| Alzheimer's disease(AD),the most frequent type of dementia,is a fatal neurodegenerative disease which mainly manifests as cognitive impairment and memory loss.With the continuous development of aging of the population,the incidence of AD increases year by year.Howerver,owing to the complex etiology of AD and its unclear pathogenesis,there is no definite treatment for AD.In recent years,it has been reported that neural stem cell transplants could improve the memory of AD mice.However,the sources of neural stem cells are limited.And the majority of neural stem cells originate from embryonic tissue,which often results a fierce debate on social ethics.Human amniotic membrane-derived mesenchymal stem cells(hAM-MSCs),as medical wastes,have the characteristics of wide sources,low immunogenicity,safety and reliability,and no social ethics dispute.Research on hAM-MSCs applied to the treatment of traumatic brain injury found that hAM-MSCs could promote the recovery of nerve function.In previous studies,hAM-MSCs were transplanted into the AD mice,and then the learning and memory abilities of mice were enhanced,which showed that hAM-MSCs have the potential to alleviate the symptoms of AD patients.It was found that hMSCs played vital roles in regulating immune functions of multiple immune cells,such as T lymphocytes,B lymphocytes,natural killer cells,dendritic cells,and so on,and then regulated immune responses.According to research,hMSCs had good therapeutic effects on graft-versus-host disease and autoimmune diseases caused by hematopoietic stem cell transplantation.MicroRNAs(miRNAs)are a class of small non-coding RNAs,which could regulate gene expression by binding specific location 3'UTR of target gene,resulting in transcriptional inhibition or degradation.miRNAs play an important regulatory role in cell differentiation,proliferation and apoptosis.A study showed that miR-21could inhibit the expression of TGFBR2 and further regulate the role of TGF signaling in the differentiation of human adiposederived stem cells(hASCs).Recently,miRNAs have been reported as biomarkers for neurodegenerative diseases and affecting the central nervous system,and it is also found that miRNA in serum has the potential to be a diagnostic biomarker for AD.Some studies found that miR-21 is an important neurotranscription factor.Moreover,serum miRNA was found to be a potential diagnostic biomarker for AD.It has been reported that miR-21 is an important neuro-transcription factor,and its high expression contributes to the recovery of neurological function.Yet,the effect and mechanism of miR-21 in AD has not been reported.The purpose of this study is to investigate the effect and mechanism of miR-21mediated hAM-MSCs on AD.Firstly hAM-MSCs separated from placenta in newborn were used as the research material.The expression and its significance of miR-21 in hAM-MSCs and the AD mice were analyzed.miR-21 overexpression and interference lentivirus vectors were constructed.And then stably transfected hAM-MSCs cells were established,exploring the effects of miR-21 on hAM-MSCs cell proliferation,cells cycle,and apoptosis,as well as the effect of miR-21 mediated hAM-MSCs on expression of TNF-?,MCP-1 and IL-10 in peripheral blood mononuclear cells.Finally,APP transgenic mice model was applied to further study in vivo the effect of miR-21 on the migration of hAM-MSCs in the brains of AD mouse.Morris water maze assay was used to detect the effect of miR-21 on learning and memory abilities of the AD mice with hAM-MSCs transplantation.Meanwhile,the effects of miR-21 on expression of surface antigens of neurons and inflammatory factors in the hippocampus tissues of AD mice transplanted intravenously of hAM-MSCs were examined.This research was mainly divided into three parts:The first part:The expression and its significance of miR-21 in hAM-MSCs and AD mice;The second part:The effect of miR-21 on the biological characteristics of hAM-MSCs and miR-21-mediated