本文選題：臍帶間充質(zhì)干細(xì)胞 + 誘導(dǎo)轉(zhuǎn)化��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2010年博士論文

【摘要】： 背景：腦出血(intracerebral hemorrhage, ICH)是指原發(fā)性腦實(shí)質(zhì)內(nèi)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(diǎn)。目前臨床上仍無(wú)有效的治療方法用于改善幸存者的神經(jīng)功能缺陷。實(shí)驗(yàn)證明神經(jīng)干細(xì)胞移植能夠促進(jìn)腦出血后神經(jīng)功能的恢復(fù)。目前,神經(jīng)干細(xì)胞主要從胚胎干細(xì)胞誘導(dǎo)或是直接從發(fā)育中和成年哺乳動(dòng)物的中樞神經(jīng)系統(tǒng)中分離培養(yǎng)獲得。但是倫理學(xué)、安全性問(wèn)題以及細(xì)胞來(lái)源和數(shù)量的有限,在一定程度上都限制了神經(jīng)干細(xì)胞的移植應(yīng)用。因此,很有必要尋找其它能夠獲得神經(jīng)干細(xì)胞的途徑來(lái)克服這些限制。研究發(fā)現(xiàn)采用堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor, bFGF)和表皮細(xì)胞生長(zhǎng)因子(epidermal growth factor, EGF)可直接誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cell, BMSC)向神經(jīng)干細(xì)胞(nerual stem cell, NSC)轉(zhuǎn)化。最近,臍帶(Umbilical cord, UC)被發(fā)現(xiàn)可以作為間充質(zhì)干細(xì)胞的理想來(lái)源,因此臍帶MSC (UC-MSC)是否可以誘導(dǎo)轉(zhuǎn)化為神經(jīng)干細(xì)胞以及獲得的神經(jīng)干細(xì)胞能否促進(jìn)腦出血后神經(jīng)功能的恢復(fù),很值得我們?nèi)パ芯俊?目的：探討體外誘導(dǎo)人UC-MSC向神經(jīng)干細(xì)胞轉(zhuǎn)化的可行性；建立大鼠腦出血模型,探討獲得的神經(jīng)干細(xì)胞移植后在大鼠腦內(nèi)的存活、分布和分化情況以及對(duì)神經(jīng)功能恢復(fù)的影響,為人UC-MSC在神經(jīng)科學(xué)領(lǐng)域的臨床應(yīng)用提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。方法：取足月妊娠剖宮產(chǎn)的新生兒臍帶,利用酶消化法和貼壁法獲得原代細(xì)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(yù)誘導(dǎo)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(dǎo)大約20天后獲得神經(jīng)干細(xì)胞(NSC derived fromUC-MSC,UC-NSC)。一方面,通過(guò)real-time RT-PCR和免疫熒光染色分別檢測(cè)mRNA和蛋白水平上Nestin、NeuroD1、Tubulin、GFAP、Galc以及Fibronectin的表達(dá)情況。另外,體外誘導(dǎo)UC-NSC向神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞分化,進(jìn)一步鑒定其是否具有神經(jīng)干細(xì)胞的特點(diǎn)。另一方面,通過(guò)流式細(xì)胞學(xué)以及成骨和成脂能力來(lái)檢測(cè)UC-NSC的細(xì)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(jìn)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(duì)神經(jīng)功能恢復(fù)的影響。建模后24小時(shí),將CM-Dil標(biāo)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(jìn)行一次神經(jīng)功能評(píng)價(jià)。于移植后3天,通過(guò)“干濕重法”進(jìn)行腦水容量分析。于移植后3天和7天,通過(guò)免疫組化染色觀察IL-1β在大鼠腦內(nèi)的表達(dá)情況；在移植后35天,制備腦冰凍切片,觀察移植細(xì)胞在腦內(nèi)的存活、分布和分化情況。此外,通過(guò)免疫熒光染色GFAP分析損傷區(qū)周圍膠質(zhì)細(xì)胞增殖情況,并測(cè)量了膠質(zhì)瘢痕的厚度。同時(shí),我們還進(jìn)行結(jié)晶紫／速蘭染色顯示其損傷區(qū)域,檢測(cè)了各組腦出血損傷體積的變化。 結(jié)果：人UC-MSC在體外可以誘導(dǎo)轉(zhuǎn)化為UC-NSC,并且獲得的UC-NSC不僅具有神經(jīng)干細(xì)胞的特點(diǎn),同時(shí)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(nèi)存活、遷移和分化為神經(jīng)元和星形膠質(zhì)細(xì)胞。與PBS對(duì)照組相比,UC-NSC移植組的腦水腫和膠質(zhì)瘢痕的發(fā)生明顯減少,且損傷區(qū)周圍IL-1β陽(yáng)性細(xì)胞也少于對(duì)照組。此外,mNSS和MLPT評(píng)分也明顯優(yōu)于對(duì)照組。 結(jié)論：1.