表皮生长因子受体介导的多肽载体系统对人喉鳞状细胞癌的基因导入研究

Reporter genes transfer into human squamous cell carcinoma of larynx with a new epidermal growth factor receptor mediated non-viral GE7 polypeptide delivery system

目的 检测一种新的以头颈肿瘤细胞表面受体为靶向的多肽基因导入系统对人喉鳞状细胞癌 (简称鳞癌 )的基因导入效果。方法 链霉亲和素标记生物素 (labeledstreptavidinbiotin ,LSAB)法检测喉癌组织标本和培养的Hep2喉癌细胞表皮生长因子受体 (epidermalgrowthfactorreceptor,EGFR)的表达。建立人喉癌裸鼠移植瘤模型 ,分别构建绿色荧光蛋白标记基因和多肽载体系统、半乳糖苷酶报告基因和多肽载体系统四元复合物 ,分别进行报告基因和标记基因的体内外导入实验。结果 EGFR在 6 5 % (15 / 2 3)喉癌组织标本呈阳性表达。Hep2细胞的EGFR亦呈阳性表达。EGFR主要位于细胞膜 ,部分位于细胞质内。绿色荧光蛋白基因转染Hep2后 4 8h显微镜下可见荧光 ,72h达高峰 ,导入率大于 80 % (78/ 97)。β 半乳糖苷酶报告基因荷人喉癌裸鼠移植瘤瘤内转染 12h即表达蓝染 ,2 4h蓝染程度加深 ,72h仍有表达。 结论 EGFR广泛存在于人喉癌细胞 ,新的多肽基因导入系统借助于EGFR可靶向性将报告或标记基因导入体内外人喉癌细胞 ,并得到高效表达 ,为头颈鳞癌的基因治疗提供了一个新的靶向高效的基因导入系统。

Objective To evaluate the efficiency of marker gene and reporter gene transfection into human squamous cell carcinoma of larynx implanted in nude mice with a new EGFR mediated non-viral polypeptide gene delivery system. Methods Labeled streptavidin biotin(LSAB) was applied to examine over-expression of epidermal growth factor receptor(EGFR) on the squamous cell carcinoma of larynx. The complex of marker gene or reporter gene and gene delivery system was prepared and subsequently inoculated with Hep 2 cells or injected into laryngeal carcinoma imptanted on nude mice. Results Over-epression of EGFR was observed on inoculated Hep 2 cells and on 65% of laryngeal carcinoma specimen examined (15/23).Positive staining was located on the surface of the cells and some of them in the cytoplasma. Green fluorescence was observed on Hep 2 cells under fluorescent microscope 48 hrs after PEGFP-C1 gene transfection and became the strongest 72 hrs after tansfection.The rate of transfection was 80%(78/97). Blue staining was noticed 12 hrs after β-gal gene transfer and it became more remarkable 24 hrs after gene transfection with X-gal staining on the implanted human laryngeal carcinoma on nude mice. Conclusions Squamous cell carcinoma of head and neck over-expressed EGFR and a non-viral GE7 polypeptide gene delivery system could transfer exogenous genes into laryngeal carcinoma cells and tissues with high efficiency and targetability both in vitro and in vivo suggesting that GE7 would be applicable as a new targeted, high efficient gene delivery system to the gene therapy of squamous cell carcinoma of head and neck.