hAM-MSCs immunoregulation;The third part:The effect of miR-21 on the migration of hAM-MSCs and expression of inflammatory factors in brain of AD mice.Main Content:The first part:The expression and its significance of miR-21 in hAM-MSCs and AD miceMethods1.hAM-MSCs were separated and cultured from the placental amnion membrane of enewborn.2.Flow cytometry assay was used to detect hAM-MSCs surface markers,and indirect immunofluorescence assay was used to detect surface antigens Vimentin and SSEA-4,identifying hAM-MSCs cells.3.qRT-PCR was used to detect the expression of miR-21 in amniotic epithelial cells and hAM-MSCs.4.qRT-PCR was used to detect the expression of miR-21 in hippocampus tissues of normal mice and AD mice.Results1.Flow cytometry analysis of the third-passage hAM-MSCs showed that the positive expression rates of CD29,CD44,CD73 and CD90 in hAM-MSCs were97.3%,96.3%,97.8%,and 98.2%,while the rates of CD34 and CD45 expression were only 0.6%and 0.84%,respectively.The immunofluorescence staining of hAM-MSCs cells showed that Vimentin and SSEA-4 were expressed in the cytoplasm of hAM-MSCs.These results showed that the high-purity hAM-MSCs with the characteristics of mesenchymal stem cells were successfully isolated.2.qRT-PCR results showed that the level of miR-21 in hAM-MSCs was significantly higher than that in amniotic epithelial cells,and the level of miR-21 in hippocampal tissues of AD mice was significantly lower than that of normal mice.The second part:The effect of miR-21 on the biological characteristics and miR-21-mediated hAM-MSCs immunoregulationMethods1.The construction of miR-21 overexpression lentiviral vector:The miR-21fragment was ligated on the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector,and then we got miR-21 overexpression lentiviral vector(pLent-miR-21).Colony PCR was used to identify the recombinant plasmid selected from individual bacterial colony cultures of the Escherichia coli.miR-21-positive clones were send to Shanghai ShengGong Biological Engineering Co.,Ltd.and were identified by sequence analysis.2.The construction of miR-21 interference lentiviral vector:The anti-miR-21fragment was ligated on the pLent-hU6-GFP-Puro vector,and we got miR-21interference lentiviral vector(pLent-miR-21).Colony PCR was used to identify the recombinant plasmid selected from individual bacterial colony cultures of the Escherichia coli.The anti-miR-21-positive clones were send to Shanghai ShengGong Biological Engineering Co.,Ltd.and were identified by sequence analysis.3.The recombinant lentivirus was packaged and the titer of the recombinant lentivirus was performed.4.Lentiviruses carring LV-anti-miR-21 or LV-miR-21 infected hAM-MSCs respectively,and we got stable hAM-MSCs-miR-21 and hAM-MSCs-anti-miR-21cells lines.5.The expression of miR-21 in hAM-MSCs,hAM-MSCs-miR-21 and hAM-MSCs-anti-miR-21 cells was detected by qRT-PCR.6.The effect of miR-21 on the proliferation of hAM-MSCs cells was detected by CCK-8 assay.7.Flow cytometry assay was used to detect the effect of miR-21 on cells cycle of hAM-MSCs cells.8.Flow cytometry assay was used to detect the effect of miR-21 on apoptosis in hAM-MSCs cells.9.The expression of TNF-?,MCP-1 and IL-10 in the co-culture supernatant of hAM-MSCs and peripheral blood mononuclear cells was detected by ELISA assay.Results1.PLent-miR-21 and pLent-anti-miR-21 colony PCR and sequencing results showed that the molecular size and sequence was in accordance with the reference sequence of NCBI.The pLent-miR-21 and pLent-anti-miR-21 vectors were established successfully.2.The titration value of LV-miR-21 and LV-anti-miR-21 lentivirus was determined as 2?10~8 TU/mL.The majority of hAM-MSCs infected with LV-miR-21or LV-anti-miR-21 lentivirus showed green fluorescence under fluorescence microscope,indicating a high efficiency of virus infection.3.Compared with hAM-MSCs group,the expression of miR-21 in hAM-MSCs-miR-21 cells was significantly increased,while the