Experimental Study Of Biological Characteristics Of Rat Bone Marrow Mesenchymal Stem Cells Transfected With Hypoxia-inducible Factor-1α Gene

Spinal cord injury with ischemia could be induced by compressed cord,traumatic spine, diseased blood-supplied artery of spinal tissues, blocked vesselafter surgical repair of thoracic and thoracoabdominal involved vessels,intense hypovolemia and so on. When the factors of ischemia were controlled,the spinal cord tissues could be restored infusion, but, its function could not getbetter, the damage after previous ischemia even get more severe. This progressis called Spinal Cord Ischemia-Reperfusion Injury （SCII）. For a long time, thehigh disability and mortality after spinal cord ischemia reperfusion injury havebeen affected the qualities of patients’ lives in a serious way, then how torecover the function of injuried neurons and improve the localmicroenvironment of them have also becoming hot issues at home and abroad.Because of the complex pathophysiological mechanism of SCII, and the difficulties of therapies, there has not been an effective therapeutic method, andthe therapeutic method has been under exploring. So far，the research of SCIIhas achieved gene and cellular level. Gene therapy and cellular transplantationtherapy are the new directions, they aim at the repairmen of function of neuronsand the improvement of microenvironment, the key procedure is the choice oftargeted genes or carried cells. These methods are hoped to get a breakthroughabout treating SCII.Recent research confirmed, hypoxia inducible factor-1α （HIF-1α）appeared and responded hypoxia at key circumstance, and regulated thetranscription of target genes with connecting to the specific subsequences ofDNA, eventually，took important parts in attenuating apoptosis of cells andremodeled microcirculation. Bone marrow mesenchymal stem cells （BMSCs）could promote the proliferation, differentiation and migration of neurons, andimprove the function and structure of injuried ones. Our objective is to try toconnect cellular transplantation with gene therapy to find a new access to treatspinal cord ischemia reperfusion injury with the help of both responsibility ofHIF-1α gene for hypoxia and function of transplantation of BMSCs.Experiment1Cultivation of Rat Bone Marrow Mesenchymal Stem Cells andInvolved Detections of HIF-1α Gene after TransfectionObjective Isolate and cultivate BMSCs of high purity, and complete thedetections after gene transfection. Methods Isolated and cultivated BMSCs by the whole bone marrow adherencemethods. We identified the BMSCs via inverted microscope,Flow Cytometer, and induction of diferentiation of cells intoosteoblasts and adipocytes; Then,we transfected the previous-constructed expression vector pcDNA3.1-HIF-1α with markedfluorescence into BMSCs via electroporation，and watched the vectorby means of fluorescence microscope.Results The identification results of cultivated cells coincided with thestandardization of BMSCs.Many red granules were observed in thenuclei of BMSCs after transfection.Conclusion The whole bone marrow adherence method could be use to isolateand obtain the BMSCs with high purity, HIF-1α gene could betransfected into BMSCs with electroporation.Experiment2Reasearch of Biological Characteristics of Rat Bone MarrowMesenchymal Stem Cells modified by Gene after HypoxiaObjective Watch the biological characteristics of BMSCs modified by HIF-1αgene after hypoxia.Methods Established the hypoxic environment induced by physical method,and Continuously cultivated the stem cells in group BMSCs-HIF-1α，group BMSCs，group BMSCs-pcDNA3.1during8days. Westernblot analysis detected the protein expression of HIF-1α gene; Flow Cytometry determined the periodicity and apoptosis of cells; MTTassay described the cell growth curve.Results According to Western Blot, HIF-1α protein was significantlyincreased in group BMSC-HIF-1α compared with the controlledgroups, group BMSCs-pcDNA3.1and group BMSCs had less proteinexpression,there was no obvious statistical significance betweenthem. Flow Cytometry showed that the PI of experimental group washigher than controlled groups, the apoptosis was lower than controlgroups obviously. MTT assay pointed out that the growth tendencyof stem cells in experimental group were better than it was in othergroups at the same condition.Conclusion The biological characteristics of bone marrow mesenchymal stemcells transfected via HIF-1α gene promoted on some extent afterhypoxia.Experiment3Effects of Biological Characteristics of Synthetic Cells afterChemical Hypoxia by CoCl₂-InducedObjective Watch the effects of biological characteristics of BMSCs modified byHIF-1α gene after CoCl₂-induced hypoxia.Method Imitated the hypoxic environment with putting hypoxia inducer-CoCl₂into culture medium, and made the final concentration ofCoCl₂150μM. Kept observing every group8days.（Group BMSCs-HIF-1α-CoCl₂, Group BMSCs-CoCl₂）. And the groups（Group BMSCs-HIF-1α、Group BMSCs） with hypoxic environmentinduced by physical method were made into controlled groups.Western blot analysis detected the protein expression of HIF-1α gene;Flow Cytometry determined the periodicity and apoptosis of cells;MTT assay described the cell growth curve, watching cells’proliferation.Results Compared with group BMSCs-HIF-1α, the expression of HIF-1αgene expression was up-regulated;the proportion of cells in G2, Sphase and PI were higher; and the proportion of cells in G1phaseand apoptosis of cells were lower in group BMSCs-HIF-1α-Hypoxia-inducible Factor-1α.Group BMSCs-CoCl₂had the similartendency with group BMSCs.Conclusion The biological characteristics of bone marrow mesenchymal stemcells transfected via HIF-1α gene promoted on some extent afterchemical hypoxia by CoCl₂-induced.