RXRα在猪前体脂肪细胞分化中的作用及其机理研究
摘要
脂肪组织不仅仅是被动的能量储存器官,也是能够分泌多种激素类物质的内分泌器官。脂肪组织的过度发育,即体脂过度沉积在人类表现为肥胖并引发其他相关疾病如糖尿病、冠心病、动脉粥样硬化、高血压、高脂血症等,在畜牧上则造成动物产品胴体品质下降等问题。体脂沉积的核心环节在于脂肪细胞的增殖和分化,因此研究脂肪细胞分化的分子机理尤为重要。猪的解剖及生理学特点与人相似,其体脂沉积模式与啮齿类动物存在显著差异,与人的更为接近,且猪是重要的畜牧养殖对象,因此以猪为实验动物研究脂肪细胞分化的机理,无论在人类医学还是动物生产方面均具有重要意义。
脂肪细胞分化过程中重要的标志基因包括维甲酸X受体(RXRα)、CCAAT增强子结合蛋白Alpha(C/EBPα)、过氧化物酶体增殖物激活受体Gamma(PPARγ)、脂肪细胞决定与分化因子-1(ADD-1/ SREBP-1)、脂肪细胞特异基因如脂蛋白脂酶(LPL)和脂肪细胞脂肪酸结合蛋白(aP2)等。其中RXR属于核激素受体超家族,它包括α、β和γ三种亚型,它们可以作为配体激活型转录因子调控多细胞动物生命的许多方面。但有关RXR的作用机制尚无定论,除了RXR作为其它核受体的配体这一途径之外,是否还存在独立的RXR调控的信号通路仍是需要深入研究的问题。研究表明,脂肪细胞中RXRα/PPARγ异源二聚体通过调控脂肪细胞特异基因的表达来影响脂肪细胞分化,但关于RXRα在脂肪细胞分化中的直接作用是否存在尚不清楚,其在猪前体脂肪细胞分化中的作用及其机理的研究则尚未见报道。
本论文以1-3日龄健康状况良好的长白仔猪为实验动物,构建猪前体脂肪细胞体外培养体系,利用SQ RT-PCR、间接免疫荧光技术检测了RXRα在猪前体脂肪细胞分化过程中的时序表达情况;利用油红O染色及提取法、GPDH活性测定、SQ RT-PCR技术研究了RXRα的配体9-cisRA对猪前体脂肪细胞分化的影响;采用载体构建、化学合成、脂质体转染、油红O染色及提取法、GPDH活性测定、SQ RT-PCR、间接免疫荧光和Western blotting等技术检测了超表达和沉默RXRα后猪前体脂肪细胞分化状况并探讨了相关机理,为进一步揭示猪前体脂肪细胞分化规律,完善猪体脂沉积理论提供资料,并为通过基因调控预防和治疗肥胖及相关疾病的方法的进一步确立提供了思路和理论依据。同时本研究也探讨了RXRα对猪前体脂肪细胞凋亡的影响。
获得的主要研究结果如下:
1. RXRα在猪前体脂肪细胞分化的2-8 d持续表达,且表达水平在此过程中无显著变化。
2. RXRα的配体9-cisRA对猪前体脂肪细胞分化具有重要的调控作用,且其作用与浓度有关。低浓度下(0-10 nmol/L)9-cisRA促进猪前体脂肪细胞分化,并且这种促进作用是通过使得脂肪细胞数目、脂肪细胞内脂肪含量、GPDH活性增加,以及上调RXRα、PPARγmRNA表达实现的;高浓度下(100 nmol/L -10μmol/L)9-cisRA则抑制前体脂肪细胞分化,并且这种抑制作用是通过使得脂肪细胞数目减少、脂肪细胞内脂肪含量及GPDH活性降低,以及下调RXRα、PPARγmRNA表达实现的。
3.超表达RXRα促进猪前体脂肪细胞分化,且这种促进作用是通过使得脂肪细胞数目、脂肪细胞内脂肪含量、GPDH活性增加,以及上调PPARγ和C/EBPαmRNA的表达实现的。
4.沉默RXRα抑制猪前体脂肪细胞分化,且这种抑制作用是通过使得脂肪细胞数目减少、脂肪细胞内脂肪含量及GPDH活性降低,以及下调PPARγ和C/EBPαmRNA的表达实现的。
5. RXRα抑制猪前体脂肪细胞凋亡。
6.运用脂质体Lipofectamine 2000转染pSUPER-RXRα干扰载体和化学合成的siRNA,结果表明pSUPER-RXRα干扰载体太大,转染效率很低,化学合成法可以有效利用Lipofectamine 2000转染猪前体脂肪细胞,且带有荧光标记的siRNA可有效用于检测转染效率及优化转染方法。
研究认为,RXRα在猪前体脂肪细胞分化过程中持续表达,并对此过程具有促进作用。其配体在不同的剂量范围内,通过活化RXRα对分化过程产生不同影响。RXRα促进猪前体脂肪细胞分化的作用与脂肪细胞转录因子PPARγ和C/EBPα有关,且此作用可能是通过与PPARγ形成异源二聚体或直接调控C/EBPα的基因表达来实现的;同时研究表明RXRα影响猪前体脂肪细胞凋亡,其影响表现为抑制作用。
Adipose tissue stores energy passively, and also acts as an endocrine organ secreting multiple hormonal materials. The over development of the adipose tissue, also expressed as excessive body fat deposition, leads to human obesity and related complications, such as diabetes, coronary artery disease, atherosclerosis, hypertension and hyperlipoidemia, and the decrease of meat quality in animal products. The key loop of fat deposition is the over proliferation and differentiation of adipocytes. Thus, it is very important to study the molecular mechanism of adipocytes differentiation. Compared with rodent animals, the anatomic and physiological characteristics, and the mode of fat deposition of pig are much similar to those of human. Additionally, pig is the main cultivated animal. Therefore, to study the mechanism of porcine adipocytes differentiation is significant not only for human medicine, but also for animal production industry.
