腺苷蛋氨酸诱导肝脏尿苷二磷酸葡萄糖醛酸转移酶活性的机制研究

发布时间：2018-08-04 14:20

【摘要】：新生儿高胆红素血症（neonatal hyperbilirubinemia，NHB）是新生儿时期的常见病，可引起多器官系统损害。尽快降低血清胆红素水平是治疗新生儿高胆红素血症的关键。目前临床常用的治疗有光疗、换血疗法、药物治疗三个方面的措施，三者各有利弊，故寻找一种能安全、经济、有效地降低血清胆红素水平的药物对治疗新生儿高胆红素血症具有突出意义。尿苷二磷酸葡萄糖醛酸基转移酶（UDP-glucuronosyltransferase，UGT）是胆红素结合的关键酶，可使脂溶性的非结合胆红素变成水溶性的结合胆红素而排出体外。 腺苷蛋氨酸（S-adenosyl-L-methionine，SAMe）是体内内源性物质，参与体内多种重要的生化反应，可作为底物参与合成半胱氨酸、牛磺酸、谷胱苷肽、辅酶A等重要物质，并作为基因供体或酶性诱导剂在人体组织三大代谢过程中起到转甲基、转硫基和丙氨化(多聚胺合成)作用。该药应用于临床已有多年，其适应证主要针对肝内胆汁郁积、肝炎高胆红素血症等黄疸，能有效降低血清结合胆红素水平，，近年来国内有报道称SAMe辅助治疗新生儿高胆红素血症有一定疗效。因此，我们从分子生物学的角度，分析SAMe对人胎肝细胞（LO2）的尿苷二磷酸葡萄糖醛酸转移酶mRNA表达和蛋白的生成、活性的影响。 第一部分 腺苷蛋氨酸诱导肝脏尿苷二磷酸葡萄糖醛酸转移酶mRNA表达的研究 目的 分别给予不同浓度、时间的SAMe干预LO2细胞，比较受干预的LO2细胞在尿苷二磷酸葡萄糖醛酸转移酶mRNA水平的变化，探讨SAMe对LO2细胞UGT的mRNA表达水平的影响及最佳的干预条件。 方法 体外培养LO2细胞，待细胞约70%铺满培养板底部，进行分组：⑴SAMe处理组，根据预实验结果加入SAMe使其终浓度分别为1、0.5、0.1mmol/L；⑵阳性对照组，予以肝酶诱导剂，终浓度为2mmol/L苯巴比妥＋5mmol/L尼可刹米；⑶阴性对照组，不作加药处理。加药培养24h、48h、72h、92h后收集细胞。通过荧光定量PCR检测各组的mRNA水平。 结果 LO2细胞的UGT1A1mRNA经一定浓度SAMe诱导后增加，其中以浓度为0.5mmol/L组最为明显，该组经SAMe诱导24h后细胞的UGT1A1mRNA开始增高（49.18±10.16，P0.05），48h达到高峰（130.69±9.23，P0.05），72h后明显下降（28.25±9.42，P0.05），此后未见明显变化；与肝酶诱导剂组比较，其诱导LO2细胞生成UGT1A1mRNA的量更多，作用时间更长（P0.05）。 结论 适当浓度的SAMe对LO2细胞的UGT1A1的mRNA表达有诱导生成作用，表达量及时效均比肝酶诱导剂好。 第二部分 腺苷蛋氨酸诱导肝脏尿苷二磷酸葡萄糖醛酸转移酶蛋白表达的研究 目的 分别给予不同浓度、时间的SAMe干预LO2细胞，比较受干预的LO2细胞在UGT蛋白表达及分泌水平的变化，探讨SAMe对LO2细胞的UGT的蛋白表达、分泌水平的影响及最佳的干预条件。 方法 体外培养LO2细胞，待细胞约70%铺满培养皿底部，进行分组：⑴SAMe处理组，根据预实验结果加入SAMe使其终浓度分别为1、0.5、0.1mmol/L；⑵阳性对照组，终浓度为2mmol/L苯巴比妥＋5mmol/L尼可刹米；⑶阴性对照组，不加药处理。加药培养24h、48h、72h、92h后收集细胞。通过western blot及ELISA检测各组的UGT1A1蛋白表达、分泌水平。 结果 LO2细胞的UGT1A1蛋白经SAMe诱导后合成和分泌增加，其中以浓度为0.5mmol/L组最为明显，该组经SAMe诱导24h后细胞的UGT1A1蛋白合成和分泌开始增高（49.18±10.16或526.18±5.21，P0.05），48h达到高峰（130.69±9.23或1278.22±9.38， P0.05），72h后明显下降（28.25±9.42或296.62±6.42，P0.05），此后未见明显变化；与肝酶诱导剂组比较，其诱导LO2细胞合成和分泌更多的UGT1A1蛋白，作用时间更长（P0.05）。 结论 适当浓度的SAMe对LO2细胞的UGT1A1蛋白的表达有诱导生成作用，表达量及时效均比肝酶诱导剂好。
[Abstract]:Neonatal hyperbilirubinemia (neonatal hyperbilirubinemia, NHB) is a common disease in the newborn period, which can cause multiple organ system damage. Reducing the level of serum bilirubin as soon as possible is the key to the treatment of neonatal hyperbilirubinemia. At present, the common clinical treatment is three aspects of phototherapy, change of blood therapy, and drug treatment, each of the three In order to find a safe, economical and effective drug to reduce the level of serum bilirubin, it is of great significance for the treatment of neonatal hyperbilirubinemia. Uridine two phosphate glucuronotransferase (UDP-glucuronosyltransferase, UGT) is the key enzyme of bilirubin binding, which can make the fat soluble non binding bilirubin into water soluble. In combination with bilirubin, the body is discharged from the body.
S-adenosyl-L-methionine (SAMe), an endogenous substance in the body, participates in a variety of important biochemical reactions in the body and can be used as a substrate for the synthesis of important substances such as cysteine, taurine, glutathione, coenzyme A and so on as a gene donor or enzyme inducer in the three major metabolic processes of human tissues. It has been used in clinical practice for many years. Its indications are mainly aimed at intrahepatic cholestasis, hyperbilirubinemia and other jaundice, which can effectively reduce the level of serum bilirubin. In recent years, it has been reported that SAMe AIDS in the treatment of neonatal hyperbilirubinemia. The effects of SAMe on the expression of uridine diphosphate glucuronosyltransferase mRNA, protein production and activity in human fetal hepatocytes (LO2) were analyzed.
Part one
Expression of glucuronosyltransferase mRNA in uridine two phosphate induced by adenosylmethionine
