本文選題：新生大鼠 + 嗅鞘細(xì)胞　； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：第一部分新生大鼠嗅鞘細(xì)胞的分離、培養(yǎng)和純化研究 目的探討新生大鼠嗅鞘細(xì)胞的分離、培養(yǎng)和純化方法,為后期的基因修飾嗅鞘細(xì)胞修復(fù)視神經(jīng)損傷研究奠定基礎(chǔ)。方法取2d內(nèi)的新生大鼠嗅球組織,在解剖顯微鏡下分離取材,原代培養(yǎng)嗅鞘細(xì)胞,運(yùn)用差速貼壁法和差速貼壁+阿糖胞苷+細(xì)胞生長刺激因子法兩種方法進(jìn)行純化,在倒置顯微鏡下觀察不同培養(yǎng)時(shí)間嗅鞘細(xì)胞的形態(tài)、數(shù)量和NGFRp75抗體免疫熒光結(jié)果,統(tǒng)計(jì)嗅鞘細(xì)胞的純度。結(jié)果嗅鞘細(xì)胞的純度分別是差速貼壁組平均為60.41%±4.32%,差速貼壁+阿糖胞苷+細(xì)胞生長刺激因子法組平均為84.98%±4.03%。兩組數(shù)據(jù)用t檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)分析,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論取材于新生大鼠,顯微鏡下分離腦膜,差速貼壁+阿糖胞苷+細(xì)胞生長刺激因子法純化,都是良好的嗅鞘細(xì)胞培養(yǎng)方法。 第二部分大鼠NEP1-40基因的擴(kuò)增及重組慢病毒載體的構(gòu)建 目的構(gòu)建攜帶大鼠NEP1-40目的基因的重組慢病毒,并檢測(cè)其體外表達(dá)目的基因的能力。方法PCR擴(kuò)增出TAT/NEP1-40編碼片段,定向克隆入pLV.Des2d.P/puro載體,Lipofectamine2000轉(zhuǎn)染293FT細(xì)胞包裝攜帶NEP1-40目的基因的重組慢病毒,測(cè)序證實(shí)為TAT/NEP1-40后大量擴(kuò)增,以基因轉(zhuǎn)移單位(GTU)法測(cè)定病毒滴度,運(yùn)用PCR和western blot分別檢測(cè)NEP1-40在293FT細(xì)胞中轉(zhuǎn)錄水平和蛋白水平的基因表達(dá)。結(jié)果成功構(gòu)建攜帶大鼠NEP1-40目的基因的重組慢病毒,并證實(shí)其在293FT細(xì)胞中可表達(dá)NEP1-40。結(jié)論攜帶大鼠NEP1-40基因的重組慢病毒可在體外表達(dá)其目的基因,為研究NEP1-40在視神經(jīng)損傷恢復(fù)中的作用奠定基礎(chǔ)。 第三部分慢病毒介導(dǎo)的NEP1-40基因在體外嗅鞘細(xì)胞中的表達(dá) 目的研究慢病毒介導(dǎo)的NEP1-40基因在體外嗅鞘細(xì)胞中的表達(dá),并初步觀察轉(zhuǎn)染了NEP1-40基因的嗅鞘細(xì)胞對(duì)視網(wǎng)膜節(jié)細(xì)胞的作用。方法用NEP1-40慢病毒載體轉(zhuǎn)染體外原代培養(yǎng)嗅鞘細(xì)胞,應(yīng)用PCR及ELISA檢測(cè)NEP1-40基因在嗅鞘細(xì)胞中轉(zhuǎn)錄水平和蛋白水平的表達(dá),并將轉(zhuǎn)基因嗅鞘細(xì)胞與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞共培養(yǎng),觀察節(jié)細(xì)胞軸突的生長情況。結(jié)果NEP1-40慢病毒載體轉(zhuǎn)染體外原代培養(yǎng)嗅鞘細(xì)胞后經(jīng)過PCR和ELISA檢測(cè),在轉(zhuǎn)錄水平和蛋白表達(dá)水平證實(shí)NEP1-40基因均有表達(dá),轉(zhuǎn)基因嗅鞘細(xì)胞與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞共培養(yǎng)時(shí)觀察到節(jié)細(xì)胞生長狀況良好。結(jié)論NEP1-40重組慢病毒載體可以轉(zhuǎn)染體外原代培養(yǎng)嗅鞘細(xì)胞,并能在細(xì)胞外表達(dá)其目的基因,促進(jìn)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞生長,為后期動(dòng)物實(shí)驗(yàn)奠定基礎(chǔ)。
[Abstract]:Part one: isolation, culture and purification of olfactory ensheathing cells from neonatal rats Objective to investigate the methods of isolation, culture and purification of olfactory ensheathing cells from newborn rats, so as to lay a foundation for the repair of optic nerve injury by gene modified olfactory ensheathing cells. Methods olfactory ensheathing cells were isolated from the olfactory bulb of newborn rats within 2 days. The olfactory ensheathing cells were cultured under anatomic microscope. The cells were purified by two methods: differential adhesion method and differential adhesion cytosine cell growth stimulating factor method. The morphology and quantity of olfactory ensheathing cells and the results of NGFRp75 antibody immunofluorescence were observed under inverted microscope, and the purity of olfactory ensheathing cells was counted. Results the purity of olfactory