脂肪来源的间充质干细胞体外转分化内皮细胞及钙调蛋白拮抗剂W7调控研究

发布时间：2018-08-07 16:46

【摘要】：研究背景：通过组织工程技术实现缺损组织或器官的修复与替代具有重要的临床意义。而理想的种子细胞是组织工程的基础。对于缺血性疾病而言,利用干细胞的多向分化潜能进行血管重构来改善局部缺血的治疗模式,是近年来许多学者关注的重点。作为候选细胞的内皮祖细胞,骨髓来源间充质干细胞,卫星细胞等,由于取材困难、获取率低、分化能力和细胞数量随体外传代培养逐渐下降等应用受限,此外更受到伦理学的质疑。脂肪来源的间充质干细胞(human adipose-derived mesenchymal stem cells, hADSCs)是从成体脂肪组织中分离出来的一类成体干细胞,具有和骨髓来源的基质干细胞相似的生物学特性和多系分化潜能,并因其不侵犯相关伦理及取材方面的优势,有望成为种子细胞的热门选择。为此,建立合理的hADSCs体外培养体系、向内皮细胞的诱导分化、以及适当载体的寻找都亟待深入研究。研究目的：第一部分研究：在无血清培养条件下,将人脂肪来源的间充质干细胞(hADSCs)分化为内皮细胞,为血管组织工程化种子细胞的来源和血管新生模型的建立提供基础。第二部分研究：以(6-氨乙基)-5-氯-1-萘磺酰胺(W7)研究钙调蛋白拮抗剂对人脂肪间充质干细胞(hADSCs)向内皮细胞分化过程中细胞表型、细胞内游离钙离子浓度变化、成血管功能以及ERK/MAPK信号通路的作用。第三部分研究：研究人脂肪来源的间充质干细胞与新型可降解多孔生物材料---聚乳酸-聚乙二醇共聚物(Poly(lactic acid)-poly(ethylene glycol), PLA-PEG)的相容性,及hADSCs在材料上转分化为内皮细胞(endothelial cells, ECs)和血管形成的能力。研究方法：第一部分：从健康人体抽脂术获得的脂肪进行hADSCs原代培养,用流式细胞术检测CD45、CD44、CD14、CD29及CD105,并分化至成骨和脂肪细胞,以免疫组化方法检测其干细胞的分化能力；然后用含40ng/ml VEGF和lOng/ml bFGF的无血清分化培养基诱导hADSCs分化为内皮细胞,在第15、30、50d通过流式细胞术检测vWF和VE-Cadherin两种特异性抗原表达,并用透射电镜检测WP小体以及成血管实验来检测是否分化为内皮细胞。第二部分：以含40ng/ml VEGF和lOng/mlbFGF的干细胞无血清分化培养基培养P3 hADSCs,将实验分为空白分化对照组(不含W7的分化培养基),A23187阳性对照组(含50μmol/L钙离子载体A23187的分化培养基)、高(30μmol/L)、中(20μmol/L)、低(10μmol/L)三个不同W7剂量的实验组,及预先将hADSCs用ERK抑制剂V0126 (40μmol/L)处理24h的高(30μmol/L)、中(20μmol/L)、低(10μmol/L)三个不同W7剂量的对照组。hADSCs加药8d后,用流式细胞术检测其vWF和VE-Cadherin表型变化,以激光共聚焦显微镜检测Fluo-3标记的细胞胞质内游离钙离子浓度变化。同时将未加抑制剂只用药物处理24h的细胞及加入抑制剂干预24h后用药物处理24h的细胞种植至Matrigel胶上,观察细胞成血管能力。利用Western blot技术分析不同浓度药物处理8d后细胞ERK和p-ERK变化。第三部分：选取孔径为60-80μm和180-200μm的PEG-PLA材料,并计算不同孔径该材料14d内的吸水率和降解率。利用本室已建立的人脂肪间充质干细胞的培养方法,将hADSCs种植在不同孔径的材料上,计算接种率。利用DAPI免疫荧光标记及扫描电镜(scanning electron microscope, SEM).观察hADSCs在材料上的生长及分布情况,并采用CCK-8法绘制hADSCs在材料上的增殖曲线。用含40ng/mlVEGF和lOng/mlbFGF的分化培养基处理材料上的hADSCs,50d后利用流式细胞术检测vWF和VE-Cadherin及免疫荧光检测F1k-1观察hADSCs在材料上分化为ECs的能力。研究结果：在第一部分中,分离得到的细胞流式细胞术分析结果为,CD45(-)CD14(-)CD44(+)CD29(+)CD105(+),表明该细胞为人脂肪来源的间充质干细胞,并且可分化成骨成脂细胞。随着分化时间的延长,vWF和VE-Cadherin的表达不断增加,并且可在电镜下观察到WP小体的出现以及在光镜下观察到分化50d的hADSCs具有成血管的能力。第二部分中,分化至8d时,与空白分化对照组相比,随着W7浓度的降低,hADSCs转分化后的细胞VE-Cadherin和vWF的表达量显著上升(P0.01),胞质内游离的钙离子浓度升高(P0.01)。药物作用24h后,药物干预组的培养细胞均有管腔样血管结构形成,空白分化对照组的细胞无血管管型形成。与空白分化对照组相比,不同药物干预组细胞ERK表达水平没有显著性改变(P0.05),而随着W7浓度的降低p-ERK表达水平明显升高(P0.01)。在第三部分中,利用已知文献的方法观察孔径分别为60-80μm和180-200μm的PEG-PLA材料14d的吸水率分别为372.73%0和245.31%,降解率为9.09%和6.25%。种植hADSCs后,SEM下观察细胞呈长梭形依附于多孔材料表面。比较不同孔径的材料对细胞的影响发现,60-80μm孔径的材料细胞接种率为99.00±0.71%,大于180-200μm孔径材料上的细胞接种率92.00±1.22%；但生长曲线却显示在180-200μm孔径材料上细胞具有更快的增殖速度。选择180-200μm孔径的材料进行EC分化实验发现,分化50d后,FCM检测vWF和VE-cadherin的阳性率分别为78.5±1.50和83.3±2.00,免疫荧光化学检测Flk-1显示大多数细胞表面均表达Flk-1。研究结论：成功从人体脂肪中分离得到间充质干细胞,并且在无血清条件下可将其分化为内皮细胞。适当浓度的钙离子拮抗剂W7可以显著促进hADSCs向内皮细胞分化,其机制为通过促进细胞内游离钙离子浓度的增加,激活细胞分化过程中的ERK/MAPK信号通路。hADSCs较适合于在孔径大小为180-200μm PEG-PLA材料上生长和增殖,并可在材料上有效分化为内皮细胞。下一步待研究工作：增加W7 10μmol/L以下浓度及30μmol/L以上浓度的多个剂量组,观察其对hADSCs转分化内皮的影响,进一步确定W7的最适剂量。尝试调节PLA-PEG共聚段中二者的共聚比或共聚物的分子量大小,以争取获得最优材料。设计动物体内试验,将该材料及细胞应用于缺血模型,从而观察其在体内的各项结果参数是否适合临床需求。
