成纤维细胞直接转分化为神经元和视网膜神经节样细胞的研究

发布时间：2018-05-08 04:23

本文选题：腺病毒 + 转分化　；参考：《复旦大学》2013年博士论文

【摘要】：再生医学的首要目的是生成可以替代受损组织的细胞,在这方面有诸多非常具有创新性的研究。2006年诱导多能干细胞的成功生成是细胞生物学以及再生医学领域里程碑式的进步,为再生医学研究清除了多年来面临的伦理障碍。最近的研究在此基础上,成功实现了一个更加大胆的理念,即一种分化成熟的细胞直接转分化为另外一种分化成熟的细胞。比如易于取材的皮肤成纤维细胞,已经成功转分化为心肌细胞,肝脏细胞,神经元等。这些转分化所得的细胞可以回植入机体,实现患者个体化细胞替代疗法,避免排斥反应。本研究拟采用非基因组整合性的腺病毒,实现皮肤成纤维细胞向神经元及视网膜神经节样细胞的转分化。第一部分利用腺病毒将小鼠成纤维细胞直接转分化为神经元中文摘要目的：利用腺病毒作为载体,过表达神经元相关转录因子,使小鼠胚胎成纤维细胞和成年成纤维细胞直接转分化为具有功能的神经元。方法：神经元相关转录因子Ascl1,Brn2,Mytl1和Ngn2分别被构建入pAd/CMV/V5-DEST7.3腺病毒载体中,通过转染293A细胞系,包装出高滴度的带有外源基因的腺病毒。C57BL/6J小鼠胚胎期E13.5天的胚胎背部成纤维细胞及成年6～8周C57BL/6J小鼠的耳尖成纤维细胞分别被不同组合的腺病毒Ascl1+Brn2+Myt11(ABM)或Ascl1+Brn2+Ngn2(ABN)感染。感染后13天到30天期间,利用免疫组织化学方法观察神经元的产生,以及不同神经元标记物的表达,并通过电流钳和电压钳方法检测转分化得到的神经元的电生理特性。最后,利用PCR及核酸杂交方法,检测腺病毒所携带的外源基因和载体片段在被感染细胞基因组内的整合情况。结果：两种腺病毒组合ABM和ABN都能将胚胎成纤维细胞直接转分化为神经元。在感染后13天对细胞进行Tuj1免疫组织化学染色,在ABN组有2.94%±0.73%的胚胎成纤维细胞转分化为神经元,在ABM组有1.64%士0.59%的胚胎成纤维细胞转分化为神经元,ABN和ABM组产生的神经元都可以发放动作电位。ABN组产生的神经元可以表达多种神经元特有的标记物,如Map2a,NeuN和Synapsin;通过更长时间的培养(20-25天),还可以检测到兴奋性神经元标记物vGLUT和抑制性神经元标记物GAD67的表达。通过与大鼠皮层神经元的共培养,转分化产生的神经元还可以检测到兴奋性突触后电流。利用ABN组合感染小鼠成年成纤维细胞,两周后同样检测到了Tuj1阳性的神经元,并且转分化所得的神经元同样具有原代培养神经元特有的电生理特性。PCR和核酸杂交结果显示,感染后的胚胎成纤维细胞的基因组中未发现外源基因及腺病毒载体片段的整合。结论：利用腺病毒作为载体,过表达Ascl1,Brn2及Ngn2,可以成功地将小鼠胚胎成纤维细胞和成年成纤维细胞直接转分化为有功能的神经元,并且所得细胞未发现外源基因及腺病毒载体片段的整合。腺病毒介导的直接转分化过程更加安全,具有更加广阔的临床应用前景。第二部分：利用腺病毒将小鼠成纤维细胞直接转分化为视网膜神经节样细胞中文摘要目的：利用腺病毒作为载体,过表达视网膜神经节细胞相关转录因子,使小鼠胚胎成纤维细胞和成年成纤维细胞直接转分化为具有功能的视网膜神经节样细胞。方法：视网膜神经节细胞发育相关基因Ascl1,Brn2,Ngn2,Ath5,Brn3b, Nf1和Pax6分别被构建入pAd/CMV/V5-DEST7.3腺病毒载体中,通过转染293A细胞系,包装出高滴度的带有外源基因的腺病毒。通过7种基因的不同分组感染C57BL/6J小鼠胚胎期E13.5天的背部成纤维细胞,利用Tuj1和视网膜神经节细胞特异性标记物的双重染色,筛选出最有效的转录因子组合。对转分化得到的视网膜神经节样细胞进行多重标记物染色,并利用膜片钳技术检测这些细胞的电生理特性。利用相同的转录因子组合感染小鼠成年成纤维细胞,检测多种视网膜神经节细胞特异性标记物的表达情况,以及转分化所得视网膜神经节样细胞的电生理特性。最后,利用PCR方法,检测腺病毒所携带的外源基因在被感染细胞基因组内的整合情况。结果：经过筛选,Asc1+Brn3b+Ngn2组合可以将小鼠成纤维细胞转分化为视网膜神经节样细胞。这种转分化所得的视网膜神经节样细胞可以同时表达多种视网膜神经节细胞特异性标记物,并且具有成熟神经元的电生理特性。不同的标记物标记的转发化效率大致一致,在3.58%-4.00%之间。Asc1+Brn3b+Ngn2组合还可以将小鼠成年成纤维细胞转分化为视网膜神经节样细胞,同样具有成熟神经元的电生理特性。通过PCR的检测验证转分化过程未发生外源基因在宿主基因组中的整合。结论：利用腺病毒作为载体,携带Asc1+Brn3b+Ngn2转录因子组合,可以有效地将小鼠胚胎成纤维细胞及成年成纤维细胞直接转分化为视网膜神经节样细胞,转分化所得的视网膜神经节样细胞具有成熟神经元的电生理特性,并且可以同时表达多种视网膜神经节细胞特异性标记物。该转分化过程快速,有效,且不伴有外源基因的插入,为青光眼及其他视神经病变的研究和治疗提供了新的思路。
[Abstract]:The primary aim of regenerative medicine is to produce cells which can replace the damaged tissues . In 2006 , there are a number of very innovative research . In 2006 , the successful generation of pluripotent stem cells was a milestone in the field of cell biology and regenerative medicine .

