目的 探讨孕及哺乳期摄入n-6/n-3多不饱和脂肪酸(PUFAs)变化对子代脑功能相关基因表达的影响.方法 选用清洁级3～4周龄C57BL/6J雌性小鼠,随机分成5组,每组分别给予n-3 PUFAs缺乏和4种不同含量n-3 PUFAs(n-6/n-3 PUFAs比值分别为15∶1、5∶1、1∶1及1∶5)饲料喂养.小鼠12～14周龄时用常规饲料喂养的雄性小鼠与之合笼交配繁殖,仔鼠断乳后继续行母鼠相同饲料喂养,随访观察至生后7d、21d和12周龄时分批处死后取脑.然后在仔鼠生后21d断乳时,随机挑选7～9只n-3 PUFAs缺乏组的仔鼠用n-6/n-3 PUFAs(5∶1)组饲料喂养,至12周龄;同时随机挑选7～9只n-6/n-3 PUFAs(5∶1)组的仔鼠,改用n-3缺乏组饲料喂养,至12周龄;剩余仔鼠用与母鼠相同的饲料喂养,至12周龄;麻醉牺牲后,取脑置于-80C条件下备用.采用实时荧光定量PCR技术测定脑皮质gfap、mbp、nse7和nr2b基因mRNA的表达.结果 在各年龄段小鼠中,饲料中添加n-3 PUFAs使各基因的表达量明显增加;不同比例的n-6/n-3PUFAs对基因表达的影响不同.孕期及哺乳期饲料缺乏n-3 PUFAs,即使断乳后的饲料中添加正常量的n-3 PUFAs,也不能使某些对脑功能起重要作用的基因的表达量升高至正常n-3 PUFAs喂养的小鼠.结果提示,孕期及哺乳期可能需要较高的n-3 PUFAs摄入(可能最优的n-6/n-3 PUFAs比例介于1∶5～1∶1之间),才能满足幼年期脑发育之需.结论 孕及哺乳期n-3多不饱和脂肪酸(PUFAs)的适量摄入,有助于维持成年期脑功能相关基因的正常表达.

In this study, we hypothesized that n-3 polyunsaturated fatty acid (PUFA) deficiency during pregnancy and lactation will make a lasting impact on brain neurogenesis and apoptosis of the adult offspring and that these harmful effects cannot be reversed by n-3 PUFA supplementation after weaning. Moreover, the underlying mechanisms may be attributable to the epigenetic changes of brain-derived neurotrophic factor (BDNF). C57BL/6J female mice were fed with n-3 PUFA-deficient diet (n-3 def) or n-3 PUFA-adequate diet (n-3 adq) throughout pregnancy and lactation. At postnatal 21 days, equal numbers of male pups from both groups were fed the opposite diet, and the remaining male pups were fed with the same diets as their mothers until 3 months of age. Feeding the n-3 adq diet to pups from the maternal n-3 def group significantly increased the n-3 PUFA concentration but did not change expressions of calretinin, Bcl2, and Bax in the hippocampus. Feeding the n-3 def diet to pups from the maternal n-3 adq group significantly reduced the n-3 PUFA concentration but did not reduce expressions of calretinin and Bcl2. Similarly, BDNF levels, especially mRNA expressions of BDNF transcripts IV and IX, were also reduced by maternal n-3 def and not reversed by n-3 PUFA supplementation after weaning. The decrease in BDNF expression by maternal n-3 def diet was associated with greater DNA methylation at special CpG sites. These results suggested that the maternal n-3 PUFA deficiency during pregnancy and lactation imprints long-term changes of brain development in adult offspring.

