| Aim: Brain is one of the organs seriously damaged by physiological aging,among which the hippocampus is deeply affected,leading to the phenomenon of aging cognitive decline,bringing non-pathological life difficulties to the elderly.In recent years,human umbilical cord mesenchymal stem cells(hUCMSCs)have been widely used in therapeutic research.micro RNA-206(miR-206)is a small RNA involved in the post-transcriptional regulation of gene expression,which is abundant in the exosomes of stem cells and is involved in the regulation of stem cell therapy.Therefore,this study aimed to explore the role of down-regulated miR-206 expression of hUCMSCs in improving d-galactose(D-gal)-induced aging cognitive decline.Methods: hUCMSCs were cultured in osteogenic and lipogenic induction media respectively,and their differentiation ability was detected.At the same time,the signature antigens on the surface of hUCMSCs were identified by flow cytometry.miR-206 inhibitors or negative control inhibitors were used to transfect hUCMSCs,and the expression of m RNA after transfection was detected by RT-q PCR,while the expression of brain-derived neurotrophic factor(BDNF)was detected by western blot(WB).DIR Iodide(Di IC18)membrane dye was used to stain hUCMSCs,and the stained cells were injected into the lateral ventricle stereotaxically.The intracerebral tracers of hUCMSCs were observed by embedding sections at 1,5 and 10 days respectively.In the aging model,male C57BL/6J which 9 weeks old could be obtained by subcutaneous injection of D-gal(500mg/kg/ d)every day for 8 weeks,and normal group was injected with the same amount of normal saline.The mice were divided into four groups: The normal group(normal group),the model group were injected with PBS(D-gal +PBS group),the Cell group was injected with untreated hUCMSCs(D-gal +Cell group),and the experimental group was injected with miR-206 inhibitors treated hUCMSCs(D-gal + Cell-anti-miR-206).The cognitive level of mice was tested by open field test,new object recognition test and Y maze animal behavior test during 10 days after operation.The protein expression of BDNF and postsynaptic compact protein 95(PSD-95)was detected by WB.Hematoxylin-eosin staining was used to observe the pyknosis of hippocampal cells.The physiological status of hippocampus cells was observed by Nissl staining.Immunohistochemistry(IHC)was used to detect the distribution and expression of early growth response factor 1(EGR-1)in hippocampus.The distribution and expression of BDNF in hippocampus were detected by immunofluorescence(IF).Result:(1)In this experiment,flow cytometry was used to detect the high expression of surface signature antigens CD44,CD90,CD105,CD29 and CD73 of hUCMSCs,while the low expression of non-surface signature antigens CD34,CD45,CD31 and HLA-DR of hUCMSCs,P <0.05;(2)The differentiation of hUCMSCs was successfully completed in osteoblast and lipogenic culture,which showed that hUCMSCs had normal differentiation ability;(3)Behavioral test showed that the learning and memory ability of mice recovered after cell transplantation;(4)No proliferation of hUCMSCs into the hippocampus was observed within 10 days after the lateral ventricle transplantation,and the cell blotting disappeared 10 days later;(5)After transfection,the expression of Mir-206 was down-regulated,the m RNA level of BDNF was not different,but the protein expression was up-regulated,and miR-206 negatively regulated the expression of BDNF;(6)BDNF expression was up-regulated in the hippocampus after cell transplantation;(7)hUCMSCs with low expression of Mir-206 were more effective in improving the physiological function of hippocampus neurons;(8)After cell transplantation,the expression of PSD-95 and Egr-1 in hippocampus was up-regulated,and hUCMSCs with low expression of miR-206 could improve neural signaling and neurogenesis in hippocampus.Conclusion: hUCMSCs with low expression of miR-206 have an effective neuroprotective effect on aging cognitive decline,providing a new intervention approach for anti-aging and cognitive improvement. |
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