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(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(tuán)（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（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 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 multiple doses, of ADM, MC and 5-FU was 0.4 g/mL, 3.0 g/mL and 10 g/mL, respectively . 分別作用24h, 48h, 72h,七、展望,胚胎干細(xì)胞的應(yīng)用研究及胚胎發(fā)育 致癌機(jī)制的研究,reference,1. Darling EM, Athanasiou KA. Retaining Zonal Chondrocyte Phenotype by Means of Novel Growth Environments. Tissue Eng. 2005 March/April;11(3-4):395-403. 2. Xie Y, Yang ST, Kniss DA. Three-dimensional cell-scaffold constructs promote efficient gene transfection: implications for cell-based gene therapy. Tissue Eng. 2001 Oct;7(5):585-98. 3. Bianco F, Basini G, Grasselli F. Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker.Domest Anim Endocrinol. 2005 Apr;28(3):308-19. Epub 2005 Jan 26. 4. Freyria AM, Cortial D, Ronziere MC, Guerret S, Herbage D. Influence of medium composition, static and stirred conditions on the proliferation of and matrix protein expression of bovine articular chondrocytes cultured in a 3-D collagen scaffold.Biomaterials. 2004 Feb;25(4):687-97. 5. 朱如里，高學(xué)純，王明麗等。 膠原海綿與兔軟骨細(xì)胞體外三維培養(yǎng)的實驗研究。中國骨傷 2002，15：7576 6. K. J. Dabos, L. J. Nelson, J. N. Plevris, M. M. Dollinger, C. Hewage, I. H. Sadler and P. C. Hayes. Microgravity environment in a rotary cell culture system (RCCS) promotes cell aggregation and maintains differentiation and proliferation of primary porcine hepatocytes. Journal of Hepatology, 1998, 28(Supplement 1) : 62. 7. H. Z. Kupchik, R. S. Langer, C. Haberern, S. El Deriny and M. OBrien. A new method for the three-dimensional in vitro growth of human cancer cells. Experimental Cell Research. (1983) 147: 454-460. 8. E. Curcio, L. De Bartolo, G. Barbieri, M. Rende, L. Giorno, S. Morelli and E. Drioli . Diffusive and convective transport through hollow fiber membranes for liver cell culture Journal of Biotechnology. (2005) 117: 309-321. 9. Kang X, Xie Y, Kniss DA. Adipose Tissue Model Using Three-Dimensional Cultivation of Preadipocytes Seeded onto Fibrous Polymer Scaffolds.Tissue Eng. 2005 Mar-Apr;11(3-4):458-68. 10. Wan-Ju Li, Richard Tuli, Xiaoxue Huang, Patrice Laquerriere and Rocky S. Tuan Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials, 2005,26(25) : 5158-5166.,11. Melvin Schindler, Ijaz Ahmed, Jabeen Kamal, Alam Nur-E-Kamal, Timothy H. Grafe, H. Young Chung and Sally Meiners. A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture. Biomaterials, 2005, 26(28) : 5624-5631. 12. Stuschke, M., V. Budach, G. Stuben, C. Streffer, and H. Sack. Heterogeneity in the fractionation sensitivities of human tumor cell lines: studies in a three-dimensional model system. Int. J. Radiat. Oncol. Biol. Phys. 1995(32): 395408. 13. Graham, C. H., H. Kobayashi, K. S. Stankiewicz, S. Man, S. J. Kapitain, and R. S. Kerbel. Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents. J. Natl. Cancer Inst. (1994)86: 975982. 14. Jaaskelainen, J., E. Lehtonen, P. Heikkila, P. Kalliomaki, and T. Timonen. Damage to multicellular human H-2 glioma spheroids incubated with LAK cells: an ultrastructural study. J. Natl. Cancer Inst. (1990) 82: 497501. 15. Lord, E. M., L. Harwell, and C. J. Koch. Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts. Cancer Res. (1993) 53: 57215726 16. Bracke, M. E., B. M. Vyncke, E. A. Bruyneel, S. J. Vermeulen, G. K. De Bruyne, N. A. Van Larebeke, K. Vleminckx, F. M. Van Roy, and M. M. Mareel. Insulin-like growth factor I activates the invasion suppressor function of Ecadherin in MCF-7 human mammary carcinoma cells in vitro. Br. J. Cancer (1993) 68: 282289. 17. Brady Kalnay, S. M., E. R. Boghaert, S. Zimmer, D. R. Soll, and R. Brackenbury. Invasion by WC5 rat cerebellar cells is independent of RSV-induced changes in growth and adhesion. Int. J. Cancer (49): 239245. 18. Kitsuki, H., M. Katano, T. Morisaki, and M. Torisu. CEAmediated homotypic aggregation of human colorectal carcinoma cells in a malignant effusion. Cancer Lett. (1995) 88: 713. 19. Abramovitch, R., G. Meir, and M. Neeman. Neovascularization induced growth of implanted C6 glioma multicellular spheroids: magnetic resonance microimaging. Cancer Res. (1995)55: 19561962. 20. Lin, K. H., S. Maeda, and T. Saito. Long-term maintenance of liver-specific functions in three-dimensional culture of adult rat hepatocytes with a porous gelatin sponge support. Biotechnol. Appl. Biochem. (1995) 21: 1927. 21. Shiraha, H., N. Koide, H. Hada, K. Ujike, M. Nakamura, T. Shinji, S. Gotoh, and T. Tsuji. Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids. Biotechnol. Bioeng. (1996) 50: 416421. 22. Nishikawa, Y., Y. Tokusashi, T. Kadohama, H. Nishimori, and K. Ogawa. Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix. Exp. Cell Res.(1996) 223: 357371. .,謝謝,1998 Blackwell Science Ltd. International Journal of Experimental Pathology 79,1-23,