本文選題：骨髓間充質(zhì)干細(xì)胞 + 分離��； 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：腦卒中引起的腦損傷是神經(jīng)外科常見的疾病，發(fā)病率和死亡率居腦血管病之首。根據(jù)原因可分為出血性腦卒中和缺血性腦卒中，無論是什么原因引起的腦損傷，神經(jīng)功能的損傷將是不可逆轉(zhuǎn)的。隨著科研和臨床研究的不斷深化和發(fā)展，抗凝治療、溶栓治療和介入治療取得了良好效果，但如何防止繼發(fā)性腦損傷的也是目前臨床面臨的難題。繼發(fā)性腦損傷腦功能的恢復(fù)有兩個(gè)關(guān)鍵因素，即血管再生與神經(jīng)細(xì)胞再生。干細(xì)胞移植和基因治療相結(jié)合，為治療腦損傷和神經(jīng)功能的恢復(fù)帶來了新的希望。 骨髓間質(zhì)干細(xì)胞（Mesenchymal stem cells，MSCs），成為近年來研究的熱點(diǎn)，由于其來源豐富，采集方便同時(shí)具有較小的免疫反應(yīng)性，目前已廣泛應(yīng)用在各個(gè)領(lǐng)域。低氧誘導(dǎo)因子1α (hypoxia-inducible factor-1α, HIF-1α）是一種重要的轉(zhuǎn)錄因子，廣泛表達(dá)于哺乳動(dòng)物體內(nèi)，參與并保持在體內(nèi)的氧平衡，在病理?xiàng)l件下對(duì)缺血缺氧反應(yīng)產(chǎn)生應(yīng)答，目前已知有70多個(gè)靶基因。在這項(xiàng)研究中，我們使用重組腺病毒HIF-1α基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞移植到局灶性腦缺血再灌注（MCAO）模型，進(jìn)一步研究HIF-1α的腦損傷保護(hù)作用。通過研究，我們發(fā)現(xiàn)，HIF-1α的可促進(jìn)下游靶基因上調(diào)，從而促進(jìn)血管新生，改善腦組織的血液供應(yīng)，減少腦組織缺血、缺氧損傷。研究同時(shí)還發(fā)現(xiàn)，HIF-1α可促進(jìn)骨髓間充質(zhì)干細(xì)胞向神經(jīng)干細(xì)胞（NSCs）分化和維持神經(jīng)干細(xì)胞的特點(diǎn)。本實(shí)驗(yàn)分為四個(gè)部分： 第一部分：骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)與鑒定 目的：通過分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞（Mesenchymal stem cells，MSCs），鑒定其生物學(xué)特性，為組織的基因治療提供實(shí)驗(yàn)依據(jù)。方法：采用密度梯度離心法分離MSCs，，倒置相差顯微鏡觀察MSCs的形態(tài)學(xué)變化；MTT測(cè)定細(xì)胞生長(zhǎng)曲線；細(xì)胞表型通過流式細(xì)胞儀進(jìn)行鑒定；利用光鏡觀察分化能力。結(jié)果：密度梯度離心法能分離出較高純度的MSCs。流式細(xì)胞儀顯示：CD29(93.2%)、CD105(92.6%)、CD45(94.68%)、CD44(94.6%)、CD73(86.4)、CD34(1.5%)、CD45(2.2%)、CD19(2.0%)、CD14(1.9%)。細(xì)胞生長(zhǎng)曲線可見細(xì)胞增殖的最高峰出現(xiàn)在第6至7d，其后細(xì)胞增殖速度迅速下降。光鏡下觀MSCs能夠在體外誘導(dǎo)分化成為成骨細(xì)胞與脂細(xì)胞。結(jié)論：采用密度梯度離心法成功的分離出高純度的MSCs是有效的方法。 第二部分重組腺病毒載體Ad. HIF-lα轉(zhuǎn)染MSCs 目的：構(gòu)建重組腺病毒Ad. HIF-lα并轉(zhuǎn)染MSCs，觀察轉(zhuǎn)染HIF-lα外源性基因及蛋白在MSCs中的表達(dá)。方法：將在目的基因HIF-lα的上游引入BamH I酶切位點(diǎn)，下游引入Xba I酶切位點(diǎn)，對(duì)質(zhì)粒pcDNA3.1/HIF-lα進(jìn)行雙酶切鑒定，測(cè)序。體外分離、培養(yǎng)MSCs，腺病毒載體與HIF-lα基因連接，轉(zhuǎn)染MSCs，用Western blot檢測(cè)HIF-lα蛋白表達(dá)。結(jié)果：雙酶切鑒定、測(cè)序和Western blot結(jié)果顯示HIF-lα基因成功轉(zhuǎn)入腺病毒載體，并且能夠在MSCs中表達(dá)。結(jié)論：成功構(gòu)建了重組腺病毒Ad. HIF-lα，并轉(zhuǎn)染到MSCs中。 第三部分：腺病毒介導(dǎo)的HIF-1α基因轉(zhuǎn)染MSCs對(duì)腦損傷后血管生成、神經(jīng)干細(xì)胞增殖分化的影響 目的：觀察低氧誘導(dǎo)因子-1α（hypoxia-inducible factor-1α, HIF-1α）基因?qū)Υ笫竽X缺血再灌注后血管生成的影響，探討HIF-1對(duì)神經(jīng)干細(xì)胞人（Neuralstem cells，NSCs)增殖與分化的作用機(jī)制。