Experimental Study On Uptake And Translocation Of Exosomes Derived From MSCs By Peripheral Nerve

Objective: Peripheral neuropathy is difficult to be treated.Developing a new efficient drug delivery system is critical for targeted therapy of peripheral neuropathy.Exosomes are a kind of nanoscale membrane vesicles produce in living cells and contain many biological activity molecules,including micro RNA,m RNA and proteins.Exosomes are a kind of natural carriers and potential drug delivers.However,the mechanisms for uptake of exosomes by peripheral nerve is remained unexplored.It will be meaningful to figure out that if they could be absorbed and transferred by peripheral nerve endings and be used as peripheral nervous system drug delivers.In present study,we aimed to investigate whether peripheral nerve terminals are able to absorb exosome derived from mesenchymal stem cells(MSCs)of rat,and provide an experimental evidence for exosomes as a treatment of peripheral nerve and a carrier of biological products.Methods:(1)Extraction and identification of MSCs;(2)Purification of Exo derived from MSCs and labeled with PKH67(appeared green fluorescence)and verification of Exo by transmission electron microscopy;(3)Co-culture of neuroblastoma cells(SHSY5Ycells or BV2 cells)with Exo derived from MSCs;(4)Rat models: In experimental group,right side sciatic nerves of the rats were transected,and the Exo were injected into the right side gastrocnemius.In control group,right side sciatic nerves of the rats were remained intact,and the Exo were injected into the similar place as the experimental group.In different time point after injection,The DRG and the traversing spinal cord segment were observed under fluorescence microscope after operation,and the fluorescence intensity of green fluorescence positive cells was statisticaled.Results:(1)MSCs were extracted successfully.(2)Under transmission electron microscope,Exo was a kind of circular membranous vesicles with an average diameter of 78.10 nm ±13.33 nm(40nm-100 nm).(3)Exo derived from MSCs labeled by green fluorescence could be uptake by neuroblastoma cells(SHSY5Y cells and BV2 cells)after co-culturing for 24 hours.(4)Green fluorescence positive particles were found in the bilateral DRG and spinal cord at the first 24 hours after gastrocnemius muscle injection,and there was no significant difference statistically in the fluorescence intensity between two side(p>0.05).(5)The green fluorescence intensity in right side DRG was statistically significant higher than that in the opposite side at the 7th day after injection(p<0.01).(6)At the same time,the gray value of anti CD63 with the immunohistochemical method was higher in the right DRG than in the left side and the difference was statistically significant.(p<0.01).(7)Green fluorescence positive particles were found in neurons of bilateral DRG and gray matter of spinal cord in rats which right sciatic nerve was cut down for 1cm at the 7th day after injection and operation,and there was no significant difference in fluorescence intensity between the two side(p>0.05).Conclusion:(1)Exo derived from MSCs can be labeled by PKH67(appeared green fluorescence).(2)Exo derived from MSCs can be ingested by neural cells.(3)Exo derived from MSCs can be transported to bilateral DRG and both sides of the spinal cord by blood.(4)Exo derived from MSCs can also be transported to bilateral DRG and spinal cord via peripheral nerve fibers.