Dissertation > Excellent graduate degree dissertation topics show

Adipose-derived mesenchymal stem cells in vitro transdifferentiation of endothelial cells and calmodulin antagonist W7 Regulation

Author: DiYuJia
Tutor: ZuoHongGang
School: Beijing Union Medical College
Course: Biomedical Engineering
Keywords: Adipose-derived mesenchymal stem cells Calmodulin antagonist Endothelial Cells Differentiation Polyethylene glycol - polylactic acid copolymer
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 50
Quote: 0
Read: Download Dissertation

Abstract

Background: defect repair and replacement of tissues or organs by tissue engineering technology has important clinical significance. Ideal seed cells for tissue engineering. For ischemic disease, pluripotent stem cells to improve the mode of treatment of ischemic vascular remodeling, is the focus of attention of many scholars in recent years. As a candidate cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, satellite cells, differentiation capacity, and the number of cells with the In vitro culture gradual decline in applications such as limited coverage difficult to obtain low, in addition to more questioned by the Ethics . Between adipose-derived mesenchymal stem cells (human adipose-derived mesenchymal stem cells, hADSCs) is from adult adipose tissue separation out of a class of adult stem cells, and bone marrow-derived mesenchymal stem cells similar biological characteristics and multi-lineage differentiation potential has and as it does not infringe the relevant ethical and drawn advantages, is expected to become a popular choice of seed cells. To this end, the establishment of a reasonable hADSCs vitro culture system, in-depth studies are urgently looking to endothelial cells induced to differentiate, as well as an appropriate carrier. Objective: The first part of the study: in serum-free culture conditions, the differentiation of human adipose-derived mesenchymal stem cells (hADSCs) to endothelial cells, to provide a basis for the establishment of vascular tissue engineering seed cells source and angiogenesis model. The second part of the study: (6 - aminoethyl) -5 - chloro-1 - naphthalenesulfonamide (W7) calmodulin antagonist table cells to endothelial cell differentiation of human adipose-derived mesenchymal stem cells (hADSCs) type, intracellular free calcium concentration changes into a blood vessel function and ERK / MAPK signal pathway role. The third part of the study: between human adipose-derived mesenchymal stem cells and new biodegradable porous biomaterials --- polylactic acid - polyethylene glycol copolymer (poly (lactic acid)-poly (ethylene glycol), PLA-PEG) compatibility, and the material on hADSCs transdifferentiation into endothelial cells (endothelial cells, ECs) and the ability of angiogenesis. Methods: Part 1: to hADSCs obtained from healthy human liposuction fat in primary culture, CD45, CD44, CD14, CD29 and CD105 detected by flow cytometry, and differentiation to osteoblasts and adipocytes by immunohistochemical was used to detect stem cells; and then serum-free differentiation medium containing 40ng/ml VEGF and long / ml bFGF induced hADSCs differentiate into endothelial cells, 15,30,50 d detected by flow cytometry vWF and VE- Cadherin specific antigen expression and detected using transmission electron microscopy the WP bodies into vascular experiment to detect whether or not to differentiate into endothelial cells. Part II: with 40ng/ml VEGF and long / mlbFGF, stem cells serum-free differentiation medium P3 hADSCs experiment is divided into blank the differentiation control group (excluding W7 differentiation medium), A23187 positive control group (containing 50 micromol / L calcium ionophore A23187 differentiation medium), and high (30 micromol / L) (20 micromol / L), low three different W7 doses of the experimental group (10μmol / L), and pre hADSCs the ERK inhibitor V0126 Processing (40μmol / L) the 24h high (30 micromol / L) (20μmol / L), low three different W7 doses of the control group (10μmol / L). after hADSCs dosing 8d, of vWF and VE-cadherin phenotypic changes detected by flow cytometry, laser scanning confocal microscope Fluo-3 labeled cytoplasmic free calcium concentration changes. At the same time is not an inhibitor only treated with drugs 24h cells and added to the the inhibitor intervention 24 hours after drug treatment 24 hours the cells were seeded into the Matrigel