The Research On Myocardin Gene-modified ASCs Transplantation To Restore ED In Diabetic Rats

BACKGROUND&OBJECTIVEStem cell transplantation therapy has been shown to have efficient curative effect on erectile dysfunction(ED)in multiple animal models and clinical studies.However,several questions were left to be answered.Firstly,the long-term effect of stem cells were not confirmed.Secondly,the transplanted cells were reported to flow away.Thirdly,more accurate method for tracking the trace of the injected stem cells were required.Finally,the underlying mechanisms were not clarified.The aim of this study was to explore the influence of Myocardin(Myocd)on Adipose-derived stem cells(ASCs)proliferation,apoptosis and differentiation.Subsequently,the Myocd gene-modified ASCs were intracavernously injected in diabetes mellitus ED(DMED)rats,aiming to prolong the residence time of the transplantation,as well as to enhance the therapeutic effect.Besides,In vivo small animal imaging was introduced for the first time to track the ASCs.METHODSFor the cell experiments,we isolated,cultured and identified the ASCs from rats,and constructed the recombinant adenovirus carrying both Luciferase and Myocd genes.Subsequently,we researched into the transfection effect of Myocd on ASCs proliferation,apoptosis and differentiation using CCK-8,EdU,Annexin V and Collagen I cell contractility assay.Confocol were applied to confirm the co-localization of Myocd and SRF,together with the fluorescence intensity of smooth muscle contractile proteins a-SMA,Calponin and the stem cell sternness marker SOX2,OCT4.For the animal studies,DMED rats were randomly devided into 4 groups,control rats,DMED plus ASCs + Ad-Luc-Myocd,DMED plus ASCs + Ad-Luc,and DMED plus PBS.Simutalneously,the in vivo small animal imaging and EdU cell tracking assay were performed to detect the transplanted ASCs.In addition,the ICP/MAP test,H&E,Massons' staining,TUNEL assay,immunohistochemistry,western blots were carried out for the examinations of the erectile function,the morphologic features,the smooth muscle and collagen content,and the apoptosis.RESULTSIn vitro,the isolated ASCs were identified with more than 99%expression of CD29 and CD90 using flow cytometry,and the abilities to differentiate into mature adipocytes and osteoblasts.CCK-8 and EdU showed reduced proliferative rate,and Collagen I cell contractility assay indicated enhanced contractile capacity in Myocd-tranferred cells.No diversity in apoptosis were observed between the two groups by Annexin V assay.PCR and Western blots suggested elevated a-SMA,Calponin,SRF and declined PCNA,SOX2,OCT4 mRNA and protein level.Consistently,overexpression of Myocd increased the fluorescence intensity of a-SMA,Calponin,SRF and decrease PCNA,SOX2,OCT4.Furthermore,Myocd and SRF were co-localized in the nucleus.In vivo,the modeling rate of DMED rats was 57.14%8 weeks after receiving streptozotocin injection.In vivo small animal imaging and EdU cell tracking assay detected the transplanted ASCs retaining in the penis within 21 days,along with higher expression of the modified cells.Meanwhile,Myocd promoted the stem cell therapeutic effect on saving the penile morphologic features,ameliorating erectile function by up-regulating ?ICP/MAP,as well as elevating a-SMA,Calponin,Bcl-2 and decreasing Collagen I,Bax,Cleaved-caspase3/caspase3.ASCs treatment also increased the protein expression of SDF-1 in the corporeal body.CONCLUTIONMyocd was capable of inhibiting ASCs proliferation,enhancing cell contractility,and inducing ASCs differentiating into the smooth muscle-like cells in both molecular expression and cell function,which was probably through both activating SRF and suppressing SOX2,OCT4.In vivo small animal imaging was more accurate in tracking stem cells after intracavernous transplantation.Gene-modified stem cells of Myocd were more efficient on ameliorating DMED,saving the penile morphological features,increasing smooth muscle content,while decreasing the Collagen expression and inhibiting cell apoptosis.In particular,the ascendant SDF-1 expression by ASCs therapy may imply its potential of recruiting and activating endogenous stem cells to the impared CCSM.