【作者】 韩桂艳；

【导师】 王姮； 邢小平； 龚凤英； 朱惠娟； 王鸥；

【作者基本信息】 中国协和医科大学 ， 内分泌临床， 2010， 博士

【摘要】 背景：维生素D除了经典的调节钙、磷代谢的作用外,近期研究发现其与免疫系统、细胞的分化增殖以及其它内分泌腺体之间有重要的关系。有研究提示低维生素D水平与代谢综合征(Metabolic syndrome, MS)间存在相关性,但结论尚不统一,是否低维生素D状态与MS的危险因素?肥胖是MS的中心环节,是否维生素D对肥胖的发生、脂质的代谢存在直接影响?目前尚不明确。目的：1)观察MS组与对照组间血25(OH)D水平的差异,及25(OH)D与MS的相关性,通过前瞻性干预研究观察MS相关指标改善后血25(OH)D水平的变化2)通过体外细胞实验探讨1,25(OH)2D3对3T3-L1前脂肪细胞增殖分化的影响。方法：1.临床研究：1)病例对照研究：收集北京平谷地区MS患者171人,血压、血糖正常的非肥胖者63人作为对照,测量人体测量学指标的同时测定生化指标,比较MS患者与对照人群血25(OH)D水平的差异,了解该组人群25(OH)D与MS间是否存在相关性。2)前瞻性干预研究：对171名MS患者饮食运动干预,部分患者给予药物干预1年,比较干预前后随着MS相关指标改善,血25(OH)D水平的变化。2.体外研究：1)以无血清培养基培养3T3-L1细胞,以MTT法观察1,25(OH)2D3对细胞增殖的影响。2)采用含胰岛素、地塞米松与IBMX的分化培养基诱导分化3T3-L1细胞,用油红O染色及甘油三酯测定量分析法观察1,25(OH)2D3对3T3-L1细胞分化的影响。3)于增殖第7天及诱导分化后第4天、第12天收集细胞,提取总RNA并逆转录为cDNA,采用实时荧光定量PCR观察1,25(OH)2D3对细胞增殖、凋亡相关基因(CyclinD1、Bax和BCL-2),维持分化相关转录因子(PPARγ、CEBP／a),以及脂质代谢相关酶(ACC1、ACS1、G3DPH、HSL)及脂肪因子(Adiponectin、Visfatin)表达的影响。结果：1)与正常对照人群相比,MS患者维生素D缺乏(<20ng／m1)所占比例较高(90.1%vs 79.4%),OR=2.192(1.1191,4.295),P=0.021。MS与BMI及年龄相关[r=1.857(1.549,2.226),P<0.0005,r=1.126(1.066,1.188),P<0.0005],未发现MS与25(OH)D及PTH间存在相关性。肥胖患者血25(OH)D水平明显低于非肥胖者(13.97±4.17 vs 15.22±4.80ng／ml,P=0.041),PTH水平两组间无统计学差异。多元线性回归分析显示,MS组血25(OH)D与TC(B=-0.189,P=0.025)、HDL-C(B=-0.188,P=0.025)水平呈负相关。干预1年后随着患者体重减轻、BMI及腰围减小、血糖降低、胰岛素抵抗减轻及甘油三酯的下降,血25(OH)D水平明显增高(14.40±4.03 vs 16.11±4.02 ng／ml,P<0.0005)。多元线性回归分析,提示A25(OH)D与ATG相关(B=0.195,P=0.014)。2)体外细胞实验发现,1,25(OH)2D3对3T3-L1细胞增殖有明显抑制作用,该作用呈剂量依赖性,10-7M、10-8M、10-9M浓度组OD值分别为对照组的39.8±4.0%、48.4±6.9%及64.3±9.7%(P<0.05)。当药物浓度等于10-10M时抑制作用明显减弱,OD值为对照组的79.8±9.9%(P<0.05)。1,25(OH)2D3可抑制增殖相关基因Cyclin D1的表达。1,25(OH)2D3可以剂量依赖性方式抑制3T3-L1细胞分化,分化第12天细胞内甘油三酯的含量降低,10-8M、10-9M浓度组较对照组分别降低13.3%和9.8%,P值分别为0.013、0.027。当浓度小于等于10-10M时抑制作用消失。1,25(OH)2D3可抑制PPARγ、C／EBPa mRNA及HSLmRNA的表达,促进G3PDH、ACC1及ACS1 mRNA的表达,且呈剂量依赖性。同时1,25(OH)2D3可明显抑制Adiponectin、Visfatin mRNA表达,随着药物浓度降低,作用减弱。结论：1)本研究发现MS组维生素D缺乏者所占比例明显高于正常人,但未发现MS与25(OH)D及PTH水平间存在相关性。干预1年后,随体重、BMI、腰围、FBG及TG降低,25(OH)D水平增高。2)1,25(OH)2D3对3T3-Ll细胞的增殖、分化有抑制作用,该作用可能通过抑制Cyclin D1、PPARγ及C／EBPa基因表达实现。同时可影响脂质代谢相关酶G3PDH、ACC1、ACS1、HSL及脂肪因子Adiponectin、Visfatin mRNA的表达。更多还原

