The Therapy Of GM-CSF-gene-transfected BMSCs On Ischemic Cerebrovascular Disease And The Mechanisms Involved

Ischemic cerebrovascular diseases (ICD), as a series of commonneurological diseases with a high mortality and disability rate, is seriouslyharmful to the human health and therefore caused large burden for thepatient and the whole society. Up to now, the main therapeutic strategy toICD is the medications treatment. However, these medications could noteffectively reverse the infarct brain tissue. Thus, searching an effectivetherapeutic strategy to repair the insulted brain to alleviate the ischemicinjury and to recover neurological impairment at the largest extent is badlyneeded.It was thought that the necrotic nervous tissue could not beregenerated by itself in the central nervous system (CNS) because of therestriction of internal ability of neurogenesis and externalmicroenvironment. Therefore, the discovery of adult neural stem cellsbroke that traditional view and brings a new light for the treatment of ICD.The most common used donor cells for neural transplantation to CNS include embryonic stem cells (ESCs), neural stem cells (NSCs), bonemarrow stromal cells (BMSCs), human umbilical cord blood stem cellsand so on. Among which, BMSCs was confirmed as adult stem cellsseveral years ago and have became the hot issue in the medical fields evenin the life science domain due to its some merits including theconvenience of obtaining and culturing, the potency of multi-directionaldifferentiation, the receptive of exogenous genes and the noneinvolvement of ethic issues.Granulocyte-macrophage colony-stimulating factor(GM-CSF) isregarded as a common multi-functional hematopoietic factor and canevoke bone marrow, promote the proliferation and differentiation ofhaemopoietic stem cells and inhibit the apoptosis of leucocytes, andtherefore increase the number of peripheral leucocytes. More recently,quite a few studies reported that GM-CSF has the neuroprotective effects;it can promote the differentiation of neural progenitors and inhibits theneural apoptosis and it can also improve the function of CNS.Our research project included the study of the neural differentiationof in vitro BMSCs and the mechanisms involved; the study of GM-CSFgene transfected BMSCs transplanted to cure the middle cerebral arteryocclusion (MCAO) rats and the mechanisms involved.Our studies include the following four parts. PartⅠThe study of GM-CSF secreting from in vitro rat BMSCs andthe expression of GM-CSF receptors on BMSCsAIMS: To investigate the level of GM-CSF secreted by in vitro ratBMSCs and the expression of GM-CSF receptor (GM-CSFR) on BMSCs.This part of study was the base experiment for the following parts.METHODS: BMSCs were isolated and identified from theSprague-Dawley rats. The expression of rat GM-CSF mRNA and proteinextracts from BMSCs were detected by RT- PCR and ELISA,respectively. The expression of GM-CSFR on BMSCs from the third tosixth passage was detected by Western blot and RT-PCR. RESULTS:RT-PCR results indicated that there were only feeble GM-CSF expressedin BMSCs from the first to sixth passage; low level of GM-CSF proteinwere detected in supernatant fluid and the extracts from BMSCs from thefirst to sixth passage by ELISA, the protein level of the extracts from thesecond passage BMSCs was higher than that of the other passages(P＜0.01), However, there were no significant difference among otherpassages (P＞0.05). Western blot and RT-PCR results showed that therewere abundant GM-CSFR expression on BMSCs, however, no significantdifference was found in the expression of GM-CSFR among BMSCs fromthe third to sixth passage (P＞0.05). CONCLUSIONS: In vitro rat BMSCssecreted GM-CSF and there were abundant GM-CSFR expression on BMSCs.PartⅡExogenous GM-CSF enhances neural differentiation ofBMSCsAIMS: To investigate the role of GM-CSF on the neural differentiation ofBMSCs and to explore its possible mechanisms. METHODS: BMSCswere isolated and identified from the Sprague-Dawley rats. To identify theeffects of GM-CSF on neural differentiation of BMSCs, cells at the fifthpassage were treated with exogenous rat-derived GM-CSF and observedthe morphological changes of BMSCs at 0h, 6h, 12h, 24h, 48h and 96h,respectively, after the treating procedures. To further characterize theGM-CSF-induced neural differentiation of BMSCs, the cells of differenttime points and the control BMSCs were tested with nestin, neuronespecific enolase (NSE) and glial fibrillary acidic protein (GFAP) byimmunocytochemistry and RT-PCR. To further explore the mechanisminvolved in GM-CSF-induced neural differentiation of BMSCs, theexpression of pCREB/CREB on different time points were measured bywestern blotting. Rat fibroblasts were used as the negative control.RESULTS: Compared with the untreated BMSCs, the GM-CSF treatedBMSCs were differentiated into the neural cells with obvious synapses,showing the morphological characteristics of neural cell and