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研究生:許志勤
研究生(外文):Chih-Chin Hsu
論文名稱:大鼠臟器組織於不同溫度下死後變化之研究
論文名稱(外文):Study on Postmortem Changes of Multiple Organs in Rats under Different Temperatures
指導教授:劉振軒劉振軒引用關係
指導教授(外文):Chen-Hsuan Liu
口試委員:張清棟林正忠廖泰慶
口試委員(外文):Ching-Dong ChangCheng-Chung LinAlbert-Taiching Liao
口試日期:2015-07-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子暨比較病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:144
中文關鍵詞:死後變化死後經過時間大鼠肉眼觀察組織學觀察免疫組織化學染色溫度
外文關鍵詞:Postmortem changespostmortem intervalratsgrosslight microscopicimmunohistochemistrytemperatures
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國內外對於動物福利及動物權日漸重視,使著許多動物醫療糾紛及虐待動物案件浮出檯面,因此動物法醫學研究開始盛興。評估死後經過時間(postmortem interval, PMI)與死後變化(postmortem changes)的觀察有密切的關係,然而,死後變化經常受到外源性或內源性因子的影響,導致PMI的錯誤評估。近期發表指出,死後變化的研究幾乎侷限於巨觀的研究,顯微鏡的觀察研究則很少。
本研究透過肉眼及組織學的觀察,配合免疫組織化學染色;嘗試於不同溫度下(25℃及35℃),進行為期0-7天的大鼠死後變化之研究,藉此了解死後經過時間與肉眼、組織學及免疫組織化學染色之間的關聯性並建立適用之表格,期望應用於實際推估法醫案例的死亡時間(time of death, TOD)。將大鼠人道犧牲後個別置放於保鮮盒內,放入恆溫(25℃及35℃)且氣流固定的培養箱內進行0-7 天的死後變化觀察。主要臟器包含腦、心臟、肝臟、脾臟、肺臟、腎臟、皮膚及背最長肌,於肉眼觀察後,皆會進行採樣並固定於10%之中性福馬林,後進行組織學及免疫化學染色的觀察,免疫組織化學染色;使用本研究室常規染色之抗體,包含CD3、CD79a、Desmin、Factor VIII、GFAP、Myoglobin、NF、SMA、Vimentin。
本研究結果證實於肉眼及組織學觀察中,大部分的變化於高溫下死後變化速率較快並具有規律性,如肉眼觀察中,皮膚變色、腹部鼓脹及臟器的軟爛程度;而屍溫、屍僵、屍水的產出及背最長肌的軟爛程度為非特異性;在脫毛,死後變化速率於低溫組快於高溫組。組織學中,僅有脾臟變化不具有特異性,其他臟器於皆為高溫組快於低溫組。此外,許多特殊變化也在本研究中發掘,例如死後變化造成的誤診,於1 dpm(day of postmortem),在小腦白質部呈現海綿樣變化,擬似病毒性疾病造成之海綿樣腦病;肝細胞內出現數量不等的囊泡,擬似脂肪肝造成之液狀小滴;肺泡腔出現嗜伊紅性且均質樣的液體,擬似肺水腫導致之液體蓄積。在免疫組織化學染色中,大部分抗體表現為陽性轉為陰性,陽性訊號的消失發生於2-3 dpm,然而在CK、desmin、GFAP、myoglobin及SMA的染色結果呈現於不同臟器、不同時段及不同溫度下的表現不一致,有些染色甚至出現交叉反應影響判讀,因此於辨識上有其困難性。
本研究將於2溫度下肉眼觀察、組織學及免疫組織化學染色中,具有顯著差異的變化統整並製作成與PMI相關之表格,期望藉由法醫解剖案例驗證本表格對於PMI估算的實用性。


Animal welfare and medical disputes are getting public attention to raise development of veterinary forensic medicine. Estimation of postmortem interval (PMI) is always correlated with observation in postmortem changes (PMCs). However, PMCs are deeply influenced by extrinsic and intrinsic factors, leading to misestimate PMI frequently. Up to date, studies explored PMCs are almost at macroscopic level, but less investigation by light microscopy.
