臺灣博碩士論文加值系統

母親孕期營養不但會影響胚胎發育過程，同時也會影響子代終身代謝功能，具有跨世代效應。油炸為廣受歡迎的烹調方式，目前已知炸油(oxidized frying oil, OFO)會改變體內脂質代謝，與活化peroxisome proliferator-activated receptor alpha (PPARα)，啟動下游基因如：acyl-Co A oxidase (ACO)與cytochrome P450 family 4A10 (CYP4A10)表現有關。本實驗室先前研究指出孕期攝食OFO飲食會增加母鼠吃仔及難產，推測原因與子代畸型有關；觀察倖存子代則發現成年後給予高油飲食誘發肥胖(Diet induced obesity, DIO)，公鼠有不易胖特質，母鼠有易胖體質。因此在本論文中探討的方向有二：(1) 探討母鼠孕期攝取OFO造成畸胎原因，並假設OFO干擾維生素A代謝；(2) 探討母鼠孕期攝取OFO影響子代成年後肥胖敏感性之機制。C57BL/6J母鼠於懷孕期間全程分別給予10% OFO或新鮮油(soybean oil, SO)飲食，部份於懷孕第十八天(day 18, d18)犧牲，進行胚胎毒理研究；其它繼續餵養使生產，仔鼠出生後一律由SO飲食母鼠哺餵，斷乳後給予chow diet至七週齡，再給予5週DIO，以供子代肥胖敏感性研究，如此依母親飲食及子代性別共計得到SO male、OFO male、SO female及OFO female四組子代。第一部份結果：孕期攝食OFO相較於SO對照，顯著增加死胎仔數、難產率及畸胎率，並且降低胎兒體重。而外觀畸形中又以臟器外露(visceral anomalies )及水腫(edema)兩組間具顯著差異。雖然孕期OFO飲食不影響母鼠肝臟維生素A含量，但顯著降低胎兒肝臟維生素A含量。以real-time PCR偵測懷孕d18母親及胚胎肝臟中維生素A代謝相關基因表現，僅發現OFO母親肝臟retinaldehyde dehydrogenase 1a1 (RALDH1a1)顯著增加，其它代謝酵素無差異。結論：孕期攝食OFO致畸胎可能與干擾母親retinoic acid生合成並迫使胚胎暴露於不適當RA濃度有關。第二部份結果：若是子代倖存至成年，OFO male相較SO male有不易胖特質，伴隨血清與組織(肝、肌肉)TG降低；而OFO female相較SO female容易肥胖，伴隨血清TG增加。懷孕d18 時OFO組母親及胚胎(不論性別)肝臟PPARα活性顯著高於SO組，肝臟的PPARα mRNA量於胚胎因母親攝取OFO而顯著增加，於成年子代卻是顯著降低。母親攝取OFO顯著增加了成年雄性子代白色脂肪的PPARα、ACO、PPAR-gamma coactivator (PGC-1α)與uncoupling protein 1(UCP-1) 表現量，雌性子代除UCP-1增加沒有上述變化，推測OFO組雄性子代抗肥胖原因部分與白色脂肪PPARα活化與生熱基因表現增加有關。結論：孕期攝取炸油飲食可能程式化子代PPARα活化效應，影響子代易胖或不易胖特質，但此效應依子代性別而異。

