【摘要】：微孔陣列(Microwell arrays)是利用微尺度技術(shù)(Microscale technologies)在不同材料(玻璃、硅、PDMS、PLGA等)表面進(jìn)行微米級(jí)加工并生成大量微孔的技術(shù),本實(shí)驗(yàn)擬應(yīng)用聚二甲基硅氧烷(polydimethylsiloxane, PDMS)為微孔支架材料制備微陣列細(xì)胞培養(yǎng)試驗(yàn)平臺(tái),對(duì)組織工程種子細(xì)胞——大鼠脂肪干細(xì)胞(Rat Adipose Stem Cells, RADSCs)在不同孔徑微孔中細(xì)胞增殖情況及形態(tài)學(xué)改變進(jìn)行研究,以期驗(yàn)證微孔陣列這一新興實(shí)驗(yàn)平臺(tái)在干細(xì)胞組織工程研究中的可行性。同時(shí),初步明確大鼠脂肪干細(xì)胞在微孔陣列三維培養(yǎng)平臺(tái)上的增殖及形態(tài)學(xué)特點(diǎn),為此平臺(tái)在干細(xì)胞及組織工程領(lǐng)域的應(yīng)用打下基礎(chǔ)。本實(shí)驗(yàn)采用聚二甲基硅氧烷(poly-dimethylsiloxane, PDMS)為微孔支架材料,在其表面加工生成40μm、60μm和80μm等不同孔徑微陣列,并將RADSCs單細(xì)胞接種其上。利用光學(xué)顯微鏡、掃描電鏡及激光掃描共聚焦顯微鏡等手段,分別對(duì)各組中細(xì)胞的貼附能力、增殖情況、形態(tài)改變進(jìn)行觀察及檢測(cè)。 結(jié)果：1、硅晶圓模板經(jīng)酸性雙氧水清洗后,再倒模后形成的PDMS微陣列支架基本符合設(shè)計(jì)要求。2、ADSCs直接種植在未經(jīng)預(yù)處理的支架上時(shí)細(xì)胞貼附能力明顯下降,而經(jīng)10%胎牛血清(Fetal Bovine Serum, FB S)培養(yǎng)基完全浸泡預(yù)處理后,細(xì)胞在PDMS表面的帖附及存活能力提高。3、體外培養(yǎng)120h時(shí),其他條件保持恒定,不同孔徑(40μm、60μm、80μm)的微孔內(nèi)細(xì)胞生長(zhǎng)總數(shù)具有顯著性差異。4、隨培養(yǎng)時(shí)間的延長(zhǎng)在40μm微孔內(nèi)生長(zhǎng)的ADSCs更傾向于鋪滿孔底,60μm和80μm微孔內(nèi)生長(zhǎng)的ADSCs更傾向于沿孔壁-孔底拐角延伸生長(zhǎng)。5、ADSCs在平面及微孔內(nèi)生長(zhǎng),伸展形成“偽足”樣及“橋”樣結(jié)構(gòu),其內(nèi)部絲狀肌動(dòng)蛋白(F-actin)排列方向與此結(jié)構(gòu)長(zhǎng)軸平行。 結(jié)論：利用微加工技術(shù)可以制作具有精細(xì)、統(tǒng)一表面結(jié)構(gòu)的微孔陣列。將大鼠脂肪干細(xì)胞(ADSCs)懸液以一定濃度種植于PDMS微孔陣列內(nèi),細(xì)胞可在微孔內(nèi)呈單個(gè)生長(zhǎng)。細(xì)胞于PDMS表面及微孔內(nèi)爬附生長(zhǎng)表現(xiàn)“偽足”樣及“橋”樣結(jié)構(gòu),其內(nèi)部F-actin纖維排列方向與此結(jié)構(gòu)長(zhǎng)軸平行。不同孔徑微孔對(duì)ADSCs增殖及形態(tài)均存在影響,其中40μm微孔較60μm和80μm微孔更適宜ADSCs生長(zhǎng)。
[Abstract]:Microwell arrayals are a technique of microscale processing on the surface of different materials (glass, silicon, PDMS, PLGA, etc.) and produce a large number of micro-pores. Polydimethylsiloxanes (polydimethylsiloxanes) are to be used in the experiment. The cell proliferation and morphological changes of rat adipose-derived stem cells (RADSCs) in different pore sizes were studied. In order to verify the feasibility of this new experimental platform in the research of stem cell tissue engineering. At the same time, the proliferation and the morphological characteristics of the adipose-derived stem cells of the rat on the three-dimensional culture platform of the micro-well array are clearly defined, and the foundation is laid for the application of the platform in the field of stem cell and tissue engineering. Polydimethylsiloxane (PDMS) was used as the material of the micro-porous scaffold, and different pore size microarrays such as 40. m u.m,60. m u.m and 80. m The adhesion, proliferation and morphological changes of the cells in each group were observed and tested by means of an optical microscope, a scanning electron microscope and a laser scanning confocal microscope. Results:1. After the silicon wafer template was cleaned by acid hydrogen peroxide, the PDMS micro-array stent formed after remolding was basically in accordance with the design requirements.2. The cell adhesion ability was significantly reduced when the ADSCs were directly planted on the unpretreated scaffold and 10% fetal bovine serum (Fetal Boven Ser.) The number of cells in the microwells with different pore size (40. mu.m,60. mu.m,80. mu.m) was significantly different from the total number of cells in the microwells with different pore sizes (40. mu.m,60. mu.m,80. mu.m). .4. The ADSCs grown in the 40 & mu; m micro-pores with the extension of the culture time tended to extend along the cell wall-bottom corner of the cell wall. The ADSCs tend to grow along the hole wall-hole bottom corner.5. The ADSCs grow in the plane and in the micro-pores to form the "pseudo-foot"-like sample and the "bridge"-like sample. The structure, the orientation of the filamentous actin (F-actin) in the structure and the long axis of the structure Parallel. Conclusion: Micro-machining technology can be used to make fine and uniform surface structure. The rat adipose-derived stem cell (ADSCs) suspension is planted in the PDMS microporous array at a certain concentration, and the cells can be in the micro-pores. The growth of the cells in the surface of the PDMS and the inside of the micro-pores shows the "pseudo-foot"-like structure and the "bridge"-like structure, and the inner F-actin fiber is arranged in the direction of the arrangement of the inner F-actin fibers. The micro-pores of 40. mu. m are more suitable for AD than 60. m u.m and 80. m
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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