【摘要】： 細胞內(nèi)膽固醇平衡的維持需要膽固醇的合成，降解，脂化和分泌的協(xié)同調(diào)節(jié)。目前認為氧化的膽固醇衍生物(氧化固醇)在膽固醇平衡中發(fā)揮重要作用。最近我們發(fā)現(xiàn)一種新的氧化固醇，3-硫酸-25-羥化膽固醇(5-cholesten-3β，25-diol3-sulphate，，25HC3S)。在原代培養(yǎng)的大鼠肝細胞中過表達一種線粒體膽固醇轉(zhuǎn)運蛋白，類固醇激素合成急性調(diào)節(jié)蛋白(StARD1)后，該氧化類固醇在細胞核及線粒體內(nèi)大量蓄積。作為一種核內(nèi)氧化固醇，其也存在于正常人肝細胞核內(nèi)。隨后，用三乙胺-三氧化硫絡合物成功地化學合成了25HC3S。在此基礎上，我們進行了肝細胞內(nèi)25HC3S的生物合成通路，調(diào)節(jié)及功能研究。 第一部分：探討了25HC3S的生物合成通路。依據(jù)5-cholesten-3β，25-diol3-sulphate(25-OH cholesterol 3β-sulfate)的分子結(jié)構(gòu)，提出了一個假設的代謝通路，該通路有兩個步驟，第一步：線粒體膽固醇27-羥化酶(CYP27A1)催化肝細胞線粒體合成25-羥化膽固醇。第二步：羥基類固醇硫酸基轉(zhuǎn)移酶催化25-羥化膽固醇為25HC3S。在第一節(jié)中，我們發(fā)現(xiàn)蛋白酶K處理線粒體后，能顯著增加27-羥化膽固醇的合成，在第二節(jié)中我們觀察到生理性的蛋白酶也可發(fā)揮類似于蛋白酶K的作用，而且嘗試著尋找是何種蛋白酶。在第三節(jié)中，我們探討了合成25HC3S的整個生物通路。 第一節(jié)：線粒體膽固醇27-羥化酶(CYP27A1)在維持細胞內(nèi)膽固醇平衡中發(fā)揮重要作用。膽固醇進入線粒體內(nèi)膜被認為是膽汁酸合成酸性通路的限速步驟。我們的實驗顯示蛋白酶K(proteinase K)處理線粒體可以顯著提高CYP27A1的特異性活性。以內(nèi)源性線粒體膽固醇為底物，蛋白酶K增加CYP27A1特異性活性5倍。當加入外源性溶解于β-環(huán)糊精(β-cyclodextrin)的膽固醇后，CYP27A1活性增加7倍以上。酶動力學實驗顯示蛋白酶K增加CYP27A1酶活性呈時間，蛋白酶K劑量及底物濃度依賴性。蛋白酶K處理將CYP27A1催化膽固醇羥化反應的Km值從400μM降到150μM。運用這個新的方法，我們觀察到與新鮮制備的大鼠肝臟線粒體相比，在大鼠原代肝細胞的分離和培養(yǎng)過程中，線粒體CYP27A1的活性逐漸降低。 第二節(jié)：我們已經(jīng)觀察到蛋白酶K顯著增加體外線粒體CYP27A1的特異性活性。而蛋白酶在機體組織細胞中無處不在，在線粒體周圍的環(huán)境中也廣泛存在。在機體的生理或病理條件下，某些蛋白酶釋放也可以影響線粒體的狀態(tài)，是否可以改變CYP27A1的酶活性?分離的肝臟細胞漿可以使CYP27A1酶活性增加2倍，煮沸滅活抑制該效應。繼而，我們觀察到分離的溶酶體釋放出的蛋白酶可以刺激27-羥膽固醇的合成。而EDTA和蛋白酶抑制劑AEBSF可以降低27-羥膽固醇生成，而二價鈣離子則可以刺激其生成。因此，某種鈣離子依賴的金屬蛋白酶和某種絲氨酸蛋白酶都有可能是刺激CYP27A1活性的蛋白酶，但我們還不能明確是何種蛋白酶，而蛋白酶又是如何發(fā)揮作用的?