台灣地處亞熱帶地區，養豬產業因夏季高溫多濕，造成豬隻生長性能及繁殖能力降低，而蒙受巨大經濟損失，因此本研究提出抗熱策略，並詳細研究高熱對豬隻生長性能與心臟生理效應，以期改善夏季熱緊迫問題，提升養豬產業價值。
首先，針對熱緊迫蛋白質誘導型表現量最大的HSP70.2基因5’端控制序列進行單核苷酸多態型(SNP)研究，並與各種生長性狀進行相關性分析。結果發現該序列中第393位核苷酸為TT或TC型的杜洛克豬隻的背脂厚度會較CC型者薄(P<0.05)；同時此位核苷酸具有Bsaw I限制酶切位。因此，我們可利用此位核苷酸多態型作為早期偵測杜洛克豬之背脂厚度之分子標記。然而，進一步的研究並未在豬隻HSP70.2結構基因發現SNP。
為發展抗熱豬隻品系，我們採用生產具有HSP70.2高表現量的基因轉殖豬為策略，並開發功能測試技術來驗證豬隻抗熱能力。利用PCR方法，全長之HSP70.2構造基因已經被選殖出來，並經詳細的定序分析工作，確認轉譯之蛋白質序列無誤。再設計以CMV為啟動子驅動HSP70.2構造基因表現，同時完成在C端融合一段綠色螢光蛋白質(GFP)序列以作為報導基因(reporter gene)。進一步結合利用基因顯微注射與胚胎移植技術，結果成功獲得兩個F0基因轉殖品系D6-11A及D6-12A；其轉殖效率為16.7%。D6-11A及D6-12A之基因轉殖套數分別為60及12；其轉殖蛋白表現量分別為6.4%及1.4%。將此品系的兩隻基因轉殖豬與另外一隻同胎非基因轉殖豬D6-01A之耳朵初級纖維母細胞分離培養以進行抗熱能力分析，結果發現D6-11A的初級纖維母細胞經加熱處理後其存活率(78.1±1.9%)與D6-12A及D6-01A的初級纖維母細胞經加熱處理後其存活率分別為60.9±1.5%及 62.9±4.5%，有顯著差異(P<0.05)。以上結果顯示，當基因轉殖豬之初級纖維母細胞中含有HSP70.2較高的蛋白質表現量時，具有較佳的細胞抗熱能力。然而，要證明豬隻的抗熱能力需要開發全豬功能測試技術。
我們進一步建立了全豬手術台加熱技術，以進行豬隻在高熱處理時生理反應之研究。將25-35公斤之HSP70.2-GFP基因轉殖豬與同胎非轉殖豬進行全豬加熱，觀察其中對熱最敏感之臟器—心臟對熱所產生之各項反應，結果發現基因轉殖豬在加熱至體溫上升5℃並維持1小時及恢復期1小時的動脈血壓，包括平均動脈壓、收縮壓及舒張壓，均比非基因轉殖豬高出約10毫米汞柱(P<0.05)；此現象表示轉殖基因HSP70.2-GFP可能參與正常血管收縮機制。另外全豬加熱會引起心電圖發生異常，如產生心室早期收縮(VPC)、ST segment 上揚及Q波變化，這些變化代表心肌發生損傷。同時，在加熱過程中，我們採集血清並測定心肌損傷標記cTnI在血清中的含量，結果發現在加熱組中當豬體溫上升3℃以上時，cTnI開始大量釋出至血清中，唯此釋出量在轉殖豬與非轉殖豬之間無顯著差異。經過18小時的恢復期，我們將所有豬隻進行犧牲採樣，並進行心臟組織切片之形態學及病理學觀察。結果發現，在兩個加熱組，無論基因轉殖豬或非基因轉殖豬，其心肌細胞微細構造都發生斷裂情形，並且在心肌組織中存有淋巴球、嗜中性白血球、嗜伊紅性白血球或巨噬細胞的浸潤作用，尤其在cTnI釋出超過0.55 ng/ml的樣本，其心肌病變更為嚴重。以上現象則表示，HSP70.2-GFP轉殖豬在心肌細胞可能表現量不足或因GFP干擾HSP70.2正常摺疊狀態，以致於失去HSP70.2的分子伴護者功能，而沒有發揮保護心肌受損之功能。
綜合以上觀察的結果，本研究發現HSP70.2基因5’端控制序列之核苷酸多態型會影響杜洛克豬隻之生長性能，而HSP70.2-GFP基因轉殖豬則可能因為表現出來的融合蛋白質摺疊狀態不正常或其蛋白質表現量不足，因而無法發揮對全身加熱所導致心肌損傷的保護功能。本研究因此也建立了檢測豬隻對高熱反應模式的手術台加熱技術，對於將來測試豬隻抗熱性能有相當大的助益。

High ambient temperature and humidity result in economic loss in pigs including growth and reproduction performances in Taiwan in subtropical area. Therefore, the strategies for thermoresistance were established and hyperthermic effects on growth performance and cardiovascular physiology were investigated exhaustively, in order to increase the economic value in pig industry.