objective
The effect of SAMe on the level of mRNA expression of UGT in LO2 cells and the optimal intervention conditions were investigated by giving SAMe LO2 cells with different concentrations and time respectively, and comparing the changes in the level of uridine two phosphorylglucuronase mRNA in the interfered LO2 cells, and the effect of SAMe on the mRNA expression level of UGT in LO2 cells.
Method
LO2 cells were cultured in vitro, and about 70% of the cells were PVE at the bottom of the culture plate to be grouped into groups: (1) SAMe treatment group, and the final concentration was 1,0.5,0.1mmol/L according to the result of pre experiment. (2) positive control group, the liver enzyme inducer was given, the final concentration was 2mmol/L phenobarbital + 5mmol/ L nibmeter, and (3) negative control group, no dosing treatment. After collecting 24h, 48h, 72h and 92h, the cells were collected, and the mRNA level of each group was detected by fluorescence quantitative PCR.
Result
The UGT1A1mRNA of LO2 cells increased after a certain concentration of SAMe, among which the concentration of 0.5mmol/L was the most obvious. After SAMe induced 24h, UGT1A1mRNA began to increase (49.18 + 10.16, P0.05), 48h reached the peak (130.69 + 9.23, P0.05). After 72h (28.25 + 9.42, P0.05), there was no obvious change thereafter; and the liver enzyme inducer group In comparison, the amount of UGT1A1mRNA induced by LO2 cells was more, and the time of action was longer (P0.05).
conclusion
Appropriate concentration of SAMe could induce the expression of UGT1A1 mRNA in LO2 cells, and the expression and aging time were better than those of liver enzyme inducers.
The second part
Expression of glucuronosyltransferase protein in hepatic uridine two phosphate induced by adenosylmethionine
objective
The effects of SAMe on the expression of UGT protein and the secretion level of the UGT in LO2 cells were compared with the changes in the level of UGT protein expression and secretion of the interfered LO2 cells. The effect of SAMe on the expression of UGT in LO2 cells and the best intervention conditions were given.
Method
LO2 cells were cultured in vitro, and about 70% of the cells were PVE at the bottom of the culture dish to be grouped into groups: (1) SAMe treatment group, and the final concentration was 1,0.5,0.1mmol/L according to the result of pre experiment. (2) positive control group, the final concentration was 2mmol/L phenobarbital + 5mmol/L Nike brake; (3) negative control group, no addition treatment. Adding drug to culture 24h, 48h, 72h, 9. The cells were collected after 2H. The expression and secretion levels of UGT1A1 protein were detected by Western blot and ELISA.
Result
The synthesis and secretion of UGT1A1 protein in LO2 cells were increased after SAMe induction, among which the concentration of 0.5mmol/L was the most obvious. After SAMe induced 24h, the UGT1A1 protein synthesis and secretion began to increase (49.18 + 10.16 or 526.18 + 5.21, P0.05), 48h reached the peak (130.69 + 9.23 or 1278.22 + 9.38, P0.05), and 72h after 72h (28.25 + 9.42) Or 296.62 + 6.42, P0.05), there was no obvious change since then, compared with the liver enzyme inducer group, it induced LO2 cells to synthesize and secrete more UGT1A1 protein, and the action time was longer (P0.05).
conclusion
Suitable concentration of SAMe could induce the expression of UGT1A1 protein in LO2 cells, and the expression and time-effect were better than those of liver enzyme inducer.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1

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