肝细胞内新的核氧化固醇—3-硫酸-25-羟化胆固醇的生物合成通路、调节及功能研究
发布时间：2019-06-17 16:18
【摘要】： 细胞内胆固醇平衡的维持需要胆固醇的合成，降解，脂化和分泌的协同调节。目前认为氧化的胆固醇衍生物(氧化固醇)在胆固醇平衡中发挥重要作用。最近我们发现一种新的氧化固醇，3-硫酸-25-羟化胆固醇(5-cholesten-3β，25-diol3-sulphate，，25HC3S)。在原代培养的大鼠肝细胞中过表达一种线粒体胆固醇转运蛋白，类固醇激素合成急性调节蛋白(StARD1)后，该氧化类固醇在细胞核及线粒体内大量蓄积。作为一种核内氧化固醇，其也存在于正常人肝细胞核内。随后，用三乙胺-三氧化硫络合物成功地化学合成了25HC3S。在此基础上，我们进行了肝细胞内25HC3S的生物合成通路，调节及功能研究。 第一部分：探讨了25HC3S的生物合成通路。依据5-cholesten-3β，25-diol3-sulphate(25-OH cholesterol 3β-sulfate)的分子结构，提出了一个假设的代谢通路，该通路有两个步骤，第一步：线粒体胆固醇27-羟化酶(CYP27A1)催化肝细胞线粒体合成25-羟化胆固醇。第二步：羟基类固醇硫酸基转移酶催化25-羟化胆固醇为25HC3S。在第一节中，我们发现蛋白酶K处理线粒体后，能显著增加27-羟化胆固醇的合成，在第二节中我们观察到生理性的蛋白酶也可发挥类似于蛋白酶K的作用，而且尝试着寻找是何种蛋白酶。在第三节中，我们探讨了合成25HC3S的整个生物通路。 第一节：线粒体胆固醇27-羟化酶(CYP27A1)在维持细胞内胆固醇平衡中发挥重要作用。胆固醇进入线粒体内膜被认为是胆汁酸合成酸性通路的限速步骤。我们的实验显示蛋白酶K(proteinase K)处理线粒体可以显著提高CYP27A1的特异性活性。以内源性线粒体胆固醇为底物，蛋白酶K增加CYP27A1特异性活性5倍。当加入外源性溶解于β-环糊精(β-cyclodextrin)的胆固醇后，CYP27A1活性增加7倍以上。酶动力学实验显示蛋白酶K增加CYP27A1酶活性呈时间，蛋白酶K剂量及底物浓度依赖性。蛋白酶K处理将CYP27A1催化胆固醇羟化反应的Km值从400μM降到150μM。运用这个新的方法，我们观察到与新鲜制备的大鼠肝脏线粒体相比，在大鼠原代肝细胞的分离和培养过程中，线粒体CYP27A1的活性逐渐降低。 第二节：我们已经观察到蛋白酶K显著增加体外线粒体CYP27A1的特异性活性。而蛋白酶在机体组织细胞中无处不在，在线粒体周围的环境中也广泛存在。在机体的生理或病理条件下，某些蛋白酶释放也可以影响线粒体的状态，是否可以改变CYP27A1的酶活性?分离的肝脏细胞浆可以使CYP27A1酶活性增加2倍，煮沸灭活抑制该效应。继而，我们观察到分离的溶酶体释放出的蛋白酶可以刺激27-羟胆固醇的合成。而EDTA和蛋白酶抑制剂AEBSF可以降低27-羟胆固醇生成，而二价钙离子则可以刺激其生成。因此，某种钙离子依赖的金属蛋白酶和某种丝氨酸蛋白酶都有可能是刺激CYP27A1活性的蛋白酶，但我们还不能明确是何种蛋白酶，而蛋白酶又是如何发挥作用的?这些都有待于进一步研究发现。这些现象也显示蛋白酶水解反应可能与控制线粒体胆固醇跨膜转运以及胆汁酸酸性合成途径有关。 第三节：提出了肝细胞中25HC3S的合成通路。大鼠肝脏，野生型和CYP27A1基因敲除小鼠肝脏分离的线粒体与胆固醇孵育进行27-羟化酶反应的实验结果显示，25-羟化胆固醇可以在肝细胞线粒体中由CYP27A1催化产生。孵育纯化的肝脏线粒体和细胞浆可以生物合成25HC3S。RT-PCR和Western-blot显示肝细胞表达羟基胆固醇硫酸基转移酶SULT281b。25HC3S，而非25HC可以反馈性降低SULT281b表达。综上，我们认为CYP27A1催化胆固醇生成25-羟化胆固醇，进而3β羟基被硫酸化，从而合成25HC3S。 第二部分：研究了催化25HC3S合成的羟基类固醇硫酸基转移酶的调节。近年来，一种新的羟基类固醇硫酸基转移酶SULT2B1b被克隆和研究。SULT281被报道表达于激素反应性器官，如胎盘、前列腺、皮肤和乳腺等。我们观察到在大鼠原代肝细胞的培养过程中，即使培养液中没有加入任何激素、血清，SULT281b表达随着培养时间的延长而显著增加。而同时其他羟基类固醇硫酸基转移酶SULT2A亚家族的SULT2A2和ST-40则显著降低。地塞米松对于SULT2A的表达具有重要性，而并不能影响SULT281b的表达。胰岛素可以显著增加SULT281b的mRNA和蛋白的表达，T4可以提高其mRNA水平，但幅度远小于胰岛素。可见，SULT281b是一种高度可调节性羟基类固醇硫酸基转移酶。 第三部分：观察了25HC3S对大鼠原代肝细胞脂质代谢的影响。加入不同剂量的25HC3S到原代培养的大鼠肝细胞中，孵育6小时后，显著抑制CYP7A1，而且该作用显著甚于25HC。25HC3S通过更有效地抑制SRBEP-1和SREBP-2mRNA表达，进而更强烈地抑制HMG辅酶A还原酶mRNA水平。25HC3S降低SREBP-1前体和活化形式的蛋白水平，而25HC升高SREBP-1前体蛋白水平，但不影响其活化蛋白表达。这些结果显示，25HC3S作为一种亲水性的氧化固醇，在大鼠原代肝细胞内脂质代谢中发挥不同于25HC的重要作用。 总之，我们认为25HC3S是一种有功能的核氧化固醇，而且可以在体内生物合成，线粒体胆固醇27-羟化酶(CYP27A1)催化肝细胞线粒体合成25-羟化胆固醇，而后羟基胆固醇硫酸基转移酶SULT2B1b催化25-羟化胆固醇为25HC3S。而且，这两步生物反应都是高度可调节的。
[Abstract]:The maintenance of intra-cell cholesterol balance requires a synergistic adjustment of the synthesis, degradation, lipotization, and secretion of cholesterol. It is believed that the oxidized cholesterol derivatives (oxysterol) play an important role in the balance of cholesterol. Recently we have found a new oxysterol,3-sulfuric acid-25-hydroxylated cholesterol (5-cholesten-3-,25-diol3-sulphoate,25 H3C). In primary cultured rat hepatocytes, a mitochondrial cholesterol transport protein, a steroid hormone synthetic acute regulatory protein (StARD1), was overexpressed in the nucleus and mitochondria. As a nuclear oxidation sterol, it is also present in the normal human liver cell nucleus. Subsequently,25 H3C was successfully synthesized with a triethylamine-sulfur trioxide complex. On this basis, we carried out the biosynthesis pathway, regulation and function of 25HC3S in the liver cells. Part 1: Discussion on 25HC3S A hypothetical metabolic pathway is proposed according to the molecular structure of 5-cholesten-3-,25-diol3-sulphate (25-OH)3-sulfinate. The pathway has two steps: the mitochondrial cholesterol,27-hydroxylase (CYP27A1), catalyzes the synthesis of the mitochondria of the hepatocytes 25. -Hydroxylation of cholesterol. Step 2: Hydroxysteroid sulfate-catalyzed 25-hydroxylated cholesterol. 