We analyzed the single nucleotide polymorphism (SNP) in 5’-flanking region of porcine heat shock protein 70.2 gene, the highly inducible form, and then analyzed the association of these SNP with growth performance in Duroc pigs. The result indicated that pigs with TT and TC genotypes at 393 nt in 5’-flanking region have thinner backfat thickness than those with CC type (P<0.05). Thus, the result suggested that the polymorphic Bsa WI site in the 5’-flanking region of porcine HSP70.2 may be used as a marker for the early selection of ultrasonic backfat thickness in Duroc pigs. However, the SNPs in structural region of porcine HSP70.2 have been not found in our further study.
In order to develop the thermoresistant breeds, the strategies were established; one was to produce the transgenic pigs overexpressing HSP70.2 gene fused with GFP gene and driven by CMV promoter, another was to set up a technique for functional test to examine resistant ability to hyperthermia. First, the whole length of porcine HSP70.2 gene was amplified by RT-PCR, and then the protein sequence translated correctly by detail sequencing. Second, two transgenic pigs were produced by microinjecting pCMV-HSP70-GFP DNA into the pronucleus of fertilized eggs. Immunoblot assay revealed the varied overexpression level (6.4% and 1.4%) of HSP70-GFP in transgenic pigs. After heating at 45℃ for 3 h, the survival rate (78.1%) of the primary fibroblast cells from the highly expressing transgenic pig exceeded that from the non-transgenic pig (62.9%). This result showed that primary fibroblasts overexpressing HSP70-GFP confer cell thermotolerance. We suggested that transgenic pigs overexpressing HSP70 might improve their thermotolerance in summer season and therefore reduce the economic lost in animal production. However, it is necessary to develop a functional test in whole body to prove the ability to resistant hyperthermia.
Furthermore, a technique, whole body hyperthermia (WBH), was set up to investigate the hyperthermic response in pig. Transgenic pigs expressing HSP70 were generated with a fusion marker of green fluorescent protein (GFP) following the gene. The offspring from F1 and F2 transgenic pigs (TG) were employed to monitor the cardiovascular response to heat stress by WBH. Both heart rate (HR) and mean arterial pressure (MAP) were substantially increased after WBH transgenic (TG) and non-transgenic (NTG) littermates compared with those in their shamed control groups. However, there was no significant difference in HR between the groups. Among the heated groups, there were significant different in arterial pressure parameters MAP, systolic or diastolic pressure when animal body temperatures were increased by up to 5℃ and prolonged for 1 hr after. Myocardial injury serum marker, cardiac troponin I (cTnI), was concomitantly monitored during WBH episodes. Notably, cTnI levels in heated groups were substantially enhanced when animal body temperature was increased by up to 3℃ (Δ3℃) and thereafter at rest timepoints during the experiment. Levels of cTnI for both NTG and TG animals were significantly enhanced for 1 hr after WBH. However, there was no significant difference in cTnI levels increased by WBH between the NTG and TG groups. The electrocardiogram (ECG) results were also monitored during the experiment; ST segment elevation, Q-wave changes, and ventricular premature complexes were initially occurred and consistently continued after Δ3℃. At 18 hr recover after heat stress, all animals were sacrificed and the hearts were harvested for pathological examination. Myofibril was fragmented, lysic, and myofibrostic and lymphocytes, eosinophils, and neutrophils infiltrated intermyofibrils in both HS groups. These pathological examination findings for the hearts were severe in the pigs releasing the cTnI level □0.55 ng/ml (Table 2). Above observations indicated that the dysfunction in chaperone of HSP70.2 may be insufficient expression or miss folding by GFP fused and then HSP70.2 can not protect cardiomyocytes from thermal injury.
In conclusion, the backfat thickness of Duroc pigs would be affect by the SNPs in 5’-flanking region of porcine HSP70.2 gene. Nevertheless, the HSP70-GFP fusion protein could not confer the protective in cardiovascular changes in transgenic pigs from heat stress which may be caused by improper folding conformation or expressed insufficiently. Finally, a novel pig model for whole body hyperthermia was established to examine the pig’s response to hyperthermia, and it is useful for testing the thermotolerance of pigs in the future.

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