方法:建立大鼠大腦中動(dòng)脈缺血再灌注(Ischemic reperfusion injury, I/R)模型，大鼠隨機(jī)分為PBS組、MSC組和Ad-HIF-1α轉(zhuǎn)染MSC (Hy-MSC組)。分別將PBS、MSCHy-MSC定位注射到I/R模型大鼠腦缺血區(qū)，于7d、14d、21d進(jìn)行神經(jīng)功能缺失評(píng)分(Neurological deficit score,NDS)并比較。免疫組化檢測(cè)HIF-1α的表達(dá)，Westentblot檢測(cè)皮層VEGF、EPO和Ang-1的表達(dá)，TUNEL檢測(cè)細(xì)胞的調(diào)亡情況。 結(jié)果:（1）Hy-MSC組在第7、14、21d的NDS優(yōu)于MSC組PBS組（P0.05）；（2）與MSC組相比, Hy-MSC組增強(qiáng)了各時(shí)間點(diǎn)HIF-1、VEGF及EPO的表達(dá)；（3）Hy-MSC組和MSC細(xì)胞數(shù)明顯，但凋亡率下降，與PBS相比細(xì)胞調(diào)亡率(P0．05)。結(jié)論: HIF-1α基因可促進(jìn)大鼠局灶性腦缺血后血管生成，同時(shí)促進(jìn)MSCs向神經(jīng)干細(xì)胞分化，并維持NSCs的特性。 第四部分HIF-1α與Wnt信號(hào)途徑的關(guān)系以及對(duì)NSCs特性的影響 目的：觀察HIF-1α對(duì)Wnt信號(hào)途徑的相互關(guān)系，以及在MSCs向NSCs分化、增殖中作用。方法:體外培養(yǎng)大鼠NSCs以及HIF-1α轉(zhuǎn)染后的骨髓間充質(zhì)干細(xì)胞（Hy-MSC）,利用免疫共沉淀觀察兩者之間存在聯(lián)系。為進(jìn)一步證實(shí)上述結(jié)果，分別加入HIF-1α與Wnt的活化劑與抑制劑，通過Westernblot觀察下游基因血管內(nèi)皮生長(zhǎng)因子（Vascular endothelial growth factor，VEGF）、促進(jìn)細(xì)胞生成素（Erythropoietin，EPO）、淋巴增強(qiáng)因子（Lymphoidenhancer factor-1，LEF-1）、β-鏈蛋白（β-catenin）表達(dá)的變化。結(jié)果：（1）阻斷HIF-1α的表達(dá)，Wnt下游基因表達(dá)減少LEF-1（P 0.01）和β-catenin（P 0.05）。（2）阻斷Wnt的表達(dá)，對(duì)HIF-1α下游基因VEGF和EPO無明顯影響。（3）阻斷Wnt的表達(dá), HIF-1α的表達(dá)無影響。結(jié)論： HIF-1α調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞向神經(jīng)干細(xì)胞分化與增殖是通過調(diào)節(jié)Wnt信號(hào)轉(zhuǎn)導(dǎo)通路實(shí)現(xiàn)的。
[Abstract]:Brain injury caused by cerebral apoplexy is a common disease in the Department of neurosurgery. The incidence and mortality rate are the first in cerebrovascular disease. According to the causes, it can be divided into hemorrhagic stroke and ischemic stroke. No matter what cause of brain injury, the damage of nerve function will be irreversible. With the continuous deepening and development of scientific research and clinical research, Anticoagulant therapy, thrombolytic therapy and interventional therapy have achieved good results, but how to prevent secondary brain injury is also a difficult problem at present. The recovery of brain function in secondary brain injury has two key factors, that is, vascular regeneration and nerve cell regeneration. Stem cell transplantation and basic therapy are combined to treat brain injury and nerve work. The restoration of energy has brought new hope.
