摘要
目的 研究血管抑素血管他丁 (angiostatin)基因转染人肝癌细胞系HCC772 1,对肿瘤细胞体外生长、周期分布、形态以及体内致瘤力的影响 ,探讨血管抑素的作用机制。方法 应用定向克隆技术构建鼠源性血管抑素血cDNA基因真核表达载体 pcDNA3 .1( + ) angio ,酶切鉴定和测序。采用脂质体基因转染技术将真核表达载体导入人肝癌细胞系HCC772 1,新霉素G4 18筛选抗性克隆 ,设置转染空载体细胞为阴性对照和未转染细胞为空白对照。通过RNA点杂交和流式细胞荧光免疫检测血管抑素的表达 ,测定细胞生长曲线 ,计算细胞倍增时间 ,流式细胞仪检测细胞周期分布。建立动物模型 ,观察转染前后肿瘤细胞致瘤力的改变。免疫组化检测肿瘤组织微血管密度 (MVD)和血管抑素体内表达。结果 成功构建带有碱基序列正确的血管抑素基因片段的重组真核表达载体 pcDNA3 .1( + ) angio。分别转染脂质体 /pcDNA3 .1( + ) angio和脂质体 /pcDNA3 .1( + )的实验组 ,HCC772 1肝癌细胞和阴性对照组经G4 18筛选 ,3 0d后均得到稳定的抗性细胞克隆 ,空白对照组在筛选 1周后全部死亡。实验组HCC772 1细胞在体内和体外均表达目的蛋白 ,而对照组细胞中无表达。与对照组相比 ,虽然实验组细胞在体外的生长速度和细胞周期分布未发生明显改变 ,但在体?
Objective To study the effect of the mouse angiostatin cDNA gene transfecting into human liver cancer cell line HCC7721 on cell growth, cell cycle phase distribution, cell morphology in vitro and tumorigenesis in vivo , and the mechanisms. Methods The gene fragment of mouse angiostatin cDNA was directly cloned into an eukaryotic expression plasmid pcDNA3.1(+) of the promoter CMV between the multicloning sites HindⅢ and XbaⅠ and confirmed the correct recombinant plasmid pcDNA3.1(+) angio through enzymatic digestion and gene sequencing. Then it was transfected into human liver cancer cell line HCC7721 with liposome, pcDNA3.1(+) as vecter control and liposome as mock control. After 30 day selection by neomycin G418, angiostatin expression at the levels of mRNA and protein in vitro were tested by RNA dot blot and FACS respectively. Cell morphology under was observed light microscope, made growth curves were made, and cell cycle distribution checked in FACS. Animal model was set up to study tumorigenesis in vivo of those cells transplanted subcutaneously in the right hind legs of nude mice BALBc. Microvessel density (MVD) was analyzed and angiostatin expression in the tumor tissues by in situ immunohistochemistry. Results The recombinant eukaryotic expression plasmid pcDNA3.1(+) angio was constructed Auccessfully. After selection of G418 for about 4 weeks there were macroscopic cell clones in the experimental group and vector control group, but no survival cells in the mock control. In vitro angiostatin were expressed in the experimental group, but not in the vector control. There weren't significant changes in cell morphology, cell growth curves and cell cycle distribution between the experimental and the control group. However, the nude mice experiment showed that tumorigenic capability of the experimental cells had been reduced greatly. Immunochemistry study showed that there were much less microvessels and conversely stronger angiostatin positive staining in
出处
《中华消化杂志》
CAS
CSCD
北大核心
2001年第10期583-586,共4页
Chinese Journal of Digestion
基金
国家自然科学基金项目 ( 3980 0 15 6)
