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系統識別號 U0026-0908201312330400
論文名稱(中文) 年齡與環孢靈素神經毒性於兒科病人之相關性
論文名稱(英文) Age is associated with cyclosporine-related neurological complications in children
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 陳俐文
研究生(英文) Li-Wen Chen
學號 S97001027
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-黃朝慶
召集委員-林秋烽
口試委員-王新台
口試委員-劉秉彥
中文關鍵字 環孢靈素  神經毒性  年齡  孩童  人類臍帶靜脈內皮細胞 
英文關鍵字 cyclosporine  neurotoxicity  age-dependent vulnerability  children  HUVECs 
學科別分類
中文摘要 環孢靈素為一有效的免疫抑制劑,廣泛運用於器官移植及許多免疫調節相關的疾病。環孢靈素目前已知會引起嚴重的神經毒性,其症狀包括意識變化、抽搐、皮質性眼盲、幻覺、妄想、失語、共濟失調、類中風症狀、及可逆性後腦病變症候群。在許多神經疾病都發現到年齡與疾病發生的機率和嚴重度有關,就環孢靈素神經毒性而言,也觀察到小孩似乎比成人還嚴重。但是在兒科病人,廣泛的年齡分布於0到18歲的族群中,年齡是否與環孢靈素神經毒性的發生及嚴重度相關,仍未被完整的探討。因此我們假設在兒科病人中,開始使用環孢靈素時年紀較小的病人群,跟年紀較大的病人相比,較易發生神經毒性,且有較嚴重的急性表現,較差的神經及發展預後,以及較顯著的神經影像異常。
由於環孢靈素會造成血管內皮細胞功能異常,其神經毒性推測應與腦部血管內皮細胞損傷有關,而人體細胞之中又以臍帶靜脈內皮細胞與腦部微血管內皮細胞最為相近,因此以人類臍帶靜脈內皮細胞建立體外研究模式,探討環孢靈素對內皮細胞的毒性與細胞年齡及老化程度的關係。我們假設環孢靈素對較年輕的內皮細胞,相對於較年老的細胞,有更顯著的細胞死亡以及內皮細胞功能異常。
臨床部分方法: 回溯國立成功大學附設醫院於中華民國77年7月至100年10月當中,使用環孢靈素且年齡在≦18歲之間的兒科病人,共183人,檢視其病歷。除了失去追蹤的4位病人之外,排除使用環孢靈素之前即存在神經疾病者以及本身疾病即會引起神經併發症者,共33人。記錄收案之146位病患的基本資料,實驗室檢查數據,以及有無發生神經毒性。以t-test分析數字變項,以費雪精確性檢定分析類別變項,以單變項及多變項精確回歸分析檢視各個變項的影響。
實驗部分方法: 第4代與第8代人類臍帶靜脈內皮細胞加入環孢靈素培養24小時, 48小時及72小時之後比較,分析乳酸脫氫酶,以Hoechst染色計算細胞死亡數量,以Annexin V及Propidium Iodide染色經由流式細胞儀檢視細胞死亡,以WST-8進行細胞增殖分析,並檢視環孢靈素對第4代與第8代細胞通透性的影響。
臨床部分結果: 在146人之中,有12人(8.2%)發生環孢靈素神經毒性。使用年齡以及使用環孢靈素之後發生高血壓,是有意義的危險因子。把所有≦18歲的病人以3歲為級距,計算不同年齡層的神經毒性發生率,發現小於6歲較易發生神經毒性。接下來針對發生環孢靈素神經毒性的病人,比較年紀較輕與年紀較大的兩群,發現年紀小於6歲者,急性表現較易發生癲癇重積症及叢發性抽搐,需較多抗抽搐藥物治療,長期預後較易發生癲癇及神經行為後遺症,於慢性期追蹤的神經影像也發現持續性頂葉萎縮。
實驗部分結果: 在環孢靈素濃度20 μM之下,經48小時後,第4代人類臍帶靜脈內皮細胞的乳酸脫氫酶相較於第8代細胞有顯著上升,以Hoechst染色計算細胞死亡數量,第4代相較於第8代有較顯著的細胞死亡,且流式細胞儀分析顯示第四代細胞呈現Annexin V及Propidium Iodide陽性之死亡細胞的比例,較第八代細胞高。而第4代細胞增殖情形,在24小時及48小時,顯著較第8代減低。以內皮細胞通透性而言,第4代細胞加入環孢靈素20 μM後的5分鐘及15分鐘,細胞通透性顯著較第8代上升。
透過本研究,我們發現年齡小於6歲以及使用環孢靈素之後發生高血壓,是環孢靈素神經毒性的危險因子。年齡小於6歲之兒童較易發生環孢靈素神經毒性,於急性期有較嚴重的神經表現,慢性期追蹤易有神經精神併發症,並於追蹤影像檢查呈現持續性頂葉萎縮。在人類臍帶靜脈內皮細胞體外模式,發現較年輕的第4代細胞加入特定濃度環孢靈素,比起第8代細胞,更易產生細胞死亡,減少細胞增殖能力,並增加內皮細胞通透性。總結,環孢靈素神經毒性的發生及嚴重度,在兒童族群中,與年紀相關;而環孢靈素對內皮細胞的毒性,在細胞死亡及屏壁功能異常方面,與細胞的代數相關。
英文摘要 Cyclosporine (CsA) is an immuno-suppressant known for its neurotoxicity. CsA-related severe neurotoxicity manifests with decreased responsiveness, seizures, hallucinations, delusions, corti¬cal blindness, aphasia, ataxia, stroke-like episodes, and posterior reversible encephalopathy syndrome (PRES). Neurologic complications of CsA seem to be more severe in children than in adults. However, whether there is age-dependent neurologic vulnerability of CsA in the pediatric population remains unknown. This study hypothesizes that younger children are more vulnerable to CsA-associated encephalopathy than older ones.