The study aims to correlate PMCs in gross examination and light microscopy, following with immunohistochemistry to estimate PMI. The rats were sacrificed by humane method, whole bodies were stored under 25℃ and 35℃ individually, and then sampled from day 0 to 7. The major organs, including the brain, heart, liver, spleen, lung, kidney, skin, and m. longissimus dorsi were observed grossly, then fixed in 10% neutral formalin for microscopy and immunohistochemistry. As for the antibodies of immunohistochemistry, including CD3, CD79a, Desmin, Factor VIII, GFAP, Myoglobin, NF, SMA, and Vimentin were used.
The results showed that postmortem rate in 35℃ was faster than 25℃, and the changes presented regularly. In gross, such as discoloration, abdominal swelling, and texture of the organs; however, the body temperature, rigor mortis, decomposition fluid, and the texture of m. longissimus dorsi were nonspecific; alopecia was faster in 25℃. In light microscopical examination, only spleen change was nonspecific, others were faster in 35℃. Some changes found in this study are worth our attention, such as in 1 dpm (day of postmortem), the spongy like change noted in the white matter might be comfused with spongiform change in the central nervous system; the intracytoplasmic vacuoles of hepatocytes should differentiate with lipid droplets of fatty liver; initiation of small amount of edematous exudates within the alveoli might be misdiagnosed in lung edema. In immunohistochemistry, most of the antibodies revealed positive to negative, and signals disappeared in 2-3 dpm; however, in CK, desmin, GFAP, myoglobin, and SMA showed variable results under different organs, dpm, and temperatures, some of them even displayed cross reaction, this leads difficulties in interpretation.
The results from gross, microscopy, and immunohistochemistry study are summarized in flow charts for the purpose of PMI estimation, and it further needs to put it into practice of forensic cases to confirm the feasibility.

中文摘要 I
Abstract III
第一章 介紹 1
前言 1
法醫科學歷史回顧 1
第二章 研究背景及文獻回顧 4
死後經過時間(Postmortem Interval, PMI) 4
組織學下的死後變化 7
第三章 材料與方法 10
實驗設計與流程 10
3.1實驗材料 11
3.2 研究方法 11
3.2.1 肉眼觀察 11
3.2.2 組織學觀察 12
3.2.3 免疫組織化學染色法 13
第四章 結果 14
4.1 實驗材料 14
4.2 溫度紀錄 14
4.3 肉眼觀察 14
4.4 組織學觀察 14
4.4.1 丘腦(Thalamus) 14
4.4.2 小腦(Cerebellum) 16
4.4.3 心臟-左心室(Left ventricle) 17
4.4.4 背最長肌(Muscle longissinus dorsi) 18
4.4.5 肝臟 19
4.4.6 脾臟 21
4.4.7 支氣管周圍類淋巴組織(Bronchus-associated lymphoid tissue, BALT) 22
4.4.8 肺臟 22
4.4.9 腎臟 23
4.4.10 皮膚 24
4.5免疫組織化學染色 25
4.5.1 CD3 及CD79a 25
4.5.2 Cytokeratin(CK) 26
4.5.3 Desmin 27
4.5.4 Factor VIII 28
4.5.5 Glial fibrillary acidic protein(GFAP) 29
4.5.6 Myoglobin 30
4.5.7 Neurofilament(NF) 30
4.5.8 Actin, alpha smooth muscle type(α-SMA) 31
4.5.9 Vimentin 32
第五章 討論 34
5.1 溫度:25℃及35℃ 34
5.1.1肉眼觀察 34
5.1.2組織學觀察 35
5.1.3免疫組織化學染色觀察 38
5.2 死後經過時間 40
5.2.1 肉眼觀察 40
5.2.2 組織學觀察 40
5.2.3 免疫組織化學染色 41
5.3 特殊臟器比較 41
5.4 肉眼變化及組織學變化 42
5.5 死後變化造成的誤診 43
第六章 表格 47
Table 1. Methods for the estimation of the postmortem interval. 47
Table 2. The recording form of gross examination. 48
Table 3. The antibodies were used for immunohistochemistry stain in the study. 49
Table 4. The materials: rats. 50
Table 5. The recording temperatures. 50
Table 6. Gross examination under 25℃. 51
Table 7. Gross examination under 35℃. 51
Table 8. Histological examination in thalamus under 25℃. 52
Table 9. Histological examination in thalamus under 35℃. 52
Table 10. Histological examination in cerebellum under 25℃. 53
