Chemerin在3T3-L1脂肪细胞分化过程中的表达改变及其与脂联素、瘦素等关系的初步研究
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摘要
目的：1.观察chemerin及其受体chemerinR基因在小鼠各脏器中的表达情况；探讨3T3-L1脂肪细胞分化成熟过程中chemerin及其受体基因表达的变化。2.探讨PPAR-γ激动剂——罗格列酮对3T3-L1脂肪细胞chemerin及其受体基因表达的影响。3.重组chemerin蛋白对3T3-L1前脂肪细胞分化的影响并观察其对脂联素、瘦素和PPAR-γ2等基因表达的影响。
方法：1.提取正常小鼠肝脏、脂肪、胃、脾脏、肾脏、心脏、骨骼肌等脏器组织中总RNA,采用半定量RT-PCR技术检测其中chemerin及chemerinR基因表达水平；体外培养3T3-L1前脂肪细胞并诱导分化,实时荧光定量PCR法检测诱导分化过程中不同天数(0、2、4、6、8、10天)chemerin和chemerinR基因的表达变化。2.3T3-L1细胞诱导分化至第10天为正常对照组；另一组在诱导分化过程中用10μM罗格列酮干预。采用实时荧光定量PCR法检测不同天数(0、2、4、6、8、10天)chemerin和chemerinR基因表达情况。3T3-L1脂肪细胞诱导成熟后,分别以不同浓度梯度(0、0.μM、1μM、10μM)的罗格列酮干预24小时后收集细胞,实时荧光定量PCR法检测chemerin和chemerinR基因的表达情况。3.3T3-L1细胞诱导分化至第10天为正常对照组；一组诱导分化过程中用罗格列酮干预；另一组予以chemerin蛋白干预。采用实时荧光定量PCR法检测第10天PPAR-γ2、C/EBPa基因以及adiponectin、leptin基因表达情况。3T3-L1细胞诱导成熟后,分别以不同浓度梯度(0、2.5ng/m1、25 ng/ml、50 ng/m1)的chemerin蛋白作用24小时后收集细胞,实时荧光定量PCR法检测PPAR-γ2、C/EBPa基因以及adiponectin、leptin基因的表达情况。
结果：1、chemerin及其受体基因在小鼠肝脏、脂肪、胃、脾脏、肾脏、心脏、骨骼肌中均有表达；chemerin及chemerinR基因低表达于3T3-L1前体脂肪细胞中,并随前体脂肪细胞诱导分化成熟表达水平呈逐渐上调趋势(P<0.05)。2、与对照组相比罗格列酮组对3T3-L1前脂肪细胞诱导分化过程中及成熟的3T3-Ll脂肪细胞chemerin和chemerinR基因均起上调作用(P<0.05)。3、与对照组相比在3T3-L1细胞诱导分化过程中第10天罗格列酮干预组、chemerin蛋白组均上调PPAR-γ2、C/EBPa、adiponectin、leptin基因的表达(p＜0.05)。3T3-L1细胞诱导分化成熟后,与对照组相比chemerin蛋白组上调了PPARγy 2、C/EBPa及adiponectin、leptin基因的表达(p<0.05)。
结论：1.chemerin及其受体基因在小鼠脂肪组织中均有表达；在3T3-L1脂肪前体细胞分化过程中,chemerin及其受体基因表达逐渐上调。2.PPAR-Y激动剂-罗格列酮上调chemerin、chemerinR基因的表达。3.chemerin蛋白上调成熟3T3-L1脂肪细胞中PPAR-γ2、C/EBPa及adiponectin、leptin基因的表达。
Objective:1.To observe the change of chemerin and chemerinR gene expression in various tissues of mice and in the process of 3T3-L1 preadipocyte differentiation.2.To explore the effects of PPAR-y agonist-rosiglitazone on chemerin and chemerinR gene expression.3.To observe the effects of recombinant mouse chemerin on 3T3-L1 preadipocyte differentiation and the change of adiponectin.leptin and PPAR-γ2 gene expression.