Maternal nutrition during pregnancy affects embryonic development and also permanently alter metabolic functions of offspring in adulthood. It has been reported that consumption of oxidized frying oil (OFO) can modulate lipid metabolism through acativation of peroxisome proliferator-activated receptor alpha (PPARα). By increasing the expression of PPARα target genes, including acyl-Co A oxidase (ACO) and cytochrome P450 family 4A10 (CYP4A10), the OFO diet up-regulates fatty acid β-oxidation. In our previous study, a higher incidence of dystocia and pups-eaten were observed in dams receiving OFO diet during pregnancy. We speculated that the malformation of embryo might be involved in these issues. Moreover, the survival offspring from OFO-dams are resisted and susceptible to obesity in male and female, respectively, after challenging with high fat diet during adulthood. The aims of this study were to investigate: 1) the teratogenic effects of dietary OFO, and 2) the underlying mechanism associated with gestational OFO diet and the suceptibility of offspring to obesity. C57BL/6J mice were given control (SO; 10% fresh soybean oil) or OFO (10 % OFO) diet throughout the whole gestational period. Portion of mice were killed on pregnancy day 18 (d18) for toxicological study. Others were raised to delivery and their pup were used for testing propensity to obesity. For this experiment, all pups was lactated by mothers receiving SO diet until weaning, fed with standard chow diet for 4 weeks, and followed by a high fat diet challenge for 5 weeks. Therefore, four groups of offspring were obtained, i.e. SO male, OFO male, SO female and OFO female according to the maternal diet during pregnancy and their respective gender. Results of Part I: Compared with SO dams, OFO dams had significantly higher number of dead fetus, incidence of dystocia and fetus with externally congenital anormalies (especially visceral anomalies and edema) and reduced birth weight. There was no difference in retinol content in liver between SO and OFO dams, but a significantly lower level in OFO fetus were observed compared to SO fetus. The mRNA levels of gene associated with retinoids metabolism were measured by qRT-PCR. No difference between the two groups were noticed except for retinaldehyde dehydrogenase 1a1 (RALDH1a1), which was significantly greater in liver of OFO dams compared with that in SO dams. We concluded that OFO-mediated interference in retinoid metabolism, by exposing the embryos to inappropriate retinoic acid concentration, partly contributes to the teratogenic effects on OFO fetus. Result of Part II: The male offspring came from OFO dams (i.e. OFO male) were resistance to DIO, along with a lowered TG level in serum and tissues, compare with the SO male. In contrast, the female offspring came from OFO dams (i.e. OFO female) were predisposed to DIO compared to SO female. In OFO female, the TG tended to be accumulated in white adipose tissue, rather than in muscle. The OFO-mediated PPARα activation was observed in livers of dams and fetus at pregnancy d18. The hepatic mRNA levels of PPARa was significantly greater in fetus, while lowered in adult offspring of OFO dams compared to their counterpart respectively. The mRNA levels of PPARa target genes (including PPARα, ACO, PGC-1a and UCP1) in the adipose tissue were significantly increased in OFO-male compared to the SO-male, while no difference was observed between OFO female and SO female, except for UCP-1. Accordingly, the resistance to DIO in OFO male is attributable to a greater PPARα activation and a thermogenic response in white adipose tissue. For Part II, we concluded that OFO diet during pregnancy might program PPARα activation in offspring, affecting the susceptibility to obesity in offspring with sexual dimorphism.

目錄
目錄 i
圖目錄 iv
表目錄 vi
縮寫對照表 vii
中文摘要 ix
Abstract xi
第一章 前言 1
第二章 文獻回顧 3
一、 發育毒理(Developmental toxicology) 3
(一) 歷史背景 3
(二) 孕期因營養素缺乏致畸胎： 4
(三) 維生素A與致畸胎 5
二、代謝程式化 (Programming) 9
(一) 背景 9
(二) 子宮內營養不良 10
(三) 代謝程式化之機制 10
三、 氧化炸油 (Oxidized frying oil, OFO) 13
(一) 炸油之化學組成 13
(二) 炸油對對動物生理影響 14
(三) 炸油與脂質代謝 15
(四) 炸油與維生素A 16
第三章 材料與方法 17
一、實驗設計與假說 17
二、氧化炸油製備 18
三、試驗飼料配製 19
四、動物飼養 19
五、檢體收集 20
六、胚胎性別鑑定 22
七、Genome DNA 萃取 22
八、SRY與DXNds3基因鑑定 23
九、組織切片 27
十、口服葡萄糖耐受試驗(Oral glucose tolerance test, OGTT) 27
十一、腹腔注射胰島素測試(Insulin tolerance test, ITT) 29
十二、血糖分析 31
十三、血清脂質分析 32
十四、肝臟脂質分析 36
十五、肝臟視網醇分析 38
十六、抽取RNA及cDNA的製備 41
十七、Real time polymerase chain reaction(同步定量PCR；qRT-PCR) 47
十八、統計分析 53
第四章 結果 54
一、 探討孕期攝取氧化炸油造成畸胎現象 54
(一) 孕期攝取炸油之胚胎毒性 54
(二) 炸油對母鼠生殖性狀之影響 54
(三) 炸油對d18胚胎存活率與外觀異常之影響 55
(四) 炸油對仔鼠內臟發育之影響 56
(五) 孕期攝食炸油對懷孕d18母鼠及胚胎肝臟retinol含量之影響 56
(六) 孕期攝食炸油對懷孕d18母鼠及胚胎肝臟retinoids代謝基因表現之影響 57
二、探討炸油造成子代肥胖敏感性之機制 77
(一) 孕期攝取炸油對子代生長影響 77
(二) 孕期攝取炸油對子代成年體脂堆積影響 77
(三) 孕期攝取炸油對子代成年血脂與肝(肌肉)脂質影響 77
(四) 孕期攝取炸油對子代成年葡萄糖耐受性及胰島素敏感性影響 78
(五) 孕期攝取炸油對母親與胚胎(懷孕第18天)肝臟PPARα活化影響 78
(六) 孕期攝取炸油對子代成年肝臟PPARα活化影響 79
(七) 孕期攝取炸油對子代成年白色脂肪PPARα活化影響 79
第五章 討論 90
一、孕期攝取OFO與畸胎 90
二、孕期攝取OFO與子代肥胖敏感性 94
第六章 結論 98
第七章 參考文獻 99

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