Col XV对脂肪细胞分化和脂代谢的影响及其机制研究

发布时间：2018-06-17 23:44

本文选题：Col + XV　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：Collagen XV(Col XV)是胶原家族的成员之一,分布在细胞基底膜表面,在动物生理和病理过程中发挥重要作用。Col XV与Col XVIII组成MULTIPLEXINs/内皮抑素产生的胶原蛋白亚类,它们结构上高度保守,在血管生成、炎症发生、肿瘤形成和脂肪代谢等方面具有类似功能。研究发现Col XVIII促进脂肪细胞分化,增加内脏脂肪的堆积,增加组织重构,维持能量稳态。Col XV在成脂分化过程中表达上调,参与组织再生、能量稳态和肥胖相关疾病的调节,然而Col XV对脂肪细胞分化的调节及其分子机制还有待研究。本研究为了明确Col XV调控脂肪细胞分化的作用机制,在脂肪细胞中超表达和干扰Col XV,得到以下结果:1.Col XV促进脂肪细胞分化和抑制脂质分解。Col XV在腹股沟白色脂肪、附睾白色脂肪的表达丰度较高,而在肩胛棕色脂肪、肝脏、肌肉和心脏等组织中的表达丰度较低,其中白色脂肪组织中Col XV的表达极显著高于棕色脂肪中的表达(P0.01)。在高脂饮食(HFD)诱导下,Col XV在腹股沟白脂、附睾白脂和棕脂中的表达均极显著提高(P0.01)。在脂肪细胞中,随着C/EBPβ、PPARγ和FABP4的表达上调,Col XV的表达极显著上升(P0.01),而CREB的表达显著下降(P0.05)。油红O和Bodipy染色发现,Col XV促进脂肪细胞分化,加速脂肪细胞内脂滴的积累。超表达Col XV在0d、4d和8d显著促进脂肪细胞分化标志基因C/EBPβ、PPARγ和FABP4的表达,干扰Col XV后作用相反(P0.05)。超表达Col XV促进脂合成基因ACCα和FASN的表达,抑制脂分解基因HSL、LPL和ATGL的表达(P0.05),干扰Col XV后上述基因表达相反。超表达Col XV降低细胞中cAMP和p-PKAThr197的水平,Forskolin逆转这种抑制作用,H89则加强这种抑制作用,干扰Col XV后则激活了cAMP/PKA信号通路。表明Col XV通过抑制cAMP/PKA信号通路促进脂肪细胞分化和抑制脂解。2.Col XV通过CREB负转录调控脂肪细胞分化和脂代谢。通过构建长度递减的Col XV启动子荧光素酶报告载体和CREB结合位点(CRE)定点突变的荧光素酶报告载体,双荧光素酶活性分析发现CREB结合在Col XV启动子上-354 bp~-334 bp处,抑制Col XV的转录活性。油红O染色发现超表达Col XV促进脂肪细胞脂质积累,干扰Col XV降低了脂质生成,CREB激动剂Forskolin处理后使脂滴消融。超表达Col XV促进C/EBPβ、PPARγ和FABP4等的基因表达,干扰Col XV抑制脂分化基因的表达,而Forskolin处理进一步降低了它们的表达。超表达Col XV促进FASN和ACCα的表达,抑制HSL的表达,Forskolin逆转了超表达Col XV对脂代谢的作用。干扰Col XV降低FASN和ACCα的表达,提高HSL的表达,Forskolin增强了干扰Col XV后的作用。表明Col XV通过CREB负转录调节它的启动子活性,进而影响脂肪细胞分化和脂代谢。3.Col XV通过DNA甲基化调节脂肪细胞分化和脂代谢。采用亚硫酸氢盐测序法发现在脂肪细胞分化后Col XV的DNA甲基化水平降低,Col XV的表达水平升高,甲基化转移酶(Dnmt1、Dnmt3a、Dnmt3b)和CpG结合蛋白3(Mbd3)的mRNA表达显著降低。在脂肪细胞分化第0 d和4 d,Col XV超表达后抑制了Dnmt1的表达,甲基转移酶抑制剂5-azacytidine(5-Aza-dC)加剧了这一作用,对Dnmt3a和Dnmt3b的作用不显著。5-Aza-dC抑制Col XV的DNA甲基化水平,促进它的mRNA表达。在脂肪细胞分化第0 d,Col XV超表达促进C/EBPβ、PPARγ的表达,5-Aza-dC显著降低C/EBPβ、PPARγ的表达;而第4 d在Col XV甲基化被抑制后Col XV上调的C/EBPβ和PPARγ的表达被进一步增强。在脂肪细胞分化第0 d和第4 d,超表达Col XV抑制HSL的mRNA表达,在Col XV甲基化水平降低后加剧了这种抑制作用。油红O结果一致,在分化的脂肪细胞中超表达Col XV促进脂沉积,干扰Col XV抑制脂沉积,Col XV甲基化被抑制后加速了脂肪细胞内脂滴形成。表明Col XV通过DNA甲基化下调来促进脂肪细胞分化和抑制脂肪细胞的分解代谢。综上所述,本试验表明Col XV通过抑制cAMP/PKA信号通路来促进脂肪细胞分化和抑制脂质分解,并证实Col XV通过CREB的负转录调控和DNA甲基化抑制来促进脂肪细胞分化和抑制脂肪细胞分解代谢。
[Abstract]:Collagen XV (Col XV), one of the members of the collagen family, is distributed on the surface of the basal membrane of the cell and plays an important role in the physiological and pathological processes of animals..Col XV and Col XVIII constitute a subclass of collagen produced by MULTIPLEXINs/ endostatin. They are highly conserved in structure, inflammation, tumor formation, and fat metabolism. The study found that Col XVIII promotes adipocyte differentiation, increases the accumulation of visceral fat, increases tissue remodeling, maintains energy homeostasis.Col XV is up-regulated during lipid differentiation, participates in tissue regeneration, energy homeostasis and regulation of obesity related diseases. However, Col XV regulates adipocyte differentiation and its molecular machine In order to determine the mechanism of Col XV regulation of adipocyte differentiation, this study overexpressed and interfered with Col XV in adipocytes. The following results were obtained: 1.Col XV promotes adipocyte differentiation and inhibits lipid decomposition.Col XV in the white fat of the groin, the expression of white fat in epididymis is higher, while in scapula Brown fat, the expression of the epididymal white fat is higher. The expression of Col XV in white fat tissue was significantly higher than that in brown fat (P0.01). The expression of Col XV in white fat in the groin, white fat and brown fat in epididymis was significantly increased (P0.01) under high fat diet (HFD). In adipocytes, with C/EBP beta, PPAR gamma in adipocytes. The