Method:1. Total RNA was extracted from several kinds of tissues (liver, adipose tissue, stomach, spleen, kidney, heart and skeletal muscle) and Semi-quantitative RT-PCR was performed in order to measure levels of chemerin and chemerinR mRNA.Culture of 3T3-L1 cells and induction of differentiation were performed in vitro. Realtime-PCR was performed in order to measure levels of chemerin and chemerinR mRNA of differential time points (0d,2d,4d,6d,8d,10d).2.3T3-L1 cells were differentiated for 10 days as the normal control group; the other group in the induction of differentiation using 10μM rosiglitazone. Real-time fluorescent quantitative PCR method was applyed to detect the expressions of chemerin and chemerinR gene. After 3T3-L1 fat cells were matured, different concentrations of rosiglitazone(0,0.1,1,10μM) interfered for 24 hours then collected the cells, real-time quantitative PCR was used to detect the expression of chemerin and chemerinR gene.3T3-L1 cells differentiated for 10 days as the normal control group; a process of induced differentiation using rosiglitazone; another group using chemerin protein intervention. Real-time quantitative PCR was used to detect the expressions of PPAR-γ2 which was in the tenth days, so were C/EBPa gene and adiponectin, leptin gene. After 3T3-L1 fat cells were matured, different concentrations of chemerin protein interfered for 24 hours then collected the cells, real-time quantitative PCR was used to detect the expression of PPAR-γ2,C/ EBPa,adiponectin, leptin gene.
Results:1. chemerin and chemerinR gene were expressed in liver, adipose tissue, stomach, spleen, kidney, heart and skeletal muscle of mice. Chemerin and chemerinR mRNA levels dramatically increased during the process of differentiation of 3T3-L1 cells after 2 days of induction. (P<0.05).2. Compared with the control group, during 3T3-L1 preadipocytes differentiation and matured 3T3-L1 adipocytes,chemerin and chemerinR gene were upregulated using rosiglitazone (P<0.05).3.In 3T3-L1 cells during differentiation for 10 days compared with the control using rosiglitazone group, chemerin protein group could raised PPAR-γ2, C/EBPa, adiponectin, leptin gene expression (p<0.05). In matured 3T3-L1 cells, compared with the control group, chemerin protein raised PPAR-γ2, C/EBPa and adiponectin, leptin gene expression (p <0.05).
Conclusion:1. chemerin and chemerinR were both expressed in adipose tissues of mice; the expressions of chemerin and chemerinR gene were gradually increasesd during the 3T3-L1 preadipocyte differentiation process.2. PPAR-y agonist Rosiglitazone upregulated the expressions of chemerin and chemerinR gene.3. chemerin protein upregulated the expressions of PPAR-γ2, C/EBPa and adiponectin, leptin gene in matured 3T3-L1 adipocytes.
方法：1.提取正常小鼠肝脏、脂肪、胃、脾脏、肾脏、心脏、骨骼肌等脏器组织中总RNA,采用半定量RT-PCR技术检测其中chemerin及chemerinR基因表达水平；体外培养3T3-L1前脂肪细胞并诱导分化,实时荧光定量PCR法检测诱导分化过程中不同天数(0、2、4、6、8、10天)chemerin和chemerinR基因的表达变化。2.3T3-L1细胞诱导分化至第10天为正常对照组；另一组在诱导分化过程中用10μM罗格列酮干预。采用实时荧光定量PCR法检测不同天数(0、2、4、6、8、10天)chemerin和chemerinR基因表达情况。3T3-L1脂肪细胞诱导成熟后,分别以不同浓度梯度(0、0.μM、1μM、10μM)的罗格列酮干预24小时后收集细胞,实时荧光定量PCR法检测chemerin和chemerinR基因的表达情况。3.3T3-L1细胞诱导分化至第10天为正常对照组；一组诱导分化过程中用罗格列酮干预；另一组予以chemerin蛋白干预。采用实时荧光定量PCR法检测第10天PPAR-γ2、C/EBPa基因以及adiponectin、leptin基因表达情况。3T3-L1细胞诱导成熟后,分别以不同浓度梯度(0、2.5ng/m1、25 ng/ml、50 ng/m1)的chemerin蛋白作用24小时后收集细胞,实时荧光定量PCR法检测PPAR-γ2、C/EBPa基因以及adiponectin、leptin基因的表达情况。
