三种不同结构的叶瓜参鞘脂对脂肪细胞分化和脂质代谢的调控作用研究

发布时间：2018-02-04 11:35

本文关键词： 叶瓜参 3T3-L1前脂肪细胞海参鞘脂分化 β-catenin 脂质代谢 AMPK　出处：《中国海洋大学》2015年硕士论文　论文类型：学位论文

【摘要】：海参鞘脂是海参体内的一类重要脂质,主要包括脑苷脂、神经酰胺及长链碱等。其中,海参脑苷脂由神经酰胺和糖基组成,神经酰胺是由长链碱与长链脂肪酸经脱水以酰胺键连接而成。研究发现,海参鞘脂具有抗肿瘤、改善阿尔茨海默症、改善糖脂代谢等功效。脂肪细胞分化及脂质代谢紊乱是导致肥胖等代谢综合征的重要因素,目前关于海参鞘脂对脂质代谢的影响已有初步报道,但其作用机制仍未阐明,且关于海参鞘脂中起作用的活性片段也没有明确的定论。本文以鞘脂含量较高的叶瓜参(Cucumaria frondosa)为实验材料,分离、制备了叶瓜参脑苷脂(Cerebroside from the sea cucumber C.frondosa, Cf-Cb)、神经酰胺(Ceramide from the sea cucumber C. frondosa, Cf-Cm)及长链碱(Long-chain base from the sea cucumber C. frondosa, Cf-LCB) 3种鞘脂,系统研究了其调控脂肪细胞分化与脂质代谢的作用及其机制,并阐明了3种叶瓜参鞘脂中起作用的活性片段。主要研究结果如下：采用传统鸡尾酒法诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,研究了3种叶瓜参鞘脂对脂肪细胞分化的影响。油红O染色结果显示,Cf-Cb、Cf-Cm和Cf-LCB均能显著抑制3T3-L1前脂肪细胞分化,且对分化早期的抑制效果最强。C/EBPα和PPARγ是脂肪细胞分化末期最重要的转录调控因子。qRT-PCR及Western blotting结果表明,Cf-Cb、Cf-Cm和Cf-LCB对C/EBPa和PPARγ的mRNA和蛋白表达均具有显著的抑制作用。WNT/β-catenin通路在脂肪细胞分化中具有重要作用,激活后显著抑制脂肪细胞分化。qRT-PCR结果表明,Cf-Cb、Cf-Cm和Cf-LCB显著地上调WNT/β-catenin通路关键基因FZ1、LRP5、LRP6、β-catenin、 CCND1和c-myc mRNA表达,但对Wnt10b及GSK3β的表达无显著性影响。Western blotting结果显示,Cf-Cb、Cf-Cm和Cf-LCB显著促进LRP6和β-catenin的蛋白表达,提高细胞核内β-catenin含量,提示3种叶瓜参鞘脂均能促进WNT/β-catenin通路的激活。21H7是β-catenin的抑制剂,可以特异性的抑制WNT/β-catenin通路的激活。结果表明,21H7干预细胞后,3T3-L1前脂肪细胞分化率,C/EBPa和PPARy的mRNA和蛋白表达均提高；而P-catenin蛋白表达,细胞核内P-catenin蛋白含量,CCND1和c-myc的mRNA表达均显著降低。Cf-Cb、 Cf-Cm和Cf-LCB均能显著改善21 H7的作用效果,进一步验证其可促进WNT/β-catenin通路的激活。综上所述,3种叶瓜参鞘脂通过激活WNT/β-catenin通路抑制3T3-L1前脂肪细胞分化。以3T3-L1成熟脂肪细胞为研究对象,研究3种叶瓜参鞘脂对脂肪细胞脂质代谢的影响。结果表明,Cf-Cb、Cf-Cm和Cf-LCB均显著降低细胞中TG含量,升高培养上清中FFA含量,提示其能抑制脂质累积,促进脂质分解。脂质合成和分解的关键酶是调控脂质代谢的重要因子,是目前重要的肥胖候选研究基因。Western blotting结果显示,Cf-Cb、Cf-Cm和Cf-LCB均能显著抑制脂质合成关键基因GPAT和FAS的蛋白表达,增加脂质分解关键基因HSL和CPT-1的蛋白表达。AMPK是细胞能量代谢的总开关,在调控细胞能量代谢中起到重要的作用。Western blotting结果表明,Cf-Cb、Cf-Cm和Cf-LCB均显著促进AMPK及ACC蛋白的磷酸化,提示其能激活AMPK通路。Compound C是AMPK蛋白的抑制剂,可以降低AMPK的活性。结果表明,Compound C干预下,TG含量、GPAT和FAS的蛋白表达均升高；而FFA含量,HSL和CPT-1蛋白表达,AMPK和ACC的磷酸化水平均降低。Cf-Cb、Cf-Cm和Cf-LCB均显著改善Compound C的作用效果,进一步验证其能激活AMPK通路。综上所述,3种叶瓜参鞘脂通过激活AMPK通路抑制脂质合成,促进脂质分解,从而改善脂肪细胞的脂质代谢。通过建立浓度相同或长链碱含量相同的3种鞘脂两个作用体系,比较研究了Cf-Cb、Cf-Cm和Cf-LCB的作用效果,以期阐明3种叶瓜参鞘脂中起作用的活性片段。结果表明,当3种鞘脂浓度相同时,其抑制3T3-L1前脂肪细胞细胞分化,促进WNT/β-catenin通路的激活,改善脂质代谢和激活AMPK通路的作用效果以Cf-LCB为最优。而当3种鞘脂长链碱含量相同时,Cf-Cb、Cf-Cm和Cf-LCB抑制3T3-L1前脂肪细胞细胞分化,促进WNT/β-catenin通路的激活,改善脂质代谢和激活AMPK通路的作用效果相当,提示3种叶瓜参鞘脂中起作用的活性片段为长链碱。本文首次系统研究了叶瓜参鞘脂对脂肪细胞分化和脂质代谢的调控作用及其机制,并进一步阐明了其中起作用的活性片段,为海参鞘脂的进一步开发利用提供了科学基础和理论依据。
[Abstract]:Sea cucumber is a kind of important sphingolipid in vivo including sea cucumber lipid, cerebroside, ceramide and long chain bases. The sea cucumber cerebroside by ceramide and glycosyl composition, ceramide is composed of long chain base and long chain fatty acid by dehydration with amide linkages and. The study found that sphingolipids with anti sea cucumber to improve cancer, Alzheimer's disease, improve glucose and lipid metabolism and other effects. Cell differentiation and lipid metabolism disorder is an important factor leading to obesity and metabolic syndrome, the sea cucumber sphingolipid effect