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研究生:余峻賢
研究生(外文):Yu, Jun-Xian
論文名稱:源自不同父系與母系遺傳組合之豬種耳朵細胞熱耐受性之差異
論文名稱(外文):Investigation on the thermotolerance of porcine ear fibroblasts derived from hybrids with different maternal and paternal genetic background
指導教授:沈朋志沈朋志引用關係吳弘毅吳弘毅引用關係
指導教授(外文):Shen, Perng-ChihWu, Hung-Yi
口試委員:王治華
口試委員(外文):Wang, zhi-hua
口試日期:2018-01-20
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物科學與畜產系所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:81
中文關鍵詞:抑凋亡因子細胞凋亡熱緊迫母系遺傳父系遺傳促凋亡因子
外文關鍵詞:anti-apoptotic factorsapoptosisheat stressmaternal geneticpaternal geneticporcinepro-apoptotic factors
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已知細胞內之細胞質與細胞核遺傳組成源自不同耐熱能力品種時,將影響該細胞之熱耐受能力,且細胞質遺傳乃屬母系遺傳。因此於本研究中將選用耐熱之純種梅山豬(Meishan, M)、不耐熱之純種杜洛克豬(Duroc, D)、M母豬與D公豬雜交之高畜黑豬(MD: KHAPS black pigs, K)、K母豬與D公豬雜交之KD、KD母豬與D公豬雜交之KDD、D母豬與K公豬雜交之DK、以及DK母豬與D公豬雜交之DKD,用以探討細胞質與細胞核遺傳源自不同豬種間耳朵細胞之耐熱能力差異。在細胞質遺傳來源之鑑定結果顯示,K、KD及KDD豬耳朵細胞之粒線體D-loop區DNA PCR-RFLP多態性均與M豬一致,而判定其細胞質均源自M豬;而DK及DKD豬之粒線體D-loop區DNA多態性則與D豬一致,因此其判定其細胞質均源自D豬。在細胞凋亡分析結果則顯示,M豬耳朵細胞經不同時距(24〜72 h)之42.0℃(24 h: 0.70 vs. 1.33%;48 h: 1.27 vs. 3.66%;72 h: 1.73 vs. 6.15%)與43.0℃(24 h: 0.76 vs. 1.21%;48 h: 1.42 vs. 3.75%;72 h: 2.01 vs. 7.53%)熱緊迫處理後之細胞凋亡率均顯著(P < 0.05)低於D豬者;且發現在相同細胞核遺傳(KD vs. DK;KDD vs. DKD)條件下,細胞質遺傳源自M豬之KD(2.37%)與KDD(2.55%)豬耳朵細胞經熱處理(42.0℃, 72 h)後之細胞凋亡率分別顯著(P < 0.05)低於細胞質遺傳源自D豬之DK(4.03%)與DKD(4.10%)者。此外,在細胞凋亡相關蛋白質相對表現結果發現,M豬耳朵細胞經熱緊迫處理(42.0℃, 72 h)後之促凋亡蛋白質相對表現量包括AIF、cytochrome c、Bax、caspase-3、caspase-8及caspase-9等均顯著(P < 0.05)低於D豬者;然在相同細胞核遺傳(KD vs. DK;KDD vs. DKD)條件下,細胞質遺傳源自M豬之KD與KDD豬耳朵細胞經熱處理(42.0℃, 72 h)後之cytochrome c、Bax、caspase-3、caspase-8及caspase-9等均分別顯著(P < 0.05)低於細胞質遺傳源自D豬之DK與DKD者;且不論細胞質遺傳源自M或D豬,此等耳朵細胞經熱緊迫處理後之caspase-3、caspase-8及caspase-9等蛋白質之相對表現量均隨細胞核之D豬遺傳比率提升而顯著(P < 0.05)增加。另方面,代表抑凋亡之Bcl-2/Bax蛋白質相對表現量比例,以細胞質遺傳源自M豬之KD與KDD豬耳朵細胞經熱緊迫處理後均顯著(P < 0.05)高於細胞質遺傳源自D豬之DK及DKD豬者;而HSP-70蛋白質之相對表現量,則以M、KD及KDD之豬耳朵細胞經熱緊迫處理後均顯著(P < 0.05)高於D、DK及DKD豬者。綜合本研究之數據說明,M豬耳朵細胞之耐熱能力確實優於D豬,且在相同細胞核遺傳條件下,細胞質遺傳源自M豬之雜交豬耳朵細胞的耐熱能力亦優於細胞質遺傳源自D豬者,但此等細胞之熱耐受能力將隨細胞核之D豬遺傳比率提升而降低,此也暗喻耐熱豬種之細胞質雖具有改善不耐熱豬種細胞核之熱耐受能力,惟不耐熱豬種之細胞核仍對細胞之耐熱能力具有不利影響。
It has known that since cytoplasm and nucleus genetic composition of intracellular derived from different thermotolerance varieties, which affect the heat resistance of cell. Furthermore, the cytoplasmic inheritance was belong with maternal inheritance. Therefore, in the present study, we will select purebred Meishan (heat-tolerance), purebred Duroc (heat-sensitivity), KHAPS black pigs (K) of crossbreed from Meishan sow and Duroc boar, KD of crossbreed from K sow and D boar, KDD of crossbreed from KD sow and D boar, DK of crossbreed from D sow and K boar, and DKD of crossbreed from DK sow and D boar. In order to explore the difference of thermotolerance of ear fibroblasts derived from various breeds of porcine with cytoplasmic and nuclear inheritance. The identification of genetic sources in cytoplasm results showed that the DNA PCR-RFLP polymorphisms of mitochondrial DNA D-loop region of ear fibroblasts derived from K, KD and KDD porcine were consistent with that of M porcine, while the cytoplasm were all determined derived from M porcine. However, the DNA polymorphisms of mitochondrial DNA D-loop region of ear fibroblasts derived from DK and DKD porcine were consistent with that of D porcine. Therefore, it was determined that cytoplasms were all derived from D porcine. The apoptotic rates of ear fibroblasts from M porcine in heat-shocked 42.0℃ (24 h: 0.70 vs. 1.33%; 48 h: 1.27 vs. 3.66%; 72 h: 1.73 vs. 6.15%) and 43.0℃ (24 h: 0.76 vs. 1.21%; 48 h: 1.42 vs. 3.75%; 72 h: 2.01 vs. 7.53%) were