【摘要】： 目的：(1)探索以電子加速器合成具有溫度敏感特性的聚異丙基丙烯酰胺凝膠(PNIPAAm)及接枝有PNIPAAm水凝膠的培養(yǎng)皿的方法。通過(guò)適當(dāng)后續(xù)處理使該培養(yǎng)皿適合細(xì)胞的生長(zhǎng)及增殖；(2)探討角膜基質(zhì)等細(xì)胞在溫敏材料PNIPAAm水凝膠上的生長(zhǎng)條件及特性,以及利用PNIPAAm水凝膠獲得的細(xì)胞片；(3)體外培養(yǎng)兔口腔黏膜上皮細(xì)胞(OMECs),探索一種較好的體外增殖OMECs的方法。 方法：(1)配制0.5%的亞甲基丙烯酰胺(MBA)和55%異丙基丙烯酰胺(NIPAAm)的異丙醇溶液70ul,加入35mm聚苯乙烯(TCP)培養(yǎng)皿中,使用電子加速器給與250kgy輻射。傅立葉紅外、掃描電鏡、原子力顯微鏡等研究輻射合成的溫敏材料的物理及化學(xué)性質(zhì)。(2)輻射合成的接枝了PNIPAAm水凝膠的溫敏培養(yǎng)皿通過(guò)后續(xù)的去離子水的浸泡、環(huán)氧乙烷消毒處理后,接種角膜基質(zhì)等細(xì)胞,置于37℃,5%CO2孵箱培養(yǎng)。通過(guò)倒置相差顯微鏡,HE染色,掃描電鏡等研究細(xì)胞在溫敏材料上生長(zhǎng)的條件及特性,以及降低溫度后獲得的細(xì)胞片的形態(tài)學(xué)結(jié)構(gòu)。(3)采用角質(zhì)細(xì)胞無(wú)血清培養(yǎng)基(K-SFM)配制的DispaseⅡ消化分離培養(yǎng)兔OMECs細(xì)胞,接種于鈣離子濃度分別為0和0.09mmol／L的K-SFM培養(yǎng)基。通過(guò)倒置顯微鏡、掃描電鏡、免疫熒光方法觀察研究,探索一種較好的體外增殖OMECs方法。 結(jié)果：(1)配制0.5%MBA和55%NIPAAm單體的異丙醇溶液,使用電子加速器給與250kgy輻射可獲得溫敏培養(yǎng)皿。傅立葉紅外顯示輻射合成了PNIPAAm,觀察發(fā)現(xiàn)有溫敏特性。掃描電鏡顯示PNIPAAm凝膠呈現(xiàn)較多的微孔結(jié)構(gòu)。原子力顯微鏡顯示溫敏材料表面輕度粗糙。(2)經(jīng)過(guò)浸泡及消毒處理后,接種角膜基質(zhì)細(xì)胞培養(yǎng)。倒置顯微鏡及掃描電鏡觀察顯示,細(xì)胞在溫敏材料上生長(zhǎng)良好,七天后細(xì)胞長(zhǎng)成片,降低溫度,能獲得細(xì)胞片；HE染色顯示細(xì)胞片無(wú)任何載體。(3)通過(guò)使用K-SFM配制的DispaseⅡ,聯(lián)合胰蛋白酶消化OMECs,制成細(xì)胞懸液接種于無(wú)血清的K-SFM培養(yǎng)基中,OMECs生長(zhǎng)旺盛,呈鵝卵石狀,細(xì)胞之間連接緊密；OMECs廣譜角蛋白表達(dá)陽(yáng)性；通過(guò)CCK8增值實(shí)驗(yàn)顯示OMECs在無(wú)鈣和低濃度鈣離子(0.09mmol／L)存在的情況下,細(xì)胞增殖無(wú)統(tǒng)計(jì)學(xué)差別。 結(jié)論：(1)使用本實(shí)驗(yàn)提供的方法,可以合成具有溫敏特性的PNIPAAm水凝膠及接枝了PNIPAAm水凝膠的培養(yǎng)皿；(2)輻射合成的接枝了PNIPAAm水凝膠的溫敏培養(yǎng)皿提供了一種獲得無(wú)任何載體的細(xì)胞片的方法；(3)本實(shí)驗(yàn)探索了一種改良的OMECs培養(yǎng)方法,使用本方法能夠在體外快捷的培養(yǎng)出單純的,能多次傳代的OMECs。
[Abstract]:Aim: (1) to explore the synthesis of temperature sensitive poly (isopropylacrylamide) gel (PNIPAAm) by electron accelerator and the grafting of PNIPAAm hydrogel into petri dish. The culture dish was suitable for cell growth and proliferation by proper follow-up treatment. (2) to investigate the growth conditions and characteristics of corneal stroma and other cells on PNIPAAm hydrogel, as well as the cell slices obtained by PNIPAAm hydrogel. (3) in vitro culture of rabbit oral mucosal epithelial cells (OMECs),) to explore a better method for proliferation of OMECs in vitro. Methods: (1) 0.5% methylene acrylamide (MBA) and 55% isopropyl acrylamide (NIPAAm) isopropanol solution 70 uls were prepared and added to 35mm polystyrene (TCP) dish. 250kgy radiation was given by electron accelerator. Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to study the physical and chemical properties of thermosensitive materials synthesized by radiation. (2) the thermosensitive petri dish grafted with PNIPAAm hydrogel was immersed in subsequent deionized water. After disinfection with ethylene oxide, corneal stromal cells were inoculated and cultured in 5%CO2 incubator at 37 鈩，

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