[Abstract]:Background: the reconstruction and replacement of tissue or organs by tissue engineering has important clinical significance. The ideal seed cells are the basis of tissue engineering. For ischemic disease, vascular remodeling by using the multidirectional differentiation potential of stem cells to improve the therapeutic pattern of ischemia is a lot of recent years. As a candidate cell, endothelial progenitor cells, bone marrow derived mesenchymal stem cells, satellite cells, and so on, are limited due to the difficulty in obtaining material, low acquisition rate, the differentiation ability and the number of cells decreasing with the gradual decline of the external transmission culture, in addition to the ethical question. Mesenchymal stem cells derived from fat (human adipose-der) Ived mesenchymal stem cells (hADSCs), a class of adult stem cells isolated from adult adipose tissue, has a similar biological and multi lineage differentiation potential with bone marrow derived stromal cells, and is expected to be a hot choice for seed cells because of its non invasion of ethical and material advantages. The induction of hADSCs in vitro, the differentiation into endothelial cells, and the search for suitable vectors are urgently needed to be further studied.
The first part of the study: the first part of the study: to differentiate human adipose derived mesenchymal stem cells (hADSCs) into endothelial cells in serum-free culture, and provide the basis for the origin of vascular tissue engineering seed cells and the establishment of a angiogenesis model. The second part studies the study of calcic eggs with (6- amethyl) -5- chloride -1- naphthalamyl sulfonamide (W7) The effect of white antagonists on the cell phenotype, intracellular free calcium ion concentration, vascular function and ERK/MAPK signaling pathway in human adipose mesenchymal stem cells (hADSCs) differentiation into endothelial cells. Third part study: the study of human adipose derived mesenchymal stem cells and new biodegradable porous biomaterials - polylactic acid poly B The compatibility of the diol copolymer (Poly (lactic acid) -poly (ethylene glycol), PLA-PEG), and the conversion of hADSCs on the material into endothelial cells (endothelial cells, ECs) and angiogenesis.
