源自不同牛種卵母細胞質與細胞核所產製體細胞核轉置牛耳朵細胞之熱耐受性差異

Investigation on the thermolerance of ear cells derived from cloned cattle produced by reconstructed embryos using ooplasm and nucleus from different bovine breeds.

李泓(Hung Lee)

指導教授 ： 沈朋志 ； 吳弘毅

國立屏東科技大學/農學院/動物科學與畜產系/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00026

摘要

本研究旨在評估台灣黃牛（Y）與荷蘭牛（H）兩牛種間，以及源自不同牛種細胞核與細胞質所產製體細胞核轉置（somatic cell nuclear transfer, SCNT）牛體細胞之耐熱能力差異性。結果顯示，於熱季台灣黃牛（38.4~38.5℃）之陰道溫度均顯著（P  0.05）低於荷蘭牛（38.8℃），且台灣黃牛之耳朵細胞經熱緊迫處理（42℃）不同時距（6~24 h）後之細胞凋亡率（6 h: 1.1%; 12 h: 1.6%; 24 h: 2.6%）均顯著（P  0.05）低於荷蘭牛者（6 h: 1.8%; 12 h: 4.0%; 24 h: 6.9%）。在不同牛種耳朵細胞之細胞凋亡及其細胞相關蛋白質表現結果發現，荷蘭牛耳朵細胞經熱緊迫處理3 h以迄24 h之AIF及Bax之蛋白質相對表現量均顯著（P  0.05）高於相同熱緊迫處理時距之台灣黃牛者；Endo-G蛋白質之相對表現量則於上述兩牛種間無顯著（P > 0.05）差異；而caspase-3、-8及-9蛋白質之相對表現量，亦以熱緊迫處理12 h及24 h之荷蘭牛耳朵細胞顯著（P  0.05）高於相同條件熱緊迫處理之台灣黃牛者；在HSP-70蛋白質之相對表現量結果顯示，荷蘭牛耳朵細胞經熱緊迫處理後無顯著（P > 0.05）提升，但台灣黃牛耳朵細胞則於熱緊迫處理12 h後顯著上升，並顯著（P  0.05）高於各熱緊迫處理組之荷蘭牛耳朵細胞。在Bcl-2蛋白質之相對表現量結果則顯示，荷蘭牛耳朵細胞經熱緊迫處理後即顯著（P  0.05）下降，惟台灣黃牛者則於熱緊迫處理後無顯著變化（P > 0.05）。在源自不同牛種細胞核與細胞質所產製體細胞核轉置（SCNT）牛耳朵細胞之耐熱能力差異比較結果顯示，卵母細胞質源自台灣黃牛之SCNT牛，不論其供核細胞源自台灣黃牛（Yo-Yd, NT4）或荷蘭牛（Yo-Hd, NT5），其等粒線體DNA多態性確實屬於亞洲牛屬（Bos indicus）；而且此等SCNT牛耳朵細胞經熱緊迫處理後之細胞凋亡率（Yo-Yd: 1.8%和Yo-Hd: 1.9%）均顯著低於（P  0.05）細胞質與核均源自荷蘭牛（Ho-Hd: 3.4%; NT1, NT2, NT3）之SCNT牛或純種H牛（4.0%）者。此外，Yo-Yd及Yo-Hd SCNT牛耳朵細胞經熱緊迫處理後之AIF、Bax、cytochrome C、caspase-3、-8及-9等蛋白質之相對表現量均顯著（P  0.05）低於熱緊迫處理之Ho-Hd SCNT牛耳朵細胞；而Endo-G蛋白質之相對表現量，則僅經熱緊迫處理之Yo-Hd SCNT牛耳朵細胞顯著（P  0.05）低於熱緊迫處理之Ho-Hd SCNT牛者；在HSP-70及HSP-27等蛋白質之相對表現量方面，則反之以經熱緊迫處理之Yo-Yd及Yo-Hd SCNT牛耳朵細胞顯著（P  0.05）高於熱緊迫處理之Ho-Hd SCNT牛者；於Bcl-2蛋白質之相對表現量則於Ho-Hd、Yo-Yd與Yo-Hd SCNT牛耳朵細胞間無顯著（P > 0.05）差異。而經熱緊迫處理之Yo-Yd（17.4%）及Yo-Hd（6.8%）SCNT牛耳朵細胞，其AIF自粒線體轉移進入細胞核之百分比亦顯著（P  0.05）低於熱緊迫處理之Ho-Hd SCNT牛者（32.8%）。綜合上述結果說明，台灣黃牛之熱耐受性確實優於荷蘭牛；而卵母細胞質源自台灣黃牛所產製之SCNT牛，其耳朵細胞之熱耐受性亦顯著優於卵母細胞質源自荷蘭牛所產製之SCNT牛者，且此等抗熱能力可能源自於細胞質內之物質。

