【作者】 赵慧茹；

【导师】 刘德敏； 于德民；

【作者基本信息】 天津医科大学 ， 生物化学及分子生物学， 2006， 硕士

【摘要】 目的:葡萄糖-6-磷酸酶(glucose-6-phosphatase,G-6-Pase)是糖代谢过程中一个重要的酶,由于它是糖异生和糖原分解的最后一步反应的限速酶,因此其基因表达水平及活性的变化直接影响到内生性糖的输出。事实上,2型糖尿病的高血糖同肝脏胰岛素抵抗所致肝糖过度输出有着紧密的关系。因此G-6-Pase是糖尿病治疗的一个潜在靶点。本实验旨在研究栎精,G-6-Pase的一种新型抑制剂,对该酶基因表达和活性的影响,为进一步探讨利用该酶的抑制剂作为糖尿病的治疗手段提供理论依据和实验基础。 方法:以胶原酶原位灌注消化的方法分离大鼠肝细胞,用含10%FBS的1640培养液进行原代培养,利用抗大鼠CK-18作为一抗,采用免疫组化方法对细胞进行鉴定。待细胞贴壁换用新鲜的含诱导剂和不同浓度抑制剂的培养液培养16小时后:1)提取细胞总RNA,采用半定量RT-PCR的方法测定G-6-pase两个亚基的mRNA的丰度;2)利用葡萄糖脱氢酶偶联反应测定酶的活性。 结果:1.采用以胶原酶原位灌注消化的方法分离的肝细胞产率及活率均比较高。细胞活率可达到85%以上。 2.免疫组化染色结果呈阳性着色(有阴性对照)。 3.大鼠肝细胞在高糖培养液(25mM Glu)中培养16小时后,G-6-Pase催化亚基和转运亚基mRNA的丰度和酶活性均明显增加。(P<0.05) 4.加入不同浓度的栎精与高糖联合培养16小时后,G-6-Pase的两个亚基的mRNA的丰度均下降(P<0.05),但没有呈现剂量依赖性的变化。 5.酶活性测定的结果与基因水平的结果相一致,加入栎精后,酶活性下降(P<0.05)。更多还原

【Abstract】 Objective:Glucose-6-phosphatase (G-6-Pase) is an important enzyme in glucose metabolism. As a rate-limiting enzyme of the last reaction of glyconeogenesis and glycogenolysis, the change of G-6-Pase in gene expression and activity will affect the output of endogenic glucose directly. Infact, insulin resistance in liver contributes to the excessive hepatic output of glucose, which is highly correlated with hyperglycemia in type 2 diabetes. So, G-6-Pase is a potent therapeutic molecular target of diabetes. The purpose of this experiment is to study the effect of quercetin on G-6-Pase gene expression and activity and provide some theoretic and exprimental basement for developing the new type medicine for DM. Methods:The hepatocytes were isolated from adult Wistar rat by perfusion of the liver with collagenase . The isolated cells were cultured in 1640 with 10% FBS. We select anti-rat CK-18 as primary antibody to identify these cells by immunohistochemistry. After cell attachment ,the hepatocytes were incubated with 25mM glucose and different concentration of quercetin for 16 hours, then 1) total RNA was extracted and levels of mRNA expression of P36 and P46 were measured by RT-PCR;2) the enzymatic activity was detected by glucose dehydrogenase-coupled reaction. Result:1. The yield and the percentage viability of the isolated cell by perfusion of the liver with collagenase are good. The percentage viability of the isolated cell is about 85%.2. The result of immunohistochemistry is positive(with negative control).3. After cultured with 25mM glucose for 16 huors, both mRNA of P36 and P46 and activity of G-6-Pase of rat hepatocyte increased significantly (p<0.05).4. Afrer incubated in medium cantaining different concentration of quercetinand glucose , the mRNA of two summits of G-6-Pase decreased (P<0.05), but it wasn’t decreased depending on the concentration of quercetin.5. Quercetin can decrease the activity of G-6-Pase (p<0.05), which is concordant with the result of gene level.Conclusion:It has a good yield and percentage viability of the isolated cell by perfusion of the liver with collagenase. Both the mRNA level of the main component of G-6-Pase P36 and p46 increased in different extent after cultured with glucose, which accord with our design. Different concentration of quercetin can inhibit the mRNA of P36 and P46, but it isn’t decreased depend on the concentration of quercetin. Quercetin can also inhibit the activity of G-6-pase (p<0.05),which is concordant with the result of gene level.更多还原