| Trauma can cause severe damage of the target cells. Infectious sepsis andnoninfectious systemic inflammatory response syndrome (SIRS) can interacteachother which will cause the dysfunction of self-repairing. What is more, the injuryof blood capillary and microcirculation disturbance caused by dysfunction ofself-repairing can result in multiple organ dysfunctions, which is deemed to be thebeginning of the multiple organ dysfunction syndromes (MODS). Nowadays MODSturn to be the most frequent cause of death in patients admitted to intensive care units.Endothelial progenitor cell (EPC) is the precursor cells of the vascular endothelialcells, with the stimulation of physiological or pathological factors, they can bemobilized to peripheral blood to repair damage from the bone marrow and othertissues. Endothelial progenitor cells, known as angioblast, both involved inangiogenesis of birth, and also involved in vascular regeneration and repair of thebody and organ damage. In1997, Asahara first demonstrated that the precursor cellscan differentiate into vascular endothelial cells in peripheral blood, and was namedendothelial progenitor cells. Recent studies show that endothelial progenitor cells isthe most important cells involved in epigenetic rational and pathologic angiogenesisafter birth, differentiating into endothelial cells in vivo, and participating in a singleheart, liver, kidney and other organ damage or organ failure repair. Thus, EPC haveimportant therapeutic role and broad clinical application prospects in the process ofpost-traumatic ischemic disease and damage repair.The Caveolin is the main nest composed of protein, also known as the integralmembrane protein. The main role of Caveolin including Caveolin-1, Caveolin-2, andCaveolin-3, is to transfer biologically molecules to the intracellular from theextracellular, and participate in a variety of signal reception and transit. Itsphysiological functions in the maintenance of the cell and out of balance, involved incell proliferation, migration, and signal to pass. Caveolin-1can directly connectedwith a variety of signaling molecules such as G protein, Ha-Has, Src tyrosine kinasefamily members (Src, of Fyn), EGF receptor, the insulin receptor, PKC and eNOS, inorder to regulate the activity state of these signaling molecules. Tan Zh found thatincreasing the expression of caveolin-1could promote proliferation anddifferentiation of EPC, and the expression of caveolin-1closely related to the functionof EPC.Further studies showed that caveolin-1is the hub of the signal transduction pathway in vivo. It could directly connect with a variety of signaling molecules such as Gprotein, Ha-Has and Src tyrosine kinase family members, regulating these signalingmolecules. Studies have shown that the expression of PI3K will decline whenCaveolin-1is removed through drug intervention, and the inhibition of Caveolin-1byβ-cyclodextrin can prevent PI3K activating. Activation of PI3K can activate thePI3K/Akt/eNOS signaling pathway. The integrity of eNOS directly impacted themobilization, proliferation and differentiation of EPC. PI3K, Akt and eNOS are veryimportant to regulate the mobilization, migration and angiogenesis function of EPC.Everaert found that the activation of PI3K can make Akt phosphorylation, which candirectly phosphorylate and activate eNOS, inducing a large number of synthesis ofNO from vascular endothelial cells. NO can promote the mobilization, migration, andangiogenesis of EPC by acting on the metal matrix protein-9(mmp-9). It caninactivate caspase-3and caspase-6and inhibit the EPC and endothelial cell apoptosisfrom a number of ways.In this study, we have successfully isolated and cultured endothelial progenitor cellsfrom different tissues (peripheral blood, bone marrow, cord blood and umbilical cord).We cultured EPC in the environment which is ischemia and lack of factor. That couldmake EPC damage, and then observed the relationship between the function and theexpression of caveolin-1in EPC. We regulated the expression intensity of caveolin-1by RNA interference, and tried to block the signal transduction pathway of caveolin-1,PI3K/Akt/eNOS. We observed the changes of function in EPC and confirmed theregulation mechanism between the expression of caveolin-1and the function of EPC.Thus we can further study the occurrence and development mechanisms of MODSfrom the view of signal transduction pathways. So, improving the function ofdamaged EPC, that can provide a firm theoretical and experimental basis for theeffectively preventing and controling MODS, and provide a new method