25HC3S. In the first section, we found that after the protease K treatment of the mitochondria, the synthesis of 27-hydroxylated cholesterol can be significantly increased, and in the second section we observed that a physiological protease can also play a role similar to that of the protease K, and try to find What kind of protease. In section III, we explored the synthesis of 25 HC3S. The entire biological pathway. Section 1: Mitochondrial cholesterol,27-hydroxylase (CYP27A1), in the maintenance of cells It plays an important role in the balance of internal cholesterol. The entry of cholesterol into the mitochondrial membrane is considered to be a gallbladder. The rate-limiting step of acid pathway was synthesized by acid-acid synthesis. Specific activity of CYP27A1. In the case of endogenous mitochondrial cholesterol as a substrate, the protease K is increased by C The specific activity of YP27A1 is 5 times. When exogenous cholesterol is added to the cholesterol-cyclodextrin (I-cyclodextrin) cholesterol, CY The activity of P27A1 is increased by more than 7 times. The K dose of the white enzyme and the concentration of the substrate were dependent on the concentration of the substrate. The Km value of the hydroxylation reaction of the CYP27A1 catalyzed by the protease K was reduced from 400. m u.M to 150. m u.M. This new method was used to observe the mitochondria C in the separation and culture of primary hepatocytes in the rat, as compared to the mitochondria of the liver of the freshly prepared rat liver. The activity of YP27A1 was gradually decreased. Section II: We have observed that the protease K is significant The specific activity of the in vitro mitochondrial CYP27A1 is increased, and the protease is in the body tissue of the body. It is ubiquitous in the environment around the mitochondria. In the physiological or pathological conditions of the body, the release of some proteases may also affect the mitochondria. State, can I change the enzyme activity of CYP27A1? The isolated liver cell slurry can make CYP2 The enzyme activity of 7A1 was increased by 2 times, and the effect was inhibited by boiling and inactivation. In turn, we observed the release of the isolated lysosomes The released protease can stimulate the synthesis of 27-hydroxycholesterol, while the EDTA and protease inhibitor AEBSF can reduce 27-hydroxycholesterol. Therefore, some kind of calcium ion-dependent metalloprotease and some kind of serine protease may be a protease that stimulates the activity of CYP27A1, but we can't make it clear. What is the protease, and the protease is, for example, What works? These are to be further studied. These phenomena also show that the hydrolysis reaction of the protease may be related to the control of the mitochondria. The trans-membrane transport of cholesterol and the acid synthesis of bile acid are related. Section III: The synthesis pathway of 25HC3S in hepatocytes was proposed. The results of the 27-hydroxylase reaction of the liver, wild type and CYP27A1 knock-out mice