expression of miR-21 in hAM-MSCs-anti-miR-21 cells was decreased significantly,suggesting that the miR-21-overexpresing and-knockdown cells lines were constructed successfully.4.CCK-8 assay revealed that miR-21 overexpression significantly promoted the proliferation of hAM-MSCs cells,while miR-21 knockdown inhibited the proliferation of hAM-MSCs cells.5.Flow cytometry assay disclosed that compared with hAM-MSCs control group,the number of hAM-MSCs-miR-21 cells at S phase was significantly increased,while the number of s-phase hAM-MSCs-anti-miR-21 cells was significantly decreased.6.Flow cytometry assay showed that miR-21 overexpression significantly inhibited apoptosis in hAM-MSCs,while miR-21 downregulation significantly promoted apoptosis in hAM-MSCs.7.ELISA assay revealed that compared with human peripheral blood mononuclear cells(PBMC),hAM-MSCs significantly inhibited the secretion of TNF-?and MCP-1,promote the secretion of IL-10 in PBMC co-cultured with hAM-MSCs.Compared with PBMC co-cultured with hAM-MSCs,miR-21overexpression enhanced the inhibitory effect of hAM-MSCs on the secretion of TNF-?and MCP-1 in PBMC co-cultured with miR-21-hAM-MSC cells,and promotive effect on the secretion of IL-10 in PBMC co-cultured with miR-21-hAM-MSCs cells.miR-21 interference reduced the inhibitory effect of hAM-MSCs on the secretion of TNF-?,MCP-1,and promotive effect on the secretion of IL-10 in PBMC co-cultured with anti-hAM-MSCs cells.The third part:The effect of miR-21 on the migration of hAM-MSCs and expression of inflammatory factors in brain of AD miceMethods1.PCR was performed to identificate the transgenic mice used in the experiment.2.Intravenous transplantation of hAM-MSCs was carried out in AD mice,and the homing and survival of hAM-MSCs in the brain of mice were observed under fluorescence microscope.3.Morris water maze assay:Positioning navigation experiment and space exploration experiment were performed to assess the learning and memory abilities of AD mice.4.The expression of GFAP,Nestin and NSE in the hippocampal tissues of the AD mice was detected by immunohistochemistry staining,qRT-PCR and Western blot.5.Western blot was used to detect the expression of proliferation-(Ki67)and apoptosis-related proteins(caspase-3,Bax,Bcl-2)as well as the expression of TNF-?,MCP-1 and IL-10 in the hippocampal tissues of mice.Results1.Compared with hAM-MSCs group,the number of green fluorescent positive hAM-MSCs was increased significantly in brain of AD mice transplanted of miR-21-hAM-MSCs by tail vein,and green fluorescent positive hAM-MSCs first increased and then decreased with the prolongation of time.In addition,the number of green fluorescent positive hAM-MSCs was decreased significantly in brain of AD mice transplanted of anti-miR-21-hAM-MSCs cells by tail vein,compared with hAM-MSCs group,and the survival time of cells was shorter.These results revealed that overexpression of miR-21 could enhance the homing and survival of hAM-MSCs in the brain of AD mice.2.Compared with saline group,after intravenous transplantation of hAM-MSCs,the number of crossing platforms of AD mice was significantly increased,and percentage of time spent in platform quadrant of AD mice was significantly increased,and the escape latency was shortened.In addition,compared with hAM-MSCs group,after intravenous transplantation of miR-21-hAM-MSCs,the number of crossing platforms of AD mice was significantly increased,and percentage of time spent in platform quadrant of AD mice was significantly increased,and the escape latency was shortened.Moreover,after intravenous transplantation of anti-miR-21-hAM-MSCs,the number of crossing platforms of AD mice was significantly decreased,and percentage of time spent in platform quadrant was significantly decreased,and the escape latency was significantly increased.It was suggested that miR-21overexpression can effectively improve the learning and memory abilities of AD mice by hAM-MSCs transplantation