【作者】 高捷；

【导师】 冯凭；

【作者基本信息】 天津医科大学 ， 内科学， 2011， 博士

【摘要】 维生素D除了经典的调节钙、磷代谢的作用外,另有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,低维生素D水平与肥胖及其引起的代谢综合征间存在相关性,也有人研究证明维生素D对鼠的脂肪细胞的增殖分化存在影响,故推断维生素D与肥胖存在关联,但结论尚不统一。维生素D对人大网膜脂肪细胞及单纯性肥胖儿童脂源性激素水平是否有影响,目前尚不明确。目的1通过体外细胞实验探讨1,25二羟维生素D[1,25(OH)2VitD]对人大网膜脂肪细胞增殖分化的影响及其对人脂肪细胞脂联素、瘦素表达的影响。2观察单纯性肥胖儿童与健康对照儿童血25羟维生素D[25(OH)VitD],脂联素、瘦素水平的差异,通过前瞻性干预观察维生素D对单纯性肥胖儿童血25(OH)VitD、脂联素、瘦素及相关指标水平的影响。方法体外研究：1)原代分离培养人大网膜前脂肪细胞,前脂肪细胞诱导分化为脂肪细胞,以细胞生长曲线及MTT法观察1,25(OH)2VitD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人大网膜前脂肪细胞,用油红0染色及甘油三酯定量分析法观察1,25(OH),VitD对人大网膜脂肪细胞分化的影响。通过免疫组化和Western blot两种方法检测脂联素、瘦素表达及1,25(OH)2VitD对其的影响。临床研究：1)病例对照研究：收集单纯性肥胖儿童46例对照组为年龄、性别与肥胖组基本配对的健康儿童46例,年龄6-14岁。测量人体测量学指标的同时测定生化指标及血25(OH)VitD、瘦素,脂联素水平,比较单纯性肥胖患儿与对照儿童生化指标及血25(OH)VitD、瘦素,脂联素水平的差异,了解该组人群肥胖与25(OH)VitD,脂联素,瘦素是否存在相关性。2)前瞻性干预研究：所有肥胖儿童除采用饮食控制、加强运动外,均接受维生素D治疗,3个月后复查,并进行治疗前后体重指数、一般生化指标、血25(OH)VitD、瘦素,脂联素水平的分析比较。结果体外实验：原代培养的人大网膜前脂肪细胞,前脂肪细胞逐渐分化为内含反光脂滴的脂肪细胞。经油红0染色后证实为脂肪细胞。经统计分析结果显示在脂肪细胞增殖过程中不同浓度1,25(OH)2VitD对体外培养增殖的大网膜脂肪细胞数及MTT比色0D值没有影响(P>0.05)。在脂肪细胞分化过程中,10-8M、10-10M1,25(OH)2VitD浓度组的细胞内脂滴较多,10-4、10-6浓度处理组细胞内脂滴明显减少,1,25(OH)2VitD是以剂量依赖的方式抑制人大网膜脂肪细胞的分化；1,25(OH)2VitD浓度为10-4M,10-6M干预组甘油三酯浓度明显低于分化组对照组(P<0.05)。10-8M,10-10M干预组甘油三酯浓度为与分化组比较无明显差别(P>0.05),提示1,25(OH)2VitD对脂肪细胞分化有剂量依赖性抑制作用。免疫组化结果显示,脂联素主要在细胞浆内弥漫性表达,瘦素主要在细胞浆内表达。未分化组瘦素也有少量表达,western blot结果显示分化组脂联素,瘦素表达水平明显高于未分化组,而10-4,10-6,10-8浓度的1,25(OH)2VitD对分化的脂肪细胞脂联素,瘦素表达水平低于分化组差异具有统计学意义(P<0.05)。临床研究：单纯性肥胖儿童体重指数,瘦素均高于健康对照组儿童差异有显著性(P<0.05),而25(OH)VitD,脂联素低于健康对照组,差异有显著性(P<0.05)；同时血糖,胆固醇,甘油三酯等在两族间无显著性差异(P>0.05)。多元逐步回归分析结果表明与体重指数相关的因素依次是25(OH)VitD,脂联素,瘦素。体重指数与瘦素成正相关,25(OH)VitD,脂联素与其成负相关。予维生素D治疗3个月后,单纯性肥胖儿童BMI,甘油三酯,胆固醇,血糖均有所下降,但没有显著性(P>0.05),表明维生素D对于血脂,血糖,及体重指数无明显改善；同时,经维生素D治疗,单纯性肥胖儿童的25(OH)VitD及脂联素水平明显升高,差异有显著性(P<0.05),瘦素也有所增高但差异没有显著性(P>0.05)。结论1,25(OH),VitD剂量依赖性的抑制脂肪细胞的分化,但对于脂肪细胞的增殖没有影响,1,25(OH),VitD剂量依赖性的抑制分化过程中人大网膜脂肪细胞脂联素,瘦素的表达。肥胖儿童存在低维生素D水平和脂源性激素分泌异常,给予肥胖儿童一定量的维生素D可以保证维生素D的供给同时可以改善脂源性激素分泌情况。更多还原