the first portion

Direct transfer of mouse fibroblast cells into neurons using adenovirus

Chinese abstract

Objective : To express neuron - related transcription factors by using adenovirus as a carrier , so that mouse embryonic fibroblasts and adult fibroblasts can be differentiated into functional neurons .

Methods : The neuron - related transcription factors A尾1 , Br2 , Mytl1 and Ngn2 were respectively constructed into pAd / CMV / V5 - DEST7.3 adenovirus vector .

Results : Both adenoviral combinations and ABN were able to transform embryonic fibroblasts directly into neurons . The neurons in ABN group were transformed into neurons by Tuj1 immunohistochemical staining . The neurons in ABN group could express various neuron - specific markers , such as Map2a , NeuN and Synapsin . The neurons in ABN group could express various neuron - specific markers , such as Map2a , NeuN and Synapsin .

Conclusion : Using adenovirus as a carrier , overexpression of A尾1 , Br2 and Ngn2 can successfully transfer mouse embryonic fibroblasts and adult fibroblasts into functional neurons , and the obtained cells do not find the integration of exogenous gene and adenovirus vector fragment . Adenovirus - mediated direct differentiation process is safer and has wider clinical application prospect .

Chinese abstract

Direct transformation of mouse fibroblast into retinal ganglion - like cells using adenovirus

Part Two :

Objective : To investigate the expression of specific markers of retinal ganglion cells and the electrophysiological properties of retinal ganglion - like cells . Methods : The expression of specific markers of retinal ganglion cells and the electrophysiological properties of retinal ganglion - like cells were determined by using the double staining of Tuj1 and retinal ganglion cell specific markers .

Results : After screening , the combination of Asc1 + Brn3b + Ngn2 could differentiate the mouse fibroblast into retinal ganglion - like cells . The retinal ganglion - like cells obtained by this differentiation could express various retinal ganglion cell - specific markers at the same time , and had the electrophysiological properties of mature neurons . The combination of Asc1 + Brn3b + Ngn2 could also transform adult fibroblasts into retinal ganglion - like cells , and also had the electrophysiological properties of mature neurons .

Conclusion : Using adenovirus as carrier and carrying Asc1 + Brn3b + Ngn2 transcription factor combination , the mouse embryonic fibroblasts and adult fibroblasts can be effectively transformed into retinal ganglion - like cells . The retinal ganglion - like cells obtained by transdifferentiation have the electrophysiological properties of mature neurons , and various retinal ganglion cell - specific markers can be expressed at the same time .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1

【共引文献】