【Abstract】 Background:Recent studies have provided new insights into the function of vitamin D. In addition to the classical effects in maintaining calcium homostasis, vitamin D has been shown to be closely associated with immune system, cell’s proliferation and differentiation, and other endocrine glands. Accumulating evidence suggests vitamin D play an important role in decreasing the risk of many chronic illnesses, including common cancers, autoimmune diseases, infectious diseases, and cardiovascular disease. Moreover, vitamin D deficiency was also found to be associated with metabolic syndrome (MS), but it was still a controversial topic. In addition, whether vitamin D deficiency can lead to the development of obesity, the basic component of metabolism syndrome, and whether it have direct effect on lipid metabolism are not clearly defined.Objective:1) To detect the association of vitamin D deficiency and metabolism syndrome in the clinical case control study. To investigate the changes in serum 25(OH)D concentration after the metabolic disorders improved, and to explore if vitamin D deficiency is a risk factor for metabolic syndrome.2) To explore the effects of 1,25(OH)2D3 on the 3T3-L1 cell proliferation and differentiation in vitro study.Methods:1.Clinical study.1) The case control study. One hundred and seventy MS patients and 63 healthy people from Pinggu district Beijing were incruited in this study. The healthy people as control group were of normal serum glucose concentration, blood pressure, and BMI. Biochemical parameters and authropometry were evaluated. And serum 25(OH)D concentrations were compared between the subjects and controls to explore the association of serum 25(OH)D and MS.2) The prospective intervention study. Life style intervention including diet instruction and exercises were conducted in the MS patients for one year, and some of them received medication therapy for hypertension and diabetes. Biochemical parameters and authropometry were reevaluated after one year intervention to explore the association of MS and serum 25(OH)D concentration.2. The in vitro study.1) In order to study the effect of 1,25(OH)2D3 on the proliferation of 3T3-L1 preadipocytes, the cell were cultured with AIMV serum-free medium for seven days. Thereafter, the 3T3-L1 cells proliferation was determined by MTT spectrophotometry.2) In order to investigate the effects of 1,25(OH)2D3 on the differentiation of 3T3-L1 preadipocytes, the cells were cultured with medium containing insulin, dexamethasone and IBMX to induce differentiation. After 12 days, red oil O staining and dye extraction as well as intracellular triglyceride assays were performed to determine the amount of intracellular lipid, which can serve as a marker of adipocyte differentiation.3) the 3T3-L1 were collected after 4 days and 8 days cultured in the differentiation medium and after 7 days cultured in the common medium. Then the total RNA was extracted and reverse transcripted into cDNA. After that, the relative mRNA levels were determined by Real-time PCR for genes listed below:gene for proliferation and apoptosis:Cyclin D1, Bax and BCL-2; transcription factors critical for the maintenance of differentiation:PPARγ、CEBP/a;critical enzymes for lipid metabolism: ACC1,ACS1,G3DPH and HSL; adipokines:adiponectin,visfatin.Results:1.Clinical study. The percent of vitamin D deficiency was higher in the patients with MS than those without MS (90.1% vs 79.4%,OR=2.192, P=0.021). MS was significantly associated with BMI and age[r=1.857(1.549,2.226), P＜0.0005, r=1.126 (1.066,1.188), P＜0.0005]. No association was found between MS and serum 25(OH)D and parathyroid hormone (PTH) concentration. The serum 25(OH)D concentration of obesity was lower than people without obesity (13.97±4.17 vs 15.22±4.80 ng/ml, P=0.041). Multiple line regression demonstrated that serum 25(OH)D concentration was negative associated with high density lipoprotein cholesterol(HDL-C)(B=-0.188, P=0.025), total cholesterol (TC)(B=-0.189, P=0.025). After one year intervention, the weight, BMI and waist circumference(WC) decreased, at the same time, the serum 25(OH)D concentration increased significantly (14.40±4.03 vs 16.11±4.02 ng/ml, P<0.0005). Multiple line regression demonstrated△25(OH)D was prospective associated with△triglyceride (TG) (B=0.195,P=0.014).2)The in vitro study.1,25(OH)2D3 inhibit 3T3-L1 cell proliferation in dose-dependent manner. When 1,25(OH)2D3 concentration was 10-7M,10-8M and 10-9M, the relative OD value was 39.8±4.0%,48.4±6.9% and 64.3±9.7%(P＜0.05). When the concentration fell to less than 10-10M, the proliferation inhibition effect decreased dramatically, with relative OD value of 79.8±9.9%(P＜0.05). Both semi-quantitative red oil O stain method and triglyceride assays showed that 1,25(OH)2D3 inhibited the differentiation of 3T3-L1 preadipocytes in dose-dependent manner. At the 12th day, the intracellular triglyceride content decreased by 13.3% and 9.8% respectivelywhen the 1,25(OH)2D3 concentration was 10-8M and 10-9M (P=0.013,P=0.027) compared with control group, and the effect disappeared when the concentration was less than 10-10M. When 3T3-L1 preadipocytes exposed to 1,25(OH)2D3 at 10-8M, the relative mRNA concentration of Cyclin Dl and transcript factors PPARy and C/EBPa reduced significently, and the critical lipid synthesis enzymes G3PDH, ACC1, and ACS1 increased dramatically, while the critical lipid catabolic enzymes HSL reduced remarkably, and the adipokines Adiponectin and Visfatin reduced significantly. When the 1,25(OH)2D3 concentration was 10-10M, the effects disappeared.Conclusion:1. The percent of vitamin D deficiency was higher in the patients with MS than those without MS. MS was positively associated with BMI and age. No association was found between MS and serum 25(OH)D and parathyroid hormone (PTH) concentration. After one year intervention, the serum 25(OH)D concentration increased following the decrease of FBG, weight,WC, BMI and TG.2.1,25(OH)2D3 can inhibit 3T3-L1 preadipocytes proliferation and differentiation in does-dependent manner, possibly through the inhibition effect it exert on the Cyclin D1 and transcription factors PPARy and C/EBPa. 1,25(OH)2D3 can stimulate the critical lipid synthesis enzymes G3PDH、ACC1、ACS1 expression and inhibit the critical lipid catabolic enzymes HSL and the adipokines Adiponectin and Visfatin expression.更多还原