细胞间接触对骨髓间充质干细胞分化为血管内皮细胞作用的实验研究

发布时间：2018-03-27 12:18

  本文选题：骨髓间充质干细胞　切入点：脐静脉内皮细胞　出处：《第三军医大学》2011年硕士论文

【摘要】：研究背景与目的: 骨髓间充质干细胞(mesenchymal stem cells, MSCs)属于成体干细胞,是来源于骨髓的除了造血干细胞外的另一类干细胞,具有干细胞的共性,即自我更新和多向分化潜能。在许多心血管疾病中进行了早期的基础和临床试验,尤其是在修复和重建血管研究方面,为防治缺血性心血管疾病提供了新的方式。血管成形及支架植入术是治疗冠状动脉及外周动脉狭窄的有效手段,但是介入术后血管内再狭窄问题较为严重,一直没有得到有效解决,其中血管内皮的损伤、修复及其功能改变起到了至关重要的作用。人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)与人动脉内皮细胞生物学特征相似,在动脉血管功能研究方面越来得到广泛的应用。利用间充质干细胞可向内皮细胞诱导分化的特性,以修复血管成形及支架植入术后的损伤血管内皮细胞,从而防治介入术后的血管内再狭窄。本研究以HUVECs与MSCs间接触共培养的方法模拟血管成形及支架植入术后的血管内环境,研究细胞间接触对骨髓间充质干细胞分化内皮细胞的作用,为MSCs修复损伤血管内皮,防治介入术后血管血管内再狭窄奠定实验基础。 研究方法: 1、分离、培养、鉴定人骨髓MSCs及HUVECs。将MSCs和HUVECs按相同密度(5×10~5/ml)等体积均匀混合构建细胞间接触模型;使用0.4μm膜孔径的Millicell Culture Plate Inserts,将5×10~5/ml MSCs种植于其下层,等密度、体积的HUVECs种植于其下层,构建细胞间非接触共培养模型作为对照。 2、ELISA检测细胞间接触共培养细胞培养48 h后培养液VEGF含量(实验组),以MSCs与HUVECs各自单独培养48h后再均匀混合的培养液VEGF含量作为对照(对照组)。共培养5天后采用免疫荧光技术检测共培养诱导后MSCs的胎肝激酶-1 ( fetal liver kinase-1, Flk-1)及血管性血友病因子( von Willebrand factor, vWF)的表达;并检测细胞间接触共培养Dil标记的乙酰化低密度脂蛋白(Dil-ac-LDL)吞噬能力,并用透射电镜观察诱导后MSCs超微结构的改变。 结果: 1、MSCs呈均一的长梭形生长。HUVECs呈小多角形、椭圆形、短梭状生长。免疫荧光鉴定MSCs的Flk-1和vWF蛋白均为阴性表达,HUVECs的Flk-1和vWF蛋白均为阳性表达。细胞间接触共培养细胞在倒置相差显微镜下观察可见两种细胞逐渐融合,细胞形态逐渐接近,不易分辨。 2、实验组VEGF含量(559.55±66.19)pg/ml均高于对照组(373.98±57.28) pg/ml(P0.05,n=3)。经免疫荧光技术鉴定,在激光共聚焦显微镜不同光源激发下细胞间接触共培养DAPI标记的MSCs细胞核发出蓝色荧光,部分细胞核发蓝色荧光的MSCs显示表达Flk-1蛋白,激光共聚焦显微镜下显示呈红色荧光,且部分Flk-1阳性MSCs开始同时表达vWF蛋白绿色荧光,非接触共培养的MSCs的Flk-1和vWF蛋白均为阴性表达。细胞间接触共培养的DAPI标记的MSCs部分细胞吞噬Dil-Ac-LDL后发出的红色荧光。透射电镜观察细胞间接触共培养诱导后MSCs超微结构可见原始未分化的MSCs核浆比例大(核浆比1.5),核不规则,形态呈多样化,有切迹,部分细胞可见2-3个核仁,细胞质中的细胞器稀少且不甚发达;而成熟的HUVECs核浆比例小(核浆比0.5),核形态较规则,核仁明显,细胞质细胞器极为丰富;分化过程的MSCs核浆比缩小,核不规则,形态多样,细胞器较为丰富;并见MSCs与HUVECs见细胞膜局部出现了电子增高的缝隙连接,并可见两者细胞融合现象。 结论: 1、HUVECs可以通过细胞间接触共培养方式诱导MSCs开始向内皮细胞分化,并具有吞噬Dil-ac-LDL能力。 2、细胞间接触体系可诱导MSCs向内皮细胞分化的机制可能与下列因素相关: 1)、细胞间直接接触促进细胞自分泌、旁分泌VEGF显著增多,促进MSCs的分化。 2)、存在细胞融合参与。3)、MSCs与HUVECs细胞间接触可能形成缝隙连接细胞间通讯。
[Abstract]:Research background and purpose:
Bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) belongs to adult stem cells are derived from bone marrow in addition to another kind of stem cells to hematopoietic stem cells, stem cells have in common, namely the self-renewal and differentiation potential. And early clinical trials in the foundation of many cardiovascular diseases, especially in the repair and reconstruction of vessels, provides a new way for the prevention and treatment of ischemic cardiovascular disease. Angioplasty and stent implantation is an effective method for treatment of coronary artery and peripheral artery stenosis, but vascular restenosis after interventional treatment is a serious problem that has not been effectively resolved, including vascular endothelial injury. Change and repair function has played a crucial role. Human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs) and the biological characteristics similar to human arterial endothelial cells, Has been widely used in the study of arterial function. Using the characteristic of MSCs can differentiate into endothelial cells in vitro, with vascular endothelial cell injury repair angioplasty and stent implantation, thus preventing after interventional intravascular restenosis. Simulation of angioplasty and stent implantation after endovascular the environment method used in this study is the HUVECs and MSCs contact Co culture, contact on the differentiation of bone marrow mesenchymal stem cells endothelial cells, repairing damaged vascular endothelial MSCs, prevention and treatment of postoperative vascular interventional endovascular restenosis experimental basis.
Research methods:
