脂肪细胞分化关键基因LXRα的生物信息学分析及表达规律
摘要
脂肪细胞分化实质是一系列基因时序表达的过程，这些基因主要有PPARr、LPL与HSL等。肝x受体α(LiverXrecePtors，LXRα)是脂类代谢过程中一种重要的核受体，研究发现LXRα在脂肪组织中高表达，并且LXRα激活后可增加一系列脂肪细胞标志基因如PPARr、LPL、瘦素等的表达。由此推断LXRα可能参与脂肪细胞的分化。细胞因子是影响脂肪细胞分化的重要因素，多项研究表明白细胞介素-6（IL-6）能够刺激脂肪分解和脂肪酸氧化，然而目前对于IL-6调控脂肪细胞分化的分子机制尚不清楚。
本试验首先对GenBank已发表的猪LXR基因进行生物信息学分析，在此理论基础上，以猪为研究对象，利用半定量RT-PCR技术研究LXRα在其各组织中的表达规律；利用不同浓度IL-6处理SD大白鼠，分别提取其脂肪组织的RNA和蛋白，通过RT-PCR和Western blotting技术检测脂肪细胞分化相关基因PPARr、LXRα、LPL与HSL的表达变化情况；最后分析IL-6调控脂肪细胞分化的可能机制，为进一步应用IL-6调控动物体脂沉积，控制人类肥胖及其相关疾病的研究提供新的靶点和理论依据。
研究结果如下：
1、用GenBank已发表猪的LXRα基因的进行蛋白质结构分析，得出猪LXRα基因编码454个氨基酸蛋白，推测分子量约502910.00 Da。并且LXRα的蛋白质二级结构为混合型，其蛋白包括2个低组分复杂性区域、1个锌指结构ZnF-C4和1个核受体超家族配体结合区（LBD）。系统发育分析发现猪的LXRα与鼠有比较近的亲缘关系。
2、以猪为研究对象，摸索出扩增猪LXRα基因的最佳条件。在退火温度为48.9℃与镁离子浓度为2mmol/L的条件下，猪LXRα基因的扩增效率最高。
3、以猪为研究对象，运用RT-PCR技术研究LXRα基因在其8个不同组织（内脏脂肪、心脏、脾脏、肝脏、肺脏、皮下脂肪、肌肉、肾脏）中的表达情况。结果表明：LXRα的表达具有组织特异性。LXRα在猪的肝脏、皮下脂肪、内脏脂肪组织中高表达，在肾脏中中等表达，而在脾脏、肺脏、肌肉、心脏中表达很少，几乎没有。并且LXRα肝脏中的表达与其他组织相比差异显著（P<0.5）。说明LXRα基因脂质代谢中发挥着重要的作用。
4、以SD大白鼠为研究对象，用不同浓度(0、0.1ug/ml、0.4ug/ml)的IL-6处理48h后，与对照组相比，PPARr、LXRα和LPL基因的转录水平显著下调，同时PPARr的蛋白水平也明显下调。然而对HSL的mRNA表达影响很小。
综上所述：IL-6抑制脂肪细胞分化相关基因PPARr、LXRα和LPL的表达，可能作为IL-6调控脂肪细胞分化的新的机制或机理。
Adipocytes differentiation is a expression process of somemarker genes really. The series of genes include PPARγ, HSL andLPL. Liver X receptorα(LXRα) gene is a member of the nuclearreceptor supper family, which play a dominant role in lipidmetabolism. LXRαis highly expressed in adipocyte. And it canenhance the expression of adipocyte specific genes. For examplePPARr, LPL and leptin. Therefore, we infer LXRαmay haveparticipated during adipocytes differentiation. Eytokines areimportant factors affecting adipocytes differentiation. Someresearches indicated IL-6 can stimulate lipolysis and fatty acidoxidation. While at present，the molecular mechanism is remainsunclear for IL-6 regulating of adipocyte differentiation.
