人脐带间充质干细胞诱导转化成神经干细胞及其移植治疗脑出血的研究

发布时间：2018-06-03 08:01

本文选题：脐带间充质干细胞 + 诱导转化　；参考：《中国协和医科大学》2010年博士论文

【摘要】： 背景：脑出血(intracerebral hemorrhage, ICH)是指原发性脑实质内血管破裂引起的出血,具有高发病率、高死亡率和高致残率的特点。目前临床上仍无有效的治疗方法用于改善幸存者的神经功能缺陷。实验证明神经干细胞移植能够促进脑出血后神经功能的恢复。目前,神经干细胞主要从胚胎干细胞诱导或是直接从发育中和成年哺乳动物的中枢神经系统中分离培养获得。但是伦理学、安全性问题以及细胞来源和数量的有限,在一定程度上都限制了神经干细胞的移植应用。因此,很有必要寻找其它能够获得神经干细胞的途径来克服这些限制。研究发现采用碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)和表皮细胞生长因子(epidermal growth factor, EGF)可直接诱导骨髓间充质干细胞(bone marrow mesenchymal stem cell, BMSC)向神经干细胞(nerual stem cell, NSC)转化。最近,脐带(Umbilical cord, UC)被发现可以作为间充质干细胞的理想来源,因此脐带MSC (UC-MSC)是否可以诱导转化为神经干细胞以及获得的神经干细胞能否促进脑出血后神经功能的恢复,很值得我们去研究。目的：探讨体外诱导人UC-MSC向神经干细胞转化的可行性；建立大鼠脑出血模型,探讨获得的神经干细胞移植后在大鼠脑内的存活、分布和分化情况以及对神经功能恢复的影响,为人UC-MSC在神经科学领域的临床应用提供理论依据和实验基础。方法：取足月妊娠剖宫产的新生儿脐带,利用酶消化法和贴壁法获得原代细胞,传4-6代后备用。在添加了bFGF、FGF8、SHH和LIF的DMEM／DF-12完全培养基中预诱导UC-MSC 6-8天,然后消化重新接种在添加了bFGF、FGF8、SHH和2%N2／B27的neurobasal media中,定向诱导大约20天后获得神经干细胞(NSC derived fromUC-MSC,UC-NSC)。一方面,通过real-time RT-PCR和免疫荧光染色分别检测mRNA和蛋白水平上Nestin、NeuroD1、Tubulin、GFAP、Galc以及Fibronectin的表达情况。另外,体外诱导UC-NSC向神经元和神经胶质细胞分化,进一步鉴定其是否具有神经干细胞的特点。另一方面,通过流式细胞学以及成骨和成脂能力来检测UC-NSC的细胞免疫表型以及向中胚层分化的能力,鉴定其是否丧失了UC-MSC的特性。此外,为了进一步研究UC-NSC的治疗潜能,我们将其移植至大鼠脑出血模型中,观察其对神经功能恢复的影响。建模后24小时,将CM-Dil标记的UC-MSC和UC-NSC移植至血肿周围。在移植后的7周每周都采用mNSS和MLPT两种方法进行一次神经功能评价。于移植后3天,通过“干湿重法”进行脑水容量分析。于移植后3天和7天,通过免疫组化染色观察IL-1β在大鼠脑内的表达情况；在移植后35天,制备脑冰冻切片,观察移植细胞在脑内的存活、分布和分化情况。此外,通过免疫荧光染色GFAP分析损伤区周围胶质细胞增殖情况,并测量了胶质瘢痕的厚度。同时,我们还进行结晶紫／速兰染色显示其损伤区域,检测了各组脑出血损伤体积的变化。结果：人UC-MSC在体外可以诱导转化为UC-NSC,并且获得的UC-NSC不仅具有神经干细胞的特点,同时也丧失了UC-MSC的特性。UC-NSC移植至大鼠脑出血模型后,能够在宿主脑内存活、迁移和分化为神经元和星形胶质细胞。与PBS对照组相比,UC-NSC移植组的脑水肿和胶质瘢痕的发生明显减少,且损伤区周围IL-1β阳性细胞也少于对照组。此外,mNSS和MLPT评分也明显优于对照组。结论：1.人UC-MSC在体外可以诱导转化为UC-NSC；2.UC-NSC可以有效地促进大鼠脑出血后神经功能的恢复。背景：脊髓损伤(spinal cord injury, SCI)是造成截瘫的主要原因,同时也是人类致残率最高的疾病之一。目前,国内外治疗SCI的方法多局限于脊柱骨脱位的复位固定和药物治疗以达到解除脊髓压迫、减轻细胞水肿和继发性损伤以及改善微循环等对症治疗的目的,但疗效不佳。细胞移植治疗SCI是近年来的研究热点。研究表明移植的细胞可在损伤部位存活、迁移且能分化为神经细胞促进神经功能的恢复。胚胎干细胞、神经干细胞、间充质干细胞、脐血干细胞以及嗅鞘细胞等均己被作为移植细胞用于脊髓损伤的神经修复治疗。特别是间充质干细胞,相对于其它细胞具有多方面的优点,因此近年来倍受研究者的关注。实验证明移植骨髓间充质干细胞能够促进大鼠脊髓损伤后神经功能的恢复。但取材困难,供体有限,易并发病毒感染以及年龄增长造成的增殖能力和多向分化能力的下降等都使骨髓间充质干细胞的临床应用受到了一定的限制。最近,作为“废弃物”的脐带(Umbilical cord,UC)被发现可以作为间充质干细胞的理想来源,相对于骨髓,从脐带中分离的MSC具有组织来源丰富、细胞原始、增殖能力强和安全无病毒感染风险等优点,因此脐带间充质干细胞(UC-MSC)是否可以成为治疗脊髓损伤的理想种子细胞,值得进我们去研究。