臺灣博碩士論文加值系統

本研究使用人腦血管周細胞與人類星形細胞依不同比例與人腦微血管內皮細胞共培養，並利用周細胞條件化培養基及星形細胞條件化培養基共培養建立出生體外人類血腦屏障(blood-brain barrier, BBB)模型。研究發現當人腦血管周細胞與人類星形細胞比例為1: 2，並利用周細胞與星形細胞條件化培養基比例為1: 1與人腦微血管內皮細胞共培養7天後transendothelial electrical resistance (TEER)值提高為319±16.67 Ω×cm2、propidium iodide穿透率下降為單層培養之39 %，且P-醣蛋白活性與人腦血管周細胞與人類星形細胞比例為1: 1及1: 6的培養模型相較之下提高84%與104%，使得體外模型更近似生體內血腦屏障，本研究透過不同人腦血管周細胞與人類星形細胞比例(1: 1, 1: 2, 1: 6)與人腦微血管內皮細胞建立的血腦屏障模型，檢測TEER值與propidium iodide穿透率測定BBB完整性及屏障功能，並利用鈣黃綠素螢光染劑滯留率測定抗藥性蛋白P-醣蛋白活性，再者配合檢測影響BBB完整性之細胞生理因子transforming growth factor beta 1 (TGF-β1)、matrix metalloproteinase-9 (MMP-9)、vascular endothelial growth factor (VEGF)濃度，實驗結果顯示與其他模型相比，當人腦血管周細胞與人類星形細胞比例為1: 2以及周細胞條件化培養基與星形細胞條件化培養基與人腦微血管內皮細胞建立之血腦屏障模型有較高的TGF-β1濃度以及較低的MMP-9、VEGF濃度，顯示建立符合生體覆蓋比例之血腦屏障模型更具代表性與研究利用性，並利用TGF-β1 receptor抑制劑SB431542、VEGF抑制劑asterric acid、MMP-9抑制劑CTT與鈣黃綠素螢光染劑滯留率，探討血腦屏障的多重抗藥性蛋白P-醣蛋白的活性與各生理因子間的交互作用，發現TGF-β1會正向調控P-醣蛋白活性，而VEGF會抑制P-醣蛋白活性。

關鍵字：血腦屏障、人腦微血管內皮細胞、人腦血管周細胞、人類星形細胞、P-醣蛋白、TGF-β1、VEGF、MMP-9

We constructed in vitro human blood-brain barrier (BBB) model using pericyte-condition medium (PCM) and astrocyte-condition medium and different ratio between human brain vascular pericytes (HBVPs) and human astrocytes (HAs) (1: 1, 1: 2, 1: 6) co-culture with human brain microvascular endothelial cells (HBMECs). After 7 days of co-culture of HBMECs, HBVPs, HAs (HBVPs:HAs=1: 2), PCM2, ACM2 (PCM: ACM= 1: 1), the transendothelial electrical resistance (TEER) increased to 319±16.67 Ω×cm2, reduced permeability of propidium iodide (PI) about 39 %. The activity of P-glycoprotein (P-gp) of the model with HBVPs: HAs= 1: 2 is higher than the model with HBVPs: HAs= 1: 1 (1.84-fold) and the model with HBVPs: HAs= 1: 6 (2.04-fold). We showed the co-culture models barrier integrity among three different ratio between HBVPs and HAs (1: 1, 1: 2, 1: 6) by TEER and propidium iodide permeability measurements and P-gp activity. The analysis of the three major barrier integrity modulators transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) displayed higher TGF-β1 activity and lower levels VEGF, MMP-9 in the coculture with HBVPs: HAs= 1: 2. It suggest constructed a in vitro model match the in vivo coverage ratio of HBVPs on HBMECs is more representative. Finally, we used the TGF-β1 inhibitor SB431542, VEGF inhibitor asterric acid, and MMP-9 inhibitor CTT to discuss the relationship between barrier integrity modulators and the P-gp activity. In our study we find that TGF-β1 can up-regular P-gp activity and VEGF can down-regular P-gp activity.

Keyword: blood-brain barrier, human brain microvascular endothelial cells, human astrocytes, P-glycoprotein, transforming growth factor-β1, ascular endothelial growth factor, matrix metalloproteinases.

