本文關(guān)鍵詞： 葉瓜參 3T3-L1前脂肪細(xì)胞 海參鞘脂 分化 β-catenin 脂質(zhì)代謝 AMPK　出處：《中國海洋大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：海參鞘脂是海參體內(nèi)的一類重要脂質(zhì),主要包括腦苷脂、神經(jīng)酰胺及長鏈堿等。其中,海參腦苷脂由神經(jīng)酰胺和糖基組成,神經(jīng)酰胺是由長鏈堿與長鏈脂肪酸經(jīng)脫水以酰胺鍵連接而成。研究發(fā)現(xiàn),海參鞘脂具有抗腫瘤、改善阿爾茨海默癥、改善糖脂代謝等功效。脂肪細(xì)胞分化及脂質(zhì)代謝紊亂是導(dǎo)致肥胖等代謝綜合征的重要因素,目前關(guān)于海參鞘脂對脂質(zhì)代謝的影響已有初步報道,但其作用機(jī)制仍未闡明,且關(guān)于海參鞘脂中起作用的活性片段也沒有明確的定論。本文以鞘脂含量較高的葉瓜參(Cucumaria frondosa)為實驗材料,分離、制備了葉瓜參腦苷脂(Cerebroside from the sea cucumber C.frondosa, Cf-Cb)、神經(jīng)酰胺(Ceramide from the sea cucumber C. frondosa, Cf-Cm)及長鏈堿(Long-chain base from the sea cucumber C. frondosa, Cf-LCB) 3種鞘脂,系統(tǒng)研究了其調(diào)控脂肪細(xì)胞分化與脂質(zhì)代謝的作用及其機(jī)制,并闡明了3種葉瓜參鞘脂中起作用的活性片段。主要研究結(jié)果如下：采用傳統(tǒng)雞尾酒法誘導(dǎo)3T3-L1前脂肪細(xì)胞分化為成熟脂肪細(xì)胞,研究了3種葉瓜參鞘脂對脂肪細(xì)胞分化的影響。油紅O染色結(jié)果顯示,Cf-Cb、Cf-Cm和Cf-LCB均能顯著抑制3T3-L1前脂肪細(xì)胞分化,且對分化早期的抑制效果最強(qiáng)。C/EBPα和PPARγ是脂肪細(xì)胞分化末期最重要的轉(zhuǎn)錄調(diào)控因子。qRT-PCR及Western blotting結(jié)果表明,Cf-Cb、Cf-Cm和Cf-LCB對C/EBPa和PPARγ的mRNA和蛋白表達(dá)均具有顯著的抑制作用。WNT/β-catenin通路在脂肪細(xì)胞分化中具有重要作用,激活后顯著抑制脂肪細(xì)胞分化。qRT-PCR結(jié)果表明,Cf-Cb、Cf-Cm和Cf-LCB顯著地上調(diào)WNT/β-catenin通路關(guān)鍵基因FZ1、LRP5、LRP6、β-catenin、 CCND1和c-myc mRNA表達(dá),但對Wnt10b及GSK3β的表達(dá)無顯著性影響。Western blotting結(jié)果顯示,Cf-Cb、Cf-Cm和Cf-LCB顯著促進(jìn)LRP6和β-catenin的蛋白表達(dá),提高細(xì)胞核內(nèi)β-catenin含量,提示3種葉瓜參鞘脂均能促進(jìn)WNT/β-catenin通路的激活。21H7是β-catenin的抑制劑,可以特異性的抑制WNT/β-catenin通路的激活。結(jié)果表明,21H7干預(yù)細(xì)胞后,3T3-L1前脂肪細(xì)胞分化率,C/EBPa和PPARy的mRNA和蛋白表達(dá)均提高；而P-catenin蛋白表達(dá),細(xì)胞核內(nèi)P-catenin蛋白含量,CCND1和c-myc的mRNA表達(dá)均顯著降低。Cf-Cb、 Cf-Cm和Cf-LCB均能顯著改善21 H7的作用效果,進(jìn)一步驗證其可促進(jìn)WNT/β-catenin通路的激活。綜上所述,3種葉瓜參鞘脂通過激活WNT/β-catenin通路抑制3T3-L1前脂肪細(xì)胞分化。以3T3-L1成熟脂肪細(xì)胞為研究對象,研究3種葉瓜參鞘脂對脂肪細(xì)胞脂質(zhì)代謝的影響。結(jié)果表明,Cf-Cb、Cf-Cm和Cf-LCB均顯著降低細(xì)胞中TG含量,升高培養(yǎng)上清中FFA含量,提示其能抑制脂質(zhì)累積,促進(jìn)脂質(zhì)分解。脂質(zhì)合成和分解的關(guān)鍵酶是調(diào)控脂質(zhì)代謝的重要因子,是目前重要的肥胖候選研究基因。Western blotting結(jié)果顯示,Cf-Cb、Cf-Cm和Cf-LCB均能顯著抑制脂質(zhì)合成關(guān)鍵基因GPAT和FAS的蛋白表達(dá),增加脂質(zhì)分解關(guān)鍵基因HSL和CPT-1的蛋白表達(dá)。AMPK是細(xì)胞能量代謝的總開關(guān),在調(diào)控細(xì)胞能量代謝中起到重要的作用。Western blotting結(jié)果表明,Cf-Cb、Cf-Cm和Cf-LCB均顯著促進(jìn)AMPK及ACC蛋白的磷酸化,提示其能激活A(yù)MPK通路。Compound C是AMPK蛋白的抑制劑,可以降低AMPK的活性。結(jié)果表明,Compound C干預(yù)下,TG含量、GPAT和FAS的蛋白表達(dá)均升高；而FFA含量,HSL和CPT-1蛋白表達(dá),AMPK和ACC的磷酸化水平均降低。Cf-Cb、Cf-Cm和Cf-LCB均顯著改善Compound C的作用效果,進(jìn)一步驗證其能激活A(yù)MPK通路。綜上所述,3種葉瓜參鞘脂通過激活A(yù)MPK通路抑制脂質(zhì)合成,促進(jìn)脂質(zhì)分解,從而改善脂肪細(xì)胞的脂質(zhì)代謝。通過建立濃度相同或長鏈堿含量相同的3種鞘脂兩個作用體系,比較研究了Cf-Cb、Cf-Cm和Cf-LCB的作用效果,以期闡明3種葉瓜參鞘脂中起作用的活性片段。結(jié)果表明,當(dāng)3種鞘脂濃度相同時,其抑制3T3-L1前脂肪細(xì)胞細(xì)胞分化,促進(jìn)WNT/β-catenin通路的激活,改善脂質(zhì)代謝和激活A(yù)MPK通路的作用效果以Cf-LCB為最優(yōu)。而當(dāng)3種鞘脂長鏈堿含量相同時,Cf-Cb、Cf-Cm和Cf-LCB抑制3T3-L1前脂肪細(xì)胞細(xì)胞分化,促進(jìn)WNT/β-catenin通路的激活,改善脂質(zhì)代謝和激活A(yù)MPK通路的作用效果相當(dāng),提示3種葉瓜參鞘脂中起作用的活性片段為長鏈堿。本文首次系統(tǒng)研究了葉瓜參鞘脂對脂肪細(xì)胞分化和脂質(zhì)代謝的調(diào)控作用及其機(jī)制,并進(jìn)一步闡明了其中起作用的活性片段,為海參鞘脂的進(jìn)一步開發(fā)利用提供了科學(xué)基礎(chǔ)和理論依據(jù)。
[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.

【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q54

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