| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 91 === 參、 英文摘要
Cholesterol is an essential component of animal cell membranes and maintains cell growth. Cell acquires cholesterol through endogenous biosynthesis or exogenous uptake. The rate-limiting reaction of the endogenous biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase). Regulation of the expression of HMG CoA reductase is by several intermediate products in the cholesterol biosynthetic pathways. Zebrafish (Danio rerio) has become a popular animal model for studying development, genetics and human diseases. We are interested in role of HMG CoA reductase in zebrafish embryo cholesterol homeostasis. We first isolated a zebrafish cDNA encoding the HMG CoA reductase involved in cholesterol biosynthesis. An HMG CoA reductase cDNA of 3066 bp, containing an open reading frame encoding 884 amino acids, shares 87﹪identical amino acids to human HMG CoA reductase. The predicted HMG CoA reductase protein consists of an amino-terminal membrane-bound region that predicted to span the endoplasmic reticulum membrane six times, and 2 N-glycosylation sites. There are two functional domains: SSD domain and catalytic domain. In order to determine which tissue and zebrafish embryo development stage expressing the HMG CoA reductase mRNA, we prepared total RNAs from adult zebrafish tissues and various stages. By using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis indicates that zebrafish HMG CoA reductase mRNA is expressed all the tissues, the liver expresses one of the highest levels of this enzyme. Zebrafish HMG CoA reductase mRNA is expressed gradually increased from 24 to 96 hpf stages. The HMG CoA reductase mRNA was detected in the zebrafish embryonic liver cell line (ZF-L). Regulation of HMG CoA reductase mRNA in the ZF-L cells was tested by lovastatin, compactin (competitive inhibitor of HMG CoA reductase), mevalonate (HMG CoA reductase product) and 25-hydroxycholesterol (25-OHC) (suppressor of cleavage of SREBPs). Reductase mRNA levels measured in extracts from ZF-L cells cultured in the presence of 25-OHC and mevalonate were found to be virtually unchanged as compared to control condition. Similar experimental with lovastatin and compactin, failed to show a drug-induced change in mRNA level. The results provide a ground-stone for further analysis the potential role of HMG CoA reductase in zebrafish and ZF-L cells cholesterol homeostasis.
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