摘要................................................................................................................................I
Abstract………………………………………………………………………………III
目錄…………………………………………………………………………………...V
圖目錄………………………………………………………………………………..XI
表目錄………………………………………………………………………………XII
第一章 研究動機與目的……………………………………………………………1
第二章 文獻回顧……………………………………………………………………3
2.1 血腦屏障(blood-brain barrier, BBB)…………………………………………3
2.1.1 The Tight junctions of blood-brain barrier…………………………….4
2.1.2 神經血管單元(neurovascular unit, NVU)……………………………5
2.1.3 腦微血管內皮細胞……………………………………………………5
2.1.4 星形細胞………………………………………………………………6
2.1.5 腦血管周細胞…………………………………………………………8
2.2 生體外BBB模型(in vitro BBB model)………………………….…..……….9
2.2.1 初代培養(primary cultures)……………………………….…………10
2.2.2 共培養模型(co-culture model)………………………………………11
2.3 ATP binding cassette (ABC) transporters…………………………………12
2.3.1 P-gp作用機制………………………………………………………13
2.4 調節BBB完整性的細胞分泌因子(secreted factor)………………………13
2.4.1 TGF-β1作用機制和抑制劑……………………………………….14
2.4.2 VEGF作用機制和抑制劑…………………………………………15
2.4.3 MMP-9作用機制和抑制劑………………………………………..16
第三章 實驗材料、儀器、原理及方法…………………………………………..17
3.1 實驗材料…………………………………………………………………...17
3.1.1 人腦微血管內皮細胞培養於transwell PET 膜表面預處理材料..17
3.1.2 人類腦血管周細胞培養於transwell PET 膜表面預處理材料….17
3.1.3 人類星形細胞培養於transwell PET 膜表面預處理材料………..18
3.1.4 人類腦微血管內皮細胞繼代培養…………………………………19
3.1.5 人類腦血管周細胞繼代培養材料…………………………………20
3.1.6 人類星形細胞繼代培養材料………………………………………21
3.1.7 人腦微血管內皮細胞與人腦血管周細胞、人類星形細胞觀察、記數與存活率計算材料………………………………………………21
3.1.8 人腦微血管內皮細胞與人腦血管周細胞、人類星形細胞保存材料……………………………………………………………………22
3.1.9 免疫螢光法材料……………………………………………………22
3.1.10 酵素連結免疫吸附法(ELIZA, enzyme-linked immunosorbent assay)材料…………………………………………………………………24
3.1.11 Calcein-AM 滯留率分析: P-gp功能鑑定材料…………………..24
3.1.12 電子顯微鏡材料……………………………………………………25
3.1.13 其他實驗器具和耗材………………………………………………26
3.2 實驗儀器…………………………………………………………………...28
3.3 實驗原理及方法…………………………………………………………...31
3.3.1 Fibronectin溶液配製………………………………………………31
3.3.2 冷凍液配製…………………………………………………………31
3.3.3 Gelatin溶液配製……………………………………………………31
3.3.4 細胞繼代培養預處理………………………………………………31
3.3.5 Transwell 培養皿上PET膜的表面預處理......................................31
3.3.6 人腦微血管內皮細胞和人腦血管周細胞、人類星形細胞培養基配製………………………………………………………………….32
3.3.7 人類腦微血管內皮細胞和人腦血管周細胞、人類星形細胞繼代培養........................................................................................................32
3.3.8 周細胞條件化培養基(pericyte-conditioned medium, PCM)............34
3.3.9 星形細胞條件化培養基(astrocyte-conditioned medium, ACM)......34
3.3.10 人類腦微血管內皮細胞和人類星形細胞共培養............................35
3.3.10.1 以星形細胞與人腦微血管內皮細胞共培養......................35
3.3.10.2 以周細胞條件化培養基和星形細胞條件化培養基、人類星 形細胞、人腦微血管內皮細胞共培養................................35
3.3.11 人類腦微血管內皮細胞和人腦血管周細胞、人類星形細胞共培養.......................................................................................................36
3.3.11.1 以人腦血管周細胞和、人類星形細胞和人腦微血管內皮細胞共培養..............................................................................36
3.3.11.2 以周細胞條件化培養基和星形細胞條件化培養基、人腦血管周細胞、人類星形細胞、人腦微血管內皮細胞共培養..........................................................................................37
3.3.12 酵素連結免疫吸附法(ELIZA, enzyme-linked immunosorbent assay)……………………..…………………………………………38
3.3.12.1 Human MMP-9 assay...........................................................38
3.3.12.2 Human VEGF assay.............................................................39
3.3.12.3 Human TGF-β1 assay……………………………………..40
3.3.13 Calcein-AM 滯留率分析: P-gp功能鑑定........................................41
3.3.13.1 蛋白質定量(BCA protein assay)檢量線.............................41
3.3.13.2 細胞蛋白質萃取溶液(ewtraction buffer)配製....................41
3.3.13.3 P-gp功能鑑定......................................................................42
3.3.14 免疫螢光法…………………………………………………………42
3.3.14.1 von Willebrand factor VIII 免疫螢光法.............................42
3.3.14.2 Glial fibrillary acidic protein (GFAP)免疫螢光法……….43
3.3.14.3 α-smooth muscle actin免疫螢光法…………………….44
3.3.14.4 ZO-1免疫螢光法………………………………………45
3.3.15 人腦微血管內皮細胞的電阻測定………………………………46
3.3.15.1 電極前處理……………………………………………..46
3.3.15.2 細胞的電阻測定……………………………………….46
3.3.16 體外BBB模型的穿透實驗…………………………………….47
3.3.16.1 Propidium iodide穿透實驗……………………………47
3.3.16.2 Propidium iodide檢量線……………………………….48
3.3.16.3 物質穿透體外BBB模型的穿透率計算………………48
第四章 結果與討論...............................................................................................49
　　4.1 周細胞條件化培養基和星形細胞條件化培養基、人類星形細胞、人腦血管周細胞與人腦微血管內皮細胞共培養對於TEER和propidium iodide穿透率的影響……………………………………………………49
4.1.1周細胞條件化培養基和星形細胞條件化培養基與HBMECs對於TEER和propidium iodide穿透率的影響…………………………49
4.1.2 周細胞條件化培養基和星形細胞條件化培養基、人類星形細胞與HBMECs對於TEER和propidium iodide穿透率的影響……….50
4.1.3 不同比例之HBVPs與HAs以及利用周細胞和星形細胞條件化培養基與HBMECs共培養對於TEER和穿透率的影響………………51
4.2 HBMECs與HBVPs、HAs繼代培養與共培養情形…………………..54
4.3 Calcein-AM滯留率分析: P-gp功能鑑定……………………………..56
4.4 細胞分泌因子TGF-β1、VEGF、MMP-9的抑制劑對於HBMECs的TEER以及propidium iodide穿透率的影響.............................................58
4.5 細胞分泌因子TGF-β1、VEGF、MMP-9的抑制劑對於calcein-AM在HBMECs滯留率的影響………………………………………………60
第五章 結論與建議……………………………………………………………......62
參考文獻……………………………………………………………………………..64

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