本文選題：皮膚干細(xì)胞 + Ngn2��； 參考：《南京醫(yī)科大學(xué)》2011年碩士論文

【摘要】：研究目的 探討皮膚源性誘導(dǎo)式多潛能干細(xì)胞(induced pluripotent stem iPS)的制備及體外定向神經(jīng)分化的機(jī)制。 研究方法 1.體外培養(yǎng)大鼠皮膚來源前體細(xì)胞(skin-derived precursors SKPs)并純化、鑒定。 2.構(gòu)建包裝含Ngn2基因質(zhì)粒的慢病毒載體并用其轉(zhuǎn)染第3代SKPs。 3.SKPs分三組:A組為慢病毒載體介導(dǎo)Neurogenin2(Ngn2)基因轉(zhuǎn)染的SKps,B組為空載體慢病毒轉(zhuǎn)染的SKps,C組為未經(jīng)處理的的SKPs。 4.誘導(dǎo)7天后,免疫熒光檢測(cè)各組細(xì)胞神經(jīng)分化效率的差異。 5.Western Blot檢測(cè)各組細(xì)胞神經(jīng)分化過程中Notch信號(hào)通路相關(guān)蛋白hes1和Dll1表達(dá)水平的差異。 研究結(jié)果 1.皮膚源性iPS可持續(xù)穩(wěn)定傳代并表達(dá)綠色熒光。 2.誘導(dǎo)培養(yǎng)基誘導(dǎo)7天后,免疫熒光結(jié)果顯示A組神經(jīng)元標(biāo)志蛋白陽性細(xì)胞率均明顯高于B組和C組,結(jié)果有均顯著統(tǒng)計(jì)學(xué)差異(P0.05)。 3.誘導(dǎo)7天后,A組細(xì)胞Dll1蛋白的表達(dá)水平明顯高于B組和C組,而Hes1蛋白的表達(dá)水平明顯顯低于B組和C組,結(jié)果均有顯著統(tǒng)計(jì)學(xué)差異(P0.01)。 研究結(jié)論 1.慢病毒介導(dǎo)Ngn2基因轉(zhuǎn)染SKPs,通過細(xì)胞程序重排技術(shù),可制備出皮膚源性iPS。 2.皮膚源性iPS向神經(jīng)細(xì)胞定向分化的效率明顯高于SKPs。 3.皮膚源性iPS高神經(jīng)分化的機(jī)制可能通過上調(diào)Dll1蛋白水平,下調(diào)hes1蛋白水平,抑制了Notch信號(hào)通路,從而促進(jìn)神經(jīng)分化。
[Abstract]:Research purpose To investigate the preparation of pluripotent stem iPS) induced by skin induced multipotential stem cells and the mechanism of directional neural differentiation in vitro. Research method 1. Rat skin derived progenitor cells (skin-derived precursors SKPs) were cultured in vitro and purified and identified. 2. The lentivirus vector containing Ngn2 gene plasmid was constructed and transfected into SKPs3. 3.SKPs was divided into three groups: group A: lentivirus vector mediated neurogenin2ngn2) gene transfection. Group B was transfected with empty vector lentivirus. Group C was untreated SKPs. 4. After 7 days of induction, the difference of neural differentiation efficiency was detected by immunofluorescence. 5.Western Blot was used to detect the expression of Notch signaling pathway related protein hes1 and Dll1 in the process of neural differentiation. Research results 1. Skin derived iPS can be continuously and stably passaged and expressed green fluorescence. 2. The results of immunofluorescence showed that the positive rate of neuronal marker protein in group A was significantly higher than that in group B and C, and there was significant difference between group A and group C (P 0.05). 3. After 7 days of induction, the expression of Dll1 protein in group A was significantly higher than that in group B and group C, while the expression level of Hes1 protein in group A was significantly lower than that in group B and group C (P 0.01). Research conclusion 1. Ngn2 gene was transfected into SKPsby lentivirus, and skin-derived iPSs could be prepared by cell program rearrangement. 2. The differentiation efficiency of skin derived iPS into neural cells was significantly higher than that of SKPs. 3. The mechanism of high neural differentiation in dermatogenic iPS may promote neural differentiation by upregulating the level of Dll1 protein and down-regulating the level of hes1 protein, thus inhibiting the Notch signaling pathway.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【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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本文編號(hào)：1925300