expressed thespecific antigen markers of the neural cell including nestin, NSE and GFAP. Nevertheless, the morphology of the rat fibroblasts in the controlswas not changed at the same inducing treatment. Western blots of theextracts showed the expression of pCREB began to increase at the 6thhour and reached a peak at the 12th hour after the treatment. However,CREB levels in the BMSCs among groups were not of significantlydifferenence. CONCLUSIONS: These findings demonstrate thatGM-CSF enhances neural differentiation of BMSCs by up-regulatingCREB phosphorylation.PartⅢThe study of pUMVC1-mGM-CSF plasmid transfected intorat BMSCs mediated by LipofectamineAIMS: To observe the transfection efficiency of plasmid into rat BMSCsmediated by Lipofectamine and the expression of exogenous GM-CSFgene. METHODS: BMSCs were isolated and identified from theSprague-Dawley rats. pUMVC1-mGM-CSF plasmid was transfected intoin vitro rat BMSCs mediated by Lipofectamine^TM2000. gWiz^TMGFPplasmid which contains green fluorescent protein (GFP) gene wastransfected into rat BMSCs at the same condition. At 0h, 6h, 12h, 24h, 48h,96h and 7d after gene transfection, the transfection efficiency wasdetermined by observation the expression of GFP with fluorescenceinverted microscope. And the expression of mRNA and protein of exogenous GM-CSF gene were detected by RT-PCR and ELISA,respectively, at the same time-points. RESULTS: GFP began to express atthe 6th hour and reached a peak at the 48th hour, which implied about 40%increase of the transfection efficiency, after gWiz^TMGFP transfection.RT-PCR and ELISA results also showed that the mGM-CSF mRNA andprotein were found to increase at the 6th hour and reach a peak at the 48thhour after the mGM-CSF gene transfection, which even lasted to the day 7with more strong expression. CONCLUSIONS: BMSCs were liable toaccept and express exogenous GM-CSF gene; plasmid transfectionmediated by Lipofectamine^TM2000 was an optimal way to transfectplasmid into BMSCs with the high transfection efficiency and long timeexpression of exogenous GM-CSF gene.PartⅣThe therapy of GM-CSF-gene-transfected BMSCs onischemic cerebrovascular diseaseAIMS: To investigate the survival and differentiation of GM-CSF-genetransfected-BMSCs in the ischemic boundary zone (IBZ) of the MCAOrats and the effects of GM-CSF-gene-transfected BMSCs transplantationon the function of nervous system after cerebral infarction and to observethe feasibility and effects of exogenous BMSCs and GM-CSF-gene-transfected BMSCs to cure the ischemic cerebrovascular disease. METHODS: 135 adult male SD rats were divided randomly into shamoperation group (n=30) and operation group (n=105), the rats in the lattergroup were again divided randomlyα-MEM group, BMSCs group andGM-CSF gene-transfected-BMSCs group. All the rats except of those insham operation group were subjected to right intraluminal MCAO.BMSCs were isolated and proliferated from the SD rats and those cells atthe third passage were infected by GM-CSF gene. At the 24th hour afterthe onset of MCAO, directed with the stereotaxic apparatus, serum-freeα-MEM media, BMSCs and GM-CSF-gene-transfected-BMSCs wereinjected into rats' IBZ, respectively. At the day 3, day 7 and day 21 aftercell transplantation, the neurological function and the infarct volume ofthe model rats were tested with the modified neurological severity score(mNSS) and TTC assay, respectively. Meanwhile, the survival anddifferentiation of transplanted cells were observed at the same time points.The cell apoptosis in the model rat's IBZ was measured with HE stainingand TUNEL methods, and the microvessel density (MVD) around IBZ andthe proliferation of in situ stem cells in the hippocampus were detectedwith immunohistochemistry way. RESULTS: Exogenous BMSCs couldsettle down and survive in the IBZ, and some of them differentiating intoneural cells. At the day 3 after cell transplantation, there were nosignificant differences of mNSS among the three groups (P＞0.05). At theday 7 and 21 after cell transplantation, however, the mNSS of GM-CSF- BMSCs group was significantly lower than that ofα-MEM group andBMSCs group (P＜0.01, P＜0.05, respectively), the infarct volume ofGM-CSF-gene-transfected-BMSCs group significantly decreased thanthose of other two groups (P＜0.01, P＜0.05, respectively), the percentageof apoptosis-positive cells in IBZ were significantly decreased (P＜0.01,P＜0.05, respectively) and the MVD in IBZ were significantly increased(both P＜0.05) in the GM-CSF-BMSCs group than those in the other twogroups, the stem cells in hippocampus were significantly increased in theGM-CSF-gene-transfected BMSCs group than those in the other twogroups (P＜0.001, P＜0.05 respectively). CONCLUSIONS: Superior tosimple BMSCs transplantation, gene-transfected-BMSCs intracerebrallytransplanting into the rats' IBZ could decrease infarction volume andpromote functional recovery of ischemic rats and therefore it could be apotential strategy to cure stroke.