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運用人類血小板及血小板微粒作為抗癌藥物傳遞系統之研究

為了解決sr400 final edition台的問題，作者吳玉雯 這樣論述：

Background:Human platelets (PLTs) and PLT-derived extracellular vesicles (PEVs) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrate

d to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLTs and PEVs targeted drug delivery systems (TDDS) should facilitate clinical developments if clinical-grade procedures can be developed.Materials and methods:PLT from therapeutic-grade PLT concentr

ate (PC; N > 50) were loaded with doxorubicin (DOX) and stored at -80 °C (PLT-DOX) with 6% dimethyl sulfoxide (frozen PLT-DOX). Surface markers and PLT functional activity of frozen PLT-DOX was confirmed by Western blot and thromboelastography (TEG), respectively. The morphology of fresh and frozen

PLT and PLT-DOX was observed by scanning electron microscopy (SEM). The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and frozen PLT-DOX triggered by various

pH and CM was determined by high performance liquid chromatography (HPLC). The cellular uptake of DOX from PLTs was observed by deconvolution microscopy. The cytotoxicities of PLT-DOX, frozen PLT-DOX, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay.We compa

red extrusion, 3 cycles of freeze and thaw (freeze-thaw), sonication, and incubation to produce PEVs from human cryopreserved PLTs. The morphology of PEVs measured by SEM. The size distribution and the amount of particles in isolated PEVs analyzed by dynamic light scattering (DLS) and nanoparticle t

racking analysis (NTA). In addition, PEVs subjected to extrusion, freeze-thaw and sonication were loaded with anti-cancer drug, DOX, by incubation for 24 h and purification with chromatography to remove unbound DOX (PEV-DOX). The encapsulation efficiency of DOX in PEVs measured by fluorospectrometry

. The surface markers and procoagulant functional activity of PEV-DOX was confirmed by Western blot and MP-PS activity assay, respectively. The cellular uptake of PEVs by three breast cancer cell lines including MCF7, MDA-MB-231 and MCF7/ADR measured by flow cytometry and ImageXpress Pico Automated

Cell Imaging System. The cytotoxicities of PEV-DOX, DOX and liposomal DOX on breast cancer cells were analyzed by CCK-8 assay.Results:15~36 × 106 molecules of DOX could be loaded in each PLT within 3 to 9 days after collection. The characterization and bioreactivity of frozen PLT-DOX were preserved,

as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEVs upon thrombin activation. The transfer of DOX from frozen PLTs to cancer

cells was achieved within 90 min, and stimulated by TF-EV and low pH. The frozen PLT-DOX formulation was 7~23-times more toxic to three cancer cells than liposomal DOX.Morphology of PEVs by SEM was spheroid. Approximate 496 PEVs/PLT and 493 PEVs/PLT could be generated by extrusion and sonication, c

ompared to 145 PEVs/PLT and 33 PEVs/PLT by freeze/thaw and incubation, respectively. The encapsulation efficiency of DOX into PEVs treated with freeze-thaw (11%) was higher than extrusion (11%) and sonication (13%) after incubation followed by purification by Sephadex G-25 chromatography measured by

fluorospectrometry. Western blot evidenced that DOX loading did not influence expression level of PEV membrane surface markers (CD41, CD42a, CD62P, CD9 and CD63). The population sizes and concentration of PEVs and PEV-DOXs by DLS and NTA was 120-150 nm and 1.2-6.2 x 1011 particles per mL, respectiv

ely. In addition, drug loading also did not increase the risk of procoagulant activity. PEVs uptake analyzed by flow-cytometry showed strong internalization by drug resistant breast cancer cell lines, MCF7/ADR, compared to MCF7 cells and MDA-MB-231 cells. Cytotoxicity data showed that higher anti-ca

ncer activity of PEV-DOX on MCF7/ADR cells than other two breast cancer cells.Conclusions:Frozen PLT-DOX and PEV-DOX can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of

PLT-based DDS.

斑尺蛾族（鱗翅目：尺蛾科、枝尺蛾亞科）的親緣關係重建與形態演化，特別關注潔尺蛾屬之系統分類與演化生態

為了解決sr400 final edition台的問題，作者魏嬗如 這樣論述：

本研究同時使用形態與分子證據試圖解析Genusa屬 (一個在許多形態與生態方面獨樹一格的屬) 在枝尺蛾亞科內的族級歸屬以及種級分類議題。此屬原隸屬於斑尺蛾族 (Hypochrosini)，並缺乏其姐妹群關係的假說。然近年的枝尺蛾亞科族級親緣關係研究顯示斑尺蛾族與Epionini、Scardamiini、Anagogini及Apeirini形成一個族級複合群 (簡稱EHSAA)，而特產非洲的Drepanogynini為整個複合群的姐妹群，故本研究須先檢驗這個複合群的單系性與內部關係，才能探索Genusa在其內部的位置，並進一步解析種級分類議題。本研究分為兩大部份：(1) 首先挑選5族28屬49

種尺蛾代表EHSAA，並以3屬6種的Drepanogynini成員作為外群進行比較形態學研究。挑選出9個形態特徵映像至演化樹上，以釐清EHSAA內部是否有形態上的分群，並針對EHSAA的雄性生殖器furca結構與其他枝尺蛾亞科各族diaphragma區域結構進行同源性的探討。接著使用來自枝尺蛾亞科28族115屬121種 (包含EHSAA的5族25屬44種資訊) 的8個分子標記 (CO1、EF-1a、Wingless、RpS5、CAD、MDH、GAPDH與IDH，共計4300bps)，使用最大然似分析與貝葉式分析重建系統發育樹以檢驗先前假說。並使用新建的演化假說推測EHSAA內各屬的分化年代與重

要的形態特徵分布、寄主植物利用以及地理分布格局；(2) 我使用來自7個國家的51隻Genusa樣本外加Celenna sp. cf. festivaria為外群重建其種間親緣關係並探討其分化年代與有效的鑑識特徵。研究結果顯示：(1) EHSAA為一單系群，然除單模之Apeirini外，其它族皆非單系群且不具備各別的鑑識特徵，因此我建議將EHSAA合併為Epionini；(2) 先前研究認為furca在Hypochrosini (或Anagogini) 與Ourapterygini (現與Ennomini合併) 為趨同演化，但我認為Ourapteryx與EHSAA所具備之結構並不相同，且並非趨同

演化的結果；(3) 先前研究認為雄性第三腹節無梳狀毛、雄生殖器具furca結構與vinculum延伸為可裝載coremata之loop為EHSAA各族之共同衍徵，但我認為除第三腹節無梳狀毛外，其它特徵應為EHSAA + Drepanogynini之共同衍徵；(4) EHSAA寄主植物利用共涉及裸子植物1科與被子植物32科，而寄主植物專一性在東方區較為強烈，此觀察吻合古典的氣候與寄主植物廣度關聯性預測；(5) 警戒性斑紋在EHSAA內部有多次獨立起源且多半集中於熱帶與亞熱帶地區；(5) EHSAA的起源可能為非洲與南歐，後再由東亞大陸與中南半島進入東南亞島嶼及澳洲；(6) 在Genusa的分類部

份，我確認此屬共有七種，內含G. semperi、G. malaysiana與G. austrovietnamica三個新種與一個疑問種G. destituta，而種間的鑑識特徵則多由雄性生殖器與幼生期形態提供。由於Genusa的姐妹群仍有疑義，且缺少許多關鍵地區的樣本，故目前尚無法確認此屬的地理起源，然其分化過程則與巽它陸塊的海平面升降與環境變遷有密切的關係。