on lipid metabolism has been reported, but the mechanism has not been clarified with, and a sea cucumber in the role of the sphingolipid active fragment is not clear conclusion. The higher content of ginseng leaf melon sphingolipids (Cucumaria frondosa) as experimental materials, separation, ginseng leaf melon cerebroside was prepared (Cerebroside from the sea cucumber C.frondosa, Cf-Cb), Ceramide (Ceramide from the sea cucumber C. frondosa, Cf-Cm) and long chain bases (Long-chain base from the sea cucumber C. frondosa, Cf-LCB) 3 kinds of sphingolipids, studied the effect and mechanism of the regulation of adipocyte differentiation and lipid metabolism, and expounds 3 kinds of active bits of leaf ginseng melon sphingolipid. The main results are as follows: induced differentiation of 3T3-L1 preadipocytes into mature adipocytes by using traditional cocktail method, the influence of 3 kinds of melon leaf ginseng sphingolipids on adipocyte differentiation. Oil red O staining showed that Cf-Cb, Cf-Cm and Cf-LCB significantly inhibited the differentiation of 3T3-L1 preadipocytes, and the inhibitory effect on the differentiation of early the strongest.C/EBP PPAR alpha and gamma is the most important stage of adipocyte differentiation and transcription factor.QRT-PCR and Western blotting results showed that Cf-Cb, Cf-Cm and Cf-LCB on C/EBPa and PPAR gamma mRNA and protein The expression inhibition of.WNT/ beta -catenin pathway significantly plays an important role in adipocyte differentiation, activation significantly inhibited the differentiation of.QRT-PCR adipocytes results showed that Cf-Cb, Cf-Cm and Cf-LCB significantly up-regulated the expression of WNT/ beta -catenin pathway key genes FZ1, LRP5, LRP6, CCND1 and beta -catenin, c-myc mRNA expression, but the expression of Wnt10b and there was no significant effect of GSK3 beta.Western blotting showed that Cf-Cb, Cf-Cm and Cf-LCB significantly promoted LRP6 and beta -catenin protein expression, increase the content of -catenin beta cells, suggesting that the 3 leaves of melon ginseng can promote the activation of.21H7 WNT/ sphingolipid beta -catenin pathway inhibitor beta -catenin, can activate specific inhibition WNT/ beta -catenin pathway. The results showed that 21H7 cells after the intervention, the rate of fat cell differentiation of 3T3-L1, mRNA and protein expression of C/EBPa and PPARy were increased; and the expression of P-catenin protein, fine In the nucleus of P-catenin protein, the expression of CCND1 and c-myc mRNA were significantly decreased in.Cf-Cb, the effect of Cf-Cm and Cf-LCB can significantly improve the 21 H7, to further verify its can promote the activation of WNT/ beta -catenin pathway. To sum up, 3 kinds of inhibition of 3T3-L1 preadipocyte differentiation activation of WNT/ beta -catenin pathway through leaf Cucumaria sphingolipids. 