本文選題：骨髓間充質(zhì)干細胞　切入點：臍靜脈內(nèi)皮細胞　出處：《第三軍醫(yī)大學》2011年碩士論文

【摘要】：研究背景與目的: 骨髓間充質(zhì)干細胞(mesenchymal stem cells, MSCs)屬于成體干細胞,是來源于骨髓的除了造血干細胞外的另一類干細胞,具有干細胞的共性,即自我更新和多向分化潛能。在許多心血管疾病中進行了早期的基礎和臨床試驗,尤其是在修復和重建血管研究方面,為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術是治療冠狀動脈及外周動脈狹窄的有效手段,但是介入術后血管內(nèi)再狹窄問題較為嚴重,一直沒有得到有效解決,其中血管內(nèi)皮的損傷、修復及其功能改變起到了至關重要的作用。人臍靜脈內(nèi)皮細胞(Human umbilical vein endothelial cells,HUVECs)與人動脈內(nèi)皮細胞生物學特征相似,在動脈血管功能研究方面越來得到廣泛的應用。利用間充質(zhì)干細胞可向內(nèi)皮細胞誘導分化的特性,以修復血管成形及支架植入術后的損傷血管內(nèi)皮細胞,從而防治介入術后的血管內(nèi)再狹窄。本研究以HUVECs與MSCs間接觸共培養(yǎng)的方法模擬血管成形及支架植入術后的血管內(nèi)環(huán)境,研究細胞間接觸對骨髓間充質(zhì)干細胞分化內(nèi)皮細胞的作用,為MSCs修復損傷血管內(nèi)皮,防治介入術后血管血管內(nèi)再狹窄奠定實驗基礎。 研究方法: 1、分離、培養(yǎng)、鑒定人骨髓MSCs及HUVECs。將MSCs和HUVECs按相同密度(5×10~5/ml)等體積均勻混合構(gòu)建細胞間接觸模型;使用0.4μm膜孔徑的Millicell Culture Plate Inserts,將5×10~5/ml MSCs種植于其下層,等密度、體積的HUVECs種植于其下層,構(gòu)建細胞間非接觸共培養(yǎng)模型作為對照。 2、ELISA檢測細胞間接觸共培養(yǎng)細胞培養(yǎng)48 h后培養(yǎng)液VEGF含量(實驗組),以MSCs與HUVECs各自單獨培養(yǎng)48h后再均勻混合的培養(yǎng)液VEGF含量作為對照(對照組)。共培養(yǎng)5天后采用免疫熒光技術檢測共培養(yǎng)誘導后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)的表達;并檢測細胞間接觸共培養(yǎng)Dil標記的乙�；兔芏戎鞍�(Dil-ac-LDL)吞噬能力,并用透射電鏡觀察誘導后MSCs超微結(jié)構(gòu)的改變。 結(jié)果: 1、MSCs呈均一的長梭形生長。HUVECs呈小多角形、橢圓形、短梭狀生長。免疫熒光鑒定MSCs的Flk-1和vWF蛋白均為陰性表達,HUVECs的Flk-1和vWF蛋白均為陽性表達。細胞間接觸共培養(yǎng)細胞在倒置相差顯微鏡下觀察可見兩種細胞逐漸融合,細胞形態(tài)逐漸接近,不易分辨。 2、實驗組VEGF含量(559.55±66.19)pg/ml均高于對照組(373.98±57.28) pg/ml(P0.05,n=3)。經(jīng)免疫熒光技術鑒定,在激光共聚焦顯微鏡不同光源激發(fā)下細胞間接觸共培養(yǎng)DAPI標記的MSCs細胞核發(fā)出藍色熒光,部分細胞核發(fā)藍色熒光的MSCs顯示表達Flk-1蛋白,激光共聚焦顯微鏡下顯示呈紅色熒光,且部分Flk-1陽性MSCs開始同時表達vWF蛋白綠色熒光,非接觸共培養(yǎng)的MSCs的Flk-1和vWF蛋白均為陰性表達。細胞間接觸共培養(yǎng)的DAPI標記的MSCs部分細胞吞噬Dil-Ac-LDL后發(fā)出的紅色熒光。透射電鏡觀察細胞間接觸共培養(yǎng)誘導后MSCs超微結(jié)構(gòu)可見原始未分化的MSCs核漿比例大(核漿比1.5),核不規(guī)則,形態(tài)呈多樣化,有切跡,部分細胞可見2-3個核仁,細胞質(zhì)中的細胞器稀少且不甚發(fā)達;而成熟的HUVECs核漿比例小(核漿比0.5),核形態(tài)較規(guī)則,核仁明顯,細胞質(zhì)細胞器極為豐富;分化過程的MSCs核漿比縮小,核不規(guī)則,形態(tài)多樣,細胞器較為豐富;并見MSCs與HUVECs見細胞膜局部出現(xiàn)了電子增高的縫隙連接,并可見兩者細胞融合現(xiàn)象。 結(jié)論: 1、HUVECs可以通過細胞間接觸共培養(yǎng)方式誘導MSCs開始向內(nèi)皮細胞分化,并具有吞噬Dil-ac-LDL能力。 2、細胞間接觸體系可誘導MSCs向內(nèi)皮細胞分化的機制可能與下列因素相關: 1)、細胞間直接接觸促進細胞自分泌、旁分泌VEGF顯著增多,促進MSCs的分化。 2)、存在細胞融合參與。3)、MSCs與HUVECs細胞間接觸可能形成縫隙連接細胞間通訊。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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