结果：1、chemerin及其受体基因在小鼠肝脏、脂肪、胃、脾脏、肾脏、心脏、骨骼肌中均有表达；chemerin及chemerinR基因低表达于3T3-L1前体脂肪细胞中,并随前体脂肪细胞诱导分化成熟表达水平呈逐渐上调趋势(P<0.05)。2、与对照组相比罗格列酮组对3T3-L1前脂肪细胞诱导分化过程中及成熟的3T3-Ll脂肪细胞chemerin和chemerinR基因均起上调作用(P<0.05)。3、与对照组相比在3T3-L1细胞诱导分化过程中第10天罗格列酮干预组、chemerin蛋白组均上调PPAR-γ2、C/EBPa、adiponectin、leptin基因的表达(p＜0.05)。3T3-L1细胞诱导分化成熟后,与对照组相比chemerin蛋白组上调了PPARγy 2、C/EBPa及adiponectin、leptin基因的表达(p<0.05)。
结论：1.chemerin及其受体基因在小鼠脂肪组织中均有表达；在3T3-L1脂肪前体细胞分化过程中,chemerin及其受体基因表达逐渐上调。2.PPAR-Y激动剂-罗格列酮上调chemerin、chemerinR基因的表达。3.chemerin蛋白上调成熟3T3-L1脂肪细胞中PPAR-γ2、C/EBPa及adiponectin、leptin基因的表达。
Objective:1.To observe the change of chemerin and chemerinR gene expression in various tissues of mice and in the process of 3T3-L1 preadipocyte differentiation.2.To explore the effects of PPAR-y agonist-rosiglitazone on chemerin and chemerinR gene expression.3.To observe the effects of recombinant mouse chemerin on 3T3-L1 preadipocyte differentiation and the change of adiponectin.leptin and PPAR-γ2 gene expression.
Method:1. Total RNA was extracted from several kinds of tissues (liver, adipose tissue, stomach, spleen, kidney, heart and skeletal muscle) and Semi-quantitative RT-PCR was performed in order to measure levels of chemerin and chemerinR mRNA.Culture of 3T3-L1 cells and induction of differentiation were performed in vitro. Realtime-PCR was performed in order to measure levels of chemerin and chemerinR mRNA of differential time points (0d,2d,4d,6d,8d,10d).2.3T3-L1 cells were differentiated for 10 days as the normal control group; the other group in the induction of differentiation using 10μM rosiglitazone. Real-time fluorescent quantitative PCR method was applyed to detect the expressions of chemerin and chemerinR gene. After 3T3-L1 fat cells were matured, different concentrations of rosiglitazone(0,0.1,1,10μM) interfered for 24 hours then collected the cells, real-time quantitative PCR was used to detect the expression of chemerin and chemerinR gene.3T3-L1 cells differentiated for 10 days as the normal control group; a process of induced differentiation using rosiglitazone; another group using chemerin protein intervention. Real-time quantitative PCR was used to detect the expressions of PPAR-γ2 which was in the tenth days, so were C/EBPa gene and adiponectin, leptin gene. After 3T3-L1 fat cells were matured, different concentrations of chemerin protein interfered for 24 hours then collected the cells, real-time quantitative PCR was used to detect the expression of PPAR-γ2,C/ EBPa,adiponectin, leptin gene.
Results:1. chemerin and chemerinR gene were expressed in liver, adipose tissue, stomach, spleen, kidney, heart and skeletal muscle of mice. Chemerin and chemerinR mRNA levels dramatically increased during the process of differentiation of 3T3-L1 cells after 2 days of induction. (P<0.05).2. Compared with the control group, during 3T3-L1 preadipocytes differentiation and matured 3T3-L1 adipocytes,chemerin and chemerinR gene were upregulated using rosiglitazone (P<0.05).3.In 3T3-L1 cells during differentiation for 10 days compared with the control using rosiglitazone group, chemerin protein group could raised PPAR-γ2, C/EBPa, adiponectin, leptin gene expression (p<0.05). In matured 3T3-L1 cells, compared with the control group, chemerin protein raised PPAR-γ2, C/EBPa and adiponectin, leptin gene expression (p <0.05).
Conclusion:1. chemerin and chemerinR were both expressed in adipose tissues of mice; the expressions of chemerin and chemerinR gene were gradually increasesd during the 3T3-L1 preadipocyte differentiation process.2. PPAR-y agonist Rosiglitazone upregulated the expressions of chemerin and chemerinR gene.3. chemerin protein upregulated the expressions of PPAR-γ2, C/EBPa and adiponectin, leptin gene in matured 3T3-L1 adipocytes.
