|Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥3,200,000 (Direct Cost: ¥3,200,000)
Microsomal triglyceride transfer protein (MTP) is recently proposed to be an important protein during the assembly of plasma very low density lipoprotein (VLDL). We have previously shown that hepatic activity of MTP was significantly lower than that of rat and that hepatic activity of acyl Co A cholesterol acyltransferase (ACAT) was almost negligible in suncus, in which fatty liver was easily induced by starvation and was easily reversed by refeeding. These observations suggest that hepatic MTP activity may be regulated by hepatic content of cholesterol ester which is the product of ACAT.
In this study, we cloned the ACAT-1 cDNA from mouse liver mRNA. The cDNA was introduced into multiple cloning site of pAdCMV Link.1, which is called as pAdCMV mACAT. We confirmed the ACAT activity in the 293 cells, which was tansfected by the pAdCMV mACAT, suggesting the expression of active ACAT protein. We obtained the adenovirus vector containing ACAT gene (AdCMV-mACAT) by cotranfection of pAdCMV mACAT with AdCMV-LacZ DNA into 293 cells. In order to test the introduction rate of the DNA into suncus liver with use of the adenovirus, we injected the AdCMVLacZ to suncus. When 3.5x10ィイD111ィエD1 particles were injected to a suncus, the introduction rate to the liver was only 15%. When particle number was increased than 3.5x10ィイD111ィエD1, all of the an animals died on 2 days after injection. When we tried to transfect the adenovirus to the primary cultured suncus hepatocytes, the introduction rate was confirmed to be 100%. The activities of ACAT and MTP in the tansfected cells and the secretion rate of VLDL from the hepatocytes are under investigation.