CAJ | 학술논문

目的用红色荧光染料PKH26标记人脐带间充质干细胞(hUCMSCs),观察PKH26标记的hU CMSCs在肝硬化大鼠体内的迁移情况. 方法 hU CMSCs经复苏培养后,用PKH26对细胞进行标记,测定标记率和单次标记后荧光可检测时长;显微镜下观察标记后细胞形态,四甲基偶氮唑盐法测定细胞生长曲线.将PKH26标记的hU CMSCs通过大鼠尾静脉分别移植给正常大鼠和肝硬化大鼠;48 h后取肝组织做冰冻切片,荧光显微镜下观察hUCMSCs在大鼠体内的迁移情况.数据采用完全随机设计两组均数t检验进行分析. 结果 PKH26对hUCMSCs的标记率为100％,标记后细胞生长状态良好,细胞形态与未标记细胞无明显差别,均为长梭形成纤维样细胞;两组细胞各时间点的吸光度值的差异亦无统计学意义(P＞0.05),细胞增殖未见明显影响;第3～5天细胞生长速度最快,为对数生长期,在对数生长期中,对照组的细胞倍增时间为(2.22±0.04)d,标记组的细胞倍增时间为(2.20±0.04)d,两组的差异无统计学意义(P=0.53).体外单次标记后荧光至少可维持20 d;移植给大鼠后,PKH26标记的hUCMSCs主要分布于硬化肝脏的汇管区、血管及假小叶周围,脾脏、肺亦有少量分布.结论 PKH26是标记hUCMSCs的理想荧光染料,PKH26标记技术可用于hU CMSCs移植治疗肝硬化时的细胞示踪研究.
목적 용홍색형광염료PKH26표기인제대간충질간세포(hUCMSCs),관찰PKH26표기적hU CMSCs재간경화대서체내적천이정황. 방법 hU CMSCs경복소배양후,용PKH26대세포진행표기,측정표기솔화단차표기후형광가검측시장;현미경하관찰표기후세포형태,사갑기우담서염법측정세포생장곡선.장PKH26표기적hU CMSCs통과대서미정맥분별이식급정상대서화간경화대서;48 h후취간조직주빙동절편,형광현미경하관찰hUCMSCs재대서체내적천이정황.수거채용완전수궤설계량조균수t검험진행분석. 결과 PKH26대hUCMSCs적표기솔위100％,표기후세포생장상태량호,세포형태여미표기세포무명현차별,균위장사형성섬유양세포;량조세포각시간점적흡광도치적차이역무통계학의의(P＞0.05),세포증식미견명현영향;제3～5천세포생장속도최쾌,위대수생장기,재대수생장기중,대조조적세포배증시간위(2.22±0.04)d,표기조적세포배증시간위(2.20±0.04)d,량조적차이무통계학의의(P=0.53).체외단차표기후형광지소가유지20 d;이식급대서후,PKH26표기적hUCMSCs주요분포우경화간장적회관구、혈관급가소협주위,비장、폐역유소량분포.결론 PKH26시표기hUCMSCs적이상형광염료,PKH26표기기술가용우hU CMSCs이식치료간경화시적세포시종연구.
Objective To observe the in vivo migration of human umbilical cord mesenchymal stem cells (hUCMSCs) labeled with the PKH26 red fluorescent dye after transplantation into rats with liver cirrhosis.Methods Frozen hUCMSCs were resuscitated and labeled with PKH26.Labeling efficiency and fluorescent maintenance time of the PKH26-1abeled cells were measured.Morphology of the labeled and unlabeled (control) cells was observed by microscopy.The cell growth curve was determined using the MTT method.The PKH26-1abeled hUCMSCs were transplanted via tail vein injection into healthy (control) rats and rats with liver cirrhosis.Migration of the PKH26-1abeled hUCMSCs observed 48 h later in frozen liver sections under a fluorescence microscope.Results The labeling ratio of PKH26 to hUCMSCs was 100％.Growth of the labeled cells was good.The cell morphology was not significantly different between the labeled and unlabeled cells; all cells were long and spindlelike.Cell proliferation was not impacted significantly by labeling.Fluorescence was maintained for at least 20 days,as detected by in vitro analysis.After transplantation into the rats,the PKH26-1abeled hUCMSCs were mainly distributed in the area surrounding the portal vein,the blood vessels,and the false lobule of the cirrhotic liver;,a small amount ofhUCMSCs were present in the spleen and lung.Conclusion PKH26 is an ideal fluorescent dye to label hUCMSCs.The PKH26 labeling technique can be used to study the migration of hUCMSCs in cirrhotic liver.