本文選題：臍帶間充質干細胞 + 誘導轉化��； 參考：《中國協(xié)和醫(yī)科大學》2010年博士論文

【摘要】： 背景：腦出血(intracerebral hemorrhage, ICH)是指原發(fā)性腦實質內血管破裂引起的出血,具有高發(fā)病率、高死亡率和高致殘率的特點。目前臨床上仍無有效的治療方法用于改善幸存者的神經功能缺陷。實驗證明神經干細胞移植能夠促進腦出血后神經功能的恢復。目前,神經干細胞主要從胚胎干細胞誘導或是直接從發(fā)育中和成年哺乳動物的中樞神經系統(tǒng)中分離培養(yǎng)獲得。但是倫理學、安全性問題以及細胞來源和數量的有限,在一定程度上都限制了神經干細胞的移植應用。因此,很有必要尋找其它能夠獲得神經干細胞的途徑來克服這些限制。研究發(fā)現采用堿性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)和表皮細胞生長因子(epidermal growth factor, EGF)可直接誘導骨髓間充質干細胞(bone marrow mesenchymal stem cell, BMSC)向神經干細胞(nerual stem cell, NSC)轉化。最近,臍帶(Umbilical cord, UC)被發(fā)現可以作為間充質干細胞的理想來源,因此臍帶MSC (UC-MSC)是否可以誘導轉化為神經干細胞以及獲得的神經干細胞能否促進腦出血后神經功能的恢復,很值得我們去研究。 目的：探討體外誘導人UC-MSC向神經干細胞轉化的可行性；建立大鼠腦出血模型,探討獲得的神經干細胞移植后在大鼠腦內的存活、分布和分化情況以及對神經功能恢復的影響,為人UC-MSC在神經科學領域的臨床應用提供理論依據和實驗基礎。方法：取足月妊娠剖宮產的新生兒臍帶,利用酶消化法和貼壁法獲得原代細胞,傳4-6代后備用。在添加了bFGF、FGF8、SHH和LIF的DMEM／DF-12完全培養(yǎng)基中預誘導UC-MSC 6-8天,然后消化重新接種在添加了bFGF、FGF8、SHH和2%N2／B27的neurobasal media中,定向誘導大約20天后獲得神經干細胞(NSC derived fromUC-MSC,UC-NSC)。一方面,通過real-time RT-PCR和免疫熒光染色分別檢測mRNA和蛋白水平上Nestin、NeuroD1、Tubulin、GFAP、Galc以及Fibronectin的表達情況。另外,體外誘導UC-NSC向神經元和神經膠質細胞分化,進一步鑒定其是否具有神經干細胞的特點。另一方面,通過流式細胞學以及成骨和成脂能力來檢測UC-NSC的細胞免疫表型以及向中胚層分化的能力,鑒定其是否喪失了UC-MSC的特性。此外,為了進一步研究UC-NSC的治療潛能,我們將其移植至大鼠腦出血模型中,觀察其對神經功能恢復的影響。建模后24小時,將CM-Dil標記的UC-MSC和UC-NSC移植至血腫周圍。在移植后的7周每周都采用mNSS和MLPT兩種方法進行一次神經功能評價。于移植后3天,通過“干濕重法”進行腦水容量分析。于移植后3天和7天,通過免疫組化染色觀察IL-1β在大鼠腦內的表達情況；在移植后35天,制備腦冰凍切片,觀察移植細胞在腦內的存活、分布和分化情況。此外,通過免疫熒光染色GFAP分析損傷區(qū)周圍膠質細胞增殖情況,并測量了膠質瘢痕的厚度。同時,我們還進行結晶紫／速蘭染色顯示其損傷區(qū)域,檢測了各組腦出血損傷體積的變化。 結果：人UC-MSC在體外可以誘導轉化為UC-NSC,并且獲得的UC-NSC不僅具有神經干細胞的特點,同時也喪失了UC-MSC的特性。UC-NSC移植至大鼠腦出血模型后,能夠在宿主腦內存活、遷移和分化為神經元和星形膠質細胞。與PBS對照組相比,UC-NSC移植組的腦水腫和膠質瘢痕的發(fā)生明顯減少,且損傷區(qū)周圍IL-1β陽性細胞也少于對照組。此外,mNSS和MLPT評分也明顯優(yōu)于對照組。 結論：1.人UC-MSC在體外可以誘導轉化為UC-NSC；2.UC-NSC可以有效地促進大鼠腦出血后神經功能的恢復。 