嵌合Neurturin在恒河猴脂肪间充质干细胞中的表达及定向诱导作用

The Expressing and Inducing Activity of Chimeric Neurturin in Rhesus Adipose Stem Cells

该研究通过重组腺病毒传递嵌合型Nerve growth factor(NGF)/Neurturin(NTN)至恒河猴脂肪间充质干细胞(rhesus adipose mesenchymal stem cells,rASCs)中,评价嵌合型NGF/NTN在诱导该细胞为神经元细胞中的作用,为进一步探讨ASC-NTN体内修复帕金森氏病猴模型潜能提供实验基础。用带有NGF/NTN嵌合基因的重组腺病毒感染rASCs,通过RT-PCR、免疫荧光及ELISA检测重组NTN在rASCs的表达及产物定位,在外源化学诱导剂协同作用下对rASCs进行诱导。rAd-NGF/NTN感染rASCs后检测到NGF/NTN转录产物,表达产物可分泌到细胞外,分泌出的NTN蛋白在体外能够促进鸡胚背根神经节长出神经突起,维持胎鼠中脑神经元的存活。rASCs在内外源性诱导因素诱导下可向神经元样细胞分化。利用rAd-NGF/NTN可将NGF-NTN传递至rASCs中并表达,rASCs能够被定向诱导为具有分泌NTN作用的神经元样细胞。这些研究结果为将来rASCs-NTN移植进入帕金森氏病猴模型进行帕金森氏病治疗提供了重要依据。

In this study, we have shown that forced expressions of chimeric Nerve growth factor （NGF）/ Neurturin （NTN） in adult rhesus adipose mesenchymal stem cells （rASCs） facilitate dopaminergic neuron differ- entiation following adenovirus vector transduction in vitro. The repair potential of ASC-NTN in gene therapy in Rhesus model of Parkinson＇s disease will be sought in the further work. In this study, rhesus ASCs were exposedto a recombinant chimeric NGF/NTN adenovirus which constructed and conserved in this lab expressing the NTN. The expression and location of chimeric NGF/NTN could be identified using RT-PCR, immunofluorescence stain- ing and ELISA technology, rASCs were induced in the microenvironment combined with other chemical inducer. We detected the NTN transcripts in rASCs. Expression product could be secreted into the extracellular. The culture of dorsal root ganglia of chick embryo test had indicated that Neurturin secreted by rASCs had positive biologicalactivity in nervous ramification forming. The ventral mesencephalic neurons of embryo rat survived with chimeric Neurturin in the culture, rASCs are able to differentiate into neuron like cells involving exogenous and endogenous factors. Our results demonstrated that rhesus adipose mesenchymal stem cells were able to specifically differenti- ate into neuron like cells by a mechanism involving NTN of the neuron developmental pathway when infected with Ad-NGF/NTN cultured in an appropriate exogenous chemical microenvironment. The operation of rASCs-NTN transplantation in Rhesus Parkinson＇s disease model would be researched in the future based on this work.