目的：建立犬脊髓损伤模型,初步探讨人UC-MSC移植对犬脊髓损伤后神经功能恢复的影响,为细胞移植治疗寻找一种具有良好应用前景的种子细胞提供理论依据和实验基础。方法：人UC-MSC来源于足月妊娠剖宫产的健康胎儿脐带,用酶消化法和贴壁法获得原代细胞,消化传代后,取P4-P6代的细胞备用。通过流式细胞学和成骨、成脂能力来检测细胞的免疫表型和多向分化能力从而进一步鉴定UC-MSC。采用闭合液压打击法制备犬脊髓损伤模型。将实验动物随机分为两组,即UC-MSC组和对照组(PBS组)。1)UC-MSC组：脊髓损伤后1周移植1×106个UC-MSC；2)对照组：脊髓损伤后1周移植同体积的PBS。分别于模型制备后1周和UC-MSC移植后1、2、4、6、8、16、24周,采用改良Tarlov评分对动物进行行为学评分。采用SIEMENS MagnetomVision超导MRI,分别于模型制备后1周、UC-MSC移植后1周及6周进行影像学检测,动态观察损伤后的脊髓。于移植24周后处死细胞移植组和对照组的实验动物,取出损伤的脊髓组织制备石蜡切片,Luxol fast blue/cresyl violet(结晶紫／速兰)染色观察组织病理改变情况。结果：流式细胞学方法检测人UC-MSC的免疫表型,结果发现其高表达CD90、CD29、CD73和CD105；不表达造血干细胞标记CD34、CD45和内皮细胞特异性标记CD31。此外,UC-MSC表达中等水平的HLA-ABC而不表达HLA-DR,提示UC-MSC具有异体移植的可行性。在特定培养条件下,UC-MSC能够成骨和成脂,说明其具有多向分化能力。以上证实移植细胞为人UC-MSC。犬脊髓损伤后,UC-MSC移植组较对照组有明显的神经功能恢复,其改良Tarlov评分具有显著差异。MRI显示UC-MSC组细胞移植后,脊髓创伤区T2WI的高信号逐渐增多,而对照组则表现为不规则高信号环绕中心低的“环征信号”。Luxol fast blue/cresyl violet染色后发现UC-MSC移植组的脊髓填充坏死区的纤维组织较对照组明显减少,而且其周边可见散在的神经元分布,无核固缩,尼氏体染色较深。结论：人UC-MSC能够促进犬脊髓损伤后的神经功能恢复。
[Abstract]:Background: intracerebral hemorrhage (ICH) refers to bleeding caused by ruptured blood vessels in the primary parenchyma, characterized by high incidence, high mortality and Gao Zhican rate. There is still no effective treatment to improve the neural function defects of the survivors. At present, neural stem cells are mainly derived from embryonic stem cells or isolated from the central nervous system of adult and adult mammals. However, ethics, security problems, and the limited number of cell sources and numbers restrict the transplantation of neural stem cells to a certain extent. It is necessary to find other ways to obtain neural stem cells to overcome these limitations. The study found that basic fibroblast growth factor (bFGF) and epidermal growth factor (epidermal growth factor, EGF) can directly induce bone marrow mesenchymal stem cells (bone marrow mesenchymal). M cell, BMSC) convert to neural stem cells (nerual stem cell, NSC). Recently, the umbilical cord (Umbilical cord, UC) is found to be the ideal source of mesenchymal stem cells. Therefore, whether the umbilical cord MSC (UC-MSC) can be induced into neural stem cells and whether the obtained neural stem cells can promote the recovery of neural function after cerebral hemorrhage, is very valuable. We'll have to study it.
