臺灣博碩士論文加值系統

熱休克反應(heat shock response；HSR)被公認為是保護細胞體甚至生物體生存的重要機制。骨骼肌是動物個體活動時不可缺少的組織，也由於長期能量的轉換與收縮放鬆是個體常受傷的組織之一，當肌肉受傷時，退化分解(degeneration)是最常見的病理變化。如何減少骨骼肌的傷害，一直是基礎及臨床醫學所關心的主題，所以本研究目的是以局部麻醉劑-bupivacaine (BPVC)來誘發骨骼肌傷害，探討熱休克反應是否可減少或避免肌肉退化分解的傷害。
實驗分為活體內(in vivo)和活體外(in vitro)二部分。活體內的實驗是將大白鼠(Wistar rat)施以體外全身加熱法，維持肛溫41.2℃持續15分鐘，在熱休克處置後24小時，以BPVC溶液作肌肉內注射，誘發腓腸肌(gastrocnemius muscle)產生傷害。在注射後1、3、6、24及48小時，取腓腸肌，作光學顯微鏡及電子顯微鏡的形態觀察；並測量血液中CK值，作為生化上測量肌肉傷害指標。活體外的實驗是取懷孕第21天胎鼠的骨骼肌分離出肌胚細胞(myoblast)，以DMEM培養液在37℃、5%CO2培養箱進行細胞培養。體外培養至第四天，肌胚細胞開始融合成多核的肌小管(myotube)。培養的細胞以保溫箱進行45℃、30分鐘的加熱處置，2小時後，利用相位差顯微鏡觀察肌胚細胞及肌小管培養於BPVC溶液中形態的變化，並以雷射細胞儀，偵測加入BPVC後，細胞內鈣離子濃度的變化。
活體內的實驗結果顯示，以光學顯微鏡觀察未經加熱組的腓腸肌，在注射BPVC後1小時，有一些肌纖維產生過度的收縮現象，6小時後，可見受影響的肌纖維，中央呈現空洞狀，顯示其內的肌原纖維已消失。在接下來的觀察，肌纖維壞死的情況越來越嚴重，在24~48小時，單核球幾乎充滿整個受傷的區域，在48小時，可見某些區域已被吞噬清除。電子顯微鏡下的形態結果顯示，注射BPVC後1小時，粒線體變的圓且腫大，有些甚至內膜已消失，部分的肌絲和Z線消失或扭曲，肌小節變形。除了組織形態的變化，CK的釋放量在注射後1小時，有非常顯著的增加，注射後3小時，CK值上升至最高峰，24和48小時後，又恢復為原來注射前的基準值。而經加熱處置後的組別，不論形態上或生化的證據皆顯示肌肉受傷的程度較輕微。活體外的實驗結果顯示，將肌胚細胞和肌小管培養於含有BPVC的培養液中，肌小管有較嚴重的傷害，經加熱前處理的組別，肌小管則只有些微的萎縮。利用雷射細胞儀偵測細胞內鈣離子濃度的變化，證實BPVC會造成細胞內鈣離子濃度上升，而經加熱前處理的細胞，細胞內鈣離子濃度沒有上升的現象。
由以上結果可以證實熱休克處置對BPVC誘發的骨骼肌傷害具有保護作用。我們推測熱休克蛋白可能是藉由作用在肌漿質網及粒線體，以降低細胞內鈣離子濃度達到保護的效果。而對於熱休克蛋白參與此保護作用的確實機制，尚待更進一步的研究。

The heat-shock response (HSR) is well documented to protect almost all living cells, as well as entire organisms, from the adverse effects of heat and other forms of stress. Skeletal muscle is indispensable while animals are in movement, but the long-term energy transfer and contraction also make it as one of the most vulnerable tissues. When skeletal muscle suffered from an injury or disease, cellular degeneration is the most common pathological characteristic. Both the severalties of the initial degeneration or its progression are highly connected with the outcome of the physiological function of muscular system. For decades, it still remains an uncertain issue for clinicians and basic researchers how to prevent or reduce the skeletal muscle injury after a trauma or in a disease entity. In the thesis, a local muscular degeneration model was established by intramuscular injection of a local anesthetic, bupivacaine (BPVC). We aimed to investigate whether previous HSR protect the muscular degenerative injury, and the possible mechanisms were also evaluated.
The whole study was divided into in vivo and in vitro experiments. Adult male Wistar rats were employed for in vivo study. Rats of experimental (pre-heated) group were subjected to whole body hyperthermia at 41.2 C for 15 minutes. Being recovering for 24 h, BPVC was injected intramuscularly. Rats of control (non-heated) group were treated identically but hyperthermia. The target muscle tissues were removed at 1, 3, 6, 24 and 48 h after the injection, and morphological changes were observed by light and electron microscope. Plasma creatine kinase (CK) levels were also measured as a biochemical marker of muscle damage. In in vitro experiment, the myoblasts, obtained from 21-day-old rats'' embryos, were used. They were cultured by DMEM medium in 37℃, 5% CO2 incubator. At 4th culture day, some myoblasts might differentiate into multinucleated myotubes. Then cells were exposed to a hyperthermic condition of 45℃ for 30 minutes. Two h later, they were treated with BPVC contained medium and morphological changes of cells were observed periodically, using a phase contrast microscope. Continuous intracellular free Ca2+ were measured before and after the addition of BPVC by confocal laser cytometry.
The results showed that hypercontraction of muscle fibers appeared in the non-heated gastrocnemius muscle 1 hour after BPVC injection. Six h later, central punch-out appearance was noticed in these affected fibers, indicating loss of myofibrils. The injured cell number and area were increasing for 24~48 hours, when the mononuclear cells occupied most of the degenerated area. Phagocytosis took place in some area 48 h after the muscle injury. As showed in the electron microscopic observation, swollen mitochondria were found 1 h after BPVC injection, some of them even lost their cristae. Part of the myofilaments and Z lines disappeared and looked like smear. In addition, the sarcomere disorganized. Significantly, these pathologic findings were absent or less severe in pre-heated rats. Furthermore, the elevation of CK was significantly obvious in non-heated rats during the experiment, 1731+957 vs. 658+125 at 3 h (p<0.05), 769+153 vs. 293+92 at 6 h (p<0.0001), respectively. These quantitative data were compatible with the severity in histo-morphological findings demonstrated above. In vitro study, the results revealed that myotubes were more sensitive to BPVC damage than myoblasts are in both groups. The myotubes shrank gradually in BPVC-contained medium while the myoblasts were less influenced. Same as in vivo study, result of less severity was obtained in cells with previous hyperthermia treatment. In confocal laser cytometry, a rapid increase of intracellular Ca2+ concentration was observed in non-heated myoblast after BPVC stimulation. However, no obvious over-shooting was accessed in pre-heated cells.
In conclusion, previous heat-shock response, highly possible the induced heat shock proteins, protects the initial damage and its ongoing degeneration caused by BPVC injection. The protective effect of HSR might come, in part, from the inhibition of the over-shooting of intracellular Ca2+ concentration. The detailed mechanisms need more advanced researches.

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………4
緒論………………………………………………………………………7
材料與方法………………………………………………………………15
第一部分：活體內的實驗………………………………………………15
第一節 實驗設計………………………………………………………15
第二節 實驗方法………………………………………………………17
第二部分：活體外的實驗………………………………………………24
第一節 實驗設計………………………………………………………24
第二節 實驗方法………………………………………………………25
結果………………………………………………………………………30
第一部分：活體內的實驗………………………………………………30
第二部分：活體外的實驗………………………………………………43
討論………………………………………………………………………51
參考文獻………………………………………………………………… 59

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