for theeffectively preventing and treating post-traumatic MODS in clinical.Part1: Isolation, culture and identification of endothelialprogenitor cells derived from different tissuesObjective: To isolate and culture endothelial progenitor cells from different tissues,and contrast the pros and cons of the function of different tissue-derived endothelialprogenitor cell, and then pick out endothelial progenitor cells with convenient tissuesource and excellent function. To provide a basis for the ultimate access to seed cells for cell transplantation or tissue engineering.Methods: Density gradient differential adherent culture and organizations explantmethod was used to culture and isolate endothelial progenitor cells from peripheralblood, bone marrow, umbilical cord blood and umbilical cord tissue. Then identifiedendothelial progenitor cells through the characteristics of cell morphology, cellularultrastructure, cell phenotype, phagocytic function of acetylated low-densitylipoprotein (Dil-Ac-LDL) and Ulex lectin-1(FITC-UEA-1) and angiogenic functionsin vitro. And to compare the function of the different tissue-derived endothelialprogenitor cell.Results: We successfully isolated, cultured and amplified endothelial progenitor cellsfrom the bone marrow, umbilical cord blood and umbilical cord tissue, but the cellsderived peripheral blood could not be passaged. Bone marrow-derived endothelialprogenitor cells can be passaged more than4generations, but the cell morphologicalwill change. Endothelial progenitor cells derived Umbilical cord blood and umbilicalcord tissue can be stablely passaged more than10generations with no significantchange in cell morphology. The cells showed colony-like growth, and thusproliferation accelerated, the spindle of differentiated cells appeared in thesurrounding. When the cells were cultured to approximately20days, they became toendothelial cell-like "cobblestone" shape. We found the presence of "Weibel-Paladebodies" which is the unique characteristics of the endothelial cell by electronmicroscopy. That are the morphological characteristics of endothelial progenitor cells.We found that three tissue-derived endothelial progenitor cells expressed CD133,CD34, CD31and KDR by flow cytometry. The expression of CD133and CD34gradually decreased with the increase of incubation time, but the expression of CD31and KDR were just the opposite. We found that more than85%of the adherent cellshave specific uptake for Dil-Ac-LDL and FITC-UEA-1by the identification ofphagocytic function. Angiogenesis in vitro experiments showed that threetissue-derived endothelial progenitor cells have angiogenesis capacity. Comparing tothe function of three tissue-derived endothelial progenitor cells, we found thatendothelial progenitor cells derived umbilical cord and umbilical cord blood are betterthan bone marrow-derived endothelial progenitor cells in maintaining thecharacteristics of progenitor cells, proliferation and ability of angiogenesis. Andcalculating in accordance with the median of each umbilical cord length30cm, we canreceive a large number of endothelial progenitor cells (approximately1.51×l07) in the first generation.Conclusions: Endothelial progenitor cells can be successfully isolated and culturedfrom bone marrow, umbilical cord blood and umbilical cord tissue, and can bepassaged amplification. The morphological features and functions of umbilicalcord-derived endothelial progenitor cells is similar with umbilical cord blood-derived.They are better than bone marrow-derived endothelial progenitor cells in maintainingthe characteristics of progenitor cells, proliferation and ability of angiogenesis.Calculating in accordance with the median of each umbilical cord length30cm, wecan receive a large number of endothelial progenitor cells (approximately1.51×l07)in primary. And culture method is simple and reliable. To prepare for the ultimateaccess to the clinical application of seed cells for cell transplantation or tissueengineering.Part2: The correlation study of the expression of Caveolin-1andthe function of endothelial progenitor cellObjective: To prove the dysfunction of endothelial progenitor cells is closely relatedto the expression of Caveolin-1.Methods: With serum-free and factor-free culture, the umbilical cord-derivedendothelial progenitor cells is damaged in vitro. Then detecte the change ofproliferation and angiogenesis ability in the cells, and the expression of Caveolin-1.And then to detect the functional changes of the proliferative capacity and ability toangiogenesis by RNA interference which inhibited the