ensheathing cells was 60.41% 鹵4.32g in differential adhesion group and 84.98% 鹵4.03g in differential adhesion cytarabine cell growth factor group. The data of the two groups were analyzed by t test, and the difference was statistically significant (P 0.05). Conclusion it is a good culture method for olfactory ensheathing cells to extract meninges from newborn rats and isolate meninges under microscope and purify cytarabine cell growth stimulating factor by differential adhesion. Part two Amplification of Rat NEP1-40 Gene and Construction of Recombinant Lentivirus Vector Aim to construct a recombinant lentivirus carrying rat NEP1-40 gene and to detect its ability to express the target gene in vitro. Methods TAT/NEP1-40 coding fragment was amplified by PCR and cloned into pLV.Des2d.P/puro vector Lipofectamine2000 to transfect the recombinant lentivirus carrying NEP1-40 target gene in 293FT cell package. The recombinant lentivirus was confirmed to be TAT/NEP1-40 by sequencing. The viral titer was determined by gene transfer unit (GTU) assay. PCR and western blot were used to detect the transcriptional and protein levels of NEP1-40 in 293FT cells. Results Recombinant lentivirus carrying rat NEP1-40 gene was successfully constructed, and NEP1-40 expression was confirmed in 293FT cells. Conclusion Recombinant lentivirus carrying rat NEP1-40 gene can express its target gene in vitro, which lays a foundation for studying the role of NEP1-40 in the recovery of optic nerve injury. The expression of lentivirus-mediated NEP1-40 gene in olfactory ensheathing cells in vitro Objective to investigate the expression of lentivirus mediated NEP1-40 gene in olfactory ensheathing cells in vitro and to investigate the effect of olfactory ensheathing cells transfected with NEP1-40 gene on retinal ganglion cells. Methods the olfactory ensheathing cells were transfected with NEP1-40 lentivirus vector in vitro. The expression of NEP1-40 gene in olfactory ensheathing cells was detected by PCR and ELISA, and the transgene olfactory ensheathing cells and retinal ganglion cells were co-cultured. To observe the growth of ganglion cell axons. Results NEP1-40 lentivirus vector was transfected into olfactory ensheathing cells in vitro and detected by PCR and ELISA. The expression of NEP1-40 gene was confirmed at the level of transcription and protein expression. It was observed that the ganglion cells grew well in co-culture of transgenic olfactory ensheathing cells and retinal ganglion cells. Conclusion Recombinant lentivirus vector of NEP1-40 can transfect olfactory ensheathing cells in vitro, express its target gene in vitro, promote the growth of retinal ganglion cells, and lay a foundation for later animal experiments.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.6

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