Quantitative Analysis Of Related Neural Molecules In The Regulation Of Neural Stem Cells

Neurodegenerative diseases are mainly characterized by the loss or death of neuronal functions.Many elegant methods have been reported for the diagnosis and treatment of neurodegenerative diseases.The regulation of"directed differentiation"of NSC can promote nerve cell renewal and repair peripheral nerve damage,which has important potential in the process intervention of neurodegenerative diseases.However,despite the great promise of NSC regulation in the treatment of neurodegenerative diseases,great challenges remain for their application.Firstly,there are many substances related to NSC regulation,sucn as ROS,metal irons and enzyme,and interact with each other.It is urgent to develop highly selectivity probes for quantifing substances closely related to NSC regulation,and further analyze the molecular mechanism of NSC regulation-related signaling pathways.Secondly,the reported regulation methods cannot precisely regulate the behavior of NSC,such as effective proliferation and directional differentiation.In addition,in vivo analysis can veritably reflect the physiological and pathological processes of the nervous system.However,the in vivo environment is complex,it is urgent to develop in vivo analysis methods that can not only regulate NSC behavior but also realize quantitative detection of related substances.In response to the key scientific issues raised above,this paper has carried out the following three aspects of work:(1)A DNA framework-based fluorescence lifetime imaging(FLIM)probe was designed and synthesized,which solved the research bottleneck of signal interference in the simultaneous detection of multiple substances,and realized the simultaneous quantitative analysis of pH and Ca²⁺in lysosomes.The Ca²⁺probes,pH-responsive molecules and lysosome-targeting molecules were co-assembled onto DNA bipyramid nanostructures,and a lysosome-targeted FLIM probe for simultaneous quantitative analysis of pH and Ca²⁺was constructed.The probe has the advantages of good selectivity,high stability and good biocompatibility.Using this probe,the simultaneous quantitative analysis and real-time imaging of pH and Ca²⁺in cell lysosomes was successfully achieved.It was found that the intracellular pH was closely related to Ca²⁺,and could jointly regulate autophagy.Meanwhile,neuronal death induced by A?protein was due to abnormal autophagy caused by changes in lysosome pH and Ca²⁺.In addition,lysosome pH and Ca²⁺could regulate the activation of NSCs to achieve anti-aging regulation.(2)To achieve precise regulation of NSC,a photoactivated acyl radical donor molecule:PAce was designed and synthesized.Rhodamine was designed as the skeleton for donor molecule.PAce was photo-activated to form acyl radicals,and simultaneously released rhodamine molecules with fluorescence emission.Therefore,PAce could controllably release of acyl radicals and target to membrane potential,which could be used for real-time monitoring of cellular redox homeostasis.Using the developed molecule,it was found that acyl radicals could accurately regulate the redox homeostasis of cells and further realize the precise regulation of NSCs directional differentiation.In addition,the directional differentiation of NSCs in the subgranular zone of hippocampal dentate gyrus(SGZ)of early Alzheimer's disease(AD)mice was successfully achieved by quantitatively releasing acyl free radicals,thereby repairing nerve injury and improving the symptoms of AD mice.(3)A fiber Raman imaging system was built,and the Raman signal collection efficiency was significantly improved by optimizing the fiber parameters.Meanwhile,a synapse-targeted acetylcholinesterase(AchE)Raman probe was designed and synthesized,which had the advantages of high selectivity,high sensitivity and good stability,and successfully realized the quantitative analysis of AchE.It was found that AchE could regulate the proliferation and differentiation of NSCs.Using the established Raman fiber imaging system and AchE Raman probe,it was found that the AchE activity in the cortex,hippocampus and amygdala of early AD mice were significantly decreased,while that in other brain regions remained basically unchanged.As for AD mice,AchE activity in each brain region was significantly decreased.In addition,by using nitric oxide stimulation to regulate AchE activity,the proliferation and directional differentiation of neural stem cells in the subgranular zone of dentate gyrus of early AD mice were regulated,and the interactions between various brain regions of mice were successfully enhanced and the treatment of early AD diseases was realized.