The Study Of HUCMSCs-derived Exosomes To Repair Spinal Cord Injury By Improving The Local Microenvironment In Rats

Objective:Through intravenous transplantation of hUCMSCs-exo in acute SCI rats,the study of its effect on the local microenvironment of acute SCI injury area and its repair effect on the chronic phase of SCI was observed.Methods:1.Extraction and identification of hUCMSCs-exo.The hUCMSCs were subcultured,and the cells were identified by flow cytometry as high-purity hUCMSCs.Subculture in serum-free medium,collect the 4th generation hUCMSCs medium supernatant,separate,extract and purify hUCMSCs-exo by ultracentrifugation.The morphology,structure and diameter of hUCMSCs-exo were observed by transmission electron microscope.The specific marker proteins CD9,ALIX of hUCMSCs-exo and negative control Golgi matrix protein GM130 were detected by Western blot.Determined of hUCMSCs-exo protein concentration by BCA method.The diameter distribution of hUCMSCs-exo particles was analyzed by Nano Sight.2.Prepare rat models of acute spinal cord contusion and experimental grouping.Modified Allen’s method was used to prepare acute spinal cord contusion models through IMPACTOR MODEL-Ⅱ,and they were randomly divided into sham operation group(Sham)and spinal cord injury group(SCI).After successful preparation,hUCMSCs-exo(200μg/100μL)was injected into the tail vein of the SCI group immediately,SCI-exo-3MA group were injected with PI3 K inhibitor,autophagy inhibitor 3-methyladenine(3-MA,15mg/kg/d)for 3 consecutive days.The experiment was divided into 4 groups: sham operation group(Sham),spinal cord injury group(SCI),spinal cord injury injection hUCMSCs-exo group(SCI-exo),spinal cord injury injection hUCMSCs-exo and 3MA group(SCI-exo-3MA),and local samples of SCI were taken on 1 day(d),3d,7d,and48 d respectively.3.The effect of hUCMSCs-exo on the local microenvironment of SCI acute injury.Western blot was used to quantitatively detect the expression of IL-1β,IL-6,and TNF-α during the acute phase of SCI at 1d,3d,and 7d,and the expression changes of local cell apoptosis indicators Bax and BCL-2 at 3d after SCI.The expression levels of autophagy indicators LC3-Ⅰ,LC3-Ⅱ,and P62 at 3d after SCI.4.The effect of hUCMSCs-exo on autophagy of injured local neurons and the mechanism of repairing spinal cord injury.The autophagy marker microtubule-associated protein light chain 3 and neuron-specific nucleoprotein double-labeled immunofluorescence detection(LC3/Neu N)were used to observe the autophagy of injured local neurons.Transmission electron microscopy was used to observe the changes of autophagosomes in neuronal cells.Western blot was used to detect the expression changes of AMPK/m TOR related indexes p-AMPK,AMPK,p-m TOR,m TOR,p-S6K1,S6K1.Double-labeled immunofluorescence detection(GAP-43/NF-200)was used to observe the reconnection of newly-born nerve fibers in the damaged area.Hematoxylin-eosin staining and Nissl staining were used to observe the number of surviving neurons in the chronic phase of SCI injury,and the changes in the area and scope of the injured area.The BBB score method was used to evaluate the effect of hUCMSCs-exo on the recovery of motor function of the hind limbs after SCI.Results:1.Gradient ultracentrifugation successfully extracted hUCMSCs-exo.Flow cytometry identified the cell marker protein to confirm that the experimental cells were hUCMSCs.Western blot detects exosomal specific marker proteins CD9,ALIX positive and intracellular protein GM130 negative.Nano Sight detects that the average particle diameter is(113.4±3.7)nm.Transmission electron microscopy detects that its shape is a round vesicle-like structure.There is no organelle structure in the center of the vesicle.2.Successfully prepared acute spinal cord contusion models.The SCI group scored by BBB were all 0 points.3.Injecting hUCMSCs-exo into the tail vein to improve the local microenvironment of acute SCI injury.Western blot detected the expression of IL-1β,IL-6 and TNF-α in the acute phase injury at 1d,3d and 7d after SCI in the SCI-exo group compared with the SCI group(P<0.05).Western blot detected that in the SCI-exo group,the expression of local cell apoptosis index Bax was down-regulated(P<0.05),and the expression of BCL-2 was up-regulated at 3d after SCI(P<0.05).Western blot detected that the ratio of autophagy index LC3-Ⅱ/LC3-Ⅰ was up-regulated(P<0.05),and the expression of P62 was down-regulated(P<0.05)at 3d after SCI.4.hUCMSCs-exo enhanced autophagy of damaged local neurons,reduced secondary damage,protected neurons,and promoted SCI repair.Immunofluorescence double-labeled Neu N/LC3 detection showed that the autophagy of neurons within 5mm of the head and tail of the SCI-exo group was enhanced(P<0.05),but it had no effect on the autophagy of neurons in the spinal cord outside 5mm of the head and tail.Transmission electron microscopy observed increased autophagosome formation in injured local neurons in the SCI-exo group(P<0.05).Western blot detected that the p-AMPK/AMPK ratio in the SCI-exo group was up-regulated(P<0.05),the p-m TOR/m TOR ratio was down-regulated(P<0.05),and the p-S6K1/S6K1 ratio was down-regulated(P<0.05).Nissl staining showed that the number of residual neurons in the injured area of the SCI-exo group was significantly increased(P<0.05).HE staining showed that the area of the injured area in the chronic phase of SCI in the SCI-exo group was reduced,and the range of injury was reduced(P<0.05).Immunofluorescence double-labeled GAP-43/NF-200 detection showed that more new nerve fibers were reconnected to the injured area in the SCI-exo group(P<0.05).The BBB score of the SCI-exo group was higher than that of the other groups(P<0.05).Conclusion:1.The hUCMSCs of the experimental cells in this study were analyzed by flow cytometry,and the cell purity was high and there were few miscellaneous cells.The differential gradient ultracentrifugation method can successfully separate and extract hUCMSCs-exo,and it has been identified that the exosomes have high purity and less impurities.2.The modified Allen’s method can successfully prepare acute spinal cord contusion models through IMPACTOR MODEL-Ⅱ,with a high repetition rate.Observing the functional recovery of hind limbs can facilitate the evaluation of nerve regeneration and nerve repair after spinal cord injury.3.Intravenous injection of hUCMSCs-exo can reduce the local inflammation in the acute phase of SCI,increase the autophagy response of the injured local cells,inhibit apoptosis,improve the local microenvironment of the acute phase of SCI,and reduce secondary damage,and provide micro-environment guarantee for repairing SCI.4.Intravenous injection of hUCMSCs-exo can activate the AMPK/m TOR pathway to increase the autophagy level of neuronal cells within 5 mm of the head and tail of the injured area,reduce neuronal apoptosis,and protect the remaining neurons in the injured area.It is the key to nerve regeneration and repair after SCI.5.Intravenous injection of hUCMSCs-exo can reduce secondary damage,retain more damaged local neuronal cells,reduce the area and scope of the damaged area,effectively promote the reconnection of neuronal axons,and promote the recovery of motor function in rats after SCI.