連續植入人類臍帶血細胞於熱中風大鼠以延長存活時間及下視丘細胞凋亡之探討

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Title:	連續植入人類臍帶血細胞於熱中風大鼠以延長存活時間及下視丘細胞凋亡之探討 Exploring Sequential Transplantation of Human Umbilical Cord Blood Cells Protect Against Hypothalamic Apoptosis and Extended Survival Time During Heatstroke in Rats
Authors:	賴怡欣
Contributors:	生物科技系周淑芬陳勝咸
Keywords:	人類臍帶血細胞連續植入熱中風細胞凋亡發炎細胞激素 Human umbilical cord blood cells Sequential transplantation Heatstroke Apoptosis Inflammation Cytokine
Date:	2013
Issue Date:	2014-03-10 20:41:02 (UTC+8)
Abstract:	背景：非常多的研究結果已經證明，hUCBCs提供了對於神經退化性疾病(如中風、顱腦損傷、脊髓損傷以及血液疾病)一個充滿希望的新治療方式。熱中風是由於過度的熱產生（> 40℃），造成多重器官功能衰竭（特別是，中樞神經系統）而危及生命。最近，我們的研究也顯示，熱中風後或先預處理人類臍帶血細胞（hUCBCs）的大鼠，可能透過減少循環性休克、腦部一氧化氮超載和缺血性腦損傷而得到改善。然而，減少細胞數的移植方式可以達到治療效果並減輕移植物抗宿主疾病（GVHD）。然而，目前在臨床上或實驗性熱中風，尚無人發表過連續性的臍帶血細胞治療。治療：麻醉大鼠，在熱中風發病後立即給予第一劑治療，以及在熱中風發病後的第12分鐘給予第二劑的治療。分組及給藥(皆為靜脈注射)如下：B）熱中風+生理食鹽水0.3毫升+生理食鹽水0.3毫升，(HS+S2)。 C）熱中風+ hUCBCs 5×105/ 0.3毫升+生理食鹽水0.3毫升，（HS+H+S）。 D）熱中風+ hUCBCs 5×105/ 0.3毫升+ hUCBCs 5×105/ 0.3毫升，（HS+H2）。 E）熱中風+ hUCBCs 1×106/ 0.3毫升，（HS+H1）。另一組大鼠在麻醉後，暴露於室溫（26 ℃），作為A）常溫控制組。將使用urethane麻醉過後的大鼠置於攝氏43 ℃的環境溫度誘導熱中風，同時全程監測生理參數變化。下視丘神經損傷和凋亡分別以蘇木精伊紅（H&E）染色和免疫組織化學（IHC）染色分別進行評估。並透過酵素連結免疫吸附法（ELISA）分析血清中炎症細胞因子（TNF-α和IL-10）的濃度。結果：當大鼠進行熱暴露後，兩劑生理食鹽水治療組（HS+S2），其存活時間是31±3分鐘。在HS+H+S, HS+H2和HS+H1，其生存時間分別為69±8，200±25和99±7分鐘。此結果顯示，與兩劑生理實驗水治療組相較，在熱中風後確實可改善存活時間。而在連續hUCBCs治療組則獲得更加顯著的生存期延長。相較於常溫控制組，兩劑生理食鹽水治療的熱中風大鼠有較低的平均動脈壓(MAP)、心臟速率(HR)，且也表現低血壓，激活炎症細胞因子和下視丘神經細胞損傷及凋亡。然而，hUCBCs治療可顯著改善熱中風所引起的上述所有反應。尤其是在連續hUCBCs治療組更是得到了顯著的改善效果。結論：我們的研究數據顯示，搶救熱中風大鼠，使用連續hUCBCs移植治療可能比單一次hUCBCs移植更能有效減少循環性休克、下視丘損傷和全身性發炎反應。 Background: Accumulating evidences have demonstrated that human umbi- lical cord blood cells (hUCBCs) provide a promising new therapeutic method against neurodegenerative diseases, such as stroke, traumatic brain injury, and spinal cord injury as well as blood disease. Heatstroke is a life-threatening di- sease defined as a hyperpyrexia (> 40�C) and multiple organ (in particular, the central nervous system [CNS]) dysfunction or failure. More recently, we have also demonstrated that post- or pretreatment by human umbilical cord blood cells may resuscitate heatstroke rats with by reducing circulatory shock, and cerebral nitric oxide overload and ischemic injury. However, less cells transplantation can achieve therapeutic effects and attenuate graft vs host disease (GVHD). Hence, sequential hUCBCs transplantation was considered and not reported in treating the clinical or experimental heatstroke. Intervention: Anesthetized rats, immediately after the onset of heatstroke and two treat were applied at onst of and 12 min after onset of heatstroke, were divided into four major groups and given the following: B) Heatstroke + Saline 0.3 ml, i.v. + Saline 0.3 ml, i.v. (HS+S2) ; C) Heatstroke+ hUCBCs 5�105/ 0.3 ml, i.v. + Saline 0.3 ml, i.v. (HS+H+S); D) Heatstroke+ hUCBCs 5�105/ 0.3 ml, i.v. + hUCBCs 5�105/ 0.3 ml, i.v. (HS+H2); E) Heatstroke+ hUCBCs 1�106/ 0.3 ml, i.v (HS+H1). Another group of rats, under urethane anesthesia, were exposed to room temperature (26 degrees C) and used as A) normothermic controls. Urethane-anesthetized animals were exposed to an ambient temperature of 43 degrees C to induce heatstroke. Their physiologic and biochemical parameters were continuously monitored. Neuronal damage and apoptosis of hypothalamus were evaluated by hematoxylin and eosin (H&E) stain and Immunohistochemical (IHC) staining respectively. Activated inflammation ( TNF-alpha and IL-10 levels in serum) was evaluated by ELISA. Measurements and Main Results: When the two saline-treated (S2) rats underwent heat exposure, their survival time values were found to be 31�3 mins. Resuscitation with intravenous H+S, H2 and H1, their survival time values were found to be 69�8 , 200�25 and 99�7 mins respectively signifycantly improved survival compared as the two saline treated group during heatstroke. And the sequential hUCBCs treating group get the more signifycantly prolonged survival. As compared with values for normothermic controls, the S2-treated heatstroke rats had lower mean arterial pressure (MAP), heart rate (HR). As compared with normothermic controls, all S2-treated heatstroke animals displayed hypotension, activated inflammation, and Hypothalamic damage and apoptosis. However, hUCBCs therapy significantly caused attenuation of all the above-mentioned heatstroke reactions. In addition, the H2-treated group got the more significantly improved during heatstroke. Conclusion: Our data indicate that sequential hUCBCs transplantation therapy may be more effective than only one hUCBCs transplantation to resuscitate victims who had a heatstroke by reducing circulatory shock , hypothalamic damage and systemic inflammation.
Relation:	電子全文公開日期：20151001，學年度：101，72頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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