本文關(guān)鍵詞： Myroilysin 神經(jīng)支架 牙髓干細(xì)胞 神經(jīng)分化　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察深海細(xì)菌酶Myroilysin水解法制備的脫細(xì)胞神經(jīng)支架對(duì)牙髓干細(xì)胞增殖及體外神經(jīng)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(xì)胞,經(jīng)有限稀釋法純化擴(kuò)增培養(yǎng),作為本實(shí)驗(yàn)中與無細(xì)胞神經(jīng)支架結(jié)合的種子細(xì)胞。2.分別運(yùn)用Myroilysin水解法和傳統(tǒng)的Sondell化學(xué)法脫除大鼠坐骨神經(jīng)內(nèi)的原生細(xì)胞制備無細(xì)胞支架。通過cck-8法測(cè)定兩種支架及其制備過程中的殘留成分對(duì)種子細(xì)胞增殖的影響。3.分別用免疫熒光染色和免疫印跡雜交實(shí)驗(yàn)western blotting檢測(cè)兩種支架對(duì)種子細(xì)胞向神經(jīng)方向分化的影響。結(jié)果:1.經(jīng)分離、純化和擴(kuò)增培養(yǎng),所獲得的人牙髓干細(xì)胞具有很強(qiáng)的增殖分裂活性,間充質(zhì)干細(xì)胞標(biāo)志性蛋白STRO-1呈陽性,神經(jīng)微管蛋白β-Ⅲtubulin經(jīng)免疫熒光染色法檢測(cè)呈陽性。2.較高濃度的Myroilysin或脫氧膽酸鈉(作為Sondell化學(xué)法的主要洗脫劑)都有使牙髓干細(xì)胞密度顯著降低的不利作用;而兩者在低濃度情況下,Myroilysin對(duì)種子細(xì)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(rùn)提取液培養(yǎng)牙髓干細(xì)胞時(shí),早期Myroilysin酶支架組的細(xì)胞增殖活性高于Sondell化學(xué)支架組,后期兩者都可達(dá)到較高的細(xì)胞密度。3.牙髓干細(xì)胞體外神經(jīng)誘導(dǎo)分為四組:單純神經(jīng)誘導(dǎo)液組、神經(jīng)誘導(dǎo)液+化學(xué)法神經(jīng)支架組、神經(jīng)誘導(dǎo)液+酶法神經(jīng)支架組,無神經(jīng)誘導(dǎo)液的普通培養(yǎng)基組作為陰性對(duì)照。免疫熒光染色顯示:三個(gè)誘導(dǎo)組的細(xì)胞干細(xì)胞標(biāo)志性蛋白STRO-1染色從有到無,微管蛋白β-III tubulin和神經(jīng)膠質(zhì)細(xì)胞標(biāo)志蛋白GFAP在誘導(dǎo)前后都有表達(dá),神經(jīng)元標(biāo)志蛋白NeuN表達(dá)量明顯增高。免疫印跡雜交實(shí)驗(yàn)顯示:GFAP在兩個(gè)支架組表達(dá)增高,NeuN只在Myroilysin支架加神經(jīng)誘導(dǎo)液組表達(dá)明顯增高。結(jié)論:深海細(xì)菌酶Myroilysin的酶活性具有自限性和時(shí)效性,在酶水天然神經(jīng)解制備脫細(xì)胞支架的過程中,酶效價(jià)呈時(shí)間依賴性降解。因此Myroilysin水解法制備的神經(jīng)支架對(duì)種子細(xì)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(xì)胞支架結(jié)構(gòu)更為膨脹疏松,在更徹底的去除組織免疫原性的同時(shí),保留了更多的原生結(jié)構(gòu),對(duì)種子細(xì)胞的漂移、附著、增殖、分化產(chǎn)生更好的地形誘導(dǎo)促進(jìn)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(hào)：1481844