3T3-L1 mature fat cells as the research object, effect of 3 kinds of melon leaf ginseng on adipocyte lipid metabolism of sphingolipids. The results showed that Cf-Cb, Cf-Cm and Cf-LCB were significantly decreased the content of TG in cell culture supernatant, increased FFA content, suggesting that it can inhibit lipid accumulation, promote lipid lipid synthesis and key enzyme decomposition. Decomposition is an important factor in the regulation of lipid metabolism, obesity is a candidate gene.Western blotting research results at present an important display, Cf-Cb, Cf-Cm and Cf-LCB can significantly inhibit lipid synthesis related genes in GPAT The expression of FAS and protein, increased lipolysis key genes HSL and CPT-1 protein expression of.AMPK is the master switch of cellular energy metabolism, play the role of the.Western blotting results showed that in the regulation of cellular energy metabolism in Cf-Cb, Cf-Cm and Cf-LCB were significantly promoted AMPK and ACC protein phosphorylation, suggesting that the activation of AMPK.Compound C pathway is an inhibitor of the AMPK protein, can reduce the activity of AMPK. The results show that the content of TG, Compound, C intervention, the expression of GPAT and FAS protein were increased; while the content of FFA, HSL and CPT-1 protein expression and phosphorylation levels of AMPK and ACC decreased.Cf-Cb, the effect of Cf-Cm and Cf-LCB were significantly improved Compound C, further validate that it could activate the AMPK pathway. To sum up, 3 kinds of inhibition of lipid synthesis in the activation of the AMPK pathway through leaf Cucumaria sphingolipid, promote lipolysis and improve lipid metabolism in fat cells through the construction. Set the same concentration or long chain alkali content of 3 kinds of sphingolipids in the same two system, a comparative study of the effect of Cf-Cb, Cf-Cm and Cf-LCB, in order to clarify the role of the 3 kinds of active fragment leaves Cucumaria sphingolipid. The results show that when the 3 kinds of sphingolipids in the same concentration, the inhibition of 3T3-L1 fat cells cell differentiation, promote the activation of WNT/ beta -catenin pathway, the effect of improving lipid metabolism and activation of the AMPK pathway in Cf-LCB is the best. When the content of long chain bases of 3 kinds of sphingolipids are same, Cf-Cb, Cf-Cm and Cf-LCB inhibited 3T3-L1 adipocyte differentiation, promote the activation of WNT/ beta -catenin pathway, considerable effect in improving the lipid the metabolism and activation of the AMPK pathway, suggesting that the 3 leaf ginseng melon in sphingolipid active fragment of long chain bases. This is the first study on the leaves of melon ginseng sphingolipids differentiation and lipid metabolism in fat cells and its regulation mechanism, and a Step toward elucidating the active fragment in which the role of, provide the scientific and theoretical basis for further development and utilization of sea cucumber sphingolipids.

【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q54

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