背景：脊髓損傷(spinal cord injury, SCI)是造成截癱的主要原因,同時也是人類致殘率最高的疾病之一。目前,國內外治療SCI的方法多局限于脊柱骨脫位的復位固定和藥物治療以達到解除脊髓壓迫、減輕細胞水腫和繼發(fā)性損傷以及改善微循環(huán)等對癥治療的目的,但療效不佳。細胞移植治療SCI是近年來的研究熱點。研究表明移植的細胞可在損傷部位存活、遷移且能分化為神經細胞促進神經功能的恢復。胚胎干細胞、神經干細胞、間充質干細胞、臍血干細胞以及嗅鞘細胞等均己被作為移植細胞用于脊髓損傷的神經修復治療。特別是間充質干細胞,相對于其它細胞具有多方面的優(yōu)點,因此近年來倍受研究者的關注。實驗證明移植骨髓間充質干細胞能夠促進大鼠脊髓損傷后神經功能的恢復。但取材困難,供體有限,易并發(fā)病毒感染以及年齡增長造成的增殖能力和多向分化能力的下降等都使骨髓間充質干細胞的臨床應用受到了一定的限制。最近,作為“廢棄物”的臍帶(Umbilical cord,UC)被發(fā)現可以作為間充質干細胞的理想來源,相對于骨髓,從臍帶中分離的MSC具有組織來源豐富、細胞原始、增殖能力強和安全無病毒感染風險等優(yōu)點,因此臍帶間充質干細胞(UC-MSC)是否可以成為治療脊髓損傷的理想種子細胞,值得進我們去研究。 目的：建立犬脊髓損傷模型,初步探討人UC-MSC移植對犬脊髓損傷后神經功能恢復的影響,為細胞移植治療尋找一種具有良好應用前景的種子細胞提供理論依據和實驗基礎。 方法：人UC-MSC來源于足月妊娠剖宮產的健康胎兒臍帶,用酶消化法和貼壁法獲得原代細胞,消化傳代后,取P4-P6代的細胞備用。通過流式細胞學和成骨、成脂能力來檢測細胞的免疫表型和多向分化能力從而進一步鑒定UC-MSC。采用閉合液壓打擊法制備犬脊髓損傷模型。將實驗動物隨機分為兩組,即UC-MSC組和對照組(PBS組)。1)UC-MSC組：脊髓損傷后1周移植1×106個UC-MSC；2)對照組：脊髓損傷后1周移植同體積的PBS。分別于模型制備后1周和UC-MSC移植后1、2、4、6、8、16、24周,采用改良Tarlov評分對動物進行行為學評分。采用SIEMENS MagnetomVision超導MRI,分別于模型制備后1周、UC-MSC移植后1周及6周進行影像學檢測,動態(tài)觀察損傷后的脊髓。于移植24周后處死細胞移植組和對照組的實驗動物,取出損傷的脊髓組織制備石蠟切片,Luxol fast blue/cresyl violet(結晶紫／速蘭)染色觀察組織病理改變情況。 結果：流式細胞學方法檢測人UC-MSC的免疫表型,結果發(fā)現其高表達CD90、CD29、CD73和CD105；不表達造血干細胞標記CD34、CD45和內皮細胞特異性標記CD31。此外,UC-MSC表達中等水平的HLA-ABC而不表達HLA-DR,提示UC-MSC具有異體移植的可行性。在特定培養(yǎng)條件下,UC-MSC能夠成骨和成脂,說明其具有多向分化能力。以上證實移植細胞為人UC-MSC。犬脊髓損傷后,UC-MSC移植組較對照組有明顯的神經功能恢復,其改良Tarlov評分具有顯著差異。MRI顯示UC-MSC組細胞移植后,脊髓創(chuàng)傷區(qū)T2WI的高信號逐漸增多,而對照組則表現為不規(guī)則高信號環(huán)繞中心低的“環(huán)征信號”。Luxol fast blue/cresyl violet染色后發(fā)現UC-MSC移植組的脊髓填充壞死區(qū)的纖維組織較對照組明顯減少,而且其周邊可見散在的神經元分布,無核固縮,尼氏體染色較深。 結論：人UC-MSC能夠促進犬脊髓損傷后的神經功能恢復。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

【引證文獻】

相關碩士學位論文 前1條

1 馮鈺珉;人臍帶間充質干細胞移植對大鼠閉合性顱腦損傷的作用研究[D];河北醫(yī)科大學;2012年

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本文編號：1972070