Objective: To explore the feasibility of transforming human UC-MSC into neural stem cells in vitro, to establish a rat model of cerebral hemorrhage, and to explore the survival, distribution and differentiation of neural stem cells in the rat brain after transplantation and the effect on the recovery of neural function, providing a theoretical basis and practical application for the clinical application of human UC-MSC in the field of Neurology. Methods: the umbilical cord of the neonates in the cesarean section of the full-term pregnancy was taken by enzyme digestion and adherence method to get the primary cells, and after 4-6 generations, the 6-8 days were preinduced in the DMEM / DF-12 complete medium of bFGF, FGF8, SHH and LIF, and then digested and reinoculated in the addition of bFGF, FGF8, SHH and 2%N2 / B27. NSC derived fromUC-MSC (UC-NSC) was obtained for about 20 days after orientation induction. On the one hand, real-time RT-PCR and immunofluorescence staining were used to detect Nestin, NeuroD1, Tubulin, GFAP, Galc, and the expression of Nestin, NeuroD1, Tubulin, GFAP, Galc, and glia cells in vitro. To further identify whether it has the characteristics of neural stem cells. On the other hand, through flow cytometry and osteogenesis and lipid ability to detect the cell immunophenotype of UC-NSC and the ability to differentiate into the mesoderm, identify whether it loses the characteristics of UC-MSC. In addition, in order to further study the therapeutic potential of UC-NSC, we transplant it to the cell. In the rat model of cerebral hemorrhage, the effects on the recovery of nerve function were observed. 24 hours after modeling, the CM-Dil labeled UC-MSC and UC-NSC were transplanted around the hematoma. Two methods of mNSS and MLPT were used for the evaluation of nerve function every week after 7 weeks of transplantation. The water capacity analysis was carried out by "dry wet weight" after 3 days after transplantation. The expression of IL-1 beta in the brain of rats was observed by immunohistochemical staining on 3 days and 7 days after implantation, and the brain frozen section was prepared on the 35 day after transplantation to observe the survival, distribution and differentiation of the transplanted cells in the brain. In addition, the proliferation of gelatin cells around the damaged area was analyzed by immunofluorescence staining GFAP and the thickness of the glial scar was measured. At the same time, we also carried out crystal violet / rapid blue staining to show the damage area, and detected the changes of the volume of cerebral hemorrhage in each group.
Results: human UC-MSC can be induced into UC-NSC in vitro, and the acquired UC-NSC not only has the characteristics of neural stem cells, but also loses the UC-MSC characteristics of.UC-NSC transplanted into the rat model of cerebral hemorrhage, and can survive in the host brain, migrate and differentiate into neurons and astrocytes. Compared with the PBS control group, UC-NSC transplantation The incidence of brain edema and glial scar was significantly decreased in the group, and the IL-1 beta positive cells around the injured area were also less than those in the control group. In addition, the scores of mNSS and MLPT were significantly better than those in the control group.
