两种AFP阳性肝癌细胞专一基因表达调控序列的构建  

Construction of Two AFPpositive Hepatoma CellspecificGene Regulatory Sequences

作　　者：许德华[1] 戈凯[1] 郑仲承[1] 刘新垣[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《生物化学与生物物理学报》1998年第4期363-368,共6页

摘　　要：构建了由人胰蛋白酶抑制剂基因的增强子（称为ＡＴ）和大鼠ａｆｐ基因启动子与沉默子（称为ｒＰＳ）组成的杂合基因表达调控序列（命名为ＡＴｒＰＳ）；还构建了１．２ｋｂ的由大鼠ａｆｐ基因增强子ＩＩ和ｒＰＳ组成的调控序列（命名为ｒＡＦＰ）。将报告基因－氯霉素乙酰转移酶基因（ｃａｔ）置于这些序列控制之下，构建成ＣＡＴ表达载体Ａ－ＴｒＰＳ－ｐＣＡＴ和ｒＡＦＰ－ｐＣＡＴ。在甲胎蛋白（ＡＦＰ）阳性的肝癌细胞或ＡＦＰ阴性的肝癌细胞、肝细胞以及非肝细胞中检测ＣＡＴ活性。结果，在ＡＦＰ阳性的肝癌细胞中两种载体都能产生ＣＡＴ活性，而在ＡＦＰ阴性的肝癌细胞、正常肝细胞和非肝细胞中均无活性，特别是在原代细胞中也得到相同结果。从而证实这些基因表达调控序列具有ＡＦＰ阳性肝癌细胞的专一表达活性。如果将ｒＡＦＰ缩短至０．６７ｋｂ，它仍然保持了很好的细胞专一性及较高的活性。Two chimerical regulation sequences for gene expression, one (called ATrPS) harboring an enhancer of human α1antitrypsin gene (AT) and a promoter and silencer ( rPS) of rat afp gene, and another (called rAFP) consisting of enhancer III of rat afp gene and its rPS , were constructed, respectively. Then, two CAT expression vectors, rAFPpCAT and ATrPSpCAT, were constructed in which the cat reporter gene was put under the control of these elements, respectively. CAT activities could were detected in the AFP positive liver cancer cells and also in those liver cancer cells, liver cells or nonliver cells whose AFP were negative. Results showed that for both constructs, the CAT activities could be found in all AFP positive hepatoma cells, however, while this activity can not be detected in all AFP negative cells. Similar results were observed in primary cell cultures. The results showed that our regulation elements of gene expression really possessed celltype specificity for AFP positive cells. The celltype specificity remains when the length of rAFP was cut short as to 0.67 kb; and the activity seemed higher. These regulatory sequences of gene expression may be used in gene therapy for primary liver cancer.

关 键 词：基因表达 调控序列 AFP 肝癌 基因治疗 

分 类 号：Q78[生物学—分子生物学] R730.59[医药卫生—肿瘤]