【摘要】：新生兒高膽紅素血癥（neonatal hyperbilirubinemia，NHB）是新生兒時期的常見病，可引起多器官系統(tǒng)損害。盡快降低血清膽紅素水平是治療新生兒高膽紅素血癥的關鍵。目前臨床常用的治療有光療、換血療法、藥物治療三個方面的措施，三者各有利弊，故尋找一種能安全、經(jīng)濟、有效地降低血清膽紅素水平的藥物對治療新生兒高膽紅素血癥具有突出意義。尿苷二磷酸葡萄糖醛酸基轉移酶（UDP-glucuronosyltransferase，UGT）是膽紅素結合的關鍵酶，可使脂溶性的非結合膽紅素變成水溶性的結合膽紅素而排出體外。 腺苷蛋氨酸（S-adenosyl-L-methionine，SAMe）是體內(nèi)內(nèi)源性物質(zhì)，參與體內(nèi)多種重要的生化反應，可作為底物參與合成半胱氨酸、�；撬帷⒐入总针�、輔酶A等重要物質(zhì)，并作為基因供體或酶性誘導劑在人體組織三大代謝過程中起到轉甲基、轉硫基和丙氨化(多聚胺合成)作用。該藥應用于臨床已有多年，其適應證主要針對肝內(nèi)膽汁郁積、肝炎高膽紅素血癥等黃疸，能有效降低血清結合膽紅素水平，，近年來國內(nèi)有報道稱SAMe輔助治療新生兒高膽紅素血癥有一定療效。因此，我們從分子生物學的角度，分析SAMe對人胎肝細胞（LO2）的尿苷二磷酸葡萄糖醛酸轉移酶mRNA表達和蛋白的生成、活性的影響。 第一部分 腺苷蛋氨酸誘導肝臟尿苷二磷酸葡萄糖醛酸轉移酶mRNA表達的研究 目的 分別給予不同濃度、時間的SAMe干預LO2細胞，比較受干預的LO2細胞在尿苷二磷酸葡萄糖醛酸轉移酶mRNA水平的變化，探討SAMe對LO2細胞UGT的mRNA表達水平的影響及最佳的干預條件。 方法 體外培養(yǎng)LO2細胞，待細胞約70%鋪滿培養(yǎng)板底部，進行分組：⑴SAMe處理組，根據(jù)預實驗結果加入SAMe使其終濃度分別為1、0.5、0.1mmol/L；⑵陽性對照組，予以肝酶誘導劑，終濃度為2mmol/L苯巴比妥＋5mmol/L尼可剎米；⑶陰性對照組，不作加藥處理。加藥培養(yǎng)24h、48h、72h、92h后收集細胞。通過熒光定量PCR檢測各組的mRNA水平。 結果 LO2細胞的UGT1A1mRNA經(jīng)一定濃度SAMe誘導后增加，其中以濃度為0.5mmol/L組最為明顯，該組經(jīng)SAMe誘導24h后細胞的UGT1A1mRNA開始增高（49.18±10.16，P0.05），48h達到高峰（130.69±9.23，P0.05），72h后明顯下降（28.25±9.42，P0.05），此后未見明顯變化；與肝酶誘導劑組比較，其誘導LO2細胞生成UGT1A1mRNA的量更多，作用時間更長（P0.05）。 結論 適當濃度的SAMe對LO2細胞的UGT1A1的mRNA表達有誘導生成作用，表達量及時效均比肝酶誘導劑好。 第二部分 腺苷蛋氨酸誘導肝臟尿苷二磷酸葡萄糖醛酸轉移酶蛋白表達的研究 目的 分別給予不同濃度、時間的SAMe干預LO2細胞，比較受干預的LO2細胞在UGT蛋白表達及分泌水平的變化，探討SAMe對LO2細胞的UGT的蛋白表達、分泌水平的影響及最佳的干預條件。 方法 體外培養(yǎng)LO2細胞，待細胞約70%鋪滿培養(yǎng)皿底部，進行分組：⑴SAMe處理組，根據(jù)預實驗結果加入SAMe使其終濃度分別為1、0.5、0.1mmol/L；⑵陽性對照組，終濃度為2mmol/L苯巴比妥＋5mmol/L尼可剎米；⑶陰性對照組，不加藥處理。加藥培養(yǎng)24h、48h、72h、92h后收集細胞。通過western blot及ELISA檢測各組的UGT1A1蛋白表達、分泌水平。 結果 LO2細胞的UGT1A1蛋白經(jīng)SAMe誘導后合成和分泌增加，其中以濃度為0.5mmol/L組最為明顯，該組經(jīng)SAMe誘導24h后細胞的UGT1A1蛋白合成和分泌開始增高（49.18±10.16或526.18±5.21，P0.05），48h達到高峰（130.69±9.23或1278.22±9.38， P0.05），72h后明顯下降（28.25±9.42或296.62±6.42，P0.05），此后未見明顯變化；與肝酶誘導劑組比較，其誘導LO2細胞合成和分泌更多的UGT1A1蛋白，作用時間更長（P0.05）。 結論 適當濃度的SAMe對LO2細胞的UGT1A1蛋白的表達有誘導生成作用，表達量及時效均比肝酶誘導劑好。
[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.
【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.1

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