1, separation, culture, identification of human bone marrow MSCs and HUVECs. of MSCs and HUVECs by the same density (5 * 10~5/ml) volume mixing construction model of contact between the cells; the use of aperture 0.4 m film Millicell Culture Plate Inserts, 5 * 10~5/ml MSCs grown on its lower density, the volume of HUVECs in plant the lower construction cell co culture model was used as control.
2, cell culture medium VEGF content after 48 h of co culture ELISA detection cell (experimental group), MSCs and HUVECs respectively after 48h culture, VEGF content of the liquid culture and mixed evenly as control (control group). After 5 days by immunofluorescence detection of co cultured MSCs after the induction of fetal liver kinase -1 Co Culture (fetal liver kinase-1, Flk-1) and von Willebrand factor (von Willebrand, factor, vWF) expression; and detection of cell-cell contact co cultured Dil labeled acetylated low density lipoprotein (Dil-ac-LDL) phagocytosis, and electron microscope after MSCs induced ultrastructural changes.
Result:
1, MSCs showed spindle shaped uniform growth of.HUVECs showed a small polygonal, oval shaped and short spindle growth. Immunofluorescence identification of MSCs Flk-1 and vWF protein were negative expression of HUVECs, Flk-1 and vWF protein were positive. There were two types of cells gradually fused cells observed under the inverted phase contrast microscope cell co culture between the cells gradually close, is not easy to distinguish.
2, the experimental group content of VEGF (559.55 + 66.19) pg/ml were higher than the control group (373.98 + 57.28) pg/ml (P0.05, n=3). The identification of immunofluorescence, stimulate co cultured DAPI labeled MSCs nuclei emit blue fluorescence under the contact between the cells by laser confocal microscopy of different light sources, some cells issued blue fluorescence MSCs the expression of Flk-1 protein by confocal laser scanning microscope showed red fluorescence, and Flk-1 positive expression of vWF protein and MSCs green fluorescence, non-contact co culture of MSCs Flk-1 and vWF protein expression was negative. Red fluorescence labeled DAPI cells were co cultured with contact MSCs phagocytosis after Dil-Ac-LDL. TEM observation of cell-cell contact Co culture induced MSCs ultrastructure changes of primitive undifferentiated MSCs high karyoplasmic ratio (karyoplasmic ratio 1.5), irregular nucleus, morphology was diverse, notch, fine There are 2-3 cell nucleoli, organelles were scarce and not very rich; and mature HUVECs karyoplasmic ratio small (karyoplasmic ratio 0.5), nuclear morphology was regular, obvious nucleolus, cytoplasm is rich; the differentiation process of MSCs reduced karyoplasmic ratio, irregular nucleus, morphological diversity, abundant organelles see MSCs and HUVECs; and cell membrane localized gap increased electronic connection, and it can be seen that both the cell fusion.
Conclusion:
1, HUVECs can induce MSCs to differentiate into endothelial cells through intercellular contact co culture and have the ability to phagocyt Dil-ac-LDL.
2, the mechanism of intercellular contact system to induce MSCs to differentiate into endothelial cells may be related to the following factors:
1), direct contact between cells promotes cell autocrine, and paracrine VEGF increases significantly, promoting the differentiation of MSCs.
2), cell fusion is involved in.3), and the intercellular communication between MSCs and HUVECs cells may form gap junctional intercellular communication.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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