Study Of The Regulatory Effect Of MiRNA On The Regulating Function Of Bone Marrow-derived Mesenchymal Stem Cells From Osteoprosis

As a major cause of bone loss in human body, osteoporosis (OP),characterized by damaged bone structure, is a kind of chronicinflammatory bone disease and this kind of disease is regulated byimmunity system. In the field of oral therapy, OP brings so manydifficulties in dental restoration, implantation and oral-maxillofacialsurgery. Mesenchymal stem cells (MSCs) have received widespreadattention for their ability of immune regulation and multipotentdifferentiation. At present, there are limited research papers focusing onOP affecting the immune regulating ability of MSCs; instead, mostresearches focused on the abnormal multipotent differentiation of MSCslike osteogenesis or adipogenesis. Recent studies have found that throughbinding target gene, microRNAs (miRNAs) could regulate themultipotent differentiation of MSCs so as to play a crucial role in thedeveloping of OP. However, whether miRNAs could regulate the immune regulatory ability of MSCs in OP is unknown.Objectives:This study is aimed to explore whether the immunomodulatoryproperty of MSCs is changed under certain pathological circumstancesand whether its regulating pathway is regulated by certain miRNAs.Furthermore, authors studied that whether suppress target miRNAs couldreverse the deteriorated immune regulatory ability of MSCs so as todeepen the understanding of OP and the immunoregulation mechanism ofMSCs aiming to offer new aspects and ideas for the disease therapy inclinic.Methods:1.8-week-old female C57BL/6J mouse were obtained to set up OVXand sham animal models. Micro-CT was applied to detect whether themodelswere successfully built up. The surface antigen markers ofmesenchymal stem cells were detected by flow cytometer.O/BMMSCsand S/BMMSCs were compared by studying their differentiationpotiential in certain inducement conditions.2. O/BMMSCs and S/BMMSCs were obtained to treat colitis and thenthe curative effectswere evaluated. After that, both of the two kinds ofBMMSCs were co-cultured with T cells to detect the ability ofapoptosis and migration for T cells. What was more, ELISA kits wereused to detect the secretion of MCP-1by T cells after co-culturing. 3. To confirm the different expression of immuneregulation relatedgenesFas and FasL in two groups. Then mirRNA data base was usedto pick out specific miRNA which could combine with Fas and FasL.In this experiment, western blot, real time-PCR and luciferaseenzymes’activity were applied.4. To analyze the immuneregulation effect of MSCs by inhibiting Let-7athrough cell transfection. Mscs were injected through tail vein ofmouse in vivo and co-cultured with T cells in vitro. The same methodswere taken as mentioned above.Result1. OVX and sham group animal models were established successfullywhich were confirmed in the aspect of trabecular number, bonemineral density, bone volume fraction as well as trabecula platespacing. Normal mesenchymal stem cells surface markers weredetected both in O/BMMSCs and S/BMMSCs groups. Cells from bothgroups have been demonstrated to have the potential of differentiatinginto osteogenic or adipogenic tissues and the S/BMMSCs showed astronger osteogenesis capacity in addition with a weaker adipogenesiscapacity.2. S/BMMSCs showed a more promising therapeutic effect incolitis micein vivo and by co-culturing with them, T cells exhibited a strongerapoptosis and migration capability by secreting a higher MCP-1lever. 3. There is no significant difference of Fas and FasL expression betweenO/BMMSCs andS/BMMSCs groups by means of gene levels whileO/BMMSCs showed lower expression in the protein level. MiRNAdatabase showed that Let-7a could specifically combined with3’UTRof mRNA such as Fas and FasL. The expression of Let-7a ofO/BMMSCs was higher than that of S/BMMSCs. Real time-PCR、westernBlot and luciferase report have demonstrated Fas and FasLwere the targetgenes of Let-7a.4. When Let-7a wasdown-regulated, O/BMMSCs could promote thesecretion of MCP-1and further promote the migration and apoptosisof T cells, thus improving the therapeutic effect on colitis finally.Conclusions:O/BMMSCs down-regulate the immunoregulation ability through Tcells mediated apoptosis pathway, affecting therapeutic effect on colitis.Fas and FasL are regulated by miRNA, which is Let-7a. Down-regulationof Let-7a in O/BMMSCs has influence on the ability of apoptosis andmigration of T cells, immunoregulation and therapeutic effect ofO/BMMSCs.