【摘要】：糖尿病是內(nèi)分泌疾病,經(jīng)長(zhǎng)期研究證實(shí)跟遺傳和環(huán)境都有關(guān),嚴(yán)重影響人體健康。合成藥物臨床治療糖尿病存在不良反應(yīng)多等問題,天然藥物與其相比具有安全性高,在疾病治療方便可使用時(shí)間長(zhǎng)的優(yōu)點(diǎn)。蘿卜硫素(sulforaphane,SFN)是一種從植物中提取的天然單體物質(zhì),有研究表明蘿卜硫素在緩解2型糖尿病患者的胰島素抵抗癥狀方面有良好效果,但是其機(jī)制仍有待深入探究。我們的實(shí)驗(yàn)證實(shí)蘿卜硫素能改善IR-Hep G2細(xì)胞胰島素抵抗,其機(jī)制可能與抑制Lipin1表達(dá)有關(guān)。肝臟是Lipin1的主要分布部位,提高藥物對(duì)肝臟的靶向性也能使藥物發(fā)揮更好的作用。此外,在實(shí)驗(yàn)中我們發(fā)現(xiàn)蘿卜硫素在高劑量情況下具有一定毒性,限制藥物使用劑量,影響了藥理作用的發(fā)揮。脂質(zhì)體作為藥物載體,具有靶向、減毒,減少藥物副作用等特殊優(yōu)點(diǎn),因此本研究將蘿卜硫素與脂質(zhì)體這種新劑型相結(jié)合,研制了蘿卜硫素脂質(zhì)體,以期提高藥物在肝臟中的靶向性,解決藥物用量限制,達(dá)到增效減毒的目的,將來能更好地服務(wù)于臨床。本課題主要分為兩個(gè)部分:1.蘿卜硫素改善Hep G2胰島素抵抗及其機(jī)制的研究采用棕櫚酸(palmitic acid,PA)建立起Hep G2細(xì)胞胰島素抵抗(insulin resistance,IR)模型,通過檢測(cè)使用藥物前后細(xì)胞培養(yǎng)基上清液中葡萄糖消耗量(glucose consumption,GC),細(xì)胞內(nèi)甘油二酯(diacylglycerol,DAG)含量的變化,孕烷X受體(Pregnane X Receptor,PXR)底物蛋白細(xì)胞色素P450 3A4酶(cytochrome 3A4,CYP3A4),脂素Lipin1蛋白表達(dá)的變化來揭示蘿卜硫素對(duì)IR-Hep G2細(xì)胞的降糖作用可能存在的機(jī)制,通過分析葡萄糖消耗量,我們發(fā)現(xiàn)模型組與對(duì)照組相比數(shù)值顯著降低,說明IR-Hep G2模型成功建立。跟模型組相比,低、中、高劑量蘿卜硫素均能增加細(xì)胞培養(yǎng)基上清中葡萄糖的消耗含量,其中高劑量組消耗量最顯著。經(jīng)甘油二酯檢測(cè)試劑盒結(jié)果顯示,跟模型組相比,低、中、高劑量蘿卜硫素能夠使IR-Hep G2細(xì)胞內(nèi)甘油二酯的含量降低。從Western blot實(shí)驗(yàn)我們得知,蘿卜硫素能夠通過抑制PXR受體底物CYP3A4蛋白的表達(dá)來抑制蛋白Lipin1的表達(dá)。提示蘿卜硫素能改善IR-Hep G2細(xì)胞胰島素抵抗,其機(jī)制可能與抑制Lipin1表達(dá)從而導(dǎo)致甘油二酯含量降低有關(guān)。2.蘿卜硫素脂質(zhì)體的制備及其質(zhì)量評(píng)價(jià)通過正交設(shè)計(jì),采用逆向蒸發(fā)法制備蘿卜硫素脂質(zhì)體并對(duì)其進(jìn)行質(zhì)量評(píng)價(jià)。使用正交實(shí)驗(yàn)方法,通過使用包封率作為評(píng)價(jià)指標(biāo)選擇用于制備蘿卜硫烷脂質(zhì)體的最合適的制劑配方。研究脂質(zhì)體的形態(tài),大小,電負(fù)性,穩(wěn)定性和其他性質(zhì)。選取了合適的組織樣品處理方法,建立了蘿卜硫素體內(nèi)分析方法并采用方法學(xué)驗(yàn)證。根據(jù)實(shí)驗(yàn)設(shè)計(jì)將小鼠分成兩組。給藥后取組織,處理后對(duì)藥物體內(nèi)分析,之后對(duì)蘿卜硫素脂質(zhì)體的組織分布性進(jìn)行初步研究。采用正交實(shí)驗(yàn)法篩選出制備蘿卜硫素脂質(zhì)體最合適的制備處方有機(jī)相/水相比例為1:3,溫度為30℃,水合時(shí)間為5min,磷脂膽固醇摩爾比6:1,按該處方得到的脂質(zhì)體包封率最高可達(dá)51.11%,所制得的蘿卜硫素脂質(zhì)體透射電鏡下為球形或類似球形單層形狀的脂質(zhì)體,粒徑均一,流動(dòng)性較好。平均粒徑為(176.733±5.443)nm,PDI為(0.197±0.032),表明脂質(zhì)體的分布專一,粒度的大小適中。其平均Zeta電位為(21.13±0.17)mv,顯示脂質(zhì)體很穩(wěn)定。在4℃條件下,脂質(zhì)體放置10天后,有少量脂質(zhì)體出現(xiàn)沉淀現(xiàn)象,一個(gè)月以后,脂質(zhì)體有部分沉淀,包封率也顯示有降低趨勢(shì),總體脂質(zhì)體體系較穩(wěn)定。HPLC法測(cè)脂質(zhì)體在小鼠體內(nèi)分布研究蘿卜硫素脂質(zhì)體在小鼠體內(nèi)肝、脾組織部位分布較多。細(xì)胞增殖實(shí)驗(yàn)證實(shí)蘿卜硫素脂質(zhì)體在Hep G2細(xì)胞中的毒性低于相同劑量游離蘿卜硫素,證實(shí)蘿卜硫素脂質(zhì)體這種劑型能使藥物減量增效。
[Abstract]:Diabetes mellitus is an endocrine disease. It has been proved by long-term research that it is related to both genetics and environment, and seriously affects human health. Natural monomers extracted from plants have been shown to be effective in alleviating insulin resistance in type 2 diabetes mellitus, but the mechanism remains to be explored. Our experiments have shown that sulforaphane can improve insulin resistance in IR-Hep G2 cells, and the mechanism may be related to the inhibition of Lipin 1 expression in liver. The viscera is the main distribution site of Lipin-1, and improving the drug targeting to the liver can also make the drug play a better role. In addition, we found that sulforaphane has certain toxicity at high doses, limiting the dosage of the drug, affecting the pharmacological effect. Liposomes as drug carriers, with targeting, detoxification and reduction. In order to improve the drug