The Study Of Microvesicle Released By Human Embryonic Stem Cell Derived Mesenchymal Stem Cells On The Repair Of Sciatic Nerve Injury In Rat

ObjectivesTo investigate the effect of the microvesicles(MVs) secreted by mesenchymal stem cells(MSCs) derived from human embryonic stem cells(hESCs) on the sciatic nerve of SD rats and the mechanism of its function. Methods1. hESC-MSC-MVs were separated and purificated of by the method of ultra speed centrifugation and identificated by electron microscope.2. SD rat sciatic nerve crush injury model was established. Rats were treated by tail vein injection of hESC-MSCs or hESC-MSC-MVs. The effect of hESC-MSCs and hESC-MSC-MVs on sciatic nerve injury in rats was valuated by sciatic nerve function index and HE staining. The miRNAs of hESC-MSCs and hESC-MSC-MVs related to sciatic nerve injury were detected by real-time quantitative PCR(Real-time).3. SD fetal rat NSCs(neural stem cells,NSCs) were isolated and cocultured with hESC-MSCs or hESC-MSC-MVs in vitro, respectively. Expression of neural specific marker GAP-43 was detected by immunofluorescence staining to evalute the effect of hESC-MSCs or hESC-MSC-MVs on the differentiation of NSCs. Results1. We successfully separated and purified the hESC-MSC-MVs.The secretory process of hESC-MSC-MVs was observed by electron microscope.2. We established a model of sciatic nerve crush injury in SD rats.Compared with the control group, the hESC-MSCs group and the hESC-MSC-MVs group’s footprint shapes were better than the PBS group. However, there were still differences in the damaged side of the hESC-MSCs group compared with the normal side, and the hESC-MSC-MVs group damaged side footprints was similar to the normal side.The hESC-MSCs and the hESC-MSC-MVs could significantly promote the recovery of hind limb and the increase of the number of regenerative nerve fibers in the injured side of model rats,significantly reduced the degree of muscle atrophy of gastrocnemius muscle. PCR Real-time detection found hESC-MSC-MVs and hESC-MSCs containing miRNA-222, mi RNA-125, miRNA-29 b, miRNA-29 c. Expression of miRNA-222 was the relatively highest.3. We successfully isolated NSCs from SD fetal rats. Compared to the control group, hESC-MSCs and hESC-MSC-MVs could promote NSCs to differentiate into neurons, and the neurites were denser and longer, and effect of the hESC-MSC-MVs was more obvious. ConclusionsThe hESC-MSC-MVs had a significant repair effect on sciatic nerve crush injury, could significantly promote the differentiation of NSCs into neurons and the link-up of nerve fibers.