The Effect Of Human Placenat Derived Mesenchymal-like Stem Cells(HPMSCs) And VEGF Accelerating Wound Healing Of Ischemic Random Skin Flaps

Tissue coloboma after trauma or tumor operation usually requires the use of the skin flap transplanting to restore. Over the years, even though designs and surgical techniques of the skin flap have improved continuously, and have tried to adopt a number of materia medica to improve the survival of skin flap, but this problem that because of the hypoxia-ischemia in remote tissue of the skin flap and venous reflux impeded after the skin flap transplanting often leading to the dead of the transplanting skip flap tissue has always plagued clinicians. The hot issue on injury and reconstruction of surgery field research is on how to improve the survival rate of over-length skin flap and expand clinical application of random skin flap. Many scholars found through research that there are two keys to solve this problem: (1) after the skin flap transplanting, repair in trauma process should be started as soon as possible, mobilize repair in trauma factor fully, speed up the repair in trauma of the skin flap transplanting section, and promote "bonding" between skin flap with the recipient site, a skin flap with the surrounding tissue(;2) speeding up reconstruction of the remote skin flap ischemia section blood circulation, resolve remote skin flap hypoxia-ischemia status. The two combined actions can promote the survival of super-ratio skin flap effectively.VEGF (Vascular endothelial growth factor) is a secreted glycoprotein, the proliferation is strong, and is easy to reach the target cells, and has the promoting effect on migration, proliferation, growth of vascular endothelial cell; and can also promote proliferation, migration of the fibroblast; and stimulates the partial reconstruction cell gathering in trauma, has a strong role in promoting repair in trauma, synthesizes and secretes extracellular matrix, has strong promoting repair in trauma. It is only known cytokines on the purpose of treatment of ischemia- hypoxia through direct effects on vascular endothelial cell surface receptors and rapid formation of new blood vessels, in the short term reconstruction blood circulation of the ischemia section. Although VEGF has an efficient ability to promote angiogenesis, but it has very short half-life and is difficult to play persistent effect with expensive price, so it is difficult to be promoted in practical applications. Therefore, looking for a suitable VEGF sustained-release system or VEGF persistent effect system is in urgent need of solution currently.In recent years, it was discovered that stem cells has important applications capacity in the field of repair in trauma. MSCs (mesenchymal stem cells) and ADSCS (adipose-derived stem cells) could be mobilized, homed and expanded to the injury point to participate in repair in trauma. the specific capacity of MSCS and ADSCS is that they can move directly to injury tissue can be confirmed in the repair in skin trauma, it is proved that MSCS has been involved in the whole process of repair in trauma, MSCS and ADSCS can home to the injury point or differentiate into phenotype of injury cells, and it also can promote the regeneration of the tissue endogenous cell by creating the enhancement of micro-environment. But at present, the fact whether the placenta-derived stem cells in repair in trauma process can home to the trauma section and play a catalytic role in repair in trauma is not reported now. Moreover, stem cells can also act as vector cells of gene therapy, transport gene to target section, exert therapeutic effects. Such as MSC etch adult stem cells can be used as gene therapy vector of multi-diseases of the central nervous system; MSC, rADSC, EPC etch can be used as therapy vector for VEGF, FGF etch cytokines to promote the establishment of collateral circulation and angiogenesis. But at present there is very rare report about HPMSC_S as gene therapy vectorIn order to explore whether HPMSC_S can home to the trauma section and can promote repair in trauma, as well as whether it can act as a vector cell of VEGF cells to deliver VEGF to trauma section, exert persistent effect of VEGF, we carried out an experiment as follows:1. To obtain HPMSCs from the placenta, this experiment separated mononuclear cells from the placenta perfusate using density gradient centrifugation, cultivated adherence, further selected independent clones to cultivate separately, purified HPMSC_S cloning from adherent cells. We've detected its general biological characteristics, and differentiated into fat, bone to identify its multidirectional differentiation potentiality through directed induction.2 In order to explore whether HPMSCs can differentiate to repair in trauma cells phenotype, we carried out the experiment on "human placenta mesenchymal stem cells (HPMSCs) inducing in vitro to differentiate into vascular endothelial ", by optimizing the endothelial nutrient fluid, after the directed inducing HPMSCs to cultivate 3 d passage, cell morphology changed ,when passage cell grew to 80% 90% confluence, expressed analogy endothelial "cobblestone"-like structure known as "endothelial-like cells", back cell body inducing 7 d or 14 d retracted gradually, enhanced a sense of three-dimension. Specific surface mark vWF of mature endothelial cell did not express in not induced HPMSC_S, but such endothelial-like cells were positive expression. Flow cytom -etry showed that the expression of CD31 was 22.39%, but it had increased compared to not induced HPMSCs; but CD34 was the same to not induced, endothelial-like cells did not express after induction. Dil-Ac-LDL uptake experiments revealed that endothelial-like cells after induction had the ability to uptake Dil-Ac-LDL. In summary, the current induced scheme can induce HPMSCs to appear endothelial cell phenotype, and its phenotypic characteristics are more obvious as the extension of the induction time, HPMSCs has the capacity to direct and different -iate into endothelial cells in vitro specific conditions.3 In order to prove that HPMSC_S has the function of gene therapy vector cell, the experiment established a pIRES2-EGFP-VEGF₁₆₅ eukaryotic expression vector, and transferred pIRES2-EGFP-VEGF₁₆₅ into HPMSC_S by liposome method successfully, and identified that HPMSC s with gene still has stem cell characteristics by multidirection induced differentiation..4 In order to explore whether HPMSC_S with pIRES2-EGFP-VEGF₁₆₅ can home to repair in trauma and promote blood circulation in ischemic section, we carried out an vitro experimental study on〝basic research on HPMSC_S of pIRES2-EGFP-VEGF₁₆₅ eukaryotic expression vector impact on the survival of ischemic skin flap〞. Through research we confirmed that HPMSC_S with gene has characteristics to home to repair in trauma section, and differentiate to repair in trauma cell and promote blood circulation of ischemic section, improve the survival of the ultra proportion skin flap.Sum up fully, this study confirms by experiment that the placenta-derived stem cells has the characteristic to differentiate to repair in trauma cell phenotype , and it can be used as vector cells of the gene therapy to deliver target genes to trauma section, play a role in promoting repair in trauma , and maintain its multi-directional differentiation potentiality; VEGF can express vector by constructed eukaryote, and transfer into HPMSCS, transport it to its target section through the vector cells , and play sufficient effect. This study further improves the theory of the stem cells family to promote repair in trauma, and also provides a new way and methods for VEGF sufficient effect.