CAJ | 학술논문

同时分离和培养兔外周血平滑肌祖细胞(SPC)和内皮祖细胞(EPC),为组织工程膀胱的构建和血管化提供种子细胞.分离新西兰兔外周血中的单个核细胞,分别进行SPC和EPC的分离和培养;同时,培养兔膀胱平滑肌细胞(BSMC)作为对照.细胞爬片,观察细胞摄取Dil-AcLDL和结合FITC-UEA-1的能力;间接免疫荧光染色观察平滑肌肌动蛋白(SMA)、结蛋白(Desmin)、KDR、eNOS和vWF的表达情况;进行细胞增殖实验,观察PDGF-BB,VEGF对SPC和EPC的增殖作用.结果发现,培养1周后出现SPC克隆和EPC克隆,SPC呈梭形,长短不一;EPC形态均一,呈典型的鹅卵石形;培养的BSMC形态均一,为长梭形,呈峰谷样形态.利用克隆环分离SPC和EPC克隆,继续培养可得到高纯度的SPC和EPC.SPC不摄取Dil-AcLDL,不结合FITC-UEA-1;SPC表达SMA、Desmin和KDR,不表达eNOS和vWF.EPC同时摄取Dil-AcLDL和结合FITC-UEA-1;EPC表达eNOS、vWF和KDR,不表达SMA和Desmin.BSMC仅表达SMA和Desmin.PDGF-BB仅能促进SPC增殖,VEGF仅能促进EPC增殖,均具有剂量依赖效应.本研究建立了同时分离和培养外周血SPC和EPC的技术,所培养的细胞具有较高的纯度,可以作为种子细胞进行组织工程膀胱及其血管化的研究和应用.
동시분리화배양토외주혈평활기조세포(SPC)화내피조세포(EPC),위조직공정방광적구건화혈관화제공충자세포.분리신서란토외주혈중적단개핵세포,분별진행SPC화EPC적분리화배양;동시,배양토방광평활기세포(BSMC)작위대조.세포파편,관찰세포섭취Dil-AcLDL화결합FITC-UEA-1적능력;간접면역형광염색관찰평활기기동단백(SMA)、결단백(Desmin)、KDR、eNOS화vWF적표체정황;진행세포증식실험,관찰PDGF-BB,VEGF대SPC화EPC적증식작용.결과발현,배양1주후출현SPC극륭화EPC극륭,SPC정사형,장단불일;EPC형태균일,정전형적아란석형;배양적BSMC형태균일,위장사형,정봉곡양형태.이용극륭배분리SPC화EPC극륭,계속배양가득도고순도적SPC화EPC.SPC불섭취Dil-AcLDL,불결합FITC-UEA-1;SPC표체SMA、Desmin화KDR,불표체eNOS화vWF.EPC동시섭취Dil-AcLDL화결합FITC-UEA-1;EPC표체eNOS、vWF화KDR,불표체SMA화Desmin.BSMC부표체SMA화Desmin.PDGF-BB부능촉진SPC증식,VEGF부능촉진EPC증식,균구유제량의뢰효응.본연구건립료동시분리화배양외주혈SPC화EPC적기술,소배양적세포구유교고적순도,가이작위충자세포진행조직공정방광급기혈관화적연구화응용.
The objective of this study was to simultaneously isolate and culture smooth muscle progenitor cell (SPC) and endothelial progenitor cell (EPC) from rabbit peripheral blood for use as cell sources in tissue engineering of bladder and vascularization. In this study, SPC and EPC were simultaneously isolated and cultured from the peripheral blood of New Zealand Rabbit, and rabbit bladder smooth muscle (BSMC) were cultured and used as smooth muscle cell positive control. The cells were cultured on coverslips and applied for uptake of Dil-AcLDL and binding of FITC-UEA-1. Indirect immunofluorescent staining was performed to characterize the cultured cells using monoclonal antibodies against smooth muscle actin (SMA) , Desmin, KDR, eNOS and vWF. Cell proliferation assay was also used to investigate the mitogenic effect of PDGF-BB and VEGF on both the SPC and EPC. Both the SPC and EPC clones emerged after one week of culture. The SPC displayed fusiform shape with different size. The EPC showed the cobblestone morphology typical of endothelial cells. The BSMC cultured from rabbit exhibited the elongated shape and hill and valleys pattern before confluence. The cluster of SPC and EPC were isolated and subcultured using cloning cylinders, after which the highly purified SPC and EPC had been obtained. The SPC display no ability of uptake of Dil-AcLDL and binding of FITC-UEA-1, while the EPC took-up Dil-AcLDL and showed FITC-UEA-1 binding affinity. Iri indirect immunofluorescent staining, the SPC demonstrated positive staining for SMA, Desmin and KDR, while negative staining for eNOS and vWF. The EPC showed positive staining for KDR, eNOS and vWF, while negative staining for SMA and Desmin. The BSMC stained only positive for SMA and Desmin. PDGF-BB had dose-dependent mitogenic effect on SPC, and VEGF had dose-dependent mitogenic effect on EPC. In conclusion, the highly purified SPC and EPC had been simultaneously isolated and cultured from rabbit peripheral blood, and can be used as cell sources for tissue engineering of bladder and vascularization.