本文關鍵詞： Myroilysin 神經支架 牙髓干細胞 神經分化　出處：《山東大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:觀察深海細菌酶Myroilysin水解法制備的脫細胞神經支架對牙髓干細胞增殖及體外神經分化的影響。材料和方法:1.使用酶消法從年輕第三磨牙牙髓組織中提取人牙髓干細胞,經有限稀釋法純化擴增培養(yǎng),作為本實驗中與無細胞神經支架結合的種子細胞。2.分別運用Myroilysin水解法和傳統(tǒng)的Sondell化學法脫除大鼠坐骨神經內的原生細胞制備無細胞支架。通過cck-8法測定兩種支架及其制備過程中的殘留成分對種子細胞增殖的影響。3.分別用免疫熒光染色和免疫印跡雜交實驗western blotting檢測兩種支架對種子細胞向神經方向分化的影響。結果:1.經分離、純化和擴增培養(yǎng),所獲得的人牙髓干細胞具有很強的增殖分裂活性,間充質干細胞標志性蛋白STRO-1呈陽性,神經微管蛋白β-Ⅲtubulin經免疫熒光染色法檢測呈陽性。2.較高濃度的Myroilysin或脫氧膽酸鈉(作為Sondell化學法的主要洗脫劑)都有使牙髓干細胞密度顯著降低的不利作用;而兩者在低濃度情況下,Myroilysin對種子細胞增殖活性的抑制明顯低于脫氧膽酸鈉。分別使用兩種不同支架的浸潤提取液培養(yǎng)牙髓干細胞時,早期Myroilysin酶支架組的細胞增殖活性高于Sondell化學支架組,后期兩者都可達到較高的細胞密度。3.牙髓干細胞體外神經誘導分為四組:單純神經誘導液組、神經誘導液+化學法神經支架組、神經誘導液+酶法神經支架組,無神經誘導液的普通培養(yǎng)基組作為陰性對照。免疫熒光染色顯示:三個誘導組的細胞干細胞標志性蛋白STRO-1染色從有到無,微管蛋白β-III tubulin和神經膠質細胞標志蛋白GFAP在誘導前后都有表達,神經元標志蛋白NeuN表達量明顯增高。免疫印跡雜交實驗顯示:GFAP在兩個支架組表達增高,NeuN只在Myroilysin支架加神經誘導液組表達明顯增高。結論:深海細菌酶Myroilysin的酶活性具有自限性和時效性,在酶水天然神經解制備脫細胞支架的過程中,酶效價呈時間依賴性降解。因此Myroilysin水解法制備的神經支架對種子細胞增殖活性的抑制作用很快消失,表現(xiàn)出良好的生物相容性。Myroilysin水解法制備的無細胞支架結構更為膨脹疏松,在更徹底的去除組織免疫原性的同時,保留了更多的原生結構,對種子細胞的漂移、附著、增殖、分化產生更好的地形誘導促進作用。
[Abstract]:Objective: to observe the effects of acellular neural scaffolds prepared by deep-sea bacterial enzyme Myroilysin hydrolysis on the proliferation and neural differentiation of dental pulp stem cells in vitro. 1. Human dental pulp stem cells were extracted from pulp tissue of young third molar by enzyme digestion. The culture was purified and amplified by limited dilution method. As seed cells combined with acellular nerve scaffolds in this experiment, the Myroilysin hydrolysis method and the traditional Sondell chemical method were used to remove the progenitor cells from the sciatic nerve of rats, respectively. Cell free stents. Effects of residual components of two scaffolds and their preparation on seed cell proliferation were determined by cck-8 method. Wester was detected by immunofluorescence staining and Western blot hybridization, respectively. N. Blotting was used to detect the effect of two scaffolds on the neural differentiation of seed cells. Purified and cultured, the obtained human dental pulp stem cells have strong proliferative and mitotic activity, mesenchymal stem cells iconic protein STRO-1 is positive. Tubulin 尾-鈪，

本文編號：1481844