組織細(xì)胞的三維培養(yǎng).ppt

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1、組織細(xì)胞的三維培養(yǎng),學(xué)生：張旭朗 導(dǎo)師：馬小軍 研究員 2005518,Seminar ,概念 三維培養(yǎng)的構(gòu)成 三維培養(yǎng)的特點 三維培養(yǎng)的方法 研究方法 三維培養(yǎng)的應(yīng)用 展望,一、三維培養(yǎng)的概念,相對于單層細(xì)胞的二維培養(yǎng)而言，利用各種方法及材料，使細(xì)胞呈空間立體方式生長，形成類似體內(nèi)組織的結(jié)構(gòu)，發(fā)揮其功能,二、三維培養(yǎng)的構(gòu)成,1、輔助物：人工軟骨1、PET聚乙烯酯2、纖維蛋白支架3、膠原支架4-5、旋轉(zhuǎn)生物反應(yīng)器6、微膠囊7、中空纖維8 2、細(xì)胞：原代組織細(xì)胞大鼠肝、軟骨、 脂肪9、成骨樣細(xì)胞、干細(xì)胞10、 腫瘤細(xì)胞11,三、三維培養(yǎng)的特點,1、生長特點：三維結(jié)構(gòu)、異質(zhì)性、生長速

2、率及氧氣、葡萄糖等梯度分布 2、組織學(xué)特點：細(xì)胞分三層（直徑300m） 可有類似組織基質(zhì)的物質(zhì)產(chǎn)生 3、生長動力學(xué)：三維培養(yǎng)細(xì)胞以G1/G0期細(xì)胞占優(yōu)勢，S期細(xì)胞較少,四、三維培養(yǎng)的方法,1、旋轉(zhuǎn)式培養(yǎng)：旋轉(zhuǎn)細(xì)胞培養(yǎng)系統(tǒng)或培養(yǎng)瓶，旋轉(zhuǎn)生物反應(yīng)器同時產(chǎn)生較多量直徑為12mm的細(xì)胞球 2、非黏附物包被的培養(yǎng)瓶或培養(yǎng)皿：瓊脂、瓊脂糖、蛋白聚糖、聚苯乙烯 3、支架,五、研究方法,1、細(xì)胞生長及形態(tài)：體積直徑 免疫組化（BrdU、PCNA、Ki-67） 流式細(xì)胞儀（DNA Hoechst33342、PI） 電鏡 2、細(xì)胞球的生理特性：微電極（pO2 or pH） NMR

3、(脂代謝) 生物體發(fā)光技術(shù)（ATP、Lactate、glucose）,六、三維培養(yǎng)的應(yīng)用,1、實驗性治療 放射效應(yīng)12：分化程度影響放療效果 藥物耐受13：培養(yǎng)方式是決定耐藥的因素之一 免疫療法14：腫瘤細(xì)胞與免疫細(xì)胞作用,2、代謝及代謝環(huán)境研究 三維培養(yǎng)的細(xì)胞可模擬腫瘤生成中的無血管階段、微轉(zhuǎn)移灶或毛細(xì)血管之間的微區(qū)域 模擬在體細(xì)胞的環(huán)境： 良好生長環(huán)境 缺氧、缺乏營養(yǎng)、廢物積聚或pH降低等協(xié)同作用“toxic enviroment” 15,,3、細(xì)胞的生物學(xué)特性 浸潤和轉(zhuǎn)移：腫瘤細(xì)胞和基質(zhì)共培養(yǎng)（E-cadherin、 ICAM-1 ）16,17 細(xì)胞黏附：研究細(xì)胞聚集過程中黏附

4、因子的作用18 血管形成： VEGF（血管內(nèi)皮生長因子）在惡性腫瘤血管形成中起主要作用19,4、實驗性組織模型 移植物以組織或器官的方式培養(yǎng)，使其在一定時間內(nèi)保持原有功能 簡單的講：使細(xì)胞自發(fā)或借助人工基質(zhì)聚集并誘導(dǎo)細(xì)胞分化、維持細(xì)胞功能 舉例：人工肝（肝細(xì)胞、纖維細(xì)胞等與明膠海綿、瓊脂、膠原共培養(yǎng)）20-22,5、胚體干細(xì)胞與擬胚體 胚胎發(fā)育到囊胚期，此時細(xì)胞分化為兩大類，即位于周邊的滋養(yǎng)層細(xì)胞和位于腔內(nèi)但居于一端的內(nèi)細(xì)胞團（inner cell mass ICM）。只有ICM將來發(fā)育成胚胎本身，把ICM從胚胎中分離出來在一定條件下培養(yǎng)，可形成具有無限增殖能力的細(xì)胞群，稱為胚胎干細(xì)胞（

5、embryonic stem cells,ES細(xì)胞） 當(dāng)ES細(xì)胞經(jīng)過2天懸滴培養(yǎng)、2天懸浮培養(yǎng)后，可形成簡單擬胚體（3D），擬胚體內(nèi)包含著來源于三個胚層的不同細(xì)胞,微囊化腫瘤細(xì)胞用于藥篩,1、抗腫瘤藥物篩選的研究現(xiàn)狀,多采用體外單層培養(yǎng)的腫瘤細(xì)胞：簡單、易操作、費用低、可大量應(yīng)用、生長方式及環(huán)境與在體腫瘤差距較大 動物實驗：費用高、生存條件苛刻、生長方式及環(huán)境與在體相似 手術(shù)切除標(biāo)本：回顧性研究、不能動態(tài)觀察 體外三維培養(yǎng)：模擬腫瘤生長方式及環(huán)境、可大量應(yīng)用、費用較低，如MCTS,腫瘤細(xì)胞在體與體外三維生長示意圖,,2、微囊化細(xì)胞三維共培養(yǎng)特點,微膠囊為半透膜 細(xì)胞呈三維立體生長 生長空間可控

6、 空間相對封閉 “被動”共培養(yǎng) 實質(zhì)與間質(zhì)細(xì)胞共培養(yǎng)-模擬體內(nèi),3、微囊化腫瘤細(xì)胞的三維培養(yǎng),材料：海藻酸鈉，聚賴氨酸，人乳腺癌 細(xì)胞系MCF-7 方法：靜電液滴法,Day 7,Day 0,Day 5,Day 2,4、藥物對三維生長細(xì)胞的抑制,藥物：絲裂霉素（MC）、阿霉素（ADM）、5氟尿嘧啶（5FU） 濃度和作用時間：0.1ppc,1ppc及10ppc Peak plasma concentration (ppc), the highest level of drug that can be obtained in the blood usually following multi

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