in the liver, wild-type and CYP27A1 knockout mice showed that 25- Hydroxylation of cholesterol can be catalyzed by CYP27A1 in the mitochondria of the liver. The cultured liver mitochondria and the cell pulp can be biosynthesized by 25-HC3S. RT-PCR and Western-blot to show the expression of the hydroxycholesterol-hydroxycholesterol-transferase SULT281b. 25H in the liver cells. The C3S, and not the 25HC, can reduce the expression of the SULT281b. In summary, we believe that the CYP27A1 catalytic cholesterol generation 25- The hydroxylated cholesterol, and the further 3-hydroxyls, are sulfated to synthesize 25 H3C. Part 2: The study of the regulation of the hydroxysteroid sulfo-transferases synthesized by the catalytic 25-HC3S In recent years, a new hydroxysteroid sulfo-transferase, SULT2B1b, is cloned and studied. ULT281 is reported to be expressed in a hormone-reactive organ, such as the placenta, the prostate, the skin, and the breast, etc. We have observed that during the culture of primary hepatocytes in rats, The addition of any hormone, serum, SULT281b expression increases significantly as the culture time is extended, while other hydroxysteroids The SULT2A2 and ST-40 of the SUL2A subfamily of the sulfo-transferase were significantly reduced. It is of importance to the expression of the SULT2A and does not affect the expression of the SULT281b. Insulin can significantly increase the SULT281b The expression of mRNA and protein, T4 can increase the mRNA level, but the amplitude is much smaller than that of the pancreas. Island. It can be seen that SULT281b is a highly adjustable hydroxysteroid sulfuric acid The third part: The effect of 25HC3S on the lipid metabolism of primary hepatocytes in rats was observed.25 HC3S of different doses were added to the primary cultured rat hepatocytes. After incubation for 6 hours, CYP7A1 was significantly inhibited and the effect was significant. The expression of SRBEP-1 and SREBP-2 mRNA was more effectively inhibited by 25 HC3S, and the level of HMG-CoA reductase mRNA was more strongly inhibited.25-H3C decreased the SREBP-1 precursor and activity. The level of protein in the form of protein, while the 25HC raised the level of the SREBP-1 precursor protein, did not affect the expression of its activated protein. These results showed that 25 H3C S As a hydrophilic oxidative sterol, it plays an important role in the lipid metabolism of primary hepatocytes of the rat, which is different from that of the 25HC. In conclusion, we think that 25-HC3S is a functional nuclear oxidative sterol and can In vivo biosynthesis, mitochondrial cholesterol 27-hydroxylase (CYP27A1) was used to catalyze the mitochondria of hepatocytes. 25-hydroxylated cholesterol, and then hydroxy-cholesterol-sulfuric acid
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
本文编号：2501112
[Abstract]:The maintenance of intra-cell cholesterol balance requires a synergistic adjustment of the synthesis, degradation, lipotization, and secretion of cholesterol. It is believed that the oxidized cholesterol derivatives (oxysterol) play an important role in the balance of cholesterol. Recently we have found a new oxysterol,3-sulfuric acid-25-hydroxylated cholesterol (5-cholesten-3-,25-diol3-sulphoate,25 H3C). In primary cultured rat hepatocytes, a mitochondrial cholesterol transport protein, a steroid hormone synthetic acute regulatory