【作者】 刘晶；

【导师】 侯俊财；

【作者基本信息】 东北农业大学 ， 食品科学， 2013， 硕士

【摘要】 共轭亚油酸（Conjugated linoleic acid，CLA）是指一组具有位置和几何异构的十八碳共轭二烯酸的总称。它广泛存在于自然界中，在反刍动物的肉制品和乳制品中含量较多。动物和人体试验已经证实CLA具有诸多生理功能，主要包括抗癌、抗动脉粥样硬化、降低胆固醇、降低体脂、减少脂肪沉积、调节骨组织代谢、强化机体免疫和抗糖尿病。基于CLA的生理功能，营养学家们努力提高乳中CLA含量，但研究发现，提高乳中CLA含量的同时，却导致奶牛乳脂降低综合症（MFD）。目前研究证实t10, c12-CLA可能是导致奶牛乳脂降低综合症（MFD）的主要原因，因此本试验采用组织块法体外培养奶牛乳腺上皮细胞，探索c9, t11-CLA和t10, c12-CLA纯品对乳腺细胞内乳脂合成及相关基因mRNA转录的影响。在分子水平上，探讨CLA对奶牛乳腺上皮细胞甘油三酯（TAG）含量及脂肪酸合成相关基因mRNA转录的影响。试验结果表明：（1）采用组织块法实现了体外培养出奶牛乳腺上皮细胞，细胞成活率较高，达到85%以上；经过多次传代和角蛋白18鉴定后，可得到纯化的奶牛乳腺上皮细胞。测定乳腺上皮细胞的相关生物学性状，试验结果为细胞生长曲线呈“S”型，符合一般细胞生长规律；细胞群体倍增时间为32.18h；细胞接种存活率最高可达87%。（2）试验结果表明，c9, t11CLA和t10, c12CLA均可显著降低乳腺上皮细胞内TAG含量（P＜0.05），且t10, c12CLA对TAG的抑制率高于c9, t11CLA。（3）气相色谱检测CLA对细胞中脂肪酸组成的影响，结果表明150μmol/L c9, t11CLA可显著提高细胞内单不饱和脂肪酸（MUFA）的比例（P＜0.05），而50μmol/L c9, t11-CLA显著增加细胞内饱和脂肪酸（SFA）的比例（P＜0.05）；细胞内MUFA的比例随着t10, c12CLA浓度的增加，呈显著下降趋势（P＜0.05）；两种CLA对多不饱和脂肪酸（PUFA）的比例都没有显著影响（P＞0.05）。（4）外源添加不同浓度的c9, t11-CLA与t10, c12-CLA直接作用于乳腺上皮细胞48h，采用实时荧光定量PCR技术，测定乳腺上皮细胞脂肪酸摄取与转运及甘油三酯合成相关基因mRNA的转录。试验结果：与对照组相比，50～150μmol/L的c9, t11CLA和t10, c12CLA均显著下调LPL基因mRNA的转录水平（P＜0.05）；50μmol/L c9, t11CLA显著下调CD36基因mRNA的转录水平（P＜0.05），150μmol/L c9, t11CLA上调它的转录水平（P＜0.05）, CD36mRNA的转录水平随t10, c12CLA浓度增加呈显著上升趋势（P＜0.05）；150μmol/L c9, t11CLA和不同浓度的t10, c12CLA均可显著上调ACBP基因mRNA的转录水平（P＜0.05）；相比对照组，c9, t11CLA与t10, c12CLA均显著下调FABP3基因mRNA的转录水平（P＜0.05）；150μmol/L的c9, t11CLA和t10, c12CLA均可显著上调AGPAT基因mRNA的转录水平（P＜0.05）。本试验采用组织块法实现了体外培养出奶牛乳腺上皮细胞，细胞接种存活率较高。试验证实CLA抑制乳腺TAG合成且影响乳腺细胞的脂肪酸合成；证明两种CLA均显著降低LPLmRNA和FABP3mRNA的转录水平，t10, c12CLA比c9, t11CLA的降低效果明显。更多还原