The main marker genes during adipocytes differentiation include RXRα, C/EBPα, PPARγ, ADD-1/ SREBP-1, LPL and aP2. RXR belongs to super family of nuclear hormone receptor, which contains three subtypes (α,βandγ), and regulates many aspects of metazoan life as ligand activated transcriptional factor. However, it is still uncertain about the role of RXR in the adipocytes differentiating process. In addition to the pathway of RXR acting as a nuclear receptor, whether there is other independent signal pathway regulated by RXR or not still needs to be further studied. It has been identified that heterodimer of RXRα/PPARγinfluence adipocytes differentiation by modulating the expression of adipocyte specific genes. However, whether RXRαhas the direct role in adipocytes differentiation or not remains unclear. Furthermore, the role and mechanism of RXRαin porcine preadipocytes differentiation has not been reported.
In this study, healthy, 1-3-old-day Landrace piglets were used as experimental animal to construct in vitro culture system of porcine preadipocytes. The time-spatial expression of RXRαduring porcine preadipocytes differentiating process was investigated with SQ RT-PCR and indirect immunofluorescence technique. Porcine preadipocytes were treated with 9-cisRA, a ligand of RXRα. Subsequently, the effects of 9-cisRA on porcine preadipocytes differentiation were detected by Oil red O staining, Oil red O extraction, GPDH activity analysis and SQ RT-PCR. The change of porcine preadipocytes differentiation and underlying molecular mechanism were checked by vector construction, chemical synthesis, liposome transfection, Oil red O staining, Oil red O extraction, GPDH activity analysis, SQ RT-PCR, indirect immunofluorescence, and Western Blotting while the overexpression or silencing of RXRαgene. The obtained results provided the information and basis for consummating the RXRαrelated adipocytes differentiation regulation theory. The influence of RXRαon the adipocyts apoptosis was studied as well in the present work.
The main results were as follows:
1. RXRαcontinuously expressed at 2-8 d during porcine preadipocytes differentiation and its expressing levels were no significant difference during the differentiating process.
2. 9-cisRA, a ligand of RXRα, plays an important regulatory role in porcine preadipocytes differentiation. 9-cisRA at lower concentration(0-10 nmol/L)promoted preadipocytes differentiation, proved to be the evidences that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα、PPARγmRNA increased. On the other hand, 9-cisRA at higher concentration(100 nmol/L -10μmol/L)repressed preadipocytes differentiation, shown as the phenomena that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα, PPARγmRNA decreased.
3. Overexpression of RXRαgene could promote porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA increased.
4. Silencing of RXRαgene could inhibit porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA decreased.
5. RXRαcould inhibit the apoptosis of porcine adipocytes.
6. Lipofectamine 2000 was used to transfect pSUPER-RXRαRNAi and chemically synthesized siRNA. It was shown that the transfection efficiency was lower with the big pSUPER-RXRαRNAi, while the chemically synthesized siRNA was effectively transfected into porcine preadipocytes by using Lipofectamine 2000. On the other hand, siRNA with fluorescence marker could be used to check transfection efficiency and optimize the transfection method.
