| Vascular disease is the world’s highest incidence of the disease, caused by trauma, atherosclerosis, vascular tumors, congenital anomalies, and the treatment of vascular graft. The vascular arterial bypass is necessary to more than100,000cases of each year in the United States, only a transplant. Therefore, the expansion of the vascular graft sources has become an important issue to people’s attention. The clinical application of vascular grafts from allogeneic vascular autologous blood vessels and artificial materials, these materials provide the conditions for the repair of vascular defects, and even extend the lives of patients, but the long-term results did not reach the desired level. Former two respectively histocompatibility self-limited sources of systemic arterial and low long-term patency of autologous vein; artificial materials such as polyester, ePTFE results in a large diameter vascular graft reliable, sure long-term patency rate, but in the small-bore (inner diameter<6mm) vascular transplantation of blood vessels in the presence of high tension, low blood flow special status after transplantation, the failure rate is very high, and more is worth noting that these artificial blood vessels did not grow and self-repair capacity. Recent years, the rapid development of tissue engineering, is a molecular biology, cell biology, bio-engineering, and clinical medicine based on the principles and methods of application of life sciences and engineering, design, construction, improvement, nurturing and maintenance living tissue to repair or rebuild the structure of tissues and organs, to maintain or improve the function of tissues and organs edge science. The use of science and technology, the vascular seed cells and scaffold materials, ideal for vascular tissue engineering is expected to build a has receptor histocompatibility good, long-term patency can maintain lumen, a self-healing capacity, long durability, plasticity strong characteristics.Tissue engineering vascular extracellular matrix as a scaffold, tissue engineering technology, vascular endothelial cells grown in the stent, and adhesion, differentiation, growth, and secretion of the ECM ultimately be used to build tissue compatibility, no immunogenicity, good durability, plasticity, and difficult to calcification, infection and thrombosis vitality vascular substitutes. Herring first reported the artificial vascular vaccination vascular endothelial cells, and the initial success obtained after a follow-up study. However, constructing tissue engineered blood vessels need the number of great seed cells, embryonic stem cells, bone marrow stem cells, adipose stem cells and other sources of stem cells are exist with certain constraints, how to make the cell’s own proliferation capacity to improve, and thus in a certain period of time to obtain more seed cells may solve vascular tissue engineering seed cells lack one of the effective way to quickly build the tissue engineered blood vessels.Although the present study, tissue engineering seed cells vascular wall cells, bone marrow mesenchymal stem cells, embryonic stem cells and endothelial progenitor cells, endothelial progenitor cells has many advantages making it the best seeds construct tissue engineered vascular cell source. Endothelial progenitor cells isolated from human umbilical cord blood culture and identification were oriented research.Part one The isolation, culture and identifying of the human umbilical cord blood endothelial progenitor cellsObjective:Isolate from fresh human umbilical cord blood endothelial progenitor cells, and its culture and identification. To adopt parcoll density gradient centrifugation isolated from umbilical cord blood endothelial progenitor cells (EPCs), the isolated cells were purified, and the use of umbilical cord blood were cultured in vitro, using flow cytometry and factor related antigen immunostaining train The cells were identified. To explore further simplified experimental procedure; how best experimental method to obtain sufficient quantities of endothelial progenitor cells, provide adequate, healthy source of endothelial progenitor cells for experimental subjects, so as to achieve the study endothelial progenitor cell transplantation in the treatment of various diseases rich cells purposes. At the same time, depth isolated human umbilical cord blood and a large number of cultured endothelial progenitor cells feasibility and clinical value.Method:Collecting the full-term cesarean producing surgical fetal cord blood in the Obstetrics and Gynecology of the First Affiliated Hospital of Bengbu Medical College, down on the The sterile conditions devolution placed in serum bottles containing50U/ml heparin, placed at4°C in the ice box to transport to the cell culture chamber. Cord blood mononuclear cells by density gradient centrifugation method. Mononuclear cells obtained is divided into two lots, fetal bovine serum (FCS-M199) M199medium and autologous serum M199medium (AS-M199) were seeded in laying someone fibronectin (HFN) and notlaying someone fibronectin dish (labeled:F (0), F (1), A (0), A (1)) for further in vitro induced differentiation as well as amplification. Observe the morphological changes of the adherent cells in the differentiation induction by immunohistochemistry, immunofluorescence and flow cytometry technology adherent cells were identified from different angles.Results:(1) when the cell culture to a week after1-2days of culture visible cord blood mononuclear cells adherent; spindle-shaped cells generally appeared three days later, after the adherent cells gradually increased, and gradually turned into a spindle; typical colony formation of spindle cells around the central round cells;10d can be observed to spindle cells, both connected to a typical line-like arrangement of the structure; cell culture to one month, interconnected cell clusters begin to connect to form network-like structure. With the increase of culture time, long spindle cells gradually shortened, and showing typical cobblestone change.