The Effect Of Cobalt Chloride In BMSCs Repairing Cognitive Function Of HIBD Rat

PartⅠExplore the best drug concentration of CoCl₂on activatingHIF-1in BMSCs and the secretion effect of HIF-1a inhibitor2-ME2in BMSCsObjective: Create a response curve of cobalt chloride （CoCl₂） drugconcentration level with HIF-1protein levels, the proliferation index MTSand the damage index LDH. Take VEGF, EPO, IGF-1protein levels aseffect Indicator to argument the secretory effect role of HIF-1stabilizerCoCl₂and inhibitor2-ME2in BMSCs and to determine the time-dose curveof BMSCs secreted protein. Explore the best drug concentration of CoCl₂on activating HIF-1in BMSCs.Methods: BMSCs was isolated from bone marrow of SD rat.After12h,24h,36h,48h administraton of CoCl₂in BMSCs，the activity（MTS）and the damage（LDH） experiment was measured in vitro. VEGF, EPO,IGF-1of the cultured medium which interfere with HIF-1stabilizerCoCl₂and inhibitor2-ME2at different time points were detected by ELISA analysis.The HIF-1α protein expression was detected by Westernblot.Results:（1）（0-100M） CoCl₂had no obvious cells toxicity whenadministration to BMSCs for12h,24h,36h,48h,（p>0.05）.（2） CoCl₂（0-100u M） had no obvious effect on cell proliferation when administrationto BMSCs for12h,24h,36h,48h,（p>0.05）.（3） The dose-responsesecreted proteins relationship after administration to BMSCs with CoCl₂:10CoCl₂can not effectively promote BMSCs to secrete VEGF within48h,25CoCl₂promote BMSCs to secret VEGF following with theincreasement of the duration of action,and reached a maximum at36h.EPOwas increased following with the dose of CoCl₂at12h and reached amaximum with100. CoCl₂（0-100M） had no obvious effect on IGF-1protein expression when administration to BMSCs for12h,24h,36h,48h,（p>0.05）.（4） HIF-1protein was detected by Western blot and theresults showed that HIF-1protein expression up to36h（.5）The secretioneffect of CoCl₂promote BMSCs growth factor can be inhibited byinhibitors of HIF-1. VEGF, EPO, IGF-1expression decreased when1M、2.5M、5M2-ME2administrationed to25M CoCl₂culturedmedium, and the VEGF,EPO, IGF-1protain expression was the lowestwhen administration to2.5M2-ME2.Conclusion:（1）（0-100M）CoCl₂had no obvious cells toxicity whenadministration to BMSCs for12h,24h,36h,48h.（2）（0-100M） CoCl₂ had no obvious effect on cell proliferation when administration to BMSCsfor12h,24h,36h,48h.（3） CoCl₂can promote BMSCs to secret growthfactors,25M CoCl₂administration to BMSCs for36h can promoteVEGF, EPO, IGF-1expression.（4） The secretion effect of CoCl₂promoteBMSCs can be inhibited by2-ME2. The best inhibitory concentration was2.5uM2-ME2. Part ⅡDiscuss the effect of Cobalt chloride in BMSCs repairingcognitive function of HIBD ratObjective: To establish the technique of intraventricular perfusion inneonatal rat and explore conditioned medium repairing cognitive functionof HIBD rat.Methods: Forty SD rats aged7d were randomly divided intoMedium control group（DMEM group，n=8）、Bone marrow mesenchymalstem cell culture supernatant group（BMSC-DMEM group，n=8）、bonemarrow mesenchymal stem cells culture supernatant effected by CoCl₂group（CoCl₂-DMEM group，n=8）、bone marrow mesenchymal stem cellsas and its culture supernatant effected by CoCl₂group（CoCl₂-DMEM+BMSCs group，n=8）、bone marrow mesenchymal stemcells culture supernatant effected by CoCl₂and2-ME2group （CoCl₂+2-ME2-DMEM group，n=8）.Collection of enriched medium andinjected into left ventricle of10-day-old rat for7days with Alzetmini-pumps. At age of7weeks, pups of each group were trained to do theMorris water maze test to evaluate spatial learning and memory abilities. Atage of8weeks, pathology was observed by HE staining, and Nissl staining;brain tissue VEGF, EPO, IGF-1level was detected by ELISA;Hippocampal slices LTP was evoked by Neurophysiological technology;AMPA receptor subunit GluR2as the key indicators of synaptic plasticitydetected by immunofluorescence.Result:（1） General situation of HIBD rats when intraventricularedperfusion for7days, feeding well, sleeping good, hair bright, behavior andmental without abnormal.（2） Morris water maze training revealed that therat in CoCl₂-DMEM group and CoCl₂-DMEM+BMSCs group hadsignificantly shortened average latency time at2to6d, and improved longterm reference memory compared with that in DMEM group （P <0.01）.（3）HE staining and Nissl staining both observed that hippocampus structureand the extent injury of the neuronal cells in DMEM group andBMSCs-DMEM group more serious compared to the CoCl₂-DMEM group.The number of abnormal cells in CoCl₂-DMEM group was much more thanCoCl₂+BMSCs-DMEM group （P <0.01）.（4） To explore the secretion ofgrowth factors in brain tissue by ELISA, VEGF, IGF-1concentration inCoCl₂-DMEM group were higher than DMEM group and BMSCs-DMEM group（p <0.01）, BMSCs can increase VEGF and IGF-1concentration.VEGF and IGF-1expression are reduced by2-ME2.（5） CoCl₂processingcan increase the rate of LTP evoked. The CoCl₂-DMEM group comparedwith DMEM group and BMSCs-DMEM group that the evoked rateincreased and the CoCl₂-DMEM+BMSCs group was the highest.（6） Toexplore the molecular mechanisms of LTP. GluR2immunofluorescenceshow that GluR2expression in DMEM group and BMSCs-DMEM grouplower than CoCl₂-DMEM group and CoCl₂+BMSCs-DMEM group （P<0.01）. CoCl₂-DMEM group expressing GluR2more thanCoCl₂+2-ME2-DMEM group but less than CoCl₂+BMSCs-DMEM group（P <0.01）.Conclusion: Conditioned medium can effectively improve thespatial learning and memory capacity in HIBD,2-ME2can inhibit theeffect.（2） CoCl₂processing conditioned medium with BMSCs can betterrepairing structural and neuronal cells damage, both have a synergisticeffect.（3）CoCl₂can promote VEGF, IGF-1and other neurotrophic factorexpression, play a Functional recovery role in HIBD rat.（4） CoCl₂processing can increase the rate of LTP evoked.（5） CoCl₂processing canregulating hippocampal synaptic plasticity from the molecular mechanisms.