引文
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[1]Hutley L, Prins JB. Fat as endocrine organ:relationship to the metabolic syndrome. Am J Med Sci,2005,330 (6):280-289.
[2]Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J. Clin. Invest,2003,112:1821-1830.
[3]Weisberg SP, McCann D, Desai M, et al. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest,2003,112:1796-1808.
[4]Yang Q, Graham TE, Mody N, et al. Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature,2005,436:356-362.
[5]吴海娅,魏丽,王琛等.高脂或高糖饮食诱导胰岛素抵抗大鼠的附睾脂肪组织视黄醇结合蛋白4表达.上海医学,2007,30：446-449.
[6]Graham TE, Yang Q, Bluher M, et al. Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. New England Journal Of Medicine,2006,354:2552-2563.
[7]Cho YM, Youn BS, Lee H, et al. Plasma retinol-binding protein-4 concentrations are elevated in human subjects with impaired glucose tolerance and type 2 diabetes.Diabetes Care,
2006,29:2457-2461.
[8]Lee DC, Lee JW, Im JA Association of serum retinol binding protein 4 and insulin resistance in apparently healthy adolescents. Metabolism-clinical And Experimental,2007,56: 327-331.
[9]Gavi S, Stuart LM, Kelly P, et al. Retinol-binding protein 4 is associated with insulin resistance and body fat distribution in nonobese subjects without type 2 diabetes. Journal Of Clinical Endocrinology And Metabolism,2007,92:1886-1890.
[10]von Eynatten M, Lepper PM, Liu D, et al.Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease. Diabetologia,2007,50:1930-1937.
[11]Lee JW, Im JA, Lee HR, et al.Visceral adiposity is associated with serum retinol binding protein-4 levels in healthy women. Obesity,2007,15:2225-2232.
[12]吴海娅,贾伟平,魏丽等.肥胖及2型糖尿病患者血清视黄醇结合蛋白4水平的变化及其临床意义.中华内分泌代谢杂志,2006,22：290-293.
[13]Jia WP, Wu HY, Bao YQ, et al. Association of serum retinol-binding protein 4 and visceral adiposity in chinese subjects with and without type 2 diabetes. Journal Of Clinical Endocrinology And Metabolism (92):3224-3229.
[14]Ng TWK, Watts GF, Barrett PHR, et al.Effect of weight loss on LDL and HDL kinetics in the metabolic syndrome:Associations with changes in plasma retinol-binding protein-4 and adiponectin levels. Diabetes Care,2007,30:2945-2950.
[15]Shea J, Randell E, Vasdev S, et al. Serum retinol-binding protein 4 concentrations in response to shortterm overfeeding in normal-weight, overweight, and obese men. American Journal Of Clinical Nutrition,2007,86:1310-1315.
[16]Haider DG, Schindler K, Prager G, et al. Serum retinol-binding protein 4 is reduced after weight loss in morbidly obese subjects. Journal Of Clinical Endocrinology And Metabolism,2007, 92:1168-1171.
[17]Cabre A, Lazaro I, Girona J, et al. Retinol-binding protein 4 as a plasma biomarker of renal dysfunction and cardiovascular disease in type 2 diabetes. Journal Of Internal Medicine,2007,262: 496-503.
[18]Ziegelmeier M, Bachmann A, Seeger J, et al. Serum levels of adipokine retinol-binding protein-4 in relation to renal function. Diabetes Care,2007,30:2588-2592.
[19]Raila J, Henze A, Spranger J, et al. Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients. Kidney International,2007,72: 505-511.
[20]Takebayashi K, Suetsugu M, Wakabayashi S, et al. Retinol binding protein-4 levels and clinical features of type 2 diabetes patients. Journal Of Clinical Endocrinology And Metabolism, 2007,92:2712-2719.
[21]Yao-Borengasser A, Varma V, Bodles AM, et al. Retinol binding protein 4 expression in humans:Relationship to insulin resistance, inflammation, and response to pioglitazone. Journal Of Clinical Endocrinology And Metabolism,2007,92:2590-2597.
[22]Janke J, Engeli S, Boschmann M, et al. Retinol-binding protein 4 in human obesity. Diabetes,2006,55:2805-2810.