Mesenchymal stem cells (MSCs), which has become a hot spot in recent years, has been widely used in various fields because of its rich sources, convenient collection and small immune response. Low oxygen inducible factor 1 alpha (hypoxia-inducible factor-1 alpha (HIF-1 a)) is an important transcription factor and is widely expressed in the field. In mammals, they participate in and maintain oxygen balance in the body and respond to ischemic anoxia response under pathological conditions. There are more than 70 known target genes. In this study, we used recombinant adenovirus HIF-1 alpha gene transfection into bone marrow mesenchymal stem cells to the focal cerebral ischemia reperfusion (MCAO) model and further study HIF-1 We have found that HIF-1 alpha can promote the up regulation of the downstream target gene, thus promoting angiogenesis, improving the blood supply of brain tissue, reducing brain tissue ischemia and hypoxia injury. The study also found that HIF-1 a can promote the differentiation and maintenance of neural stem cells from bone marrow mesenchymal stem cells to neural stem cells (NSCs). The characteristics of the cell. This experiment is divided into four parts:
Part one: isolation, culture and identification of bone marrow mesenchymal stem cells
Objective: to identify the biological characteristics of bone marrow mesenchymal stem cells (Mesenchymal stem cells, MSCs), and to provide experimental basis for the gene therapy of tissue. Methods: MSCs was separated by density gradient centrifugation, and morphological changes of MSCs were observed by inverted phase contrast microscope, and cell growth curve was measured by MTT; cell phenotype passed through flow. Results: the density gradient centrifugation can separate the high purity MSCs. flow cytometer: CD29 (93.2%), CD105 (92.6%), CD45 (94.68%), CD44 (94.6%), CD73 (86.4), CD34 (1.5%), CD45 (2.2%), CD19 (2%), CD14 (1.9%). The cell growth curve can see the peak of cell proliferation peak. The cell proliferation rate decreased rapidly after sixth to 7d. MSCs could be induced to differentiate into osteoblasts and fat cells in vitro under light microscope. Conclusion: the successful separation of high purity MSCs by density gradient centrifugation is an effective method.
The second part is recombinant adenovirus vector Ad. HIF-l alpha transfected into MSCs.
Objective: to construct recombinant adenovirus Ad. HIF-l alpha and transfect MSCs, and to observe the expression of HIF-l - alpha exogenous gene and protein in MSCs. Methods: the BamH I enzyme cutting site was introduced into the upstream of HIF-l alpha of the target gene, and the Xba I enzyme cutting site was introduced into the downstream, and the plasmid pcDNA3.1/HIF-l alpha was identified by double enzyme cutting, sequenced and cultured in vitro. The virus vector was connected with the HIF-l alpha gene, transfected to MSCs and detected the expression of HIF-l alpha protein with Western blot. Results: double enzyme digestion identification, sequencing and Western blot results showed that HIF-l a gene was successfully transferred to adenovirus vector and could be expressed in MSCs. Conclusion: the recombinant adenovirus Ad. HIF-l alpha was successfully constructed and transfected into MSCs.
The third part: the effect of adenovirus mediated HIF-1 MSCs gene transfection on angiogenesis and proliferation and differentiation of neural stem cells after brain injury.
Objective: To observe the effect of hypoxia inducible factor -1 alpha (hypoxia-inducible factor-1 alpha, HIF-1 a) gene on angiogenesis after cerebral ischemia-reperfusion in rats and to explore the mechanism of HIF-1 on the proliferation and differentiation of neural stem cells (Neuralstem cells, NSCs). Methods: to establish the middle cerebral artery ischemia reperfusion (Ischemic reperfusion injury) in rats. I/R model, rats were randomly divided into PBS group, MSC group and Ad-HIF-1 alpha transfected MSC (Hy-MSC group). PBS, MSCHy-MSC were injected into the ischemic area of rat model of I/R model, and 7d, 14d, and 21d were compared. The expression of -1 and TUNEL were used to detect the apoptosis of the cells.
Results: (1) NDS in group Hy-MSC was better than group MSC in group MSC (P0.05); (2) compared with group MSC, Hy-MSC enhanced the expression of HIF-1, VEGF and EPO at all time points; (3) the number of Hy-MSC groups and cells was obvious, but apoptosis rate decreased and cell apoptosis rate was compared with those of MSC. Conclusion: the gene can promote the blood vessels of focal cerebral ischemia in rats. It also promotes the differentiation of MSCs into neural stem cells and maintains the characteristics of NSCs.
The fourth part is the relationship between HIF-1 alpha and Wnt signaling pathway and its effect on NSCs characteristics.
Objective: To observe the relationship between HIF-1 alpha and Wnt signal pathway, and to differentiate and proliferate from MSCs to NSCs. Methods: bone marrow mesenchymal stem cells (Hy-MSC) after transfection of NSCs and HIF-1 a were cultured in vitro, and the relationship between them was observed by immunoprecipitation. In order to further confirm the above results, HIF-1 A and Wnt were added to the above results. Activator and inhibitor, the expression of Vascular endothelial growth factor (VEGF) was observed by Westernblot, and the expression of cytokine (Erythropoietin, EPO), lymphatic enhancement factor (Lymphoidenhancer factor-1, LEF-1), beta chain protein (beta -catenin) was changed. Gene expression reduced LEF-1 (P 0.01) and beta -catenin (P 0.05). (2) blocked the expression of Wnt and had no obvious effect on the HIF-1 alpha downstream gene VEGF and EPO. (3) blocking the expression of Wnt, the expression of HIF-1 a was not affected. Conclusion: HIF-1 alpha modulate the differentiation and proliferation of bone marrow mesenchymal stem cells to neural stem cells by regulating Wnt signal transduction pathway. Yes.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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1 呂苗苗;HIF-lα基因修飾骨髓間充質(zhì)干細(xì)胞對(duì)腦損傷的保護(hù)作用及其機(jī)制的研究[D];第四軍醫(yī)大學(xué);2012年

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