Conclusion: 1. human UC-MSC can be induced to transform into UC-NSC in vitro, and 2.UC-NSC can effectively promote the recovery of neurological function after intracerebral hemorrhage in rats.
Background: spinal cord injury (SCI) is the main cause of paraplegia, and is one of the most disabling diseases. At present, the treatment of SCI at home and abroad is mostly limited to the reduction and fixation of spinal dislocation and drug treatment to relieve spinal cord compression, reduce cell edema and secondary injury, and improve microcirculation. SCI is a research hotspot in recent years. Cell transplantation is a research hotspot in recent years. Studies have shown that the transplanted cells can survive in the damaged areas, migrate and can differentiate into neural cells to promote the recovery of nerve function. The neural repair therapy used as a transplanted cell for spinal cord injury, especially mesenchymal stem cells, has many advantages relative to other cells, so it has attracted more and more attention in recent years. It has been proved that transplantation of bone marrow mesenchymal stem cells can promote the recovery of nerve function after spinal cord injury in rats. Umbilical cord (UC), a "waste", has recently been found to be an ideal source of mesenchymal stem cells, relative to bone marrow, from the bone marrow. The MSC isolated from the umbilical cord has the advantages of rich tissue, primitive cells, strong proliferation and safety without the risk of virus infection. Therefore, whether umbilical cord mesenchymal stem cells (UC-MSC) can become ideal seed cells for the treatment of spinal cord injury is worthy of our study.
Objective: to establish a model of canine spinal cord injury (SCI), and to explore the effect of human UC-MSC transplantation on the recovery of nerve function after spinal cord injury in dogs, and provide theoretical and experimental basis for the treatment of a kind of seed cells with good prospects for the treatment of cell transplantation.
Methods: human UC-MSC was derived from the healthy fetal umbilical cord of the cesarean section of full term pregnancy. The original cells were obtained by enzyme digestion and adherence method. After digestion, the cells were taken for P4-P6 generation. The immunophenotype and multidirectional differentiation ability of the cells were detected by flow cytology and osteogenesis, and the ability of lipid formation was used to identify the closed hydraulic pressure of UC-MSC.. The experimental animals were randomly divided into two groups: group UC-MSC and control group (group PBS).1) UC-MSC group: 1 x 106 UC-MSC after spinal cord injury; 2) control group: 1 weeks after spinal cord injury, the same volume of PBS. was transplanted to 1 weeks after the preparation of the model and 1,2,4,6,8,16,24 weeks after the UC-MSC transplantation, and the modified Tarlov was used. SIEMENS MagnetomVision superconducting MRI, 1 weeks after the preparation of the model, 1 weeks and 6 weeks after UC-MSC transplantation, was performed to observe the spinal cord after the injury. After 24 weeks of transplantation, the cell transplantation group and the control group were killed and the injured spinal cord tissue was taken out to prepare the paraffin section, Luxo L fast blue/cresyl violet (crystal violet / rapid blue) staining was used to observe histopathological changes.
Results: flow cytometry was used to detect the immunophenotype of human UC-MSC, and it was found that it expressed high expression of CD90, CD29, CD73 and CD105, and did not express hematopoietic stem cell marker CD34, CD45 and endothelial cell specific marker CD31., UC-MSC expression of HLA-ABC but not HLA-DR, suggesting that UC-MSC has the feasibility of allograft. Under the condition of culture, UC-MSC was capable of osteogenesis and lipid formation, indicating that it had a multidirectional differentiation ability. It was confirmed that after the injury of human UC-MSC. canine spinal cord, the UC-MSC transplantation group had obvious neurological function recovery compared with the control group, and the improved Tarlov score was significantly different from that of.MRI in the UC-MSC group after cell transplantation, and the high signal by T2WI in the spinal cord wound area. In the control group, while the control group was stained with the irregular high signal circling center "ring sign".Luxol fast blue/cresyl violet, it was found that the fibrous tissue in the necrotic region of the spinal cord of the UC-MSC transplantation group was significantly lower than that of the control group, and the distribution of scattered neurons in the surrounding area was visible, and the Nissl body staining was deeper without nuclear condensation.
Conclusion: human UC-MSC can promote neurological function recovery after spinal cord injury in dogs.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【引证文献】