關鍵字

細胞核與細胞質來源組成 ； 荷蘭牛 ； 熱耐受性 ； 體細胞核轉置牛 ； 台灣黃牛

並列摘要

The objectives of this study were to compare the thermotolerance of somatic cells from Holstein (H) and Taiwan yellow cattle (Y), as well as the somatic cells derived from SCNT cattle reconstructed with recipient ooplasm and nucleus from different bovine breeds. Results showed that the vaginal temperatures of Taiwan yellow cattle (38.4~38.5℃) were significantly (P  0.05) lower than Holstein (38.8℃) during the hot season. Moreover, the apoptosis rates of ear cells derived from Taiwan yellow cattle (6 h: 1.1%; 12 h: 1.6%; 24 h: 2.6%) were significantly (P  0.05) lower than those of cells derived from Holstein (6 h: 1.8%; 12 h: 4.0%; 24 h: 6.9%) after heat shock treatment with different durations. At the protein level, the relative abundances of AIF and Bax proteins in ear cells derived from Holstein were higher than (P  0.05) that of ear cells derived from Taiwan yellow cattle after heat shock for 3h to 24 h treatments. The relative abundances of Endo-G protein in ear cells were no different (P > 0.05) between Holstein and Taiwan yellow cattle after heat shock. The relative abundances of caspase 3, caspase 8 and caspase 9 proteins in ear cells derived from Holstein were higher than (P  0.05) that of ear cells derived from Taiwan yellow cattle after heat shock for 12h and 24 h treatments, respectively. The relative abundance of HSP 70 protein in ear cells derived from Holstein were no increased (P > 0.05) at the duration of heat shock, but the relative abundance of HSP 70 protein were significantly increased (P  0.05) in ear cells derived from Taiwan yellow cattle after heat shock with 12h treatment, which were all higher than (P  0.05) that of heat shocked Holstein ear cells. The relative abundances of Bcl-2 protein in ear cells derived from Holstein were significantly decreased (P  0.05) after heat shock, but in ear cells derived from Taiwan yellow cattle were no different (P > 0.05) after heat shock with different duration. In addition, the DNA polymorphism of mtDNA D-loop region in ear cells derived from SCNT cattle reconstructed with Y ooplasm and donor nuclei from either Y (Yo-Yd, NT4) or H (Yo-Hd, NT5), which were all the Bos indicus types. Moreover, the apoptosis rate of heat-shocked ear cells derived from Yo-Yd (1.8%) and Yo-Hd (1.9%) SCNT cattle were significantly lower (P  0.05) than those of cells derived from SCNT cattle reconstructed with H ooplasm and donor nuclei (Ho-Hd: 3.4%; NT1, NT2, NT3) and Holstein (4.0%). At the protein level, the relative abundances of AIF, Bax, cytochrome C, caspase-3, caspase-8 and caspase-9 proteins in ear cells derived from Yo-Yd and Yo-Hd SCNT cattle were significantly lower than (P  0.05) those of cells derived from Ho-Hd SCNT cattle after heat shock. The relative abundance of Endo-G protein in ear cells derived from Yo-Hd SCNT cattle was significantly lower than (P  0.05) that of cells derived from Ho-Hd SCNT cattle after heat shock In contrast, the relative abundances of HSP 70 and HSP 27 proteins in ear cells derived from Yo-Yd and Yo-Hd SCNT cattle were higher than (P  0.05) those of cells derived from Ho-Hd SCNT cattle. The relative abundances of Bcl-2 protein in ear cells were similar among (P  0.05) ear cells derived from Ho-Hd, Yo-Yd and Yo-Hd SCNT cattle. However, the rates of AIF-positive nucleus in ear cells derived from Yo-Yd (17.4%) and Yo-Hd (6.8%) SCNT cattle were lower than (P  0.05) those of cells derived from Ho-Hd SCNT cattle (32.8%). In conclusion, the thermotolerance of ear cells derived from Taiwan yellow cattle and SCNT cattle reconstructed with cytoplasm derived from Y were higher than those of cells derived from H. Therefore, the cytoplasm may be a major determinant for thermal sensitivity in bovine ear cells.

並列關鍵字

Composition of cytoplasm and nucleus ； Holstein ； Somatic cell nuclear transfer ； Taiwan yellow cattle ； Thermotolerance

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