Table 11. Histological examination in cerebellum under 35℃. 54
Table 12. Histological examination in heart under 25℃. 55
Table 13. Histological examination in heart under 35℃. 55
Table 14. Histological examination in m. longissimus dorsi under 25℃. 56
Table 15. Histological examination in m. longissimus dorsi under 35℃. 56
Table 16. Histological examination in liver under 25℃. 57
Table 17. Histological examination in liver under 35℃. 58
Table 18 Histological examination in spleen under 25℃. 59
Table 19 Histological examination in spleen under 35℃. 59
Table 20. Histological examination in BALT under 25℃. 60
Table 21. Histological examination in BALT under 35℃. 60
Table 22. Histological examination in lung under 25℃. 61
Table 23. Histological examination in lung under 35℃. 61
Table 24. Histological examination in kidney under 25℃. 62
Table 25. Histological examination in kidney under 35℃. 63
Table 26. Histological examination in skin under 25℃. 64
Table 28. The immunohistochemical result of CD3 under 25 and 35℃. 66
Table 29. The immunohistochemical result of CD79a under 25 and 35℃. 66
Table 30. The immunohistochemical result of CK under 25℃. 67
Table 31. The immunohistochemical result of CK under 35℃. 68
Table 32. The immunohistochemical result of desmin under 25 and 35℃ 69
Table 33. The immunohistochemical result of factor VIII under 25℃. 70
Table 34. The immunohistochemical result of factor VIII under 35℃. 70
Table 35. The immunohistochemical result of GFAP under 25℃. 71
Table 36. The immunohistochemical result of GFAP under 35℃. 71
Table 37. The immunohistochemical result of myoglobin under 25 and 35℃ 72
Table 38. The immunohistochemical result of Neurofilament under 25℃. 73
Table 39. The immunohistochemical result of Neurofilament under 35℃. 73
Table 40. The immunohistochemical result of smooth muscle actin under 25℃. 74
Table 41. The immunohistochemical result of smooth muscle actin under 35℃. 75
Table 42. The immunohistochemical result of vimentin under 25℃. 76
Table 43. The immunohistochemical result of vimentin under 35℃. 77
Table 44. The leptomeningeal breakdown in thalamus with under 35℃. 78
Table 45. The leptomeningeal breakdown in cerebellum with under 35℃. 79
Table 46. The flow chart of postmortem changes in still-air under 25℃ from 0-7 dpm. 80
Table 47. The flow chart of postmortem changes in still-air under 35℃ from 0-7 dpm. 80
第七章 圖次 81
Figure 1. 6 aspects of forensic scene-5W1H 81
Figure 2. Time frame of the TOD (Time of death) and PMI (Postmortem interval). 81
Figure 3. Cell death: Apoptosis vs. necrosis. 82
Figure 4. Histological findings in apoptosis and necrosis. 82
Figure 5. The reparation of the study. 83
Figure 6. Examples for histological examination in structure observation. 83
Figure 7. Examples for histological examination in cells observation. 84
Figure 8. Histological findings of structure in thalamus. 85
Figure 9. Histological findings of cells in thalamus under 25℃. 86
Figure 10. Histological findings of cells in thalamus under 35℃. 87
Figure 11. Histological findings of structure in cerebellum. 88
Figure 12. Histological findings of molecular layer in cerebellum. 89
Figure 13. Histological findings of purkinje cell layer & granule cell layer under 25℃. 90
Figure 14. Histological findings of purkinje cell layer & granule cell layer under 35℃. 91
Figure 15. Histological findings of white matter in cerebellum under 25℃. 92
Figure 16. Histological findings of white matter in cerebellum under 35℃. 93
Figure 17. Histological findings of structure in heart. 93