【Abstract】 Recent studies have provided new insights into the function of vitamin D. In addition to the classical effects on regulating metabolism of Calcium and phosphorus, vitamin D has been shown to be closely associated with immune system, cells differentiation and proliferation, and other endocrine glands. Low Vitamin D levels have long been linked to obesity and metabolic syndrome it causes. There is also inference about the connection between Vitamin D and obesity based on the research done showing the effects of vitamin D on preadipocyte proliferation and differentiation of human omental adipose cells. Yet no definite evidence is given about the effects of Vitamin D on human omental adipose cells and hormone level of obese children with Simple obesity.Objective:1.To explore the effects of l,25(OH)2VitD on the proliferation and differentiation of human omental adipose cells and the effects of1,25(OH)2VitD on human secreting of adipocyte adiponectin and leptin in vitro study.2. To detect the differences between the simple obesity group and the healthy control group in1,25(OH)2VitD adiponectin, and leptin, and to investigate effects of Vitamin D on the obese children with simple obesity in serum concentration of1,25(OH)2VitD, adiponectin, leptin and other related targets with prospective intervention study.Methods:1) human omental preadipocytes were isolated and cultured, which became adipocytes during induced differentiation, to observe the effects of1,25(OH)2VitD on preadipocyte proliferation by growth curve and MTT method.2) human omental preadipocyte cells were induced to differentiate by using differentiation medium with insulin, dexamethasone and IBMX to observe the effects of1,25(OH)2VitD on preadipocyte differentiation by quantitative analysis of oil red O dyeing and triglyceride. The expression of adipokine adiponectin and leptin and the effects of1,25(OH)2VitD on the result was detected by immunohistochemisty and Western blot. Clinical study:1) case-control study:46cases of children with simple obesity were gathered from clinic and health screening in the Affiliated Hospital of North China Coal Medical College. Control group consist of children aged between6to14, matched in age and gender with the children in simple obesity group biochemical indicator, the serum concentrations of adiponectin and leptin, and serum concentration of25(OH)VitD, as well as authropometry index were detected between obese children with Simple obesity and control group to evaluate whether25(OH)VitD was interrelated with adiponectin, leptin and obesity2) Prospective intervention study:all obese children received treatment with vitamin D in addition to diet control and exercise for three month. Then comparative analysis was made in the differences in BMI(body mass index), general biochemical indicator and fat source sex hormones such as adiponectin and leptin before and after the treatment.Results:Vitro experiment: The primary cultured human omental preadipocytes differentiated into adipocytes containing reflective lipid drops tested by the oil red O dyeing. Statistics showed that1,25(OH)2VitD had no influence on the number and MTT of omental preadipocytes cultured in vitro(P>0.05) in proliferation. Immunohistochemistry results demonstrated adiponectin was in diffuse expression in cytoplasm. Western blot results showed that the expression level of differentiation group was significantly higher than that of non-differentiation group. While expression level of adiponectin from differentiated adipocyte decreased gradually when treated with10-6-10-41,25(OH)2VitD with statistic significance (P<0.05) Immunohistochemistry results also showed that leptin was mainly expressed in cytoplasm with a little expression of leptin in the undifferentiated group (IOD1867.56). Western blot results demonstrated that10-6-10-41,25(OH)2VitD has inhabitation effect on differentiated adipocyte leptin (P<0.05)Clinical study:Level of BMI and leptin of obese children with simple obesity was higher than that in the healthy control group(P<0.05), whereas level of adiponectin and25(OH)VitD was lower than normal subjects(P<0.05), with statistic significance. But there was no significant difference in blood sugar, cholesterol and triglyceride(P>0.05). Multiple linear regression analysis indicates that correlation factors with BMI were25(OH)VitD adiponectin, and then leptin. BMI and adiponectin were in positive correlation, BMI and leptin,25(OH)VitD negative. After treated with vitamin D for three months,25(OH)VitD, blood sugar, cholesterol and triglyceride of children with simple obesity decreased with no significant difference, which showed vitamin D did not improve indices of blood fat, blood sugar and BMI. While the25(OH)VitD and adiponectin increased obviously and leptin decreased obviously, with significant difference.(P<0.05).Conclusion:Vitamin D has no effects on adipocyte proliferation, but inhibits adipocyte differentiation in suitable density. Vitamin D in certain density can inhibit the expression of adiponectin and leptin in the process of adipocyte differentiation. Obese children with low level of vitamin D are in lipid disorders, which can be improved if treated with proper amount of vitamin D.更多还原