In this study，at first, we analyzed the the pig LXRαbybioinformatics methods which has been published on gene bank. Then healthy pigs were used as experimental animal. The mRNAdistribution profile of the porcine LXRαgene in eight tissues (heart,liver, subcutaneous adipose, kidney, visceral adipose, spleen, lung,Muscle) from the pig sample were examined by RT-PCR. Then the SDrats were incubated with different IL-6 concentrations for varioustimes Afterthat, RNA and protein were extracted from adiposetissue. Further, gene expression of PPARr, LXRα, LPL snd HSL weredetermined with RT-PCR and Western blot to resolve the possiblemechanisms of IL-6 regulating of adipocyte differentiation. It willprovide available information for control of human obesity and thetherapy of metabolic diseases.
The main results were as follows:
1. Protein structure analysis suggested that that the relativemolecular weight of LXRαreceptor is 502910.00 D, coding 454Amino Acids, LXRαprotein is mainly consists of three functionaldomains: a Ligand-binding Domain, a DNA-binding Domain, and aZinc fingerr C4 region. phylogenetic result showed that the LXRαgenes in Mammalia clade were very conserved.
2. When the annealing temperature is 48.9℃and MgCl2concentration is 2mmol/L, the pig LXRαgene was amplificatedefficiently.
3. The mRNA distribution profile of the porcine LXRαgene in eighttissues (heart, liver, subcutaneous adipose, kidney, visceral adipose,spleen, lung, Muscle) from the pig sample were examined by RT-PCR.The results showed that the LXRαgene was expressed specificity.The porcine LXRαgene highly expressed in liver, visceral adiposetissue, subcutaneous adipose tissue and kidney. While remarkablelower in heart, lung, spleen and muscle. The highest of LXRαexpression levels were found in liver tissue. The results suggestedthat LXRαhas a close correlation with lipid metabolism.
4. Then the SD rats were incubated with different IL-6concentrations (0, 0.1ug/ml, 0.4ug/ml) for 48h. The result of RT-PCRshow that IL-6 significantly inhibits the mRNA expression ofadipogenic marker genes PPARr, LXRαand LPL. But HSL mRNAlevels were not changed. The effect of IL-6 on expresion of PPARγwas detected by Western blot. All these test support the downregulation of protein expression for PPARγby IL-6.
These results showed that different concentrations of IL-6 couldinhibit adipocyte differentiation by regulating the expressions ofLXRα, LPL and PPARγ.
本试验首先对GenBank已发表的猪LXR基因进行生物信息学分析，在此理论基础上，以猪为研究对象，利用半定量RT-PCR技术研究LXRα在其各组织中的表达规律；利用不同浓度IL-6处理SD大白鼠，分别提取其脂肪组织的RNA和蛋白，通过RT-PCR和Western blotting技术检测脂肪细胞分化相关基因PPARr、LXRα、LPL与HSL的表达变化情况；最后分析IL-6调控脂肪细胞分化的可能机制，为进一步应用IL-6调控动物体脂沉积，控制人类肥胖及其相关疾病的研究提供新的靶点和理论依据。
研究结果如下：
1、用GenBank已发表猪的LXRα基因的进行蛋白质结构分析，得出猪LXRα基因编码454个氨基酸蛋白，推测分子量约502910.00 Da。并且LXRα的蛋白质二级结构为混合型，其蛋白包括2个低组分复杂性区域、1个锌指结构ZnF-C4和1个核受体超家族配体结合区（LBD）。系统发育分析发现猪的LXRα与鼠有比较近的亲缘关系。
2、以猪为研究对象，摸索出扩增猪LXRα基因的最佳条件。在退火温度为48.9℃与镁离子浓度为2mmol/L的条件下，猪LXRα基因的扩增效率最高。
3、以猪为研究对象，运用RT-PCR技术研究LXRα基因在其8个不同组织（内脏脂肪、心脏、脾脏、肝脏、肺脏、皮下脂肪、肌肉、肾脏）中的表达情况。结果表明：LXRα的表达具有组织特异性。LXRα在猪的肝脏、皮下脂肪、内脏脂肪组织中高表达，在肾脏中中等表达，而在脾脏、肺脏、肌肉、心脏中表达很少，几乎没有。并且LXRα肝脏中的表达与其他组织相比差异显著（P<0.5）。说明LXRα基因脂质代谢中发挥着重要的作用。
4、以SD大白鼠为研究对象，用不同浓度(0、0.1ug/ml、0.4ug/ml)的IL-6处理48h后，与对照组相比，PPARr、LXRα和LPL基因的转录水平显著下调，同时PPARr的蛋白水平也明显下调。然而对HSL的mRNA表达影响很小。
