- 學位論文
脂源性幹細胞對燒燙傷引發脊髓運動神經元凋亡的保護作用探討
Assessment the protective effects of adipose-derived stem cells against burn-induced spinal cord motor neuron apoptosis
摘要
嚴重燒燙傷常會導致高代謝狀態，在傷口癒合後出現後續的肌肉無力及萎縮的情況，這個症狀通常會持續超過幾個月以上，進而限制病患恢復的情況。然而，燒傷對於相應的脊髓皮節的影響仍然未知，本研究利用動物燙傷模式探討燒傷是否誘導脊髓腹角運動神經元的細胞凋亡，再者幹細胞在治療一些神經性病變，有抑制神經元細胞凋亡，我們也將探討脂源性幹細胞是否具有神經保護效果，分析細胞治療的可行性。 在第一部分研究，先建立三度燙傷大鼠模式，使用加熱金屬板在實驗大鼠的右後足底引發三度燙傷，燙傷面積約1％體表面積，在燒傷後4和8週將大鼠犧牲，分析這種燙傷模式誘導腰椎腹角運動神經元、坐骨神經(利用S100標記坐骨神經中的許旺細胞)、及腓腸肌細胞凋亡的情況。 研究結果發現：在燙傷後，腰椎腹角運動神經元出現細胞凋亡增加的現象；坐骨神經染色中S100和末端脫氧核苷酸轉移酶雙陽性細胞也有顯著增加的現象；腓腸肌細胞去神經萎縮面積，在燒傷後顯著增加。這部分的研究結果，建立一種可行的動物模型，來評估燒燙傷後引起的肌肉萎縮，用以找出潛在的治療方法及後續的機轉探討。 在第二部分的研究中，我們利用先前所建立的三度燒傷大鼠模型，評估脂源性幹細胞的治療效果，我們從大鼠腹股溝的脂肪墊取得脂肪組織，培養出所需要的脂源性幹細胞，在燙傷後第4週，將這些脂源性幹細胞注射到右後足底，並在4週後 (燙傷後第8週)，分析其腰脊髓、坐骨神經、及注射處皮膚的免疫螢光染色，包括末端脫氧核苷酸轉移酶測定、胱天蛋白酶-3、胱天蛋白酶-9、S100和CD90，腓腸肌利用蘇木素-伊紅染色，觀察其組織學的變化。 結果發現: 燙傷後在其相對應的皮節脊髓腹角運動神經元中，胱天蛋白酶-3陽性、胱天蛋白酶-9陽性、和TUNEL陽性細胞，均有顯著增加；此外，也觀察到在坐骨神經的許旺細胞有減少的情形，和腓腸肌細胞去神經萎縮的情形增加；不過這種情形在脂源性幹細胞移植後，明顯減少神經細胞凋亡及肌肉去神經萎縮的情形，顯示幹細胞治療可能有神經保護的作用。 根據上述研究的結論，我們認為脂源性幹細胞具有臨床上的潛力，可能可應用於治療燙傷引起的運動神經元細胞凋亡及肌肉萎縮。
並列摘要
Severe burns result in hypercatabolic state and concomitant muscle atrophy that persists for several months, thereby limiting patient recovery. However, the effects of burns on the corresponding spinal dermatome remain unknown. We used an experimental model of burn injury to investigate whether burns induce apoptosis of spinal cord ventral horn motor neurons (VHMNs). Furthermore, stem cells have therapeutic effect for preventing apoptosis in several neuropathological conditions, we also examined the neuroprotective effect of adipose-derived stem cells (ASCs). The first part of our study established full-thickness burn injury rat model. 1% total body surface area (TBSA), third-degree hindpaw burn injury rats were euthanized 4 and 8 weeks after burn injury. The apoptosis profiles in the ventral horns of the lumbar spinal cords, sciatic nerves, and gastrocnemius muscles were examined. The Schwann cells in the sciatic nerve were marked with S100. The gastrocnemius muscles were harvested to measure the denervation atrophy. Local hindpaw burn injury induces apoptosis in VHMNs and Schwann cells in sciatic nerve, which causes corresponding gastrocnemius muscle denervation atrophy. In the second study, we used the same burn injury model which was established and assessed the treatment effect of ASCs. The ASCs were transplanted into the injured hind paw at 4 weeks after burn injury. The lumbar spinal cord, sciatic nerve, and hindpaw skin were processed for immunofluorescent staining at 4 weeks after transplantation, including terminal deoxynucleotidyl transferase (TUNEL) assay, caspase-3, caspase-9, CD90 and S100, and the gastrocnemius muscle was evaluated through the use of hematoxylin and eosin staining. Caspase-3 positive, caspase-9 positive and TUNEL-positive cells were significantly increased in the corresponding dermatome spinal cord VHMNs after burn injury. Moreover, the decrease of Schwann cells in sciatic nerve and the increase of denervation atrophy in gastrocnemius muscle were observed. ASCs transplantation significantly attenuated apoptotic death of VHMNs and the area of muscle denervation atrophy in the gastrocnemius muscle fibers. The conclusion of these two studies, we demonstrated that burn injury induced VHMN apoptosis and subsequently caused muscle atrophy, and revealed the potential neuroprotective of ASCs.