Study On The Effect Of MicroRNA-138 Regulating Bone Marrow Mesenchymal Stem Cell Transplantation In The Treatment Of Pelvic Floor Dysfunction Diseases In Rats

Pelvic floor dysfunction(PFD)refers to the physiological and anatomical changes related to the dysfunction of pelvic floor muscles,ligaments and fascia.The main manifestations of PFD are pelvic organ prolapse(POP),stress urinary incontinence(SUI),chronic pelvic pain,sexual dysfunction and fecal incontinence.Recently,it has been found that Bone Marrow Mesenchymal Stem Cells(BMSCs)with multidirectional differentiation and secretory tissue factor function play an important role in the repair and regeneration of damaged tissues and have become seed cells of tissue engineering.fibulin-5(FBLN5)is a member of the family fibulin extracellular matrix proteins widely distributed in tissues rich in elastic fibers.Studies have reported that PFD models can be constructed by knockout FBLN5 genes,which further shows that they play a vital role in the development of PFD.This study found that,micro RNA-138(mir-138)can target to FBLN5 and inhibit the expression of elastin.In vitro experiments show that,Suppressing mir-138 expression increases BMSCs activity,And can improve the ability of BMSCs to secrete elastin.In addition,build PFD rat model,the effects of BMSCs transplantation with different treatments on pelvic floor dysfunction in rats were evaluated by urodynamic tests such as leak point pressure(LPP)and conscious bladder pressure test(CMG)..Turns out,inhibition of mir-138 expression in BMSCs can improve LPP and CMG experimental results in rats.Therefore,considering the combination of mir-138 and biological tissue engineering may be a potential therapeutic target for therapeutic PFD.ObjectiveThrough changing the expression of mir-138 in BMSCs,the proliferative activity of BMSCs was analyzed by CCK-8 experiment,and investigate the regulation effect of mir-138 on BMSCs.Subsequently,PFD rat model was established and injected through its tail with different treated BMSCs,using leak point pressure(LPP)and sober bladder pressure test(CMG)to evaluate the efficacy of BMSCs transplantation in the treatment of PFD rat model.Further provide certain experimental basis and theoretical basis for PFD clinical treatment.Materials and methods1.Density gradient centrifugation was used to collect rat BMSCs,cell adhesion was used to purify and subculture,morphological changes were observed by inverted microscope,and surface specific antigen was identified by flow cytometry.and induce its adipogenic osteogenic differentiation to identify its multidirectional differentiation potential.2.mir-138 target genes were predicted by target gene prediction library,and double luciferase reporter gene was used to detect the correctness of target gene.3.Liposome 2000 was transfected into rat BMSCs mir-138mimics、mimics NC、mir-138inhibitor、inhibitors NC,the transfection efficiency was detected by Western blot,the proliferative activity of BMSCs was detected,and the expression of mir-138、FBLN-5、elastin in transfected cells was detected by RT-q PCR and Western blot.4.PFD rat model was established.A random selection of 6 month old female SD rats as control group(Twelve in the group),Six month old female SD rats were randomly divided into 4groups of 12 animals each,500μL of a solution containing 8×10~5BMSCs or modified BMSCs were injected into via tail:(1)PFD+saline group;(2)Group PFD+BMSCs;(3)Group PFD+BMSCs antagomir-NC;(4)Group PFD+BMSCs mir-138antagomir.Treatment until day 7,The conscious bladder pressure test(CMG)and leak point pressure test(LPP)were carried out in the above five groups of experimental animals.Results1.Separation and purification of BMSCs.by density gradient centrifugation and cell adhesion Microscopically,the adherent cells were vortex-like and slender.the positive rates of CD29、CD44、CD73 and CD90 expression of isolated mesenchymal stem cells were as high as 99.56%,98.24%,72.38%and 99.45%,respectively,and the negative expression CD45 hematopoietic cell markers was detected by flow cytometry.The cells can be induced into adipocytes and osteoblasts.2.Dual luciferase reporter gene assay analysis demonstrated that mir-138 can targeted inhibit FBLN5.3.The results of PT-q PCR and western blot show that,after mir-138 mimics transfection,The m RNA and protein expression of endogenous FBLN5 in BMSCs were obviously inhibited;while the expression of FBLN5 m RNA and proteins in transfected mir-138 inhibitor was significantly increased.In addition,CCK-8experiments show that,Transfection mir-138 mimics inhibited BMSCs proliferation,The BMSCs proliferation ability of transfection mir-138 inhibitor was enhanced.Compared to mimics NC,After the transfection of mir-138 mimics,FBLN5 and elastin expression levels were significantly reduced.However,high expression of FBLN5 can reverse the inhibition of mi R-138.Therefore,mir-138 can be shown to target BMSCs FBLN5 to inhibit elastin expression,affect BMSCs proliferation.4.PFD rat model was successfully established.Compared with saline-treated PFD rats,There was little difference in bladder pressure peak and LPP between PFD+BMSCs groups,while the bladder pressure peak,bladder void volume and LPP of rats in PFD+BMSCs mir-138 antagomir group increased significantly.The expression of elastin and FBLN5 increased in both PFD+BMSCs and PFD+BMSCs antagomir-NC groups,and the PFD+BMSCs mir-138 antagomir group increased more obviously than the first two groups.These results indicate that,mir-138antagomir transfected BMSCs transplantation can significantly alleviate the PFD symptoms of rats.Conclusion1.Inhibition of mir-138 expression can promote elastin expression by targeting FBLN5 and enhance BMSCs proliferative activity.2.The urodynamic results of PFD rats can be effectively improved by transplantation of mir-138 antagomir modified BMSCs.