via tail vein,which can improve AD symptoms of mice.3.Compared with saline group,after intravenous transplantation of miR-21-hAM-MSCs,the expression of Ki67 and Bcl-2 was significantly increased in the hippocampal tissues of AD mice,while the expression of Caspase-3 and Bax was significantly decreased.After intravenous transplantation of miR-21-hAM-MSCs,the expression of Ki67 and Bcl-2 was significantly increased in the hippocampal tissues of AD mice,compared with control group,while the expression of Caspase-3 and Bax was significantly decreased.Moreover,the levels of Ki67 and Bcl-2 were significantly decreased in the hippocampal tissues of AD mice transplanted of anti-miR-21-hAM-MSCs cells by tail vein,whereas the expression of Caspase-3 and Bax were significantly increased.4.Immunohistochemical staining,qRT-PCR and Western blot analyses showed that compared with saline group,the expression of GFAP,Nestin and NSE in hippocampal tissues of AD mice transplanted of hAM-MSCs by tail vein was significantly elevated.In addition,miR-21 overexpression promoted the expression of GFAP,Nestin and NSE in hippocampal tissues of AD mice transplanted of hAM-MSCs by tail vein.However,miR-21 interference significantly inhibited the expression of GFAP,Nestin and NSE in hippocampal tissues of AD mice transplanted of hAM-MSCs by tail vein.5.Compared with saline group,in hippocampal tissues of AD mice transplanted of hAM-MSCs by tail vein,the expression of TNF-?and MCP-1 was significantly reduced,while the expression of factor IL-10 was significantly increased.Moreover,in the hippocampal tissues of AD mice transplanted of hAM-MSCs via tail vein,miR-21 overexpression significantly inhibited the expression of TNF-?and MCP-1,and promoted the expression of factor IL-10.miR-21 downregulation significantly promoted the expression of TNF-?and MCP-1,while the expression of IL-10 was significantly decreased in hippocampal tissues of AD mice transplanted of hAM-MSCs by tail vein.Conclusion1.The hAM-MSCs with high purity were obtained in the study.The expression of miR-21 in hAM-MSCs was significantly increased than that in human amniotic epithelial cells,and the expression of miR-21 in hippocampal tissues of AD mice was significantly decreased,compared with normal mice.2.miR-21 overexpression significantly promoted the proliferation of hAM-MSCs cells,increased the cell proportion of S phase,and inhibited apoptosis.In addition,miR-21 overexpression enhanced the inhibitory effect of hAM-MSCs on the secretion of TNF-?,MCP-1,and promotive effect on the secretion of IL-10 in PBMC co-cultured with hAM-MSCs.miR-21 interference inhibited the proliferation of hAM-MSCs cells,promoted its apoptosis,and decreased the proportion of cells at S stage.Moreover,miR-21 interference reduced the inhibitory effect of hAM-MSCs on the secretion of TNF-?,MCP-1,and promotive effect on the secretion of IL-10 in PBMC co-cultured with hAM-MSCs.3.Animal studies showed that miR-21 overexpression enhanced the positive effect of transplantation of hAM-MSCs by tail vein on the expression of Ki67 and Bcl-2 in hippocampal tissues of AD mice,and also strengthened the inhibitory effect of transplantation of hAM-MSCs by tail vein on the expression of Caspase-3 and Bax.miR-21 overexpression enhanced the positive effect of transplantation of hAM-MSCs by tail vein on the expression of GFAP,Nestin and NSE in hippocampal tissues of AD mice,which might promote nerve repair and regeneration.In addition,miR-21overexpression could enhanced the inhibitory effect of transplantation of hAM-MSCs by tail vein on the expression of proinflammatory factor TNF-?and MCP-1 in hippocampus tissues of AD mice,and promotive effect on the expression of inflammatory factor IL-10,which might reduce the local inflammatory response and neuronal injury,effectively improve the learning and memory ability of AD rats,and improve AD symptoms of mice. |
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