目的：：观察给予母孕期及哺乳期小鼠添加不同含量n-3多不饱和脂肪酸( PUFAs )饲料喂养,对成年期仔小鼠脑神经细胞凋亡的影响。方法：使用6-8周龄清洁级C57 BL/6 J雌性小鼠,随机分为5组,每组10只,分别给予小鼠缺乏n-3 PUFAs的饲料、n-6/n-3 PUFAs比值为15：1、5：1、1：1的饲料和高含量鱼油的n-3 PUFAs饲料( n-6/n-3 PUFAs比值为1：5)喂养。小鼠12-14周龄时进行雌雄合笼交配繁殖,仔小鼠断乳后继续行母小鼠相同饲料喂养,选取生后3m成年仔小鼠用于实验。取小鼠脑进行组织固定,采用免疫组织化学技术对脑组织海马区神经细胞凋亡情况进行分析。结果：与缺乏n-3 PUFAs组小鼠相比,n-3 PUFAs饲料喂养组,尤其是n-6/n-3 PUFAs比值(5：1)和(1：1)组小鼠海马CA3区神经细胞抗凋亡蛋白Bcl2表达明显增加,而致凋亡蛋白Bax表达则显著降低。但高含量鱼油的n-3 PUFAs喂养组小鼠(即n-6/n-3 PUFAs 1：5组)与缺乏n-3 PUFAs组小鼠相比,未表现出显著性差异。结论：孕期及哺乳期小鼠饲料中添加n-3 PUFAs,尤其是n-6/n-3 PUFAs比值在5~1：1时有助于减少成年仔小鼠脑组织神经细胞凋亡的发生,而过高含量n-3 PUFAs摄入则未对小鼠脑凋亡发生起到促进作用。

OBJECTIVE:although n-3 polyunsaturated fatty acids (PUFAs) play crucial roles in brain development and function, neither the optimal level of n-3 PUFAs nor the optimal ratio of n-6/n-3 PUFAs in the maternal diet are well defined. In this study, we investigated the effects of dietary n-6/n-3 PUFA ratios during pregnancy on neurogenesis and apoptosis in the brains of mouse offspring. Metods: female C57BL/6J mice were fed one of three diets with high, medium and low ratios of n-6/n-3 PUFAs (15.7:1, 6.3:1, 1.6:1), as well as a high fish oil diet with a n-6/n-3 ratio of 1:5.7; an n-3 PUFA-deficient diet served as control. The feeding regimens began two months before mouse conception and continued for the duration of the pregnancy. The neurogenesis and apoptosis of hippocampal CA3 area in the offspring were detected.RESULTS:compared to the n-3 PUFA-deficient diet, n-3 PUFA-containing diets, particularly those with n-6/n-3 PUFA ratios of 6.3:1 and 1.6:1, significantly increased both phosphorylation of histone H3 at ser 10 (p-H3ser10) and calretinin-positive cells in hippocampus CA3 of the offspring. Furthermore, increased expression of Bcl2 protein, decreased expression of Bax protein, and reduced caspase 3 activity and numbers of TUNEL apoptotic cells were found in the three diets with high, medium and low n-6/n-3 PUFA ratios. However, there were no differences in any of these parameters between the high fish oil diet group and the n-3 PUFA-deficient diet group.CONCLUSIONS:these data suggest that a higher intake of n-3 PUFAs with a lower ratio of n-6/n-3 PUFAs of between about 6:1 to 1:1, supplied to mothers during pregnancy, may benefit brain neurogenesis and apoptosis in offspring. However, excessive maternal intake of n-3 PUFAs may exert a negative influence on brain development in the offspring.

目的 探讨生命早期n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids,n-3 PUFAs)营养状态对成年期脑神经元发生及凋亡的影响.方法 使用20只3-4w龄清洁级C57BL/6J雌性小鼠,随机分为两组(10只/组),分别给予n-3 PUFAs缺乏和n-3 PUFAs饲料喂养;12-14 w龄时与雄性小鼠合笼交配繁殖.仔鼠生后21d断乳时,随机挑选20只n-3 PUFAs缺乏组仔鼠用n-3.PUFAs饲料喂养,挑选等量n-3 PUFAs饲料组仔鼠用n-3 PUFAs缺乏饲料喂养,剩余仔鼠用与母鼠相同饲料继续喂养,至仔鼠3月龄时结束实验.小鼠麻醉处死后取全脑组织,采用脂肪酸甲酯化-气相色谱分析对脑脂肪酸进行测定;采用免疫组织化学技术对海马CA3区B淋巴细胞瘤-2蛋白(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)和钙视网膜结合蛋白(Calretinin,CR)表达进行分析.结果 与n-3 PUFAs缺乏饲料持续喂养的仔鼠相比,孕期和哺乳期n-3 PUFAs缺乏而断乳后给予n-3 PUFAs饲料喂养的仔鼠,成年后脑组织n-3 PUFAs含量明显升高(P＜0.05),但Bcl-2、Bax和CR的表达量均未发生显著性变化,仍然低于n-3 PUFAs饲料持续喂养仔鼠的水平.孕期和哺乳期n-3 PUFAs饲料喂养而断乳后给予n-3 PUFAs缺乏饲料喂养的仔鼠,成年后脑组织n-3PUFAs含量较n-3 PUFAs饲料持续喂养的仔鼠明显降低(P＜0.05);Bcl-2和CR的表达量高于n-3 PUFAs缺乏饲料持续喂养的仔鼠(P＜0.05).结论 孕期和哺乳期保证适量n-3 PUFAs的摄入,有助于成年期脑神经元的发生,并减少神经元凋亡.

