Dichek D A, Bratthauer G L, Beg Z H, Anderson K D, Newman K D, Zwiebel J A, Hoeg J M, Anderson W F
Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Somat Cell Mol Genet. 1991 May;17(3):287-301. doi: 10.1007/BF01232823.
We have achieved in vivo expression of recombinant low-density-lipoprotein (LDL) receptors in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for the human disease familial hypercholesterolemia. A retroviral vector was constructed containing the human LDL receptor cDNA and was used to stably transduce primary skin fibroblasts from WHHL rabbits. The integrity and function of the introduced LDL receptor was established by immunoprecipitation, by a fluorescent LDL binding assay, and by the ability of the transduced cells to suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase activity in response to exogenous cholesterol. Autologous transduced fibroblasts were reimplanted into donor rabbits; in vivo LDL receptor expression and the survival of the transduced cells were analyzed by immunohistochemistry and by LDL binding assays performed on cells recovered from the implants. LDL receptor-bearing cells could be identified on tissue sections and recovered from implants for up to four weeks. Total and LDL cholesterol levels decreased significantly after implantation of the transduced cells; however, control experiments indicated that the decreases were not mediated through the recombinant LDL receptor. While in vivo stable expression of recombinant LDL receptors in Watanabe rabbits is possible, consequent changes in lipid levels must be interpreted with caution. This system of site-specific in vivo expression of recombinant LDL receptors permits further evaluation of the role of LDL receptor-gene replacement in the therapy of hypercholesterolemia.
我们已在渡边遗传性高脂血症（WHHL）兔体内实现了重组低密度脂蛋白（LDL）受体的表达，该兔是人类疾病家族性高胆固醇血症的动物模型。构建了一种包含人LDL受体cDNA的逆转录病毒载体，并用于稳定转导WHHL兔的原代皮肤成纤维细胞。通过免疫沉淀、荧光LDL结合试验以及转导细胞响应外源性胆固醇抑制3-羟基-3-甲基戊二酰辅酶A还原酶活性的能力，确定了导入的LDL受体的完整性和功能。将自体转导的成纤维细胞重新植入供体兔体内；通过免疫组织化学以及对从植入物中回收的细胞进行LDL结合试验，分析体内LDL受体的表达和转导细胞的存活情况。在组织切片上可以识别出带有LDL受体的细胞，并且在长达四周的时间内都可以从植入物中回收这些细胞。植入转导细胞后，总胆固醇和LDL胆固醇水平显著降低；然而，对照实验表明，这种降低并非通过重组LDL受体介导。虽然在渡边兔体内稳定表达重组LDL受体是可能的，但脂质水平的相应变化必须谨慎解释。这种重组LDL受体的位点特异性体内表达系统允许进一步评估LDL受体基因替代在高胆固醇血症治疗中的作用。