gel, cells were observed to vascular capacity. The Western blot technical analysis of different concentrations of drugs for 8d cell ERK and p-ERK change. Part III: Select a pore size of 60-80μm and 180, 200, PEG-PLA materials, and calculate the water absorption and degradation rate of different pore size of the material within 14 days. This room has been established human adipose mesenchymal stem cell culture method, the hADSCs planting material of different pore sizes, the vaccination rate was calculated. DAPI immune fluorescence labeling and scanning electron microscopy (scanning electron microscope, SEM). Observed hADSCs in the growth and distribution of materials and the growth curve of CCK-8 method to draw hADSCs in the material. Differentiation medium containing 40ng/mlVEGF and long / mlbFGF, processing materials hADSCs 50d flow cytometry vWF and VE-cadherin and immunofluorescence detection Flk-1 observed hADSCs of of material ability to differentiate into ECs. Results: In the first part, the separation of the cells obtained by flow cytometry analysis for CD45 (-) CD14 (-) CD44 () CD29 () CD105 () indicates that the cells for human adipose-derived mesenchymal stem cells, and adipogenic cells can differentiate into bone. With the differentiation of the extension of time, increasing vWF and VE-cadherin expression, and to WP bodies appear at the electron microscope and observed under light microscope to the differentiation 50d hADSCs vascular capacity. Second differentiation to 8d, compared with blank differentiation control group, with decreasing W7 concentration hADSCs transdifferentiation of cells VE-cadherin and vWF expression levels increased significantly (P lt; 0.01), cytoplasmic intracellular free calcium concentration (P lt; 0.01). 24 hours after drug effects, drug intervention group cultured cells have luminal-like vascular structure formation, vascular tube formation blank differentiation of cells of the control group. Compared with the blank differentiation control group, cell ERK expression levels of different drug intervention group there was no significant change (P gt; 0.05) was significantly higher (P lt; 0.01) With W7 reduce the concentration of p-ERK expression levels. In the third part, the use of the known literature viewing aperture were 60-80μm and 180-200μm 14d water absorption of PEG-PLA materials were 372.73% and 245.31%, the degradation rate of 9.09% and 6.25%. The planting hADSCs SEM observed under the fusiform cells attached to the surface of the porous material. Compare different pore material effect on the cell found ,60-80μm aperture material cell coverage was 99.00 ± 0.71%, 92.00 ± 1.22% greater than the rate of the cells were seeded on 180-200μm aperture material; growth curve at 180 - 200μm aperture on the proliferation of cells having a faster speed. 180-200μm aperture materials for EC differentiation was found that differentiation after 50 days, FCM detection of vWF and VE-cadherin-positive rate to 78.5 ± 1.50 and 83.3 ± 2.00, immunofluorescence detection Flk-1 shows the surface of most cells Expression of Flk-1. The conclusion of the study: successfully isolated from human fat-derived mesenchymal stem cells under serum-free conditions can be differentiated into endothelial cells. Appropriate concentration of calcium antagonist W7 can significantly promote the hADSCs to the endothelial cells, the mechanism by promoting the increase of intracellular free calcium concentration, the activated cell differentiation in the ERK / MAPK signal pathway. the more suited hADSCs a pore size of 180-200μm PEG-PLA materials on the growth and proliferation effectively differentiate into endothelial cells can be used in the materials. Next to be studied: increased In W7 10μmol / L concentration and more than 30 micromol / L concentration of multiple-dose group, observe the endothelial of hADSCs transdifferentiation, to further define the optimal dose of W7. Try adjusting both segments of the PLA-PEG copolymer copolymerization ratio or size of the molecular weight of the copolymer to Be to obtain the optimal material. Design animal in vivo tests, the material and cell used in the ischemic model, thereby observe whether the results of in vivo parameters suitable for clinical needs.

Adipose-derived mesenchymal stem cells in vitro transdifferentiation of endothelial cells and calmodulin antagonist W7 Regulation

Abstract

Related Dissertations