Research methods: the first part: hADSCs primary culture obtained from healthy human liposuction, CD45, CD44, CD14, CD29 and CD105 were detected by flow cytometry and differentiated into osteoblasts and adipocytes, and the differentiation ability of stem cells was detected by immunohistochemical method; then the serum free differentiation culture containing 40ng/ml VEGF and lOng/ml bFGF was used. The culture medium induced hADSCs to differentiate into endothelial cells. Two specific antigens of vWF and VE-Cadherin were detected by flow cytometry in 15,30,50d. The WP corpuscles and vascular experiments were used to detect the differentiation into endothelial cells by transmission electron microscopy. The second part was serum-free differentiation and culture of the stem cells containing 40ng/ml VEGF and lOng/mlbFGF. The base culture of P3 hADSCs was divided into blank differentiation control group (without W7 differentiation medium), A23187 positive control group (including 50 micron mol/L calcium carrier A23187 differentiation medium), high (30 mu mol/L), medium (20 mu mol/L), low (10 micron), three different W7 dosages, and hADSCs ERK inhibitor (40 mu) pretreatment High (30 u mol/L), medium (20 mol/L), and low (10 u mol/L) three different W7 doses of.HADSCs added 8D, the phenotypic changes of vWF and VE-Cadherin were detected by flow cytometry. The change of intracellular free calcium concentration in the cytoplasm of the Fluo-3 labeled cells was detected by laser confocal microscopy. And after adding inhibitor to 24h, the cells of 24h were grown on Matrigel glue to observe the vascular capacity of the cells. The Western blot technique was used to analyze the changes of ERK and p-ERK in the cells after 8D treatment with different concentrations. The third part: select the PEG-PLA material with a pore size of 60-80 u m and 180-200 micron m. Water absorption and degradation rate. Using the culture method of human adipose mesenchymal stem cells established in this room, hADSCs was planted on different pore size materials to calculate the inoculation rate. The growth and distribution of hADSCs on the material were observed by DAPI immunofluorescent labeling and scanning electron microscopy (scanning electron microscope, SEM), and the CCK-8 method was used. The proliferation curve of hADSCs on material. HADSCs on the material was treated with 40ng/mlVEGF and lOng/mlbFGF differentiation medium. 50D was used to detect vWF and VE-Cadherin and immunofluorescence detection F1k-1 by flow cytometry to observe the ability of hADSCs to differentiate into ECs on the material.
Results: in the first part, the results of cell flow cytometry, CD45 (-) CD14 (-) CD44 (+) CD29 (+) CD105 (+), indicate that the cells are mesenchymal stem cells derived from human fat and can differentiate into osteoblast. With the extension of differentiation, the expression of vWF and VE-Cadherin is increasing and can be used in electron microscopy. The appearance of WP body and the ability of hADSCs to differentiate 50D under light microscopy were observed under the light microscope. In the second part, the expression of VE-Cadherin and vWF in the cells after hADSCs transdifferentiation was significantly increased (P0.01), and the concentration of free calcium ions in the cytoplasm was raised as compared with the blank differentiation control group. High (P0.01). After the drug action of 24h, the cultured cells in the drug intervention group had a lumen like vascular structure, and the cells in the blank differentiation control group had no vascular type formation. Compared with the blank differentiation control group, the expression level of ERK was not significantly changed in the different drug intervention groups (P0.05), but with the decrease of W7 concentration, the expression level of p-ERK was obvious. Increase (P0.01). In the third part, the water absorption of PEG-PLA material 14d with the pore size of 60-80 m and 180-200 micron m, respectively, was 372.73%0 and 245.31%, the degradation rate was 9.09% and 6.25%. planted hADSCs, and the cells were observed on the surface of the porous material with a long shuttle shape under SEM, and the cells with different pore sizes were compared to the cells. The results showed that the inoculation rate of the material cells with 60-80 m aperture was 99 + 0.71%, and the cell inoculation rate on the material larger than 180-200 mu m was 92 + 1.22%, but the growth curve showed that the cells had faster proliferation rate on the 180-200 micron m pore material. The material selected for the aperture of 180-200 mu m was found by EC differentiation experiment, and after the differentiation of 50D, the FCM examination was found. The positive rates of vWF and VE-cadherin were 78.5 (+ 1.50) and 83.3 (+ 2.00) respectively. Flk-1 was detected by immunofluorescence.
It is concluded that mesenchymal stem cells can be isolated from human body fat and differentiate into endothelial cells in serum-free conditions. The appropriate concentration of calcium antagonist W7 can significantly promote the differentiation of hADSCs into endothelial cells. The mechanism is to activate the cell differentiation process by promoting the increase of intracellular free calcium concentration. The ERK/MAPK signal pathway.HADSCs is more suitable for growth and proliferation on the size of 180-200 micron m PEG-PLA, and can effectively differentiate into endothelial cells on the material.
The next step to study: to increase the concentration of W7 10 mu below mol/L and more than 30 mol/L concentration, observe its effect on the hADSCs transdifferentiation endothelium and further determine the optimum dosage of W7. Try to adjust the copolymerization ratio or the molecular weight of the copolymer in the PLA-PEG copolymerization segment, in order to obtain the optimal material. In the internal test, the material and cells were applied to the ischemic model to observe whether the parameters of the results in vivo were suitable for clinical needs.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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