(2) adherent cell culture to a week after the observation, the cells covered with fibronectin medium than unpaved fibronectin cell, but there is no significant change in the number of cells of two different medium(3) immunohistochemistry:the negative expression of CD31and vWF antigen immunohistochemistry freshly isolated mononuclear cells; mononuclear cells to14d after adherent cell CD31by FCS-M199cultured positive expression ratefor:(88.95±7.23)%; the vWF positive expression rate compared:(75.01±9.42)%. Through AS-M199culture adherent cells CD31positive expression rate was:(86.79±7.53)%, the vWF positive expression rate compared:(75.31±7.14)%.(4) Immunofluorescence staining:cell culture to7d, with the phagocytosis of DiⅠ-acLDL (+), show red fluorescence; combination of FITC-UEA-Ⅰ (+), show green fluorescence; double staining (+) show yellow fluorescence. FCS-M199adherent cells double staining positive rate (86.56±6.81)%, AS-M199of adherent cells double staining positive rate (84.47±7.25)%.(5) Flow cytometric analysis Hint:one week when measured adherent cell surface markers CD133, CD34, and KDR, respectively:(29.35±6.43)%,(52.79±8.01)%,(80.67±6.82)%; the analysis shows that in twenty days when the CD133-positive rate of0, CD34, and VEGFR2expression rate (61.48±7.65)%,(91.28±7.98)%, respectively.Conclusion:(1) mononuclear cells were isolated from umbilical cord blood by density gradient centrifugation, and then induced by factors such as VEGF, bFGF, VEGFR-2, detecting with CD133and CD34markers can obtain more purified endothelial progenitor cells.(2) By adding fibronectin promotes endothelial cell adherence. The number of adding fibronectin cell group is significantly more than the number of a group without fibronectin. (3) Autologous serum M199medium (AS-M199) can replace fetal calf serum M199medium (FCS-M199) to culture EPCs, and achieve better results.(4) CD133was highly expressed in7days, in20days rare expression, at when we believed that endothelial progenitor cells shift to mature endothelial cells. Therefore, CD133can be used as an important indicator of endothelial cells and endothelial progenitor cells differentiated. Part Two Preparation of improving freeze-thaw method de-cell stentObjective:Improved freeze-thaw de-cellular vascular stent technology, and use histological staining and electron microscopy and other methods to compare stent prepared by two different methods. Further to find a safe and effective de-cellular method, find a simple, sterilization, the low-cost, low residual method of manufacturing de-cellular vascular scaffold. Simplify experimental procedures and provide adequate, healthy source of vascular stents for vascular tissue engineering experiments.Method:Collecting the full-term cesarean producing surgical fetal cord blood in the Obstetrics and Gynecology of the First Affiliated Hospital of Bengbu Medical College, down on the The sterile conditions devolution placed in serum bottles containing50ml PBS, placed at4℃in the ice box to transport to the cell culture chamber, remove the connective tissue of the surface of the umbilical vein and vascular adventitia, after PBS solution cleaning prepare the material as the following three methods:(1) The traditional method:Place15ml plastic vials in precooling1hour, then in the refrigerator at4℃and-80℃refrigerator for2h,37℃water bath for30minutes to rewarm. Do as the above3times.(2) The modified method:Place15ml plastic vials in1hour, pre-cooling in the refrigerator at4℃, quickly immersed-196°C liquid nitrogen for20min, remove the specimens and placed in100ml sterile bottle containing50ml of sterile distilled water, placed in37°C constant temperature water bath, repeated oscillation, melted for30minutes;3times in the same method, then detecting.(3) Untreated:directly prepare for detecting.Results:(1) The general form of observation:the untreated group, milky white, pale yellow wall, good elasticity, no collapse, smooth surface and in the lumen. The traditional repeated freezing and thawing method group material is in plain colors, wall slightly collapse, smooth surface and in the lumen. Improved repeated freezing and thawing method group’s material is in plain colors, no apparent collapse of the wall, smooth surface and in the lumen. Three have no obvious appearance distinction of gross form.(2) HE staining:HE staining to observe:the cells are rich in the whole layer of the not-off arterial wall, the lumen cells arranged in a monolayer inner surface of endothelial cells, medial smooth muscle cell which is slender showed polarity arrangement. The material of vascular endothelial cells and smooth muscle cells in the traditional repeated freezing and thawing treatment was significantly deformed, broken; visible nucleus deformation, stain, fragmentation; vascular material wall layer and tube cavity surface in the improved repeating freezing and thawing treatment are rare of residual cells, wavy structures of wall fibers are preserved largely intact.(3) Electronic microscopy scanning results:untreated group endometrial complete, smooth, complete endothelial cell coverage. The traditional repeated freezing and thawing treated endothelial cells rupture, discontinuing; subendocardial collagen structure remained intact. Improved repeated freezing and thawing method vascular surface is rare of endothelial cells covering, the stent surface coarse collagen fiber bundle structure preserved, material structure dense.Conclusions:Using the modified repeated freezing and thawing de-cellular method, the stent cell component can be removed completely within a short period of time, and has no significant effect on the collagen structure of the material. Relative to repeated freezing and thawing process, improved de-cellular vascular stent repeated freezing and thawing method provides a new way of thinking and methods for the rapid and efficient preparation of future de-cellular vascular stent. However, further test needs to be done on its resistance to blood flow and long-term patency rate. |
PDF Full Text Request