targeting in the liver, solve the drug dosage limitation, achieve the purpose of synergism and detoxification, and serve the clinical better in the future. This topic is mainly divided into two parts: 1. Radish. Improving Insulin Resistance and Its Mechanisms in Hep G2 Cells Using Palmitic Acid (PA) to Establish Insulin Resistance (IR) Model of Hep G2 Cells. Glucose Consumption (GC) and Diacyl Glycerol (DAG) in the Supernatant of Cell Medium before and after the Use of the Drug were detected. Changes in the content of pregnane X Receptor (PXR) substrate protein, cytochrome 3A4 (CYP3A4), and lipoprotein Lipin 1 protein expression were used to reveal the possible mechanism of glucose-lowering effect of sulforaphane on IR-Hep G2 cells. By analyzing glucose consumption, we found that the model group compared with the control group values. Compared with the model group, low, medium and high doses of sulforaphane could increase the glucose consumption in the supernatant of the cell culture medium, especially in the high dosage group. Western blot showed that sulforaphane could inhibit the expression of Lipin-1 by inhibiting the expression of CYP3A4 protein, the substrate of PXR receptor. It suggested that sulforaphane could improve insulin resistance in IR-Hep G2 cells, and the mechanism might be related to the inhibition of Lipin-1 expression leading to the decrease of glycerol-2-ester content. 2. The preparation and quality evaluation of sulforaphane liposomes were carried out by orthogonal design. The sulforaphane liposomes were prepared by reverse evaporation method and evaluated by quality evaluation. The optimum formulation for preparing sulforaphane liposomes was selected by orthogonal experiment using entrapment efficiency as evaluation index. The morphology, size, electronegativity, stability and other properties of the mice were studied. The method of in vivo analysis of sulforaphane was established and validated by methodology. The mice were divided into two groups according to the experimental design. Orthogonal experiment was used to select the optimum preparation formula for sulforaphane liposomes. The ratio of organic phase to water was 1:3, the temperature was 30 C, the hydration time was 5 min, the molar ratio of phospholipid to cholesterol was 6:1, and the encapsulation efficiency of liposomes was up to 51.11%. The average particle size was (176.733 (+5.443) nm and the PDI was (0.197 (+0.032), indicating that the liposomes were well distributed and moderate in size. The average Zeta potential was (21.13 (+0.17) mv, indicating that the liposomes were stable. At 4 (?) C, there were few liposomes after 10 days of storage. After one month, the liposome was partially precipitated and the entrapment efficiency showed a decreasing trend. The overall liposome system was stable. The distribution of sulforaphane liposomes in mice liver and spleen was detected by HPLC. The cytotoxicity of Hep G2 cells was lower than that of free radish sulfur at the same dose, which proved that the dosage form of radish sulfur liposome could reduce the dosage and increase the efficacy of the drug.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R283.6;R285

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