protein (StARD1), was overexpressed in the nucleus and mitochondria. As a nuclear oxidation sterol, it is also present in the normal human liver cell nucleus. Subsequently,25 H3C was successfully synthesized with a triethylamine-sulfur trioxide complex. On this basis, we carried out the biosynthesis pathway, regulation and function of 25HC3S in the liver cells. Part 1: Discussion on 25HC3S A hypothetical metabolic pathway is proposed according to the molecular structure of 5-cholesten-3-,25-diol3-sulphate (25-OH)3-sulfinate. The pathway has two steps: the mitochondrial cholesterol,27-hydroxylase (CYP27A1), catalyzes the synthesis of the mitochondria of the hepatocytes 25. -Hydroxylation of cholesterol. Step 2: Hydroxysteroid sulfate-catalyzed 25-hydroxylated cholesterol. 25HC3S. In the first section, we found that after the protease K treatment of the mitochondria, the synthesis of 27-hydroxylated cholesterol can be significantly increased, and in the second section we observed that a physiological protease can also play a role similar to that of the protease K, and try to find What kind of protease. In section III, we explored the synthesis of 25 HC3S. The entire biological pathway. Section 1: Mitochondrial cholesterol,27-hydroxylase (CYP27A1), in the maintenance of cells It plays an important role in the balance of internal cholesterol. The entry of cholesterol into the mitochondrial membrane is considered to be a gallbladder. The rate-limiting step of acid pathway was synthesized by acid-acid synthesis. Specific activity of CYP27A1. In the case of endogenous mitochondrial cholesterol as a substrate, the protease K is increased by C The specific activity of YP27A1 is 5 times. When exogenous cholesterol is added to the cholesterol-cyclodextrin (I-cyclodextrin) cholesterol, CY The activity of P27A1 is increased by more than 7 times. The K dose of the white enzyme and the concentration of the substrate were dependent on the concentration of the substrate. The Km value of the hydroxylation reaction of the CYP27A1 catalyzed by the protease K was reduced from 400. m u.M to 150. m u.M. This new method was used to observe the mitochondria C in the separation and culture of primary hepatocytes in the rat, as compared to the mitochondria of the liver of the freshly prepared rat liver. The activity of YP27A1 was gradually decreased. Section II: We have observed that the protease K is significant The specific activity of the in vitro mitochondrial CYP27A1 is increased, and the protease is in the body tissue of the body. It is ubiquitous in the environment around the mitochondria. In the physiological or pathological conditions of the body, the release of some proteases may also affect the mitochondria. State, can I change the enzyme activity of CYP27A1? The isolated liver cell slurry can make CYP2 The enzyme activity of 7A1 was increased by 2 times, and the effect was inhibited by boiling and inactivation. In turn, we observed the release of the isolated lysosomes The released protease can stimulate the synthesis of 27-hydroxycholesterol, while the EDTA and protease inhibitor AEBSF can reduce 27-hydroxycholesterol. Therefore, some kind of calcium ion-dependent metalloprotease and some kind of serine protease may be a protease that stimulates the activity of CYP27A1, but we can't make it clear. What is the protease, and the protease is, for