Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.
Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.
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通过丝氨酸蛋白酶抑制剂复合物受体将基因转移到肝癌细胞系中。
DOI：
10.1152/ajpgi.1997.273.2.g545
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Davis,PB
中科院分区：
文献类型：
--
作者：
Ziady,AG;Perales,JC;Ferkol,T;Gerken,T;Beegen,H;Perlmutter,DH;Davis,PB
在肝细胞上表达的丝氨酸蛋白酶抑制剂酶复合物受体（SECR）与α 1-抗胰蛋白酶（α 1-AT）和其它丝氨酸蛋白酶抑制剂中的保守序列结合。由基于人α 1-AT的SECR结合基序共价偶联到聚-L-赖氨酸的合成肽（C1315）组成的分子缀合物用于将报告基因引入培养的肝癌细胞系中。该缀合物将DNA浓缩成直径为18-25 nm的球状颗粒。当用含有pGL 3的SECR定向复合物转染时，表达受体的Hep G2细胞，而不是不表达受体的Hep G2细胞，在转染后4天表达538，731 +/-144，346整合光单位/mg蛋白的峰值荧光素酶活性。游离肽以剂量依赖性方式抑制摄取和表达。与聚赖氨酸或LC-磺基-N-琥珀酰亚胺基-3-（2-吡啶二硫基）丙酸酯取代的聚赖氨酸缩合的DNA复合物无效。用编码人因子IX的质粒转染在表达SECR的Hep G2（高）和HuH 7细胞中产生表达，但在缺乏受体的Hep G2（低）细胞中不产生表达。用C1315肽标记的Hep G2（高）、HuH 7（10-14%）和Hep G2（低）细胞的比例分别为9-31%、0.6-3.4%，当转染lac Z基因时，只有这些细胞表达β-半乳糖苷酶。SECR介导的基因转移系统具有高效、特异性的摄取和报告基因的高水平表达，值得进一步研究。
The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.