缺血性腦中風為臨床上最常見的中風類別，其主要原因為腦血管栓塞所導致。目前最常用來治療腦中風的方式為使用血栓溶解藥物，但腦中風區域的神經細胞在中風後即開始壞死，且需要長期的修復時間，僅用血栓溶解藥物無法對於病患神經功能給予太大的改善。為了提升神經功能的新生及修復，細胞療法已被認為是具展望性的治療方式，但因細胞植入後缺乏有效的結構支持以及腦內發炎的環境限制了細胞療法的療效；先前研究已證實三維幹細胞球體可透過保存細胞間及細胞外基質的連結而提高其臨床應用潛力，並可有效治療下肢缺血的小鼠，但目前針對缺血性腦中風尚未有任何研究。在本篇研究中，我們建立了以間葉幹細胞與內皮細胞共培養的三維幹細胞球體系統，並與傳統二維培養幹細胞的療法進行比較，分析其治療效益。在體外實驗中，我們以免疫螢光染色證明三維幹細胞球體可藉由分泌更多的促神經生長、促存活及血管新生因子以達成神經保護的作用；除此之外，我們發現三維幹細胞球體的條件培養液可有效提升神經細胞的存活、分化及促進血管新生。在動物實驗中，我們在小鼠腦中風後將三維幹細胞球體施打到小鼠腦受損區域，並分析植入細胞的留存情形。實驗結果顯示，相較於二維培養的幹細胞，三維幹細胞球體可完整滯留於損傷區域。透過動物行為及腦受損區域染色，可觀察到小鼠的運動行為明顯獲得改善，且腦受損區域有明顯縮小。由組織切片免疫染色結果可知，三維幹細胞球體可有效抑制腦受損區域星狀膠質細胞及微膠細胞之活化，並能夠促進腦內源性神經幹細胞的聚集而加速神經再生，以及促進腦損傷區域周圍的血管新生。綜合以上結果，三維幹細胞球體可促進神經保護作用並促進神經再生及血管新生，期望未來可將三維幹細胞球體應用於缺血性腦中風的細胞療法，提升臨床治療效果。

Ischemic stroke is one of the most frequent causes of death and disability globally. The cause of ischemic stroke is due to the plaque rupture of atherosclerotic arteries which supply the brain. To date, no treatment is available that would grant functional recovery when administered in the post-ischemic phase. Therefore, the novel therapeutic strategies for enhancing the repair of neural structures and the recovery of functions are urgently warranted. In this work, we aim to employ a stem cell-based approach for treating ischemic stroke. Three-dimensional (3D) spheroids of human mesenchymal stem cells and human umbilical vein endothelial cells, which are capable of secreting neuroprotective agents and inducing therapeutic angiogenesis, are constructed using a methylcellulose hydrogel system. Our in vitro study reveals that the developed 3D stem cell spheroids can secrete several neurotrophic and pro-survival, pro-angiogenic factors, thus holding great neuroprotective and neovascularization potential. In animal study, a surgically established mouse model of ischemic stroke is employed for evaluating the therapeutic efficacy of the fabricated 3D stem cell spheroids. Our in vivo study indicates that transplantation of 3D stem cell spheroids into mice with ischemic stroke significantly promotes functional recovery, with the potential of reducing lesion area. Moreover, 3D stem cell spheroids may induce a conducive environment for neural repair through the downregulation of macrophage recruitment and astrocyte activity into peri-infarct tissue, facilitating neurogenesis and angiogenesis. These experimental data highlight the fabrication of 3D stem cell spheroids may represent a novel therapeutic approach of stem cell based therapy for ischemic stroke.

