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Experimental Research about Effect of Cell-cell Contact to Mesenchymal Stem Cells Differentiate into Vascular Endothelial Cells

Author: PengJin
Tutor: ChiLuXiang
School: Third Military Medical University
Course: Internal Medicine
Keywords: Bone marrow mesenchymal stem cells Umbilical vein endothelial cells Cell contact Differentiation VEGF Ultrastructure
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 37
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Abstract

Background and purpose: bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) are stem cells derived from the bone marrow of the addition to the hematopoietic stem cells of another type of stem cell, stem cells commonalities that self-update and multiple differentiation potential. Early basic and clinical trials carried out in a number of cardiovascular diseases, especially in the repair and reconstruction of vascular research provides a new way for the prevention and treatment of ischemic cardiovascular disease. Angioplasty and stenting is an effective means of treatment of coronary and peripheral artery restenosis after interventional treatment of intravascular more serious problem has not been effectively resolved, including vascular endothelial damage, repair and its functional changes played a a crucial role. Human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs) with human arterial endothelial cell biological characteristics similar in the arteries features research increasingly widely used. The use of mesenchymal stem cells to endothelial cells induced differentiation characteristics angioplasty and stent implantation to repair damage endothelial cells, and thus the prevention of restenosis after vascular intervention. The method study HUVECs and MSCs coculture analog angioplasty and intravascular stent implantation environment of contact between the cells of the endothelial cells of bone marrow mesenchymal stem cell differentiation, vascular endothelial repair MSCs, prevention and treatment intravascular interventional postoperative vascular restenosis experimental basis. Methods: separation, cultivation and identification of human bone marrow MSCs and HUVECs were. MSCs and HUVECs volume at the same density (5 × 10 ~ 5/ml), etc. are uniformly mixed to build the cell contact model; Millicell Culture Plate Inserts, using a 0.4μm membrane pore size of 5 × 10 ~ 5/ml MSCs planted thereon underlayer , isodense volume HUVECs planting its lower build non-contact cell co-culture model as a control. ELISA to detect cell contact between the co-culture after 48 h of cell culture medium VEGF content (experimental group), MSCs and HUVECs individually VEGF levels in culture 48h after uniform mixing of culture medium as a control (control group). 5 days after co-culture with immunofluorescence co-culture of MSCs after induction of fetal liver kinase -1 (fetal liver kinase-1, Flk-1) and von Willebrand factor (von Willebrand factor, vWF) expression; detection contact between cells co-cultured Dil labeled acetylated low density lipoprotein (Dil-ac-LDL) phagocytosis, and observed by transmission electron microscopy after induction of MSCs ultrastructural changes. The results: 1 of MSCs was uniform long spindle growth. HUVECs were small polygonal, oval, short fusiform growth. Identified by immunofluorescence MSCs Flk-1 and vWF protein were negative expression in HUVECs Flk-1 and vWF protein were positive expression. The contact between the cells co-cultured cells were observed under an inverted phase contrast microscope visible both cell gradually fusion, cell morphology close gradually become indistinguishable. 2, VEGF levels in the experimental group (559.55 ± 66.19) pg / ml higher than that in the control group (373.98 ± 57.28) pg / ml (P lt; 0.05, n = 3). Technical appraisal by immunofluorescence laser confocal microscope the different excitation coculture DAPI labeled MSCs cell nucleus emits blue fluorescence MSCs part cell nuclear blue fluorescence expression of Flk-1 protein, laser scanning confocal microscope Showing the red fluorescence, and Flk-1 positive MSCs expressing both the green fluorescence of the vWF protein, non-contact co-culture of MSCs in the Flk-1 and vWF protein were negative. The cell contact cells co-cultured with DAPI-labeled MSCs part swallowed a red fluorescent Dil-Ac-LDL. The TEM observation cell contact between MSCs after co-culture induced ultrastructure visible the original undifferentiated MSCs nuclear cytoplasm ratio (nuclear cytoplasm ratio gt; 1.5), irregular nuclear morphology diversification notch some cells visible 2 small (nuclear cytoplasm ratio lt; 0.5) -3 nucleolus organelles in the cytoplasm is sparse and not very developed; while mature HUVECs nuclear cytoplasm ratio, nuclear morphology than the rule, prominent nucleoli, cytoplasmic organelles extremely rich; differentiation process MSCs nuclear pulp than narrow, irregular nuclei varied shapes, abundant organelles; see MSCs and HUVECs see cell membrane localized gap junctions increased electronic and visible both cell fusion phenomenon. Conclusion: 1, HUVECs were by the contact between cells co-cultured MSCs induced endothelial cell differentiation and to have swallowed Dil-ac-LDL ability. Contact system cells can induce MSCs to endothelial cell differentiation mechanism may be related to the following factors: 1), intercellular direct contact to promote cell autocrine, paracrine VEGF was significantly increased, and promote the differentiation of MSCs. 2), the presence of cell fusion participation. 3) contact of MSCs with HUVECs cells may form gap junction intercellular communication.

Experimental Research about Effect of Cell-cell Contact to Mesenchymal Stem Cells Differentiate into Vascular Endothelial Cells

Abstract

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