, the positive rate of markers was calculated, and the growth and proliferation activity of SPIO labeled cells were detected by MTT.
3. the magnetic resonance signals of labeled cells were detected by external magnetic resonance GRE T2*WI and SE T2WI imaging.
4. labeled cells were transplanted into the spinal cord of rats, and magnetic resonance TSE T2WI imaging was used to track the transplanted magnetic labeled cells in vivo.
Result:
The positive rate of 1. cell markers increased with the prolongation of incubation concentration and time. Under the condition of 22.4 mu g Fe/ml concentration and 24h-pll, the labeling rate of cells reached 94.1%, the concentration increased to 44.8 mu Fe/ml, 24h-pll, and the positive rate no longer increased; at 5.6 mu g, 11.2 mu g, 22.4 mu gFe/ml three concentration (incubating 12h), increasing PLL could significantly increase the labeling rate; in 24h+ Under the condition of PLL, cell growth was obviously affected, and most of the cells were necrotic and shedding.
2. below 22.4 mu gFe/ml concentration, cell growth and proliferation activity were not significantly affected, with a concentration of 44.8 gFe/ml (incubation of 24h), and two of them decreased significantly.
After 3. 22.4 g Fe/ml SPIO labeling 24h, in vitro magnetic resonance imaging showed that both GRE T2*WI and SE T2WI showed low signal, and as the number of cells increased, the signal decreased continuously, and there was a statistical difference with the unlabeled cell group, and the signal intensity was linearly related to the number of cells.
After the 4. cell transplantation, the MRI examination showed that the marked cell injection point was obviously low signal, while the unlabeled cell injection point signal slightly reduced.14d could still be traced to the low signal of the labeled cells. The spinal specimen Prussian blue and nuclear solid red staining, and the marked cell injection point had a large number of SPIO positive cells, but not marked by the marked cell injection point. A small number of positive cells were observed at the point of cell injection.
Conclusion:
1. superparamagnetic iron oxide nanoparticles can effectively label human umbilical cord mesenchymal stem cells and do not affect cell growth and proliferation activity.
2. in vitro magnetic resonance imaging of GRE T2*WI and SE T2WI can produce characteristic low signal transformation, and its signal intensity is linearly correlated with cell number.
3. magnetic resonance TSE T2WI imaging can track transplanted magnetic labeled cells in vivo, lasting more than 2W.
The effect and mechanism of third human umbilical cord mesenchymal stem cells transplantation on spinal cord injury
Objective:
Objective to study the effect of human umbilical cord mesenchymal stem cells transplantation on spinal cord injury and to explore its mechanism.
Method:
1.36 SD healthy adult female rats were randomly divided into 12 rats in the sham operation group. The rats were only treated with T9-T11 laminectomy and no spinal cord injury; 12 rats in the control group were treated with T10 segment spinal cord injury and DMEM/F12 in the spinal cord after the injury; the experimental group was treated with T10 segment spinal cord injury, and fifth generation of human umbilical cord mesenchymal stem cells were injected into the spinal cord after the injury in the spinal cord.
2. after injury, 1D, 1W, 3W, 5W, 7W, 8W were used to evaluate the behavioral function of BBB, and the expression of GDNF, BDNF and NT-3 were observed by cell immunofluorescence staining.
3. immunofluorescence staining was used to observe the migration and differentiation of umbilical cord mesenchymal stem cells in the spinal cord of 1m after 2m.
4. the expression of GAP-43, NF-200 and GFAP in spinal cord was observed by immunohistochemical staining after 2m.
Result:
1. the exercise function of the sham operation group was basically normal after 1W, and the control group and the experimental group were gradually restored with the extension of time, and recovered obviously in the 3W after the injury. The BBB score of the experimental group was significantly higher than that of the control group after 5W.
The results of 2. cell immunofluorescence staining showed that BDNF, nestin was not expressed, GDNF, and NT-3 weak expression.ELISA detected the experimental group GDNF, NT-3 content was significantly higher than the control group, but there was no significant difference between the two groups of BDNF.
3. 2m after transplantation, human umbilical cord mesenchymal stem cells live in the host spinal cord and migrate longitudinally, the distance reaches 5mm, and a large number of hNu staining positive cells can be found in the injured area. After transplantation, 1M, 2m does not see the direction differentiation of umbilical cord mesenchymal stem cells to neurons, oligodendrocytes and astrocytes.
4. after transplantation, 2m, GFAP immunohistochemical staining showed that the expression of gray matter and white matter GFAP in the area around the spinal cord injury of the control group was significantly higher than that of the experimental group and the sham operation group. The degree of spinal cord injury in the control group was heavier than the experimental group, and the GFAP formed a dense glial scar, and the NF-200 immunohistochemical staining fruit showed the NF-200 positive God around the spinal cord injury area of the control group. The fiber length was significantly shorter than the experimental group, and the results of GAP-43 immunohistochemical staining showed that the GAP-43 positive cells around the spinal cord injury area in the experimental group were significantly higher than those in the control group, and there were more typical conical structure of the regeneration axon growth, and the control group did not see the typical growth cone.
Conclusion:
1. umbilical cord mesenchymal stem cells can survive in the host spinal cord after transplantation and migrate along the longitudinal axis of the spinal cord. But they can not be seen in the direction of the neuron, oligodendrocytes and astrocytes.
2. after transplantation, umbilical cord mesenchymal stem cells can secrete GDNF and NT-3 to promote the motor function score of rats with spinal cord injury and improve their behavioral function.
3. the transplantation of umbilical cord mesenchymal stem cells can inhibit the formation of glial scar after spinal cord injury and promote the regeneration of nerve fibers.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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