Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3952
標題: 以光纖螢光感測定量細胞族群或組織階層的活動
Fiber-optical biosensing platform for quantifying cell population or tissue level activities
作者: 范書毓
Fan, Shu-Yu
關鍵字: 光纖感測平台
Fiber-optic sensor platform
活體細胞檢測
螢光活體校正
立體細胞分佈監測
In vivo cell assays
Fluorescent-based in vivo calibration
3-dimension cell distribution monitoring in vivo
出版社: 生醫工程研究所
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摘要: 本研究建立一套以光纖感測結合微量流體輸送系統於活體中監測細胞族群或組織階層生理標的物的活性。以光纖感測器進行活體細胞階層檢測常因組織中靠近感測端的細胞密度或分佈狀況不同,無法有效建立評估特定生理活性的基準閥值,這項問題無法以現行的校正法或生理模型模擬消除。 我們以兩種螢光物分別與細胞結合來解決這項立體空間分佈所引起的問題。第一種 (空間分佈指示) 螢光物對對照組和實驗組細胞有相同的反應,其訊號不會因細胞該特定生理活性高低而呈現的不同的結果,可被用來做為感測端周邊微環境的細胞密度或分佈狀況的參考基準。第二種 (生理活性表現指示) 螢光物將反應實驗組細胞中該特定生理活性的高低。每個細胞“單位個體”的特定生理活性強度將以第二項螢光訊號除以第一項螢光訊號的比值來修正感測端微環境的影響,呈現對照組和實驗組間的差異。 本研究中先以仿生立體細胞分佈系統中測定化療藥品cyclophosphamide (CPA) 所引起的人類乳癌細胞MCF-7細胞凋亡來顯示這項光纖感測平台於監測細胞族群或組織階層活性的可行性和潛能。在第一階段的開發中,25mM CPA可使平面 (2D) 細胞培養環境MCF-7細胞產生明顯的細胞凋亡情形(細胞存活實驗,MTT),增加定量細胞FM1-43總染色量 (螢光吸附實驗) 及流式細胞儀分析下平均個別細胞FM1-43親和量,但是對定量細胞吸附的非對稱naphthalene indocyanine衍生螢光物 (Cpd.B) 數量則無明顯影響 (螢光吸附實驗)。第二階段實驗中,於模仿活體立體 (3D) 空間分佈人類乳癌細胞MCF-7模式下,光纖感測平台以200μm內直徑光纖傳輸激發光及接收感測端周邊微環境螢光動態變化,經325μm內徑毛細管定點傳遞750nl 300μm Cpd.B 先偵測仿組織系統內細胞分佈狀態 (密度),再藉由毛細管定點傳遞螢光物750nl 160μm FM1-43經校正細胞分佈狀態後呈現癌細胞受化療藥物cyclophosphamide (CPA) 作用後的凋亡情形。當細胞密度超過107cells/ml,Cpd.B於立體 (3D) 細胞分佈狀態下,其動態螢光變化的峰值增加比率與細胞密度呈正相關,但與CPA誘發細胞凋亡的狀態無統計上的關聯性,證實可做為細胞於活體立體 (3D) 空間分佈狀態的校正指標,不受細胞凋亡程度的影響;而螢光物FM1-43,其動態螢光變化的峰值增加比率與細胞密度和細胞凋亡程度均呈正相關。在細胞密度於107-108cells/ml間,CPA引發細胞凋亡動態螢光變化峰值增加比率為控制組 (未經CPA作用) 峰值增加比率的2.1-3.3倍。當控制組與實驗組細胞先經Cpd.B偵測組織內細胞分佈狀態 (密度),再加入螢光物FM1-43呈現細胞凋亡情形,各細胞密度控制組FM1-43動態變化峰值增加比率除以Cpd.B動態變化峰值增加比率的比值維持於0.71-1.58之間,各細胞密度實驗組峰值比值增加比率則落在3.62-10.68之間,此比值可呈現不受細胞密度影響的細胞凋亡指標。此研究初步成果也可驗證此光纖感測器平台具有即時監控活體內細胞族群 (或組織階層活動) 的潛能。
A fiber-optical biosensing platform in coordination with localized fluidic delivery to perform topical cell-scale assay inside living tissue or organs in vivo. The deviation from the cell density and spatial configuration in the vicinity of sensor probe can cause error in threshold value determination, which is difficult for the conventional extrinsic catheter fiber-optic designs to predict or calibrate for in vivo applications. The strategy for correcting / calibrating the difference from the spatial issue is to stain the cells with two fluorescent agents. The 1st fluorophore (indicator) will has a known effect on all cells which is not affected by the conditions (“normal / control” or “treated” with desired physiological changes) of the cells; the signal from the indicator will be considered as “baseline” reflecting each independent measurement with specific density and configuration of the cells in the vicinity of sensor tip. The 2nd fluorophore (reporter) will exhibit level of physiological change on “treated” cells. The significance of the physiological changes on individual cell will be evaluated by the ratio of the two fluorescent signals (reporter / indictor) to report the normalized deviation between “control” and “treated” cell population. In this research, we applied the fiber-optic sensor platform in monitoring chemotherapeutic, cyclophosphamide (CPA), induced 3D-distributed MCF-7 cell (human breast carcinoma cell line) apoptosis for verifying the feasibility and capability of the system in monitoring cell population or tissue level activities in vivo. In the 1st stage of the development, 25mM CPA was found to enhance apoptosis of cancer cells in cell viability (MTT) assay under 2D cell culture, increase the adsorption amount of fluorophore FM 1-43 on apoptotic cells, but had no effect on the adsorption amount of indocyanine derivatives on apoptotic cells. In the 2nd stage of study, the fiber-optic sensing platform monitored the kinetics of fluorescence changes around the micro-environment of sensor tip with a 200μm i.d. optical fiber, conveying the excitation and returning emission, and a 325μm i.d. microcapillary initially delivering fluorophore, 750nl 300 μm naphthalene asymmetric indocyanine derivative (Cpd.B), in indicating the spatial distribution (density) of cells in the tissue-mimic system, following by delivering fluorophore, 750nl 160μM FM1-43, in demonstrating the apoptotic activity induced by CPA after calibrated with the previous Cpd.B reported cell distribution indicating signal. When cell density exceeded 107cells/ml, the increase percentage of peak values in dynamic fluorescent change pattern of Cpd.B were in proportion to cell densities, but not correlated statistically with CPA-induced apoptotic activities. However, the increase percentages of peak values in dynamic fluorescent change pattern of FM 1-43 were in proportion to both cell densities and CPA-induced apoptotic activities. In the cell density between 107-108cells/ml, the increase percentage of peak values in dynamic fluorescent change pattern from FM 1-43 interacted with CPA-induced apoptotic cells were 2.1-3.3 folds of peak values from FM 1-43 interacted with control cell. When sequentially interacted with Cpd.B and FM 1-43, the ratios from increase percentage of peak values of FM 1-43 divided by increase percentage of peak values of Cpd.B were around 0.71-1.58 in the control cells with different densities, while the ratios from CPA-induced apoptotic cells were around 3.62-10.68. The value of ratio could be applied as indication of apoptotic activity without interference of spatial distribution of cells. The preliminary result verified the feasibility and capability of the system in monitoring cell population or tissue level activities in vivo.
URI: http://hdl.handle.net/11455/3952
其他識別: U0005-0102201313392000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0102201313392000
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