【作者】 沈振斌；

【导师】 秦新裕；

【作者基本信息】 复旦大学 ， 外科学， 2004， 博士

【摘要】 胆囊结石是一种常见病,在我国自然人群发生率约为10%左右,也就是1.3亿人左右。目前尚没有有效的预防方法,有症状后往往需要手术治疗,花费了大量的医疗资源。因此了解胆囊结石形成的机制有助于为预防和非手术治疗该疾病提供依据。由于胆固醇不能随意跨越肝细胞的胞膜,胆固醇的分泌又并非完全与磷脂的分泌耦连,位于肝细胞膜表面的胆固醇转运体可能是调节胆固醇分泌的关键环节。ABC跨膜转运体家族中的ABCG5和ABCG8在小肠和肝脏中有高表达,产物位于细胞膜形成管腔的一端,与胆固醇逆向转运有关,可能参与肝脏向胆汁排泄固醇的过程,两者的确切作用尚不十分清楚。本研究通过转基因的方法将小鼠ABCG5和ABCG8基因导入人肝肿瘤细胞系7721中,观察细胞胆固醇转运能力的改变,从而对上述基因功能作出判断。同时对胆固醇性结石病人中ABCG5和ABCG8与结石形成的关系作了初步的探讨。一、ABCG5和ABCG8在胆固醇转运中的作用验证ABCG5和ABCG8在胆固醇转运中的作用。通过脂质体介导将含ABCG5或ABCG5和ABCG8的质粒导入7721细胞系,通过抗生素G418、zeocin筛选获得新的细胞系7721-G5与7721-DG。用RT-PCR及Western blot等方法证明细胞系7721-G5与7721-DG中分别含有ABCG5,ABCG5和ABCG8。7721、7721-G5、7721-DG细胞系分别用[3H]标记的胆固醇、游离胆固醇、卵磷脂、无脂血清作用使细胞内胆固醇饱和,随后用牛磺胆酸钠/卵磷脂受体系统进行胆固醇外运实验。每60分钟测定培养液中[3H]标记的胆固醇含量计算外运的胆固醇数量。研究发现转染后含有ABCG5和ABCG8的7721-DG细胞系胆固醇转运能力明显高于其余两株细胞系,而仅含有ABCG5的7721-G5细胞系胆固醇转运能力无明显变化,提示ABCG5与ABCG8具有促进胆固醇外运的作用,两者必须形成异二聚体才能行使这种作用。此外,用myc和HA标记后并不影响ABCG5和ABCG8发挥功能。二、ABCG5和ABCG8与胆固醇性结石形成的关系 <WP=5>探讨ABCG5和ABCG8与结石形成的关系。测定结石组病人结石胆固醇含量,确定为胆固醇性结石。比较禁食情况下胆固醇性结石病人和胃癌病人中血脂、脂蛋白差异,采用实时PCR方法比较胆固醇性结石病人与胃癌病人ABCG5和ABCG8 mRNA的差异。两组病例血脂、脂蛋白比较无明显差异。胆固醇性结石组ABCG5/beta-actin和ABCG8/beta-actin均高于胃癌组(为17.6%+9.8%vs13.5%+6.2%,P<0.01;18.9%+7.1%vs16.5%+4.2%,P<0.01),说明胆固醇性结石病人在基础情况下ABCG5和ABCG8有较高的转录水平,意味着产生更多胆固醇跨膜转运蛋白。本实验提示胆固醇性结石的形成与肝脏ABCG5和ABCG8高表达,向胆汁中转运较多的胆固醇有关。小结:本研究认为ABCG5和ABCG8在肝细胞胆固醇外运中发挥重要作用,两者需形成二聚体行使功能。胆固醇跨膜转运是胆固醇向胆汁排泄的关键步骤,其速度决定结石的形成,由于ABCG5和ABCG8行使肝细胞跨膜转运胆固醇的功能,因此两者在肝细胞中高表达是结石形成的关键。更多还原

【Abstract】 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 <WP=7>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.更多还原