ω-3脂肪酸去饱和酶在肠道乳酸菌中的重组表达及对小鼠脂肪酸谱的影响

ω-3 polyunsaturated fatty acids (ω-3 PUFA) have many beneficial effects, sunch as energy support,immunomodulatory, lipid-lowering and anticoagulant effect. Furthermore, it can also be used in prevention and treatment of cancer. However, ω-6 polyunsaturated fatty acids have the opposite and adverse affect in many respects, such as pro-inflammation, and promote tumor proliferation and other harmful effects. Supplementation of ω-3 PUFA and reduction of ω-6 PUFA intake has become a broad consensus. ω-3 fatty acid desaturase is the key enzymes which can convert the ω-3 PUFA to ω-6 PUFA. This study will construct recombinant Lactobacillus which can express ω-3 fatty acid desaturase. In vitro, the function of the combined enzyme will be testified by bio-molecular and gas chromatography-tandem mass spectrometry method. Then administrate the recombinant Lactobacillus to the intestine of mice, where it turns omega-6 PUFA into omega-3 PUFA during the fermentation process. Record the changes of fatty acids profiles in the diet and mouse tissues. Intestinal administration of recombinant lactobacillus which express ω-3 fatty acid desaturase has potential value in dairy production (yogurt), animal husbandry (production of eggs and meat riched in ω-3 PUFA) and bio-pharmaceutical field (disease prevention and clinical treatment by supplementing probiotics and ω-3 PUFA). The success of this study will provide a good animal model to explore ω-3PUFAs's effects on disease treatment.

ω-3多不饱和脂肪酸（ω-3 PUFA）有提供能量、降脂、免疫调节、防癌及抑制肿瘤等有益作用。而ω-6多不饱和脂肪酸（ω-6 PUFA）在很多方面则具有相反的不利影响，如促炎、促肿瘤增殖等有害作用。补充ω-3 PUFA并减少ω-6 PUFA的摄入已成为广泛共识。 ω-3脂肪酸去饱和酶是ω-6 PUFA转化为ω-3 PUFA的关键酶。本课题拟在乳酸菌中基因重组构建表达ω-3脂肪酸去饱和酶，通过分子生物学及气相色谱串联质谱等技术，体外检测并验证该重组表达蛋白酶的功能。再经小鼠肠道给予重组表达该酶的乳酸菌，观察其对食物及粪便中ω-3/ω-6比例的影响，并探索小鼠各器官组织脂肪酸谱的变化情况。本研究在乳制品生产（酸奶），畜牧业生产（富含ω-3 PUFA蛋类及肉食品）及医药领域（补充益生菌及ω-3 PUFA）具有潜在应用价值，并可为研究ω-3脂肪酸对各种疾病小鼠的干预作用提供较好的动物模型。

ω-3多不饱和脂肪酸（ω-3 PUFA）有提供能量、降脂、免疫调节、防癌及抑制肿瘤等有益作用。而ω-6多不饱和脂肪酸（ω-6 PUFA）在很多方面则具有相反的不利影响，如促炎、促肿瘤增殖等有害作用。补充ω-3 PUFA并减少ω-6 PUFA的摄入已成为广泛共识。ω-3脂肪酸去饱和酶是ω-6 PUFA转化为ω-3 PUFA的关键酶。本课题拟在乳酸菌中基因重组构建表达ω-3脂肪酸去饱和酶，通过分子生物学及气相色谱串联质谱等技术，体外检测并验证该重组表达蛋白酶的功能。再经小鼠肠道给予重组表达该酶的乳酸菌，观察其对食物及粪便中ω-3/ω-6比例的影响，并探索小鼠各器官组织脂肪酸谱的变化情况。.本研究成功将Fat-1基因与Mistic基因融合表达，实现了在大肠杆菌中高效表达真核膜整合蛋白ω-3脂肪酸去饱和酶Fat-1。成功构建Fat-1在三种乳酸菌中的表达载体，并观察到在乳酸菌NZ900中对添加亚油酸有脂肪酸谱的改变。本研究仍需进一步研究。.成功构建pLenti-CMV-Fat-1过表达慢病毒载体，成功筛选了稳定转染Fat-1基因的各种细胞系，脂肪酸谱检测发现Fat-1基因可以使得细胞中明显增加ω-3/ω-6的比率，从而证实Fat-1基因可以在真核细胞中使得ω-6脂肪酸改变为ω-3脂肪酸。我们进一步实验，在小鼠AKT/Ras癌基因激活的小鼠肝癌细胞株，以pLenti-CMV-GTP空载体病毒作为对照，Fat-1基因可以明显增加ω-3/ω-6的比率，进而抑制肝癌细胞的增殖和克隆形成，抑制癌基因AKT/Ras信号通路。 .