Taken together, RXRαexpresses in the duration of porcine preadipocyte differentiation and enhances this process. Its ligand 9-cisRA influences the differentiation via RXR in different manners with different treating dosage. The effects of RXRαon the porcine preadipocyte differentiation closely relate to its influence on the expression of PPARγand C/EBPαgenes, and these effects might be due to its formation to heterodimer with PPARγor its directly regulation of the expression of C/EBPαgene. It is also concluded that RXRαmight influence the apoptosis of porcine preadipocytes, and this influence appears to be an inhibition effect.
脂肪细胞分化过程中重要的标志基因包括维甲酸X受体(RXRα)、CCAAT增强子结合蛋白Alpha(C/EBPα)、过氧化物酶体增殖物激活受体Gamma(PPARγ)、脂肪细胞决定与分化因子-1(ADD-1/ SREBP-1)、脂肪细胞特异基因如脂蛋白脂酶(LPL)和脂肪细胞脂肪酸结合蛋白(aP2)等。其中RXR属于核激素受体超家族,它包括α、β和γ三种亚型,它们可以作为配体激活型转录因子调控多细胞动物生命的许多方面。但有关RXR的作用机制尚无定论,除了RXR作为其它核受体的配体这一途径之外,是否还存在独立的RXR调控的信号通路仍是需要深入研究的问题。研究表明,脂肪细胞中RXRα/PPARγ异源二聚体通过调控脂肪细胞特异基因的表达来影响脂肪细胞分化,但关于RXRα在脂肪细胞分化中的直接作用是否存在尚不清楚,其在猪前体脂肪细胞分化中的作用及其机理的研究则尚未见报道。
本论文以1-3日龄健康状况良好的长白仔猪为实验动物,构建猪前体脂肪细胞体外培养体系,利用SQ RT-PCR、间接免疫荧光技术检测了RXRα在猪前体脂肪细胞分化过程中的时序表达情况;利用油红O染色及提取法、GPDH活性测定、SQ RT-PCR技术研究了RXRα的配体9-cisRA对猪前体脂肪细胞分化的影响;采用载体构建、化学合成、脂质体转染、油红O染色及提取法、GPDH活性测定、SQ RT-PCR、间接免疫荧光和Western blotting等技术检测了超表达和沉默RXRα后猪前体脂肪细胞分化状况并探讨了相关机理,为进一步揭示猪前体脂肪细胞分化规律,完善猪体脂沉积理论提供资料,并为通过基因调控预防和治疗肥胖及相关疾病的方法的进一步确立提供了思路和理论依据。同时本研究也探讨了RXRα对猪前体脂肪细胞凋亡的影响。
获得的主要研究结果如下:
1. RXRα在猪前体脂肪细胞分化的2-8 d持续表达,且表达水平在此过程中无显著变化。
2. RXRα的配体9-cisRA对猪前体脂肪细胞分化具有重要的调控作用,且其作用与浓度有关。低浓度下(0-10 nmol/L)9-cisRA促进猪前体脂肪细胞分化,并且这种促进作用是通过使得脂肪细胞数目、脂肪细胞内脂肪含量、GPDH活性增加,以及上调RXRα、PPARγmRNA表达实现的;高浓度下(100 nmol/L -10μmol/L)9-cisRA则抑制前体脂肪细胞分化,并且这种抑制作用是通过使得脂肪细胞数目减少、脂肪细胞内脂肪含量及GPDH活性降低,以及下调RXRα、PPARγmRNA表达实现的。
3.超表达RXRα促进猪前体脂肪细胞分化,且这种促进作用是通过使得脂肪细胞数目、脂肪细胞内脂肪含量、GPDH活性增加,以及上调PPARγ和C/EBPαmRNA的表达实现的。
4.沉默RXRα抑制猪前体脂肪细胞分化,且这种抑制作用是通过使得脂肪细胞数目减少、脂肪细胞内脂肪含量及GPDH活性降低,以及下调PPARγ和C/EBPαmRNA的表达实现的。
5. RXRα抑制猪前体脂肪细胞凋亡。
6.运用脂质体Lipofectamine 2000转染pSUPER-RXRα干扰载体和化学合成的siRNA,结果表明pSUPER-RXRα干扰载体太大,转染效率很低,化学合成法可以有效利用Lipofectamine 2000转染猪前体脂肪细胞,且带有荧光标记的siRNA可有效用于检测转染效率及优化转染方法。
研究认为,RXRα在猪前体脂肪细胞分化过程中持续表达,并对此过程具有促进作用。其配体在不同的剂量范围内,通过活化RXRα对分化过程产生不同影响。RXRα促进猪前体脂肪细胞分化的作用与脂肪细胞转录因子PPARγ和C/EBPα有关,且此作用可能是通过与PPARγ形成异源二聚体或直接调控C/EBPα的基因表达来实现的;同时研究表明RXRα影响猪前体脂肪细胞凋亡,其影响表现为抑制作用。
Adipose tissue stores energy passively, and also acts as an endocrine organ secreting multiple hormonal materials. The over development of the adipose tissue, also expressed as excessive body fat deposition, leads to human obesity and related complications, such as diabetes, coronary artery disease, atherosclerosis, hypertension and hyperlipoidemia, and the decrease of meat quality in animal products. The key loop of fat deposition is the over proliferation and differentiation of adipocytes. Thus, it is very important to study the molecular mechanism of adipocytes differentiation. Compared with rodent animals, the anatomic and physiological characteristics, and the mode of fat deposition of pig are much similar to those of human. Additionally, pig is the main cultivated animal. Therefore, to study the mechanism of porcine adipocytes differentiation is significant not only for human medicine, but also for animal production industry.