expression of Col XV and the expression of FABP4 increased significantly (P0.01), while the expression of CREB decreased significantly (P0.05). Oil red O and Bodipy staining found that Col XV promoted adipocyte differentiation and accelerated the accumulation of lipid droplets in adipocytes. Interference with Col XV is opposite (P0.05). Overexpression of Col XV promotes the expression of ACC alpha and FASN in lipid synthesis genes, inhibits the expression of lipid decomposition genes HSL, LPL and ATGL (P0.05), and the expression of these genes is opposite after interfering Col. The effect of Col XV activates the cAMP/PKA signaling pathway. It indicates that Col XV promotes adipocyte differentiation by inhibiting the cAMP/PKA signaling pathway and inhibits the lipo.2.Col XV to regulate adipocyte differentiation and lipid metabolism through CREB negative transcription. The mutated luciferase reporter carrier, double luciferase activity analysis found that CREB combined with -354 bp~-334 BP at the promoter of Col XV promoter, and inhibited the transcriptional activity of Col XV. Oil red O staining found that the overexpression of Col XV promoted lipid accumulation in fat cells, interfering Col and reducing lipid formation. The expression of Col XV promotes the expression of C/EBP beta, PPAR gamma and FABP4, which interferes with the inhibition of Col XV to inhibit the expression of lipid differentiation genes, while Forskolin treatment further reduces their expression. The expression of alpha, enhanced the expression of HSL, Forskolin enhanced the effect of interference with Col XV. It showed that Col XV regulates its promoter activity through CREB negative transcription, and then affects adipocyte differentiation and lipid metabolism.3.Col XV through DNA methylation to regulate adipocyte differentiation and lipid metabolism. After the differentiation of adipocytes, a hydrogen sulfite sequencing method was used to detect the differentiation of adipocytes after the differentiation of adipocytes. The level of DNA methylation in Col XV decreased, the expression level of Col XV increased, and the mRNA expression of methyltransferase (Dnmt1, Dnmt3a, Dnmt3b) and CpG binding protein 3 (Mbd3) decreased significantly. The effect on Dnmt3a and Dnmt3b did not significantly inhibit the DNA methylation level of Col XV and promote its mRNA expression. In the adipocyte differentiation zeroth D, Col XV overexpression promoted the expression of C/EBP beta and PPAR gamma, which significantly reduced the expression of beta and gamma, while fourth Further enhancement. In the adipocyte differentiation zeroth D and fourth D, the overexpression of Col XV inhibits the mRNA expression of HSL, and increases this inhibitory effect after the reduction of Col XV methylation level. The result of the oil red O results is consistent, and the overexpression of Col XV promotes lipid deposition in the differentiated adipocytes, interfering Col to inhibit lipid deposition, and accelerates the inhibition after inhibition methylation is suppressed. The formation of lipid droplets in adipocytes indicates that Col XV can promote adipocyte differentiation and inhibit the catabolism of adipocytes through the downregulation of DNA methylation. To sum up, this experiment shows that Col XV promotes adipocyte differentiation and inhibits lipid decomposition by inhibiting the cAMP/PKA signaling pathway, and that Col XV is regulated by CREB negative transcriptional regulation and DNA methyl. Inhibition can promote adipocyte differentiation and inhibit adipocyte catabolism.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.2

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