example, What works? These are to be further studied. These phenomena also show that the hydrolysis reaction of the protease may be related to the control of the mitochondria. The trans-membrane transport of cholesterol and the acid synthesis of bile acid are related. Section III: The synthesis pathway of 25HC3S in hepatocytes was proposed. The results of the 27-hydroxylase reaction of the liver, wild type and CYP27A1 knock-out mice in the liver, wild-type and CYP27A1 knockout mice showed that 25- Hydroxylation of cholesterol can be catalyzed by CYP27A1 in the mitochondria of the liver. The cultured liver mitochondria and the cell pulp can be biosynthesized by 25-HC3S. RT-PCR and Western-blot to show the expression of the hydroxycholesterol-hydroxycholesterol-transferase SULT281b. 25H in the liver cells. The C3S, and not the 25HC, can reduce the expression of the SULT281b. In summary, we believe that the CYP27A1 catalytic cholesterol generation 25- The hydroxylated cholesterol, and the further 3-hydroxyls, are sulfated to synthesize 25 H3C. Part 2: The study of the regulation of the hydroxysteroid sulfo-transferases synthesized by the catalytic 25-HC3S In recent years, a new hydroxysteroid sulfo-transferase, SULT2B1b, is cloned and studied. ULT281 is reported to be expressed in a hormone-reactive organ, such as the placenta, the prostate, the skin, and the breast, etc. We have observed that during the culture of primary hepatocytes in rats, The addition of any hormone, serum, SULT281b expression increases significantly as the culture time is extended, while other hydroxysteroids The SULT2A2 and ST-40 of the SUL2A subfamily of the sulfo-transferase were significantly reduced. It is of importance to the expression of the SULT2A and does not affect the expression of the SULT281b. Insulin can significantly increase the SULT281b The expression of mRNA and protein, T4 can increase the mRNA level, but the amplitude is much smaller than that of the pancreas. Island. It can be seen that SULT281b is a highly adjustable hydroxysteroid sulfuric acid The third part: The effect of 25HC3S on the lipid metabolism of primary hepatocytes in rats was observed.25 HC3S of different doses were added to the primary cultured rat hepatocytes. After incubation for 6 hours, CYP7A1 was significantly inhibited and the effect was significant. The expression of SRBEP-1 and SREBP-2 mRNA was more effectively inhibited by 25 HC3S, and the level of HMG-CoA reductase mRNA was more strongly inhibited.25-H3C decreased the SREBP-1 precursor and activity. The level of protein in the form of protein, while the 25HC raised the level of the SREBP-1 precursor protein, did not affect the expression of its activated protein. These results showed that 25 H3C S As a hydrophilic oxidative sterol, it plays an important role in the lipid metabolism of primary hepatocytes of the rat, which is different from that of the 25HC. In conclusion, we think that 25-HC3S is a functional nuclear oxidative sterol and can In vivo biosynthesis, mitochondrial cholesterol 27-hydroxylase (CYP27A1) was used to catalyze the mitochondria of hepatocytes. 25-hydroxylated cholesterol, and then hydroxy-cholesterol-sulfuric acid
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
【引证文献】
相关硕士学位论文 前1条
1 孙海林;LPS处理对猪背最长肌能量代谢的影响及其机制研究[D];南京农业大学;2015年
本文编号：2501112
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