OBJECTIVEThe role of epigenetic modifications on leptin expression during the development of obesity has not been clearly determined. This study aimed to investigate changes in the expression of DNA methyltransferases (DNMTs) at the leptin promoter and their effect on gene transcription during the development of obesity.METHODSUsing a high-fat diet (HFD)-induced obese (DIO) mouse model, we examined adipose expression of leptin, its promoter associated DNMTs and the methyl CpG-binding domain protein 2 (MBD2) at different time points after HFD feeding.RESULTSThe leptin expression levels in epididymal fat were significantly increased after feeding the mice a HFD for 4, 8, 12 and 18 weeks (w), as opposed to feeding them a standard diet (SD). However, the CpG promoter methylation fractions were significantly reduced at 8 w with a decreased association of MBD2 and DNMT1, and increased at 12 w and 18 w with an increased association of MBD2, DNMT3A and DNMT3B, after HFD feeding. Additionally, the binding of RNA polymerase II was increased at 8 w and decreased at 18 w after HFD feeding compared with SD feeding.CONCLUSIONSThese data indicate that time-specific changes in promoter associated DNMTs may be associated with the regulation of leptin expression, indicating that a complex and dynamic epigenetic mechanism underlies aberrant leptin expression during the development of obesity.

目的 观察母孕期及哺乳期不同含量n-3多不饱和脂肪酸(PUFAs)饲料对成年期仔鼠脑神经细胞凋亡的影响.方法 使用6～8w龄清洁级C57 BL/6J雌性小鼠,随机分为5组,每组10只,分别给予n-3 PUFAs缺乏和3种不同比例n-6/n-3 PUFAs(n-6/n-3 PUFAs比值分别为15∶1、5∶1、1∶1)饲料及1种高含量鱼油n-3 PUFAs饲料(n-6/n-3 PUFAs比值为1∶5)喂养.小鼠12 ～ 14 w龄时雌雄合笼交配繁殖,仔鼠断乳后继续行母鼠相同饲料喂养,选取生后3 m成年仔鼠用于实验.取脑进行组织固定,采用免疫组织化学技术对脑组织海马区Bcl-2和BaX表达进行定量分析.结果 与n-3 PUFAs缺乏组相比,n-3 PUFAs饲料喂养组,尤其是n-6/n-3 PUFAs比值(5∶1)和(1∶1)组小鼠海马CA3区神经细胞抗凋亡蛋白Bcl-2表达明显增加(P＜0.05),而致凋亡蛋白Bax表达则显著降低(P＜0.05).但高含量鱼油n-3 PUFAs喂养组(即n-6/n-3PUFAs 1∶5组)与n-3 PUFAs缺乏组相比未表现出显著性差异.结论 孕期及哺乳期添加n-3PUFAs,尤其是n-6/n-3 PUFAs比值在5～1∶1之间时有助于减少成年仔鼠脑组织神经细胞凋亡的发生,而过高含量n-3 PUFAs摄入则未对脑凋亡发生起到积极作用.

