The Role Of ABCG5 And ABCG8 In Liver Cholesterol Transport And The Formation Of Cholesterol Gall Stone

Gallbladder stone is such a common and costly disease that it happens in ten percent of Chinese people. That is about 130,000,000 people. Till now there is no effective method for preventing its occurrence. When symptoms appear, operation will be the best choice. Researches in the mechanism of gallbladder stone formation will provide great understanding in preventing and treating the disease. Cholesterol is not able to across the liver cell plasma membrane freely. Its secretion is depend on the secretion of phospholipid. But their secretion are not tightly coupled. Cholesterol secretion may be finely tuned by transporters on plasma membrane, which determines the formation of lithogenesis bile. ABCG5 and ABCG8 are new members of ABC transporter family, which are highly expressed in intestine and liver. They located in canalicular membrane and involve in cholesterol reverse transport. They may act as transporters in sterol secretion into bile, but their exact function still need to be determined. In this research, ABCG5 cDNA and ABCG8 cDNA were transfected into 7721 cell line alone or together. The changes of cholesterol efflux were observed to evaluate the role of ABCG5 and ABCG8. The relationship between expression of ABCG5 and ABCG8 in cholesterol gall stone patients and stone formation is established.The role of ABCG5 and ABCG8 in cholesterol transportTo determine the role of ABCG5 and ABCG8 in liver cholesterol transport, mouse ABCG5 cDNA alone or ABCG5 and ABCG8 cDNA together were transfected into 7721 cell line through lipofectamine 2000. New cell lines 7721-G5 containing mouse ABCG5 cDNA and 7721-DG mouse ABCG5 and ABCG8 cDNA outcame through G418 and zeocin screening. The existence of ABCG5 and ABCG8 were proved by RT-PCR and Western blot. 7721, 7721-G5,7721-DG cells were all saturated with free cholesterol and radioactive labeled [3H]-cholesterol. Cholesterol efflux began when cells cultured with taurocholate sodium/egg PC acceptors. Every 60 minutes aliquots of medium were detected for [3H]-cholesterol by liquid scintillation techniques. 7721-DG cells showed increased cholesterol efflux than other two kinds of cells, while 7721-G5 cells had little change in cholesterol efflux. That means ABCG5 and ABCG8 functions as cholesterol transporters and promote cholesterol efflux under the circumstance they form heterodimer. Besides, ABCG5 or ABCG8 tagged with myc or HA won't change its function much.The relationship between ABCG5, ABCG8 and cholesterol gall stone formationTo establish the relationship between ABCG5, ABCG8 and cholesterol gall stone formation, components of stones were analysed to identify cholesterol stone. Serum lipids and lipoproteins were compared in cholesterol gall stone group and gastric cancer group. There are no obvious differences. ABCG5 and ABCG8 mRNA were detected in both two groups by real-time PCR. ABCG5/beta-actin and ABCG8/beta-actin were elevated obviously in cholesterol gall stone group. (17.6%+9.8%vs13.5%+6.2%, P<0.01; 18.9%+7.1%vs16.5%+4.2%, P<0.01) ABCG5 and ABCG8 are relatively highly induced in gall stone group and more cholesterol transporters are produced to secrete cholesterol. In this research it shows that cholesterol gall stone formation is the result of high expression of ABCG5 and ABCG8 in liver and high secretion of cholesterol into bile.