荧光标记肿瘤转移体内模型的建立

目的 建立一种带荧光标记的肿瘤转移模型并探讨其应用价值.方法 人胃癌MGC-803细胞和小鼠宫颈癌U14细胞进行体外传代培养,转染pEGFP-N1质粒,24h检测转染效率,用无限稀释法筛选稳定表达绿色荧光蛋白(GFP)的单克隆细胞株MGC-803-GFP和U14-GFP.分别体内移植于BALB/c-nn裸鼠和C57BL/6J小鼠,观察成瘤潜伏期,绘制体内生长曲线,利用活体荧光成像系统活体连续观察GFP在体内的表达,观察肺部和淋巴结的转移情况,计算转移率.免疫组织化学检测与转移相关的分子CD44和E-cadherin在移植瘤中的表达.结果 MGC-803细胞和U14细胞pEGFP-N1转染24 h后的转染效率分别为30%和60%.获得了GFP(+)的单克隆MGC-803-GFP细胞株和U14-GFP细胞株.MGC-803-GFP在BALB/c-nu裸鼠和U14-GFP在C57BL/6J小鼠移植成瘤的潜伏期分别是3～5 d和2～4 d,成瘤率均为100%.利用活体荧光成像系统连续观察,可以清楚直观地观察表达GFP的MGC-803-GFP移植瘤在体内的生长,接种后第60天处死全部荷瘤鼠,解剖后仅有1只可见同侧腋窝下淋巴结的转移.接种U14-GFP后,分别在第28、37和52天观察了荷瘤小鼠肿瘤转移的发展过程.在U14-GFP原发瘤体积达到≥5 cm3时,肺部和淋巴结转移率分别为67%和100%.在MGC-803-GFP和U14-GFP的移植瘤中都可以检测到CD44的表达,而无E-cadherin的表达.结论 成功建立了表达绿色荧光蛋白单克隆细胞株MGC-803-GFP和U14-GFP,体内移植建立了荧光标记的肿瘤转移模型.该模型可用于可视化肿瘤体内的研究.

Establishment of green-fluorescent protein expressing tumor metastasis models

Objective To establish a green-fluorescent protein (GFP) labeled tumor metastasis model and to evaluate its biological characteristics.Methods Human gastric carcinoma cell MGC-803 and murlne cervical carcinoma cell U14 were transfected with the plasmid pEGFP-N1 and the efficiency of transfection was assessed 24 h Iater.Limited dilution was employed to screen and establish menoclonal cell strains,MGC-803-GFP and U14-GFP.The two fluorescent tumor cell stains were transplanted into BALB/c-nu mice and C57BL/6J mice respectively.The latency period of tumor mass appearance and the growth curve in vivo were documented.The tumor growth and metastasis were evaluated in vivo by the Viviperception Fluorescence Imagining System(VFIS).Expressions of CD44 and E-cadherin in tumor tissue were monitored by immunohistochemistry.Results The efficiency of pEGFP-N1 transfeetion of MGC-803 cells and U14 cells were 30%and 60%,respectively.Monoclonal GFP(+)cell strains-MGC-803-GFP and U14-GFP were established.The latency periods of tumor formation of MGC-803-GFP and U14.GFP were 3-5 days and 2-4 days,respectively.Their tumorigenicity rates were 100%in both.The tumor growth of MGC-803-GFP was well defined by the VFIS. Only one mouse was shown to harbor lymphatic metastasis by VFIS.60 days after transplantation.The metastasis process of U14-GFP wag depicted through VFIS on 27.37 and 52 days post-transplantation.The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor vo]ume was ≥5 cm3. CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry.Conclusions Successfully established two monoclonal tumor cell strains stably expressing GFP:MGC-803-GFP and U14-GFP.Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.