目錄
摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 XI
第一章、緒論 1
1-1、腦中風 1
1-2、腦中風成因與分類 1
1-3、缺血性腦中風病理進程及機制 2
1-3-1、急性期(acute phase) 2
1-3-2、亞急性期(sub-acute phase) 3
1-3-3、慢性期(chronic phase) 3
1-4、腦水腫 5
1-4-1、血管性腦水腫(vasogenic edema) 5
1-4-2、細胞毒性水腫(cellular edema) 5
1-5、現今通用治療方法 6
1-6、缺血性腦中風的細胞療法 6
1-6-1、預處理細胞(preconditioning) 9
1-6-2、基因工程(gene modification) 9
1-6-3、生醫材料共同移植系統(biomaterial co-administration) 9
1-6-4、細胞外囊泡(extracellular vesicle, EV) 10
1-7、細胞療法之細胞種類 10
1-7-1、神經幹細胞(neural stem cell, NSC) 10
1-7-2、間葉幹細胞(mesenchymal stem cell, MSC) 11
1-7-3、內皮細胞(endothelial cell, EC) 11
1-8、三維幹細胞球體於細胞療法的效用 12
1-8-1、甲殼素(chitosan) 13
1-8-2、甲基纖維素(methylcellulose) 13
1-9、研究動機與實驗目的 16
第二章、材料和方法 18
2-1、細胞培養 18
2-2、甲基纖維素水膠及三維幹細胞球體製備 19
2-3、細胞免疫螢光染色 20
2-3-1、二維幹細胞螢光染色 20
2-3-2、三維幹細胞球體螢光染色 20
2-4、條件培養液(Conditioned medium, CM)製備 21
2-5、細胞存活/死亡染色(Live/Dead Staining) 22
2-6、細胞存活率分析(CCK-8 assay) 22
2-7、傷口癒合試驗(Wound healing assay) 23
2-8、管狀生成試驗(Tube formation) 23
2-8-1、二維細胞及三維幹細胞球體的血管管狀生成實驗 23
2-8-2、內皮細胞的管狀生成實驗 24
2-9、酵素連結免疫吸附分析法(ELISA) 25
2-10、 小鼠腦中風動物模式建立(middle cerebral artery occlusion, MCAO) 26
2-11、組織處理 27
2-12、小鼠腦受損面積分析 28
2-12-1、結晶紫染色(cresyl violet staining) 28
2-12-2、MRI影像分析 28
2-13、小鼠動物行為分析(Cylinder test & Grid test) 29
2-14、組織免疫螢光染色 30
2-15、統計分析 31
第三章、實驗結果 32
3-1、三維幹細胞球體型態和特性 32
3-1-1、型態 32
3-1-2、細胞外基質(extracellular matrix)分布 32
3-1-3、營養因子(trophic factor)分布 33
3-1-3、細胞裂解後營養因子含量 35
為了更驗證在螢光染色觀察中三維幹細胞球體所含有的營養因子含量，我們在收集固定細胞量的二維培養幹細胞及三維幹細胞球體後，以ELISA分析二維幹細胞及三維幹細胞所含有的腦內源性神經營養因子(BDNF)及血管內皮生長因子(VEGF)的含量。 35
3-1-4、血管管狀生成能力 36
3-2、三維幹細胞球體的促神經細胞存活能力 38
3-3、三維幹細胞球體的促神經細胞突增生(neurite regrowth)能力 40
3-4、三維幹細胞球體的促血管新生能力 42
3-6、細胞植入留存能力分析(cell engraftment) 45
3-7、小鼠動物行為變化 46
3-8、小鼠腦中風受損區域分析 48
3-9、組織免疫螢光染色 51
3-9-1、發炎因子分布(GFAP&IBA-1) 51
3-9-2、神經再生(neurogenesis) 54
3-9-3、血管新生(angiogenesis) 56
第四章、討論 57
第五章、結論 60
參考文獻 61