這些都有待于進一步研究發(fā)現(xiàn)。這些現(xiàn)象也顯示蛋白酶水解反應可能與控制線粒體膽固醇跨膜轉(zhuǎn)運以及膽汁酸酸性合成途徑有關(guān)。 第三節(jié)：提出了肝細胞中25HC3S的合成通路。大鼠肝臟，野生型和CYP27A1基因敲除小鼠肝臟分離的線粒體與膽固醇孵育進行27-羥化酶反應的實驗結(jié)果顯示，25-羥化膽固醇可以在肝細胞線粒體中由CYP27A1催化產(chǎn)生。孵育純化的肝臟線粒體和細胞漿可以生物合成25HC3S。RT-PCR和Western-blot顯示肝細胞表達羥基膽固醇硫酸基轉(zhuǎn)移酶SULT281b。25HC3S，而非25HC可以反饋性降低SULT281b表達。綜上，我們認為CYP27A1催化膽固醇生成25-羥化膽固醇，進而3β羥基被硫酸化，從而合成25HC3S。 第二部分：研究了催化25HC3S合成的羥基類固醇硫酸基轉(zhuǎn)移酶的調(diào)節(jié)。近年來，一種新的羥基類固醇硫酸基轉(zhuǎn)移酶SULT2B1b被克隆和研究。SULT281被報道表達于激素反應性器官，如胎盤、前列腺、皮膚和乳腺等。我們觀察到在大鼠原代肝細胞的培養(yǎng)過程中，即使培養(yǎng)液中沒有加入任何激素、血清，SULT281b表達隨著培養(yǎng)時間的延長而顯著增加。而同時其他羥基類固醇硫酸基轉(zhuǎn)移酶SULT2A亞家族的SULT2A2和ST-40則顯著降低。地塞米松對于SULT2A的表達具有重要性，而并不能影響SULT281b的表達。胰島素可以顯著增加SULT281b的mRNA和蛋白的表達，T4可以提高其mRNA水平，但幅度遠小于胰島素�？梢�，SULT281b是一種高度可調(diào)節(jié)性羥基類固醇硫酸基轉(zhuǎn)移酶。 第三部分：觀察了25HC3S對大鼠原代肝細胞脂質(zhì)代謝的影響。加入不同劑量的25HC3S到原代培養(yǎng)的大鼠肝細胞中，孵育6小時后，顯著抑制CYP7A1，而且該作用顯著甚于25HC。25HC3S通過更有效地抑制SRBEP-1和SREBP-2mRNA表達，進而更強烈地抑制HMG輔酶A還原酶mRNA水平。25HC3S降低SREBP-1前體和活化形式的蛋白水平，而25HC升高SREBP-1前體蛋白水平，但不影響其活化蛋白表達。這些結(jié)果顯示，25HC3S作為一種親水性的氧化固醇，在大鼠原代肝細胞內(nèi)脂質(zhì)代謝中發(fā)揮不同于25HC的重要作用。 總之，我們認為25HC3S是一種有功能的核氧化固醇，而且可以在體內(nèi)生物合成，線粒體膽固醇27-羥化酶(CYP27A1)催化肝細胞線粒體合成25-羥化膽固醇，而后羥基膽固醇硫酸基轉(zhuǎn)移酶SULT2B1b催化25-羥化膽固醇為25HC3S。而且，這兩步生物反應都是高度可調(diào)節(jié)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
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R341

【引證文獻】

相關(guān)碩士學位論文 前1條

1 孫海林;LPS處理對豬背最長肌能量代謝的影響及其機制研究[D];南京農(nóng)業(yè)大學;2015年

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