| Objective Human induced pluripotent stem cells(hiPSC)were differentiated into neural stem cells(iNSC),and MSC / iNSC composite microsphere culture system was established.In the rat penile corpus cavernosal injection to treat erectile dysfunction which because of cavernouse never injury(CNI),further more observing the treatment effect and exploring the treatment mechanismand.To provide new cell therapy methods for the treatment of CNI ED and provide ideas for clinical transformation.Methods Part Ⅰ: characterization of neural stem cells(iNSC): human induced pluripotent stem cells(hiPSC)were differentiated into neural stem cells by germ system;the expression of neural stem cell markers was detected by immunofluorescence staining technology to verify the success of differentiation;Flow cell technology to identify the surface markers of frozen human adipose mesenchymal stem cells(hADSC);Green fluorescent protein(GFP)lentivirus was transfected with hADSC to facilitate the formation of composite microspheres to observe the distribution of stem cells;The distribution of stem cells was observed by confocal laser scanning microscope(CLSM);Observe the morphology of composite spheres cultured with different cell density,and count the diameter of cell spheres to provide the basis for cell density selection during treatment;Real-time quantitative polymerase chain reaction(RT-qPCR)detects the gene expression in the composite microspheres,detects the cytokine changes,and also provides a basis for cell density selection during treatment;Immunofluorescence(IF)technology to detect the expression of composite microsphere extracellular matrix and cytokines,to detect whether there is abundant protein expression;Cell scratch experiments were used to verify whether the conditioned medium promoted nerve cells and endothelial cell growth;The CCK8(Cell Counting Kit-8)method was used to test whether the conditioned medium was protected against the nerve cells and endothelial cells under hypoxia.Part Ⅱ: 24 male 10-week-old SD(Sprague Dawley)rats,randomly divided into 6 control groups and 18 model groups,which established a rat model of cavernosal nerve injury erectile dysfunction;After successful mold making,18 rats were randomly divided into three groups,injured group,hADSC cell microsphere treatment group,iNSC + hADSC composite cell pellets group,6 in each group,including the same volume of PBS,hADSC cell microsphere and iNSC + hADSC composite cell pellets,the injected cell pellets contained 1×106cells for each rat.After 28 days of administration,the intracavernosal pressure and the mean arterial pressure of the right common carotid artery were measured;Modified Masson trichrome staining method to detect the smooth muscle and collagen in rat penile tissue;Immunofluorescence staining technique detected α-smooth muscle actin(α-SMA)expression in rat penile tissue;Immunohistochemistry(IHC)technique evaluated neural nitric oxide synthase(nNOS)changes in rat penile tissue;nNOS expression in rat penile tissue was detected by western blotting technique(WB).ResultsPart Ⅰ: Differentiation and composite microsphere characterization of neural stem cells(iNSC):(1)Human induced pluripotent stem cells(iPSC)marked with mRuby2 gene,after adding small molecule Y-27632,the first angel cells showed significantly long spindle shape;on the third day,the undifferentiated hiPSC was observed;after the first suspension culture,the inner dense and smooth neural rings;the neural stem cells(iNSC)were formed after secondary suspension culture and adherent culture.(2)The results of stem cell marker detection by immunofluorescence technology showed that the neural stem cell marker protein Nestin(nestin)of the cells differentiated from hiPSC differentiation was positive,which confirmed the successful differentiation,and the stem cell pluripotency markers SOX2 and Oct4 were positive,indicating that the differentiated cells still had multi-directional differentiation potential.(3)The results of hADSC surface markers by flow cytometry showed that the surface antigens CD73,CD90,CD146,and CD45 and HLA-DR,indicating that hADSC met the identification criteria of mesenchymal stem cells,and the properties of the cells did not change after resuscitation by cryopreservation.(4)Under an inverted fluorescence microscope,the red fluorescent protein expressed by mRuby2 gene is shown as dark red;while hADSC transfected with GFP lentivirus appears as green,indicating successful lentiviral transfection.(5)At the number of 2000-5000 cells,respectively 201.88 ± 10.80 μ m,218.44 ±15.97μm,231.69±5.88,309.34±23.81μm,with increasing cell density on suspension.(6)After mixing iNSC and hADSC in 1:1 ratio(6 × 104/ml),the scanning results of the lower layer of laser confocal microscope showed that iNSC and hADSC stem cells were evenly distributed in the microsphere,and some similar cells clustered into cell clusters.(7)Compared with hADSC,VEGF,PLGF,HGF,b FGF,BDNF,GDNF,and the expression was stable at the composite microsphere density of 6 × 104/ml.(8)The immunofluorescence results of cytokines and extracellular matrix in the composite microspheres showed that the composite cell spheres contained rich extracellular matrix(collagen,fibronectin,lamectin)and cytokines(brain-derived nerve growth factor,etc.).(9)The cell scratch results show that the compound microsphere conditioned medium can significantly promote the growth of nerve cells(HT22)and endothelial cells(HUVEC).(10)Composite microsphere-conditioned medium can protect cells and reduce cell damage caused by hypoxia.Part Ⅱ:(1)The ICPmax / MAP ratio of rats in hADSC microspheres group and compound cell microspheres group was significantly higher than that of the injured group,and the ratio of compound cells was higher than that in hADSC microspheres group(P <0.05),and there was no significant difference with the normal group(P> 0.05),indicating that the compound cell microsphere could greatly improve the erectile function of rats.(2)Compared with the rats in the injured group,the smooth muscle / collagen ratio and α-SMA ratio in the hADSC microsphere group and compound cell microsphere group were significantly higher,indicating that the stem cell microspheres helped to restore the penile smooth muscle tissue of the damaged rats,while the ratio of compound cell microspheres was higher than that of the hADSC group(P<0.05),indicating that the treatment effect of compound cell microsphere was better.(3)Immunohistochemistry results show that compared with the injured group,the hADSC microsphere group and compound cell microsphere group,the number of nNOS increased significantly,indicating that the stem cell microbulb helps to restore the damaged rat penile nerve,and the ratio of compound cell microsphere group is higher than the hADSC microsphere group(P <0.05),indicating that the recovery effect is better.(4)Western blot results showed that compared with the damage group,nNOS protein expression in the two stem cell microsphere treatment groups was significantly increased,and nNOS protein expression in the compound cell microsphere group was higher than that in the hADSC alone group(P <0.05).Conclusions(1)The iPSC can successfully differentiate into iNSC and still has multidirectional differentiation potential;compound cell microsphere can enhance paracrine function,facilitate cell growth and improve the ability to cope with harsh growth environment.(2)Compound cell microsphere can improve the smooth muscle content of the penile tissue in rats with cavernosal nerve injury,significantly restore the damaged penile tissue,restore the endothelial and nerve function,and then improve the penile erectile function. |
PDF Full Text Request