本文關(guān)鍵詞： 嗅鞘細(xì)胞 PRGD 蛋白吸附 親水性 細(xì)胞親和性　出處：《武漢理工大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 近年來，利用嗅鞘細(xì)胞移植治療脊髓神經(jīng)損傷已成為最為引人注目的方法，但是單純用細(xì)胞移植來修復(fù)脊髓損傷，缺乏理想的載體，細(xì)胞存活時(shí)間短，且不能很好地遷移，從而影響了神經(jīng)修復(fù)和功能恢復(fù)。因此，為嗅鞘細(xì)胞選擇合適的支架材料是亟待解決的問題。這些合成材料需要具有生物相容性好、對(duì)嗅鞘細(xì)胞的黏附性強(qiáng)的特點(diǎn)，，同時(shí)細(xì)胞黏附在其表面以后能夠維持很好的活性并持續(xù)生長。 本研究比較了幾種分離與純化嗅鞘細(xì)胞的方法，優(yōu)選了改良的NASH法作為實(shí)驗(yàn)方法，從新生大鼠的嗅球提取嗅鞘細(xì)胞，并對(duì)其進(jìn)行了純化，采用GFAP、P~(75)以及S-100免疫組化染色法鑒定其特性及純度，結(jié)果顯示采用改良的NASH法提取和分離的嗅鞘細(xì)胞的數(shù)量最多，且純度最高，達(dá)80％以上，是一種經(jīng)濟(jì)而有效的方法，為本課題的研究提供了嗅鞘細(xì)胞。 本研究合成了聚乳酸(PLA)和聚(羥基乙酸-L-賴氨酸-乳酸)，簡稱PLGK，以及將PLGK接枝了RGD，合成了PRGD材料，選用1mg／mL的血清白蛋白(BSA)與三種材料作用，通過Bradford法，分別在不同的作用時(shí)間，用酶標(biāo)儀測定并計(jì)算三種材料表面的蛋白吸附量，結(jié)果顯示PLA在最初的2h，蛋白吸附量較PLGK和PRGD多，隨：著時(shí)間的延長，PRGD吸附蛋白的量有所增加，4小時(shí)后，高于其他兩者；將三種材料PLA、PLGK和PRGD分別與5％的BSA37℃震蕩孵育12h，取出干燥后，采用FT-IR測試三種材料表面，通過比較三種材料與BSA作用前后的光譜圖，分析材料對(duì)BSA的吸附情況，這一結(jié)果與蛋白吸附結(jié)果一致。 分別在三種材料PLA、PLGK和PRGD表面滴加一滴去離子水，在常溫下采用ZAP高溫顯微鏡觀測，并攝像，圖像處理系統(tǒng)測試水接觸角，比較三種材料的親／疏水性。通過測試水接觸角，三種材料的親水性由強(qiáng)到弱依次為PRGD＞PLGK＞PLA。 將純化后的嗅鞘細(xì)胞分別接種于PLA、PLGK和PRGD材料表面，共培養(yǎng)3天后，通過MTT檢測OD值來評(píng)價(jià)材料表面細(xì)胞活性；細(xì)胞計(jì)數(shù)法計(jì)算細(xì)胞粘附率；環(huán)境掃描電鏡以及S-100染色后熒光顯微鏡觀察細(xì)胞在材料表面的生長狀態(tài)，結(jié)果顯示，材料PRGD表面細(xì)胞數(shù)目多，活性好，細(xì)胞粘附率高，細(xì)胞生長良好。 綜上所述，材料PRGD的親水性強(qiáng)，蛋白吸附性能好，嗅鞘細(xì)胞在其表面黏附性高，細(xì)胞生長活性強(qiáng)，生長狀態(tài)良好；材料PRGD是一種細(xì)胞親和性強(qiáng)、生物相容性好的材料。
[Abstract]:In recent years, olfactory ensheathing cell transplantation has become the most attractive method for the treatment of spinal cord nerve injury, but only with cell transplantation to repair spinal cord injury, lack of ideal carrier, cell survival time is short. Therefore, the selection of appropriate scaffold materials for olfactory ensheathing cells is an urgent problem. These synthetic materials need to have good biocompatibility. The cell adhesion to olfactory ensheathing cells is strong, and cell adhesion on its surface can maintain good activity and continue to grow. In this study, several methods of separation and purification of olfactory ensheathing cells were compared. The improved NASH method was selected as the experimental method to extract and purify olfactory ensheathing cells from olfactory bulb of newborn rats. The results showed that the number of olfactory ensheathing cells extracted and isolated by modified NASH method was the highest. The highest purity of above 80% is an economical and effective method, which provides olfactory ensheathing cells for this study. Polylactic acid (PLA) and poly (glycolic acid-L-lysine-lactic acid) (PLGK) were synthesized, and PLGK was grafted onto PRGD. One mg / mL of serum albumin (BSA) was used to react with three kinds of materials. By Bradford method, the reaction time was different. The amount of protein adsorbed on the surface of the three materials was measured and calculated by enzyme labeling instrument. The results showed that the amount of protein adsorbed on the surface of PLA was more than that of PLGK and PRGD at the first 2 h, and with the prolongation of the time. The amount of PRGD adsorbed protein increased after 4 hours, and was higher than that of the other two. The three materials PLAGK and PRGD were incubated at 5% BSA37 鈩

本文編號(hào)：1475350