目的 探讨生命早期n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids, n-3 PUFAs）营养状态对成年期脑源性神经营养因子（bdnf）表达及其启动子区DNA甲基化的影响。方法 使用3-4 w龄清洁级C57BL/6J雌性小鼠，随机分为两组（10只/组），分别给予n-3 PUFAs缺乏和n-3 PUFAs饲料喂养；12-14 w龄时与雄性小鼠合笼交配繁殖。仔鼠生后21 d断乳时，随机挑选20只n-3 PUFAs缺乏组仔鼠用n-3 PUFAs饲料喂养，挑选等量n-3 PUFAs饲料组仔鼠用n-3 PUFAs缺乏饲料喂养，剩余仔鼠用与母鼠相同饲料继续喂养，至仔鼠3 m龄时结束实验。小鼠麻醉牺牲后取脑组织，采用实时荧光定量PCR技术测定大脑皮质bdnf剪切体II、IV、VI和IX 的mRNA表达。应用亚硫酸盐修饰直接测序法（BSP）和半巢式PCR检测具有差异表达的bdnf剪切体（IV和IX）启动子区DNA甲基化水平。 结果 ① mRNA表达：与n-3 PUFAs缺乏饲料持续喂养组相比，孕期和哺乳期n-3 PUFAs缺乏而断乳后给予n-3 PUFAs饲料喂养的仔鼠，成年后脑皮质区bdnf IV表达量未见明显改变，仍低于持续n-3PUFAs饲料喂养组。与含n-3 PUFAs饲料持续喂养组仔鼠相比，孕期和哺乳期n-3 PUFAs饲料喂养而断乳后给予n-3 PUFAs缺乏饲料喂养的仔鼠，成年后脑组织bdnf IV和IX表达也未见明显降低，仍高于持续n-3 PUFAs饲料喂养组。②甲基化改变：与n-3 PUFAs缺乏饲料持续喂养组相比，孕期和哺乳期n-3 PUFAs缺乏而断乳后给予含n-3 PUFAs饲料喂养，仔鼠成年后各位点甲基化水平未见明显改变；孕期及哺乳期n-3 PUFAs饲料喂养而断乳后给予n-3 PUFAs缺乏饲料喂养bdnf IV启动子区CG7、9、12位点，bdnf IX启动子区CG1、4和5位点甲基化水平明显降低。 结论 孕期和哺乳期保证适量n-3 PUFAs的摄入，有助于维持成年期脑组织中bdnf剪切体的正常表达，其机制可能与基因启动子区DNA甲基化改变有关。

探讨孕及哺乳期摄入n-6/n-3 PUFAs变化对子代脑功能相关基因表达的影响，使用清洁级C57BL/6J 3-4周龄雌性小鼠，随机分为5 组，分别给予n-3 PUFAs缺乏和4种不同含量n-3 PUFAs（n-6/n-3 PUFAs 比值分别为15:1、5:1、1:1及1:5）饲料喂养。小鼠12-14周龄时用常规饲料喂养的雄性小鼠与之合笼交配繁殖，仔鼠断乳后继续行母鼠相同饲料喂养，随访观察至生后7 d、21 d和3 m时分批处死后取脑。然后在仔鼠生后21d断乳时，随机挑选7-9只n-3 PUFAs缺乏组的仔鼠用n-6/n-3 PUFAs（5:1）组饲料喂养，至3m龄；同时随机挑选7-9只n-6/n-3 PUFAs（5:1）组的仔鼠，改用n-3缺乏组饲料喂养，至3m龄；剩余仔鼠用与母鼠相同的饲料喂养，至3m龄，麻醉牺牲后，取脑置于-80℃条件下备用。采用实时荧光定量PCR技术测定脑皮质gfap、mbp、nse、gad65、gad67、nr2b基因mRNA的表达。在各年龄段小鼠中，饲料中添加n-3 PUFAs使各基因的表达量明显增加；不同比例的n-6/n-3PUFAs对基因表达的影响不同。孕期及哺乳期饲料缺乏n-3PUFAs，即使断乳后的饲料中添加正常量的n-3 PUFAs，也不能使某些对脑功能起重要作用的基因的表达量升高至正常n-3 PUFAs喂养的小鼠。结果提示，孕期及哺乳期可能需要较高的n-3 PUFAs摄入（可能最优的n-6/n-3 PUFAs比例介于1:5~1:1之间），才能满足幼年期脑发育之需。保证生命早期n-3 PUFAs的适量摄入，有助于维持成年期脑功能相关基因的正常表达。