The Role And Mechanism Of Smooth Muscle Progenitor Cell Promotes Smooth Muscle Regeneration In Tissue-engineered Bladder

Chapter 1 IntroductionThe development of tissue engineering technology and introduction to the medical field has brought great hope to transplantation medicine and surgical reconstruction in the 21st century.In the past two decades,a great breakthrough has been made in the field of tissue engineering bladder research.Bladder smooth muscle plays an important role in the storage and urination function of urinary bladder.In current,there is still lack of ideal seeds for bladder smooth muscle cells.For such a purpose,this topic was performed under the supervision of Dr.Dai Yutian to seek an ideal seed cells to promote regeneration of bladder smooth muscle,blood vessels and recovery of bladder function.In addition,the possible mechanism of this progress was explored.Chapter 2 Isolation,culture and identification of smooth muscle progenitor cellsIn this study,rabbit peripheral blood mononuclear cells(PBMNC)were separated by density gradient centrifugation,cultured in EGM-2 medium added with platelet-derived growth factor-BB(PDGF-BB)and transforming growth factor-?1(TGF-?1),and induced to differentiate into smooth muscle progenitor cells(SPCs).Rabbit bladder smooth muscle cells(RBSMCs)were isolated and cultured from rabbit bladder by enzymatic method.SPCs were identificated by means of flow cytometry,immunofluorescence and real-time PCR technology.RBSMCs were served as positive control.One single cell assay was conducted to evaluate the proliferation potential of SPCs.In addition,contraction activity of SPCs was assessed by stimulating SPCs with carbachol.In the results,SPCs and BSMCs were successfully isolated and cultivated.SPCs were satined positive for SMA,Desmin,Calponin,SM22 and SMMHC,which are characteristic markers of smooth muscle cell.In addition,SPCs could express stem/progenitor cell markers-KDR,CD 133,CD29 and CD90.Furthermore,gene expression of smooth muscle has also been detected.SPCs maintain self-renew ability of progenitor cells.Carbachol induced contraction capability of SPCs is further evidence that SPCs has the characteristics of smooth muscle cells.In conclusion,the cultured SPCs with high proliferation potential and high purity,possessing the characteristics of smooth muscle cells,could be used as cell sources for tissue engineering bladder.Chapter 3 Smooth muscle progenitor cells cultured from peripheral blood promote proliferation,migration and scrape wound closure of bladder smooth muscle cellsSPCs and RBSMCs isolated and cultured as the protocols of previous chapter underwent serum starvation for 24h.Then the supernatant of SPCs and RBSMCs was collected and neutralized with PDGF-BB and TGF-?1 antibodies.RBSMCs proliferation,migration and scrape wound closure assay were conducted to explore the role of SPCs on RBSMCs.In addition,qualification of PDGF-BB in the supernatant of SPCs and RBSMCs was detected by enzyme linked immunosorbent assay(ELISA),while expression of PDGF-B mRNA in SPCs and RBSMCs was evaluated by real-time PCR.In the results,SPCs supernatant could significantly promote proliferation,migration and wound closure ability of RBSMCs than RBSMCs supernatant(p<0.05).After SPCs supernatant neutralized with PDGF-BB,the promotion effect was obviously inhibited.There was no obvious influence on the promotion effect after SPCs supernatant neutralized with TGF-?1.Amount of PDGF-BB secreted by SPCs was siginificantly higher than that of RBSMCs(p<0.05).PDGF-B mRNA expression level of SPCs was also siginificantly higher than that of RBSMCs(p<0.05).This study showed that SPCs could promote proliferation,migration and wound closure ability of RBSMCs through secreting PDGF-BB by paracrine method.This result further supports the application of SPCs as cell source in tissue engineering.Chapter 4 In vitro evaluation of fluorescent dye labeled smooth muscle progenitor cells and in vitro construction of SPC-BAMSPCs were isolated and cultured from the peripheral blood of New Zealand Rabbit,and were labeled with CM-DiI.Labeling rate was detected with fluorescence microscope and flow cytometry study.After cells cultured on coverslips,indirect immunofluorescent staining was performed to characterize the cultured cells using monoclonal antibody against alpha-smooth muscle actin(a-SMA),Calponin and Proliferating Cell Nuclear Antigen(PCNA).The impact of CM-Dil on the growth of SPCs were examined by trypan blue exclusion,cell adhesion assay,cell proliferation assay and cell migration assay.In addition,labeled SPCs were seeded into porcine BAM to construct cell-matrix complex in vitro.Growth state of SPCs into porcine BAM was detected by histological staining.In the results,SPCs began to growth after four days of culture and clones emerged after one week.The labeling rate of SPCs labeled with CM-Dil was about 96%as detected with fluorescence microscope and flow cytometry study.After continuous passage culture for two weeks,the labeling rate was more than 40%.In indirect immunofluorescent staining,the labeled SPCs showed positive staining for?-SMA,Calponin and PCNA.There was no decrease of cell survival rate of labeled RSPCs.The labeled SPCs still had the equal capability of adhesion,proliferation and migration with normal SPCs.After seeded on porcine BAM,labeled SPCs were found to growth quickly into BAM by histological staining.PDGF-BB could promote the ingrowth of SPCs in dose-dependent pattern.In conclusion,CM-DiI can effectively and easily label SPCs cultured in vitro.CM-DiI has no impact on cell phenotype and cell capability of adhesion,proliferation and migration of SPCs.Labeled SPCs could growth quickly into BAM.PDGF-BB has a promoting effect on SPCs ingrowth in dose-dependent pattern.Chapter 5 Functional and histological recovery of tissue engineering bladder reconstructed by bladder acellular matrix seeded with smooth muscle progenitor cellsIn this study,we used porcine BAM with well-preserved bioactive factors,high porosity and appropriate pore size as scaffold material,and used SPCs that were isolated and cultured from rabbit peripheral blood as cell source,to reconstruct partial bladder of rabbit.We observe the effect of SPCs on the tissue regeneration and functional recovery of tissue engineering bladder.In the control group,the rabbits received bladder partial replacement with the BAM scaffold only.Our results showed that the urothelium was multilayered in the marginal zone and central zone of tissue engineered neo-bladder in both the control and the experimental group at 1 month after sugery.At each time point,the smooth muscle bundle density,microvascular density as well as regenerated nerve fiber in experimental group were significantly higher than that in the control group(p<0.05).However,there was no significant difference in regenerated nerve fiber between these two groups(p>0.05).The recovery of bladder function in the experimental rabbits was more excellent than that in the control ones at each time point after sugery(p<0.05).Moreover,SPCs were also directly involved in the regenerated bladder smooth muscle tissues.This research indicated that SPCs can be used as cell source for bladder reconstruction to promote smooth muscle regeneration,vascularization and nerve regeneration as well as functional recovery of tissue engineering bladder.Conclusion of this doctoral programIn this study,we successfully isolated and cultured SPCs from rabbit peripheral blood.Cultured SPCs were comprehensively identificated from cellular,protein and gene level,respectively.We found that SPCs can secret PDGF-BB,and then promote proliferation,migration and scrape wound closure of RBSMCs.CM-DiI can effectively and easily label SPCs cultured in vitro.Labeled SPCs could growth quickly into BAM.Cultured SPC-BAM can be used to construct urinary bladder.SPCs can be used as cell source for bladder reconstruction to promote smooth muscle regeneration,vascularization and nerve regeneration as well as functional recovery of tissue engineering bladder.In addition,SPCs can be directly involved in the regenerated bladder smooth muscle tissues.Furthermore,SPCs can promote bladder regeneration through paracrine method.