significantly (P < 0.05) lower than those of cells derived from D porcine with various durations (24-72 h), moreover, genetic in the nucleus (KD vs. DK; KDD vs. DKD) under the same conditions, the apoptotic rates of KD (2.37%) and KDD (2.55%) ear fibroblasts of cytoplasmic inheritance derived from M porcine were significantly (P < 0.05) lower than those of DK (4.03%) and DKD (4.10%) cells of cytoplasmic inheritance derived from D porcine after heat shock (42.0℃, 72 h), respectively. In the protein level, the relative abundances of pro-apoptotic proteins inducing AIF, cytochrome c, Bax, caspase-3, caspase-8 and caspase-9 proteins in ear fibroblasts derived from M porcine were significantly (P < 0.05) lower than those of cells derived from D porcine after heat shock (42.0℃, 72 h). However, genetic in the nucleus (KD vs. DK; KDD vs. DKD) under the same conditions, the relative abundances of cytochrome c, Bax, caspase-3, caspase-8 and caspase-9 proteins in KD and KDD ear fibroblasts of cytoplasmic inheritance derived from M porcine were significantly (P < 0.05) lower than those of DK and DKD cells of cytoplasmic inheritance derived from D porcine after heat shock (42.0℃, 72 h), respectively. Therefore, whether the cytoplasmic inheritance was derived from M or D porcine, the relative abundances of caspase-3, caspase-8 and caspase-9 proteins in those of ear fibroblasts were all significantly (P <0.05) increased in the genetic ratio of D porcine in the nucleus increased after heat shock. Moreover, the relative abundances of Bcl-2/Bax protein ratios in KD and KDD ear fibroblasts of cytoplasmic inheritance derived from M porcine were significantly (P < 0.05) higher than those of DK and DKD cells of cytoplasmic inheritance derived from D porcine after heat shock. In contrast, the relative abundances of HSP-70 protein in ear fibroblasts derived from M, KD and KDD were significantly (P < 0.05) higher than those of cells derived from D, DK and DKD after heat shock. In conclusion, the thermotolerance of ear fibroblasts derived from M porcine is indeed better than those of cells derived from D porcine, moreover, genetic in the nucleus under the same conditions, the thermotolerance of crossbreed porcine ear fibroblasts of cytoplasmic inheritance derived from M porcine are also better than those of cytoplasmic inheritance derived from D porcine, however, the heat resistance of those cells will decrease with the genetic ratio of D porcine in the nucleus increased. Therefore, this also implies that although the cytoplasm of thermotolerance porcine can be improved heat resistance from nucleus of thermosensitive porcine, but the nucleus of thermosensitive porcine still have an adverse effect on the heat resistance of the cells.
摘要 I
Abstract III
謝誌 VI
目錄 VIII
圖表目錄 X
壹、前言 1
貳、文獻回顧 4
一、不同豬品種之發展 4
(一)梅山豬(Meishan, M) 4
(二)杜洛克豬(Duroc, D) 4
(三)高畜黑豬(KHAPS black pigs, K) 5
二、熱緊迫環境在不同豬品種之生理反應差異 6
(一)熱緊迫對不同豬種繁殖性能之影響 6
(二)熱緊迫對豬體細胞及胚發育能力之影響 7
三、熱緊迫環境對不同牛品種生理反應之影響 8
(一)熱緊迫對不同牛種體細胞之影響 8
(二)熱緊迫對不同牛種胚發育能力之影響 9
(三)胚及體細胞之細胞質及細胞核源自不同牛種時之熱耐受差異 10
四、細胞凋亡之研究發展 11
五、細胞凋亡訊息傳遞途徑之種類 12
(一)外源性細胞凋亡路徑(extrinsic apoptosis pathway) 12
(二)內源性細胞凋亡路徑(intrinsic apoptosis pathway) 13
六、細胞凋亡相關調控基因之功能 15
(一)凋亡誘導因子(apoptosis-inducing factor, AIF) 15
(二)細胞色素C(cytochrome c, Cyt c) 16
(三)Bcl-2蛋白家族 17
(四)熱休克蛋白質(heat shock proteins, HSPs) 19
(五)半胱氨酸天冬氨酸蛋白酶(cysteinyl asparate-specific
proteinases, caspases)家族 21
參、研究內容 23
研究一:源自不同母系雜交豬種耳朵細胞於熱緊迫處理後細胞凋亡之差異 23
一、材料與方法 23
二、結果 30
三、討論 36
研究二:源自不同母系雜交豬種耳朵細胞於熱緊迫處理後,其相關細胞凋亡相對蛋白質表現量之差異 40
一、材料與方法 40
二、結果 45
三、討論 57
肆、結論 62
伍、參考文獻 63
作者簡介 81
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