圖目錄
圖一、大腦威力氏環(circle of willis)結構[5]。 2
圖二、缺血性腦中風之病理進程[7]。 3
圖三、缺血性腦中風所誘發的病理機制[5]。 4
圖四、缺血性腦中風的神經發炎(neuroinflammation)進程[23]。 4
圖五、血管性腦水腫和細胞毒性水腫[26]。 5
圖六、細胞療法的治療潛力[33]。 7
圖七、細胞植入過程中所造成的破壞示意圖[43]。 8
圖八、提升細胞療法的改善方式[46]。 8
圖九、透過將細胞預處理於低氧環境及經藥物作用影響的細胞存活機制[51]。 9
圖十、間葉幹細胞的治療潛力[65]。 11
圖十一、內皮細胞的治療潛力[69]。 12
圖十二、甲殼素之結構及三維幹細胞成球過程[78]。 13
圖十三、甲基纖維素成膠示意圖及分子結構[79]。 14
圖十四、甲基纖維素水膠於乾燥及水和狀態下的物理結構[79]。 14
圖十五、運用三維幹細胞球體治療下肢缺血的小鼠[84]。 15
圖十六、實驗架構圖。 17
圖十七、細胞球體系統形成及三維幹細胞製備示意圖。 19
圖十八、條件培養液製備及收集示意圖。 22
圖十九、傷口癒合實驗製備示意圖。 23
圖二十、二維幹細胞及三維幹細胞球體管狀生成製備示意圖。 24
圖二十一、內皮細胞管狀生成實驗示意圖。 25
圖二十二、(a)動物實驗分組；(b)動物實驗觀察流程。 27
圖二十三、Cresyl violet染色流程圖。 28
圖二十四、三維幹細胞球體型態與組成。 32
圖二十五、三維幹細胞球體之細胞外間質染色結果。 33
圖二十六、(a)三維幹細胞與二維幹細胞的血管生長及促存活因子表現圖； 34
圖二十七、二維幹細胞及三維幹細胞球體的BDNF、VEGF含量。 35
圖二十八、二維幹細胞與三維幹細胞球體於(a)正常培養環境；(b)OGD環境下管狀結構生成能力。 37
圖二十九、(a)以條件培養液培養SH-SY5Y細胞之細胞存活/死亡染色結果；(b)以條件培養液培養SH-SY5Y細胞之存活率定量分析。 39
圖三十、以條件培養液培養SH-SY5Y細胞之傷口癒合遷移之(a)定性；(b)定量結果。 40
圖三十一、量測二維幹細胞及三維幹細胞球體之條件培養液於正常環境及OGD環境下所含有的BDNF含量。 41
圖三十二、量測二維幹細胞及三維幹細胞球體之條件培養液於正常環境及OGD環境下所含有的IGF-1含量。 42
圖三十三、量測二維幹細胞及三維幹細胞球體之條件培養液於正常環境及OGD環境下所含有的VEGF含量。 43
圖三十四、以條件培養液培養HUVEC細胞的管狀生成能力(a)定性；(b)定量結果。 45
圖三十五、二維幹細胞與三維幹細胞球體於植入損傷區周圍後其1小時、2天、7天之結果。 46
圖三十六、小鼠動物行為變化：(a) cylinder test；(b) grid test。 47
圖三十七、以MRI分析小鼠腦損傷之(a)定性；(b)定量結果。 49
圖三十八、以cresyl violet分析小鼠腦損萎縮區域之(a)定性；(b)定量結果。 50
圖三十九、星狀細胞(astrocyte)於損傷區域周圍分布之(a)3天；(b)7天；(c)14天之結果。 52
圖四十、微小膠質細胞(microglia)於中風後3天分布於損傷區域周圍之(a)定性；(b)定量結果。 53
圖四十一、腦內源性神經幹細胞於非腦損傷半球之分布。 54
圖四十二、腦內源性神經前驅細胞於腦損傷周圍區域分布之(a)3天；(b)14天結果。 55
圖四十三、血管內皮細胞於中風後14天於腦損傷周圍區域分布之結果。 56
圖四十四、graphical abstract。 60

表目錄
表一、細胞染色一級抗體列表。 21
表二、細胞染色二級抗體列表。 21
表三、小鼠動作行為分析計算比較表。 29
表四、組織染色一級抗體列表。 30
表五、組織染色二級抗體列表。 30

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