综上所述：IL-6抑制脂肪细胞分化相关基因PPARr、LXRα和LPL的表达，可能作为IL-6调控脂肪细胞分化的新的机制或机理。
Adipocytes differentiation is a expression process of somemarker genes really. The series of genes include PPARγ, HSL andLPL. Liver X receptorα(LXRα) gene is a member of the nuclearreceptor supper family, which play a dominant role in lipidmetabolism. LXRαis highly expressed in adipocyte. And it canenhance the expression of adipocyte specific genes. For examplePPARr, LPL and leptin. Therefore, we infer LXRαmay haveparticipated during adipocytes differentiation. Eytokines areimportant factors affecting adipocytes differentiation. Someresearches indicated IL-6 can stimulate lipolysis and fatty acidoxidation. While at present，the molecular mechanism is remainsunclear for IL-6 regulating of adipocyte differentiation.
In this study，at first, we analyzed the the pig LXRαbybioinformatics methods which has been published on gene bank. Then healthy pigs were used as experimental animal. The mRNAdistribution profile of the porcine LXRαgene in eight tissues (heart,liver, subcutaneous adipose, kidney, visceral adipose, spleen, lung,Muscle) from the pig sample were examined by RT-PCR. Then the SDrats were incubated with different IL-6 concentrations for varioustimes Afterthat, RNA and protein were extracted from adiposetissue. Further, gene expression of PPARr, LXRα, LPL snd HSL weredetermined with RT-PCR and Western blot to resolve the possiblemechanisms of IL-6 regulating of adipocyte differentiation. It willprovide available information for control of human obesity and thetherapy of metabolic diseases.
The main results were as follows:
1. Protein structure analysis suggested that that the relativemolecular weight of LXRαreceptor is 502910.00 D, coding 454Amino Acids, LXRαprotein is mainly consists of three functionaldomains: a Ligand-binding Domain, a DNA-binding Domain, and aZinc fingerr C4 region. phylogenetic result showed that the LXRαgenes in Mammalia clade were very conserved.
2. When the annealing temperature is 48.9℃and MgCl2concentration is 2mmol/L, the pig LXRαgene was amplificatedefficiently.
3. The mRNA distribution profile of the porcine LXRαgene in eighttissues (heart, liver, subcutaneous adipose, kidney, visceral adipose,spleen, lung, Muscle) from the pig sample were examined by RT-PCR.The results showed that the LXRαgene was expressed specificity.The porcine LXRαgene highly expressed in liver, visceral adiposetissue, subcutaneous adipose tissue and kidney. While remarkablelower in heart, lung, spleen and muscle. The highest of LXRαexpression levels were found in liver tissue. The results suggestedthat LXRαhas a close correlation with lipid metabolism.
4. Then the SD rats were incubated with different IL-6concentrations (0, 0.1ug/ml, 0.4ug/ml) for 48h. The result of RT-PCRshow that IL-6 significantly inhibits the mRNA expression ofadipogenic marker genes PPARr, LXRαand LPL. But HSL mRNAlevels were not changed. The effect of IL-6 on expresion of PPARγwas detected by Western blot. All these test support the downregulation of protein expression for PPARγby IL-6.
These results showed that different concentrations of IL-6 couldinhibit adipocyte differentiation by regulating the expressions ofLXRα, LPL and PPARγ.
引文
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