EFFECT OF CATIONIC CHOLESTEROL DERIVATIVES ON GENE-TRANSFER AND PROTEIN-KINASE-C ACTIVITY
EFFECT OF CATIONIC CHOLESTEROL DERIVATIVES ON GENE-TRANSFER AND PROTEIN-KINASE-C ACTIVITY
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DOI：
10.1016/0005-2736(92)90316-e
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发表时间：
1992-11-09
期刊：
影响因子：
--
通讯作者：
HUANG, L
中科院分区：
文献类型：
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作者：
FARHOOD, H;BOTTEGA, R;HUANG, L
合成了四种不同的胆固醇阳离子衍生物，它们含有叔胺基或季胺基，有或没有琥珀基间隔臂。在洗涤剂混合胶束溶液中测定了它们抑制蛋白激酶C （PKC）活性的能力。含有季氨基头基的衍生物是有效的抑制剂(K(i)约。12和59 μ m)的PKC和衍生物含有叔氨基头基约。抑制性降低4-20倍。脂质体含有二油基磷脂酰乙醇胺（DOPE）和阳离子胆固醇衍生物的等摩尔混合物，测试了小鼠L929细胞dna介导的转染活性。具有较低PKC抑制活性和琥珀基间隔臂的衍生物活性最高。叔胺衍生物N，N-二甲基乙基内二胺基琥珀酰胆固醇的转染活性依赖于作为辅助脂质的DOPE；含有二油基磷脂酰胆碱及其衍生物的脂质体活性较低。从脂质体/DNA比、复合物剂量和细胞孵育时间等方面对该阳离子脂质体试剂的转染方案进行了优化。该脂质体试剂可有效转染多种贴壁细胞系，且无明显细胞毒性。然而，转染活性受到血清成分的强烈抑制。
Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (K(i) approx. 12 and 59 muM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethyl-enediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.