CAJ | 학술논문

采用荧光染料CM-DiI标记兔外周血平滑肌祖细胞(SPC),评价其生物可行性.分离和培养 SPC,采用 CM-DiI进行标记,通过荧光显微镜和流式细胞仪检测标记效果和传代后的细胞标记率.同时,进行细胞爬片培养后免疫荧光染色,观察标记后细胞表达平滑肌肌动蛋白(α-SMA)、钙调节蛋白(Calponin)和增殖细胞核抗原(PCNA)的情况.进行台盼蓝排斥实验、细胞粘附实验、细胞增殖实验和细胞迁移实验,观察标记后的 SPC存活率以及粘附、增殖和迁移能力的变化.结果发现,培养4 d左右可见 SPC开始生长,一周后出现 SPC 克隆.通过免疫荧光和流式细胞术分析,CM-DiI标记率为96%左右,连续传代2周后标记率仍在40%以上.标记后的细胞可表达α-SMA、Calponin和PCNA.同时发现,标记后的细胞存活率无明显降低,细胞粘附能力、增殖能力和迁移能力无明显改变.本研究证明采用 CM-DiI 标记 SPC 操作简便,效果好,不改变细胞表型,不影响细胞的存活率和粘附、增殖及迁移能力,可作为 SPC的荧光示踪剂.
채용형광염료CM-DiI표기토외주혈평활기조세포(SPC),평개기생물가행성.분리화배양 SPC,채용 CM-DiI진행표기,통과형광현미경화류식세포의검측표기효과화전대후적세포표기솔.동시,진행세포파편배양후면역형광염색,관찰표기후세포표체평활기기동단백(α-SMA)、개조절단백(Calponin)화증식세포핵항원(PCNA)적정황.진행태반람배척실험、세포점부실험、세포증식실험화세포천이실험,관찰표기후적 SPC존활솔이급점부、증식화천이능력적변화.결과발현,배양4 d좌우가견 SPC개시생장,일주후출현 SPC 극륭.통과면역형광화류식세포술분석,CM-DiI표기솔위96%좌우,련속전대2주후표기솔잉재40%이상.표기후적세포가표체α-SMA、Calponin화PCNA.동시발현,표기후적세포존활솔무명현강저,세포점부능력、증식능력화천이능력무명현개변.본연구증명채용 CM-DiI 표기 SPC 조작간편,효과호,불개변세포표형,불영향세포적존활솔화점부、증식급천이능력,가작위 SPC적형광시종제.
Smooth muscle progenitor cell(SPC)is a novel cell source with the capability of proliferating,migrating,and differentiating into smooth muscle cell.In our previous study,SPC had been successfully isolated from the peripheral blood of New Zealand Rabbit and demonstrated the potential of being used as a smooth muscle cell source for tissue engineering of urinary bladder.However,it is important and necessary to investigate whether SPCs can survive,pro-liferate,migrate,differentiate into smooth muscle cells and integrate into the host tissue after they are seeded into a scaffold and implanted for bladder augmentation.Cell tracer technique may help resolve this problem,which can be used to trace the cell fate after transplantation.It is important to select an appropriate cell tracer with a high labeling efficiency but without the side effects on cells.CM-DiI is such a fluorescent dye that has being used widely for cell la-beling in tissue engineering.Therefore,the obj ective of this study was to evaluate the feasibility of using CM-DiI as a tracer to label SPC cultured from rabbit peripheral blood.In this study,SPCs were isolated and cultured from the pe-ripheral blood of New Zealand Rabbit and labeled with CM-DiI according to the manufacturer’s instructions.Labeling rate was evaluated by fluorescence microscope and flow cytometry study.After cultured on coverslips,SPCs were detected by indirect immunofluorescent staining using monoclonal antibody against alpha-smooth muscle actin(α-SMA),Calponin and Proliferating Cell Nuclear Antigen(PCNA).The impact of CM-DiI on the growth of SPC was examined by trypan blue exclusion,cell adhesion,cell proliferation and cell migration assays.Our results demonstrated that SPCs emerged after four days of culture and could formed clones after one week in culture.The labeling rate using CM-DiI was about 9 6% as detected with fluorescence microscope and flow cytometry study.After continuous passage culture for two weeks,the labeling rate was still greater than 40%.In indirect immunofluorescent staining,SPCs showed positive staining forα-SMA,Calponin and PCNA after labeling.There were no significant differences in cell survival,adhesion,proliferation or migration between labeled and unlabeled SPCs.In conclusion, CM-DiI labeling didn’t change the phenotypes of SPCs.No side effects on the capabilities of SPCs adhesion, proliferation or migration were noted.Our results support the use of CM-DiI as an effective and safe cell tracer in SPC-based tissue engineering of bladder.