Effects Of GDNF Modified Human Umbilical Cord Blood Cells Combined With Brain Blood Barrier Permeabilizer On Stroke In Spontaneous Hypertensive Rats

Effects of GDNF modified human umbilical cord blood cells combined with brain blood barrier permeabilizer on stroke in spontaneous hypertensive ratsBackgroundIn present, studies about treatments for cerebrovascular ischemic-reperfusion injury are focused on stem cell transplantation and gene therapy. Stem cell transplantation has becoming a new strategy for neurological disorders including stroke in recent years. Stem cells can be differentiated into all the major cellular phenotypes of the brain including neurons, oligodendrocytes, and glial cells. This multipotent feature makes stem cells a potentially neural source of transplantable cells capable of reconstituting the damaged neuronal circuitry that occurs in numerous diseases including stroke. Stem cells such as haemopoietic stem cell, mesenchymal stem cell and many others has been found rich in umbilical cord blood cells, and experiments in vitro found nucleated cell in umbilical cord blood cell has the potency to differentiate to neural cells, signifing a perspective of application using this kind of cells to treat neuropathy. The Nucleared cell in umbilical cord blood cell is primitive, easy to obtain, able to differentiate and proliferate, immature immunogenicity, fit to heterogeneity transplantation, and umbilical cord blood cells also have extensive source, and retain undifferentiated phenotype during culture in vitro, can differentiate into neural cells by induction.Several previous studies found there were significantly increases in GDNF expression in ischemic induced injuried area in animals with middle cerebral artery occlusion (MCAO), suggesting GDNF might be likely to contribute to this neuroprotective mechanism during early period after transplanted stem cell from bone marrow in the ischemic brain.One resent study demonstrated that transplantation of human bone marrow stromal cells genetically modified to secreted BDNF has been shown effective in MCAO animals. It is suggested that there are some possible advangtages of gene therpy combined with stem cell transplantation. Nowadays, methods to increase level of GDNF in cebral ischmic area have been known as following two: injection through skull purified GDNF directly, and another was transfecting GDNF directly into brain cells. The both were possible to damage brain tissue.The blood-brain barrier (BBB) would limit systemically transplanted hUCBCs migrated into injured brain, which decreased survival of cells at damaged site and partially observed behavioral recovery. Rescent studies revealed the possibility that modulation of the BBB could be used to augment the migration and survival and neurobehavioral effects of hUCBCs.Based on the previous studies, the present study designed GDNF modified human umbilical cord blood cell combined with brain blood barrier permeabilizer to treat spontaneous hypertensive rats with stroke. We investigate the differentiat ability and effectiveness of umbilical cord blood cell, concentrations of GDNF in damaged brain after combined treatments in spontaneous hypertensive rats with MACO. This study was the first to explore the feasibility and and the efficacy of the combination of stem cell transplantation, gene therapy and brain blood barrier permeabilizer to treat stroke in animal.AIM: To investigate the effects of GDNF modified hUCBCs on ischemic reperfusion in spontaneous hypertensive rats(SHR)METHODS: construct eukaryotic vector contains of glial cell line-derived neurotrophic Factor gene; transfect GDNF gene into hUCBCs; the focal cerebral ischemic-reperfusion injury model in spontaneous hypertensive rats was made by suture embolic method. The animal models were assigned into 6 groups according to the treatment: 1 normal saline + normal saline; 2 normal saline+ manitol; 3 pEGFP transfected hUCBCs + normal saline; 4 pEGFP transfected hUCBCs+ manitol; 5 pEGFP-GDNF transfected hUCBCs+ normal saline; 6 pEGFP-GDNF transfected hUCBCs+ manitol. Migration of transplatated cells was determined by counting GFP positive cells under fluorescence microscope; differentiation of transplatated cells was assayed by immunofluorescence and immunohistochemistry; GDNF level was estimated by ELISA; cerebral infarct area was judged by TTC staining, the neurological deficit was evaluated with the score of neurological function (mNSS).Result:(1) Brain blood barrier permeabilizer- manitol enhanced transplantedcell entering into brain, application of manitol itself in short term has no obvious effect on cerebral ischemic-reperfusion injury.(2) Transplanted cells transfected with pEGFP-GDNF secrate GDNF in vivo, and enhanced cell differentiate into neural cell in vivo.(3) Compared with control group(NS+NS), transplantation of pEGFP transfected hUCBCs could decrease infarcted volumn, promote recovery in neural function; transplantation of pEGFP-GDNF transfected hUCBCs could significantly decrease infarcted volumn, improve recovery in neural function even more (P<0.01).CONCLUSION(1) manitol as a brain blood barrier permeabilizer could increase transplanted cells migration into cerebral ischemic area.(2) Transplated hUCBCs transfected with GDNF could increase GDNF level in situ.(3) Transplated hUCBCs are able to differentiated into neural cells,increased level of GDNF may improve the differentiation of grafted cells.(4) Transplantation of GDNF transfected hUCBCs could decrese damages of cerebral ischemic-reperfusion injury and improve neurological function.