【Abstract】 Conjugated linoleic acids（CLAs）refer to a group of position and geometric isomers ofoctadecadienoic acid with conjugated double bonds. They exist widely in nature, ruminant meatand milk products are the main sources. Animal and human studies have confirmed that CLA had avariety of physioligical functions, mainly inculding anti-cancer, anti-atherosclerosis, loweringcholesterol level, reduction in body fat, decreasing fat deposition, modulation of bone tissuemetabolism, strengthening the body’s immunity and antidiabete.Based on the physiological functions of CLA, nutritionists strived to improve the CLAcontent of milk. But the studies found that with the increased CLA content in milk, it can led tomilk fat depression (MFD). The present studies confirmed that t10, c12-CLA was the major causethat can led to MFD in dairy cows, so bovine mammary gland epithelial cells were cultured in vitroby tissue blocks in this experiments and explore the pure c9, t11-CLA and t10, c12-CLA affect thetranscription of mRNAs of genes related to milk fat synthesis in mammary gland epithelial cells.At the molecular level, probe the effect of CLA on triacylglycerol (TAG) contents andtranscription of mRNAs of genes related to fatty acid synthesis in bovine mammary glandepithelial cells. The results showed that:(1) This experiment was used the tissue blocks to culture dairy cow mammary gland epithelialcells in vitro, and cell survival rate was higher, more than85%. After several passages andidentification of cytokeratin18, we can obtain the purified bovine mammary gland epithelial cells.Determination of biological characters of bovine mammary gland epithelial cells, the resultsshowed that the cell growth curve was “S” type, in line with the general cell growth pattern, cellpopulation doubling time was32.18h, cell planting survival efficiency was up to87%.(2) The results showed that c9, t11-CLA and t10, c12-CLA both significantly reduced TAGcontents in mammary gland epithelial cells (P<0.05), and the TAG inhibition ratio of t10, c12-CLAwas higher than c9, t11-CLA.(3) Study the effect of CLA on fatty acid composition in bovine mammary gland epithelial cellsby gas chromatograph, the results showed that150μmol/L c9, t11-CLA can significantly increasethe proportions of monounsaturated fatty acid (MUFA)(P<0.05),50μmol/L c9, t11-CLAsignificantly increased saturated fatty acid (SFA) proportions (P<0.05). With the increased concentrations of t10, c12-CLA, the proportions of MUFA had a downward trend (P<0.05). Thetwo CLA isomers didn’t affect the proportions of polyunsaturated fatty acid（PUFA）in bovinemammary gland epithelial cells (P＞0.05).(4) The bovine mammary gland epithelial cells were directly treated with differentconcentrations of exogenous c9, t11-CLA and t10, c12-CLA for48h, and then the transcription ofmRNAs of genes related to the uptake and transport of fatty acid and TAG synthesis in themammary gland epithelial cells was determined using the real-time fluorescence-basedquantitative PCR technology. The results showed that50-150μmol/L c9, t11-CLA and t10,c12-CLA both significantly downregulated LPL mRNA transcription level, compared with thecontrol group (P<0.05).50μmol/L c9, t11-CLA significantly downregulated the transcription levelof CD36(P＜0.05), but150μmol/L c9, t11-CLA significantly upregulated (P<0.05), and CD36mRNA transcription level was significantly increased with the increased concentrations of t10,c12-CLA (P<0.05).150μmol/L c9, t11-CLA and different concentrations of t10, c12-CLA bothsignificantly upregulated the transcription of ACBP gene (P<0.05). c9, t11-CLA and t10, c12-CLAboth significantly downregulated the transcription of FABP3, compared with the control group(P<0.05).150μmol/L c9, t11-CLA and t10, c12-CLA both significantly upregulated AGPAT mRNAtranscription level (P<0.05).This experiment was achieved the tissue blocks to culture bovine mammary gland epithelialcells in vitro, and cell planting survival efficiency was higher. The results indicated that CLAinbibited TAG accumulation and affected fatty acid synthesis in bovine mammary gland epithelialcells. The two CLA isomers both significantly downregulated the mRNA transcription levels ofLPL and FABP3, but the effect of t10, c12-CLA was apparent than that of c9, t11-CLA.更多还原