摘要
目的建立稳定自发绿色荧光的高、低转移人肝癌细胞株并明确鉴定。方法脂质体转染法将绿色荧光蛋白(GFP)基因分别转染至高、低转移人肝癌细胞株MHCC97-H／L内,经 G418反复筛选并单克隆化,得到两株新细胞株MHCC97-HG／LG,对新细胞株GFP表达稳定性、生物学特性、体内外侵袭转移能力及肿瘤血管生成等进行鉴定。结果 MHCC97-HG／LG在体外培养或裸鼠体内接种36 d后均能稳定表达绿色荧光。MHCC97-HG肝、肺转移率均为100％,较 MHCC97-LG及其父系体内外侵袭转移能力显著增强(P<0．01),其原位肿瘤微血管密度较 MHCC97-LG显著增加[(121．0±15．9)／HP vs(87．0±14．2)／HP,P<0．01]。体视荧光显微镜可实时观察裸鼠体内MHCC97-HG／LG种植瘤生长、转移及血管形成情况。结论 MHCC97-HG／LG及其动物模型在肝癌转移机制、肿瘤血管生成等诸多研究领域可能有较广阔的应用前景。
Objective To establish stable green fluorescent protein （GFP）-expressing metastatic human hepatocellular carcinoma （HCC） cell lines with different metastatic potentials for long-term in vivo studies of metastasis and angiogenesis. Methods pIRES2-EGFP vector containing an enhanced GFP gene was transfected into MHCC97-H and MHCC97L, HCC ceLl lines with different metastatic potentials. The stability of GFP expression, basic biological characteristics, invasion abilities in vitro and spontaneous metastasis in vivo of the new ceLl lines （MHCC97-HG and MHCC97-LG） were studied. Microvessel density （MVD） of orthotopic implanted tumors was compared by anti-CD31 immunohistochemical staining, and real-time angiogenesis and metastasis of GFP-transfected tumors were detected by intravital fluorescent microscopy. Results The GFP-transfected ceLl lines stably expressed green fluorescence in the absence of G418 over a 36-day period. Compared with the parental cell lines, they exhibited no distinct differences in biological characteristics. MHCC97-HG showed more aggressive invasion and spontaneous metastatic behaviors than MHCC97-LG, and even its parental cell line （P 〈 0.01）. MVD levels induced by MHCC7-HG orthotopic implanted tumors were significantly higher than MHCC97-LG [ （ 121.0 ± 15.9）/HP vs （87.0 ± 14.2） / HP, P 〈 0.01 ]. Real-time angiogenesis and sequential steps of metastasis could be detected dearly under intravital fluorescent microscopy. Conclusion These two stable GFP-expressing HCC cell lines could be useful models for monitoring metastasis and angiogenesis of HCC.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第5期531-533,i0001,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项自(30500594)
关键词
肝细胞癌
绿色荧光蛋白
转移
血管形成
Carcinoma, hepatocellular
Green fluorescent protein
Metastasis
Angiogenesis