Figure 18. Histological findings of cardiomyocytes in heart under 25℃. 94
Figure 19. Histological findings of cardiomyocytes in heart 35℃. 95
Figure 20. Histological findings of structure in m. longissimus dorsi. 96
Figure 21. Histological findings of myocytes in m. longissimus dorsi under 25℃. 97
Figure 22. Histological findings of myocytes in m. longissimus dorsi under 35℃. 98
Figure 23. Histological findings of structure in liver under 25℃. 99
Figure 24. Histological findings of structure in liver under 35℃. 100
Figure 25. Histological findings of cells in liver under 25℃. 101
Figure 26. Histological findings of cells in liver under 35℃. 102
Figure 27. Histological findings of structure in spleen. 103
Figure 28. Histological findings of cells in spleen. 104
Figure 29. Histological findings of structure and cells in BALT. 105
Figure 30. Histological findings of structure in lung. 106
Figure 31. Histological findings of bronchi/bronchiole region in lung. 107
Figure 32. Histological findings of alveoli in lung. 108
Figure 33. Histological findings of renal cortex. 109
Figure 34. Histological findings of renal medulla. 110
Figure 35. Histological findings of structure in skin. 111
Figure 36. Histological findings of cells in skin. 112
Figure 37. Immunohistochemical results of CD3 & CD79a in spleen. 113
Figure 38. Immunohistochemical results of CD3 & CD79a in BALT. 114
Figure 39. Immunohistochemical results of cytokeratin in skin. 115
Figure 40. Immunohistochemical results of cytokeratin in liver. 116
Figure 41. Immunohistochemical results of cytokeratin in spleen. 117
Figure 42. Immunohistochemical results of cytokeratin in lung. 118
Figure 43. Immunohistochemical results of cytokeratin in renal cortex. 118
Figure 44. Immunohistochemical results of cytokeratin in renal medulla. 119
Figure 45. Immunohistochemical results of desmin in heart. 120
Figure 46. Immunohistochemical results of desmin in m. longissimus dorsi. 121
Figure 47. Immunohistochemical results of factor VIII in liver. 122
Figure 48. Immunohistochemical results of factor VIII in spleen. 123
Figure 49. Immunohistochemical results of factor VIII in lung. 124
Figure 50. Immunohistochemical results of factor VIII in kidney. 125
Figure 51. Immunohistochemical results of GFAP in thalamus. 126
Figure 52. Immunohistochemical results of GFAP in white matter. 127
Figure 53. Immunohistochemical results of myoglobin in heart. 128
Figure 54. Immunohistochemical results of myoglobin in m. longissimus dorsi. 129
Figure 55. Immunohistochemical results of NF in thalamus. 130
Figure 56. Immunohistochemical results of NF in white matter. 131
Figure 57. Immunohistochemical results of SMA in skin. 132
Figure 58. Immunohistochemical results of SMA in liver. 132
Figure 59. Immunohistochemical results of SMA in spleen. 133
Figure 60. Immunohistochemical results of SMA in lung. 133
Figure 61. Immunohistochemical results of SMA in kidney. 134
Figure 62. Immunohistochemical results of vimentin in skin. 134
Figure 63. Immunohistochemical results of vimentin in liver. 135
Figure 64. Immunohistochemical results of vimentin in spleen. 136
Figure 65. Immunohistochemical results of vimentin in lung. 136
Figure 66. Immunohistochemical results of vimentin in renal cortex. 137
Figure 67. Immunohistochemical results of vimentin in renal medulla. 137
Figure 68. The leptomeninges breakdown in thalamus and cerebellum. 138
第八章 參考文獻 139
附錄一、相關案例屍體解剖報告 141




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