人UC-MSC在體外可以誘導(dǎo)轉(zhuǎn)化為UC-NSC；2.UC-NSC可以有效地促進(jìn)大鼠腦出血后神經(jīng)功能的恢復(fù)。 背景：脊髓損傷(spinal cord injury, SCI)是造成截癱的主要原因,同時(shí)也是人類致殘率最高的疾病之一。目前,國(guó)內(nèi)外治療SCI的方法多局限于脊柱骨脫位的復(fù)位固定和藥物治療以達(dá)到解除脊髓壓迫、減輕細(xì)胞水腫和繼發(fā)性損傷以及改善微循環(huán)等對(duì)癥治療的目的,但療效不佳。細(xì)胞移植治療SCI是近年來(lái)的研究熱點(diǎn)。研究表明移植的細(xì)胞可在損傷部位存活、遷移且能分化為神經(jīng)細(xì)胞促進(jìn)神經(jīng)功能的恢復(fù)。胚胎干細(xì)胞、神經(jīng)干細(xì)胞、間充質(zhì)干細(xì)胞、臍血干細(xì)胞以及嗅鞘細(xì)胞等均己被作為移植細(xì)胞用于脊髓損傷的神經(jīng)修復(fù)治療。特別是間充質(zhì)干細(xì)胞,相對(duì)于其它細(xì)胞具有多方面的優(yōu)點(diǎn),因此近年來(lái)倍受研究者的關(guān)注。實(shí)驗(yàn)證明移植骨髓間充質(zhì)干細(xì)胞能夠促進(jìn)大鼠脊髓損傷后神經(jīng)功能的恢復(fù)。但取材困難,供體有限,易并發(fā)病毒感染以及年齡增長(zhǎng)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(zhì)干細(xì)胞的臨床應(yīng)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(xiàn)可以作為間充質(zhì)干細(xì)胞的理想來(lái)源,相對(duì)于骨髓,從臍帶中分離的MSC具有組織來(lái)源豐富、細(xì)胞原始、增殖能力強(qiáng)和安全無(wú)病毒感染風(fēng)險(xiǎn)等優(yōu)點(diǎn),因此臍帶間充質(zhì)干細(xì)胞(UC-MSC)是否可以成為治療脊髓損傷的理想種子細(xì)胞,值得進(jìn)我們?nèi)パ芯俊?目的：建立犬脊髓損傷模型,初步探討人UC-MSC移植對(duì)犬脊髓損傷后神經(jīng)功能恢復(fù)的影響,為細(xì)胞移植治療尋找一種具有良好應(yīng)用前景的種子細(xì)胞提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。 方法：人UC-MSC來(lái)源于足月妊娠剖宮產(chǎn)的健康胎兒臍帶,用酶消化法和貼壁法獲得原代細(xì)胞,消化傳代后,取P4-P6代的細(xì)胞備用。通過(guò)流式細(xì)胞學(xué)和成骨、成脂能力來(lái)檢測(cè)細(xì)胞的免疫表型和多向分化能力從而進(jìn)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(shí)驗(yàn)動(dòng)物隨機(jī)分為兩組,即UC-MSC組和對(duì)照組(PBS組)。1)UC-MSC組：脊髓損傷后1周移植1×106個(gè)UC-MSC；2)對(duì)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(píng)分對(duì)動(dòng)物進(jìn)行行為學(xué)評(píng)分。采用SIEMENS MagnetomVision超導(dǎo)MRI,分別于模型制備后1周、UC-MSC移植后1周及6周進(jìn)行影像學(xué)檢測(cè),動(dòng)態(tài)觀察損傷后的脊髓。于移植24周后處死細(xì)胞移植組和對(duì)照組的實(shí)驗(yàn)動(dòng)物,取出損傷的脊髓組織制備石蠟切片,Luxol fast blue/cresyl violet(結(jié)晶紫／速蘭)染色觀察組織病理改變情況。 結(jié)果：流式細(xì)胞學(xué)方法檢測(cè)人UC-MSC的免疫表型,結(jié)果發(fā)現(xiàn)其高表達(dá)CD90、CD29、CD73和CD105；不表達(dá)造血干細(xì)胞標(biāo)記CD34、CD45和內(nèi)皮細(xì)胞特異性標(biāo)記CD31。此外,UC-MSC表達(dá)中等水平的HLA-ABC而不表達(dá)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(shuō)明其具有多向分化能力。以上證實(shí)移植細(xì)胞為人UC-MSC。犬脊髓損傷后,UC-MSC移植組較對(duì)照組有明顯的神經(jīng)功能恢復(fù),其改良Tarlov評(píng)分具有顯著差異。MRI顯示UC-MSC組細(xì)胞移植后,脊髓創(chuàng)傷區(qū)T2WI的高信號(hào)逐漸增多,而對(duì)照組則表現(xiàn)為不規(guī)則高信號(hào)環(huán)繞中心低的“環(huán)征信號(hào)”。Luxol fast blue/cresyl violet染色后發(fā)現(xiàn)UC-MSC移植組的脊髓填充壞死區(qū)的纖維組織較對(duì)照組明顯減少,而且其周邊可見(jiàn)散在的神經(jīng)元分布,無(wú)核固縮,尼氏體染色較深。 結(jié)論：人UC-MSC能夠促進(jìn)犬脊髓損傷后的神經(jīng)功能恢復(fù)。
[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(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

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

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本文編號(hào)：1972070