The main marker genes during adipocytes differentiation include RXRα, C/EBPα, PPARγ, ADD-1/ SREBP-1, LPL and aP2. RXR belongs to super family of nuclear hormone receptor, which contains three subtypes (α,βandγ), and regulates many aspects of metazoan life as ligand activated transcriptional factor. However, it is still uncertain about the role of RXR in the adipocytes differentiating process. In addition to the pathway of RXR acting as a nuclear receptor, whether there is other independent signal pathway regulated by RXR or not still needs to be further studied. It has been identified that heterodimer of RXRα/PPARγinfluence adipocytes differentiation by modulating the expression of adipocyte specific genes. However, whether RXRαhas the direct role in adipocytes differentiation or not remains unclear. Furthermore, the role and mechanism of RXRαin porcine preadipocytes differentiation has not been reported.
In this study, healthy, 1-3-old-day Landrace piglets were used as experimental animal to construct in vitro culture system of porcine preadipocytes. The time-spatial expression of RXRαduring porcine preadipocytes differentiating process was investigated with SQ RT-PCR and indirect immunofluorescence technique. Porcine preadipocytes were treated with 9-cisRA, a ligand of RXRα. Subsequently, the effects of 9-cisRA on porcine preadipocytes differentiation were detected by Oil red O staining, Oil red O extraction, GPDH activity analysis and SQ RT-PCR. The change of porcine preadipocytes differentiation and underlying molecular mechanism were checked by vector construction, chemical synthesis, liposome transfection, Oil red O staining, Oil red O extraction, GPDH activity analysis, SQ RT-PCR, indirect immunofluorescence, and Western Blotting while the overexpression or silencing of RXRαgene. The obtained results provided the information and basis for consummating the RXRαrelated adipocytes differentiation regulation theory. The influence of RXRαon the adipocyts apoptosis was studied as well in the present work.
The main results were as follows:
1. RXRαcontinuously expressed at 2-8 d during porcine preadipocytes differentiation and its expressing levels were no significant difference during the differentiating process.
2. 9-cisRA, a ligand of RXRα, plays an important regulatory role in porcine preadipocytes differentiation. 9-cisRA at lower concentration(0-10 nmol/L)promoted preadipocytes differentiation, proved to be the evidences that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα、PPARγmRNA increased. On the other hand, 9-cisRA at higher concentration(100 nmol/L -10μmol/L)repressed preadipocytes differentiation, shown as the phenomena that the number of adipocytes, fat content in adipocytes, GPDH activity and the expression of RXRα, PPARγmRNA decreased.
3. Overexpression of RXRαgene could promote porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA increased.
4. Silencing of RXRαgene could inhibit porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, GPDH activity, and the abundance of PPARγand C/EBPαmRNA decreased.
5. RXRαcould inhibit the apoptosis of porcine adipocytes.
6. Lipofectamine 2000 was used to transfect pSUPER-RXRαRNAi and chemically synthesized siRNA. It was shown that the transfection efficiency was lower with the big pSUPER-RXRαRNAi, while the chemically synthesized siRNA was effectively transfected into porcine preadipocytes by using Lipofectamine 2000. On the other hand, siRNA with fluorescence marker could be used to check transfection efficiency and optimize the transfection method.
Taken together, RXRαexpresses in the duration of porcine preadipocyte differentiation and enhances this process. Its ligand 9-cisRA influences the differentiation via RXR in different manners with different treating dosage. The effects of RXRαon the porcine preadipocyte differentiation closely relate to its influence on the expression of PPARγand C/EBPαgenes, and these effects might be due to its formation to heterodimer with PPARγor its directly regulation of the expression of C/EBPαgene. It is also concluded that RXRαmight influence the apoptosis of porcine preadipocytes, and this influence appears to be an inhibition effect.
引文
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