expression of Caveolin-1inendothelial progenitor cells.Results: The serum-free and factor-free culture can make endothelial progenitor cellsdamage, and the damage gradually increased with time. The capacity of angiogenesisalso significantly reduced, and the expression of Caveolin-1was significantlydecreased compared with the normal group (P <0.01). The expression of Caveolin-1was decreased with the aggravated damage of the endothelial progenitor cell. Theseillustrated that the functional changes of endothelial progenitor cells is closely relatedto the expression of Caveolin-1. Using RNA interference technology to inhibit theexpression of Caveolin-1in umbilical cord-derived endothelial progenitor cells, thecell proliferation was markedly decreased in the group of RNA interference (P <0.05),and the capacity of angiogenesis also significantly reduced. The results showed thatthe inhibition of caveolin-1mRNA expression can inhibit the growth and angiogenesis of endothelial progenitor cells.Conclusions: When the endothelial progenitor cell was damaged, the functionalchanges of proliferation and angiogenesis is closely related to the expression ofCaveolin-1. The capacity of growth and angiogenesis was significantly reduced whenthe mRNA expression of caveolin-1was inhibited in endothelial progenitor cells. Theresults showed that the expression of caveolin-1can regulate the function ofendothelial progenitor cells. Through the positive regulation of caveolin-1we canimprove the function of endothelial progenitor cells which was damaged, and providea firm theoretical basis and experimental basis for the effective prevention andtreatment of MODS.Part3: Mechanism of Caveolin-1on functional regulation ofendothelial progenitor cellsObjective: To explore that whether Caveolin-1regulated the function of endothelialprogenitor cells by the PI3K/Akt/eNOS signal transduction pathways.Methods: Using β-cyclodextrin, LY294002and triciribine which is specific inhibitorof caveolin-1, PI3K and Akt, we blocked the signal transduction pathway inendothelial progenitor cells in vitro system. Then observated the expression ofphosphorylated Akt and NO (indirectly reflected eNOS) which are the signaltransduction pathway downstream products, and observated the functional changes ofgrowth and angiogenesis in endothelial progenitor cell. To confirm whetherCaveolin-1regulated the function of endothelial progenitor cells by thePI3K/Akt/eNOS signal transduction pathways.Results: The expression of phosphorylated Akt and NO reduced when β-cyclodextrinand LY294002inhibited caveolin-1and PI3K. And when triciribine inhibited Akt, theexpression of eNOS decreased. This showed that β-cyclodextrin blocked caveolin-1,then inhibited Akt phosphorylation by preventing PI3K activation, thereby affectedthe activation of eNOS resulting in the reduction of NO. Three suppression groupcompared to the control group, the capacity of proliferation and angiogenesisdecreased in endothelial progenitor cells(P＜0.05). The results showed thatCaveolin-1regulated the function of endothelial progenitor cells by thePI3K/Akt/eNOS signal transduction pathways. Of course, caveolin-1may alsoregulated the function of endothelial progenitor cells by other signal transductionpathways. And this requires further study. Conclusions: When endothelial progenitor cells was impaired, the expression ofCaveolin-1reduced and the capacity of proliferation and angiogenesis decreased inendothelial progenitor cells. The reduction of Caveolin-1affected the activation ofPI3K which decreased Akt phosphorylation and reduced the activation of eNOS, andthen the production of NO reduced. This result in that the capacity of proliferation andangiogenesis decreased in endothelial progenitor cells, thereby affecting the bodyrepair.In short, when some diseases (such as ischemia, trauma, and even MODS) madeendothelial progenitor cells damage, the reduction of Caveolin-1affected theactivation of PI3K which decreased Akt phosphorylation and reduced the activation ofeNOS, and then the production of NO reduced. This result in that the capacity ofproliferation and angiogenesis decreased in endothelial progenitor cells, therebyaffecting the body repair. So we can further study the occurrence and developmentmechanisms of MODS from the view of signal transduction pathways. And improvingthe function of damaged EPC, that can provide a firm theoretical and experimentalbasis for the effectively preventing and controling MODS, and provide a new methodfor the effectively preventing and treating post-traumatic MODS in clinical.
|
PDF Full Text Request