The Research Of A New Type Of Peripheral Nerve Tracer For Quantum Dots

Objective :Peripheral nerve injury has always been one of the thorny diseases in the field of hand and foot microsurgery.Early diagnosis is an important method to improve the effect of the peripheral nerve injury.At present,the fluorescent nanoparticle is an ideal material for early neural tracer,but the fluorescent particle is commonly used to emit single color wave and its length closes to the ultraviolet region.Therefore,the biological tissue has strong auto-fluorescence effect,and during observation the contrast effect is not obvious.Quantum dots have quantum size effect,whose emission spectrum can be adjusted by changing their size,giving off different colors of fluorescence.It has the advantage of combining with functional proteins through different linking reactions after the surface modification.Hence,we use it as a fluorescent marker,and use neuron specific cholera toxin B subunit as ligand to solve the neural specificity,and synthesize the new nerve tracer through EDC reaction.Methods :Under room temperature,merge a certain concentration of CTB with QD in proper proportion,then add EDC to promote reaction.Filter it and put it into the centrifugal ultrafiltration to achieve centrifugation,and finally the synthesis of CTB-QD is finished.The fluorescence spectra of CTB,QD and CTB-QD are measured,then absorb spectra and the Zeta potential.The transmission electron microscope is used to observe CTB-QD.CTB-QD was added to the prepared PC12 cells and separately cultured for 1,2,4 and 8 hours.Then the cell imaging is observed by confocal microscope,and DAPI nuclear fluorescent dye is used to conduct double staining.A certain amount of CTB-QD is injected at the proximal end of adult rats' sciatic nerve.One week after the operation,the rats are lavaged and fixed with 4% paraformaldehyde.The sciatic nerve,L4 dorsal root ganglion and L4 segment spinal cord which have been injected are taken out for further study.Conduct frozen sections on them,then examine them directly by confocal microscope.Results:The PL spectra and absorption spectra of CTB and CTB-QD are obviously different.There is a slight difference between the emission peak positions of QD and CTB-QD.The PL spectrum of centrifugal solution shows that there is no CTB-QD leakage and the centrifugal separation is more complete.The average value of the CTB potential is +7.1mV,while the value of the CTB-QD potential is-37.8mV,and the Zeta potential of CTB and CTB-QD has changed significantly.Under electron microscope,the maximum particle size of CTB-QD is 9.07 nm,while the minimum particle size is 5.74 nm,and the average particle size is 7.16 Nm.Most of the samples are distributed between 7-7.5nm,accounting for 27% of the total,whose diameter is not very different from that of QD.CTB-QD enters the PC cells within 1 hour.Around 2 hours,CTB-QD is partially distributed.After 4 hours,the number of cells entering the cells is saturated.The brightness gradually becomes stable and clear.As for the part of cell imaging,there is no significant difference between 8 hours and 4 hours.CTB-QD's specificity is mixed in the cell membrane and does not enter the nucleus.One week after the operation,CTB-QD is found at the sciatic nerve injection point,and CTB-QD is also found at the dorsal root ganglion of the L4 segment and the anterior horn of the L4 segment of the spinal cord.Conclusion :The spectral and potential changes of CTB and CTB-QD indicate that CTB is successfully combined with QD,and CTB-QD still has good fluorescence properties and participates in axonal transport in neurite.The conjugate can be specifically associated with the GM1 binding site on the cell body of the neuron and can be used for clear cell imaging after 4 hours.One week after the operation,the conjugate could be found at the injection point segment,the corresponding dorsal root ganglion and the anterior horn of the spinal cord,which is same as the anatomical pathway.CTB-QD can be used for peripheral nerve retrograde tracing,and the synthesis and observation method is simple,intuitive and convenient.It not only has significant meaning to monitor the repairing effect of peripheral nerve and improve the effect of treatment,but also lays a foundation for improving the clinical diagnosis and treatment of peripheral nerve injury.