| Part one Extraction,Culture,and Identification of Endothelial Progenitor Cells as well as Identification of CytokinesObjective: To observe the extraction,culture and identification of endothelial progenitor cells,and identify the secretory factors of endothelial progenitor cells.Methods: Mononuclear cells from the tibia and femur of rats were collected and cultured for 14 days.Cell growth was observed,and cell growth curve was drawn.The rat CD133 was labeled with FITC,and the PE labeled rat CD34 was added to the cell culture solution and was measured by flow cytometry,and compared with the blank control group that also added two types of homotypic antibodies.Dil labeled acetylated LDL and FITC labeled agglutinin 1 were added to cell culture solution to add cell culture solution to the cell culture medium to observe and photograph under the laser confocal microscope,and compared with the blank control group that also added two types of same type antibody.The content of vascular endothelial growth factor(VEGF),brain derived neurotrophic factor(BDNF)and insulin like growth factor(IGF)in cell culture fluid was measured by ELISA kit for third,seventh,fourteenth days.Results: in vitro cultured rat endothelial progenitor cells proliferation exuberant,biological stability.The typical endothelial progenitor cells were detected by flow cytometry and double fluorescence staining.The VEGF,BDNF and IGF were detected in the cell culture medium,and the content increased with the increase of culture time.Conclusion: the extraction,culture and identification of EPCs in vitro can provide the best transplantation concentration for animal experiments,and it also lays a foundation for the study of EPCs transplantation.EPCs itself can secrete many cytokines that contain angiogenesis and trophic neurons.Part two Is the effect of hyperbaric oxygen intervention on macrophage activation and neuroprotection in rats with spinal cord injuryObjective: To observe the quantity changes and neuroprotective effects of 2 kinds of macrophage subtypes after hyperbaric oxygen intervention on SCI in rats.Methods: 10 SCI rats were randomly divided into 3 groups.1)the blank control group(n=5).2)normal air intervention was used to intervene SCI rats(NBA,1ATA,21% oxygen,n=5).Hyperbaric oxygen intervened in SCI rats group(2.8ATA under 100% oxygen,n=5).The BBB scores of the three experimental rats were measured at 1 weeks,2 weeks and 4 Wednesday.The content of M1/M2 subtype macrophages in the spinal cord of three groups of experimental rats was measured by immunohistochemical method,and the three group of experimental rats were wrapped around the axon for seventh days,fourteenth days and twenty-eighth days with Lux's blue(LFB)staining.Out of the myelin sheath.Results: HB0 T promoted the formation of M2 macrophages,and HBO intervention significantly increased the myelin content.Functional recovery after spinal cord injury in ratsConclusion: hyperbaric oxygen intervention can promote the recovery of limb motor function in rats,especially to promote the recovery of hind limb motor function.Part three Is endothelial progenitor cell transplantation combined with hyperbaric oxygen to repair spinal cord injury in ratsObjective: To observe the effect of endothelial progenitor cell transplantation combined with hyperbaric oxygen(HBO)on vascular regeneration and hind limb function in the injured area of spinal cord of rats and its molecular biological mechanism.Methods: 28 adult female SD rats were randomly divided into 4 groups,7 rats in each group.1)there was no intervention in group SCI after successful modeling.2)spinal cord injury+endothelial progenitor cell group(group SCI+EPCs).After successful modeling,EPCs was injected at the site of spinal cord injury.3)spinal cord injury+hyperbaric oxygen group(group SCI+ HBO): SCI rats were treated with hyperbaric oxygen.4)combined group(group SCI+EPCs+HBO): SCI rats were treated with hyperbaric oxygen after injecting EPCs locally.The motor function was assessed by BBB score before transplantation,1 days,3 days,1 weeks,2 weeks,3 weeks and 4 weeks after transplantation.4 weeks after transplantation,TUNEL method was used to determine the apoptotic status of SCI nerve cells in the experimental group,and the pathological changes of the pathological tissue of SCI were observed by HE staining.The RT-PCR and Western blot methods were used to detect MMP9/2,synaptophysin(Synaptophysin,Syn)and transforming growth factor-beta 1(transforming grow)in the injured area of the spinal cord and the surrounding tissue.The expression change of th factor-beta 1,TGF-beta 1 gene and protein.Results: after 1 weeks of transplantation(after cell transplantation),the combined group of the hind limb motor function score(BBB score)was superior to the SCI+EPCs group and the SCI+HBO group.The SCI+EPCs group and the SCI+HBO were higher than the SCI group,and there were significant statistical differences(P<0.05)in each group.The number of TUNEL apoptotic cells was the lowest in the combination group,the SCI+EPCs group and the SCI+HBO group.Two,SCI group was the highest,and each group had significant statistical difference(P<0.05).After HE staining,it was observed that the recovery degree of spinal cord injury area in combined group was better than that in group SCI+HBO and SCI+EPCs.MMP9/2 and TGF-beta 1 gene and protein expression: the combination group was the lowest,the SCI+EPCs group and the SCI+HBO group were second,the SCI group was the highest,each group had significant statistical difference(P<0.05).The gene and protein expression of synaptophysin(Synaptophysin,Syn)were higher in the combination group,second in group SCI+EPCs and in group SCI+HBO,and the highest in SCI group.There were significant statistical differences in each group(P<0.05).Conclusion: hyperbaric oxygen intervention plays a role in the neuroprotection of spinal cord injury.The intervention of endothelial progenitor cell transplantation combined with hyperbaric oxygen can promote the repair of nerve function and axon regeneration in rats with spinal cord injury,and improve the motor function of the rats. |
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