CsA would lead to endothelial dysfunction, which is supposed to cause neurotoxicity. The clinical phenomenom of age-dependency was examined by human umbilical vein endothelial cells (HUVECs) in vitro model because HUVECs were most similar to human brain microvasculature endothelial cells among human endothelium.
Clinical part methods. Pediatric patients (age <18 years) who received CsA in National Chen Kung University Hospital between 1988 and 2011 were retrospectively reviewed for CsA-related neurologic complications. Of the 183 children who received CsA during the study period, 4 were lost to follow-up and 33 were excluded because of pre-existing brain malformations or underlying diseases leading to cental nervous system complications. Thus, 146 children were enrolled for evaluation of CsA-related neurologic complications. The demographic factors, laboratory data, and blood pressures between the neurotoxicity and non-neurotoxicity groups were compared using the t-test for numerical variables and by the Fisher’s exact test for categorical variables. Univariate and multivariate analyses of exact logistic regression were conducted to examine the independent effects of each variable on the occurrence of CsA neurotoxicity.
Experimental part methods. Passage 4 (HUVEC-P4) and passage 8 (HUVEC-P8) cells were exposed to CsA for 24, 48, and 72 hours, then analyzed for cell proliferation with WST-8 assay, cell death with lactate dehydrogenase detection, Hoechst staining, annexin V/propidium iodide (PI) staining, and endothelial permeability with sodium fluorescein permeability test.
Clinical part results. Twelve of the enrolled 146 patients (8.2%) developed CsA-induced encephalopathy. Compared to the non-neurotoxicity group, the neurotoxicity group was significantly younger upon starting CsA treatment. Regression analysis showed that age <6 years and hypertension after CsA application were significantly associated with CsA encephalopathy. Younger children were prone to have more severe seizures requiring multiple anti-epileptic drugs in the acute stage and worse neuro-behavioral outcome than older children. Follow-up neuroimaging showed parietal cerebral atrophy in all examined children <6 years of age.
Experimental part results. Compared to the HUVEC-P8, the HUVEC-P4 showed more cytotoxicity with evidences of higher levels of lactate dehydrogenase, higher percentages of cell death by Hoechst staining, and more positive Annexin V/PI-stained cells by flow cytometry. HUVEC-P4 also showed decreased proliferation after 24- and 48-hour treatment of 20 μM CsA. The HUVEC-P4 also had significant increases of cell permeability after 5 minutes and 15 minutes of CsA treatment compared to the HUVEC-P8.
In conclusion, our clinical study showed that among the children receiving CsA treatment, the children younger than 6 years old had a higher risk of developing CsA encephalopathy, more severe acute presentations, and worse long-term neurologic outcomes than the children older than 6 years. The experimental study using HUVECs showed that the passage 4 cells had increases of cytotoxicity and permeability, and decreases of proliferation under 20 μM CsA compared to the passage 8. These findings suggested a developmental vulnerability of CsA neurotoxicity in the pediatric population, and passage-dependent CsA susceptibility in HUVECs.
論文目次 中文摘要 1
Abstract 4
Acknowledgement 7
Contents 8
Table Contents 9
Figure Contents 10
Abbreviation Index 11
Introduction 12
Materials and Methods 17
Results 24
Discussion 30
Conclusion 37
References 38
Tables 42
Figures 49
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