动物造模,充质干细胞 z-bo 2020-09-11 348

　　Introduction: Peripheral nerve injury usually limits nerve regeneration or leads to incomplete functional recovery. In the field of regenerative medicine, mesenchymal stem cells (esophageal stem cells) are considered as cell reservoirs because they can differentiate into specific cells. MSCS also secretes biologically active molecules to help establish a regenerating microenvironment after injury. In view of these characteristics, mesenchymal stem cells are considered a promising tool for repairing and regenerating nerve damage. The regenerative potential of MSC therapy is mediated by the cis-oblique effect. Compared with umbilical cord stem cells (bmcs) and fat-derived mesenchymal stem cells, human umbilical cord medullary stem cells (hucMSC) produce more quasi-clinical effects. Accumulated evidence shows that hucMSC can promote axon regeneration after peripheral nerve injury. Guo et al. reported that the transplantation of hucMSCs promotes axon regeneration and functional recovery of peripheral nerve repair through oblique movements. In addition, hucMSC can also reduce the allogeneic or heterologous immune response of transplanted animal models through its immunosuppressive ability. Therefore, hucMSC secretes soluble factors, promotes and supports tissue repair through the antagonistic mechanism, and plays an important role in regenerative medicine. Extracellular vesicles (EVS) are one of the recently discovered quasi-clinical mechanisms of mesenchymal stem cells. Electric vehicles include exosomes, microvesicles and apoptotic bodies. These are nanomembrane fragments secreted by different types of cells (such as stem cells). EV delivers a variety of proteins, lipids, cytokines, transcription factors and other biological substances to target cells. They are resistant to freezing and thawing processes, avoid the problems associated with bone marrow mesenchymal stem cell transplantation, and have low immunogenicity, so they can be used for allogeneic therapy. Electric vehicles derived from MSC are easy to store, highly secure, and have huge potential applications in regenerative medicine. Recent studies on animal models have shown that EV has great potential as a cell-free therapy. MSC-EV treatment can enhance the functional recovery of rats with ischemic stroke, and promote axon remodeling, neurogenesis and angiogenesis. Treatment with EV derived from human bone marrow stromal cells can improve nerve regeneration after stroke and prevent immunosuppression in mice after ischemia. Our previous studies have shown that BMSC-derived EV promotes nerve regeneration in rats with sciatic nerve contusion. However, the effect of hUCMSC-EV on sciatic nerve transection has not been studied. In this study, a rat model of sciatic nerve transection was established. We have observed that hUCMSC-EVs can improve motor function recovery and nerve regeneration defects, and reduce nerve atrophy. hUCMSC-EVss accumulate in nerve defects, down-regulate pro-inflammatory cytokines (interleukin [IL]-6 and IL-1β), and up-regulate anti-inflammatory cytokines (IL-10). hUCMSC-EV may contribute to nerve regeneration in rats with sciatic nerve defect. Therefore, hUCMSC-EVss is a very promising strategy for nerve regeneration and peripheral nerve injury treatment after nerve transection.

Materials and methods: 3-4 weeks old, weighing 220-230 grams, male SD rats. Isolation and characterization of hucMSC: Isolate and culture human umbilical cord mesenchymal stem cells (hucMSC) according to the above method. The experiments conducted in this study used 3 to 5 generations of huc MSC. As described above, perform Alizarin Red and Oil Red O staining. These experiments show that hucMSC has the ability to distinguish between bone formation and fat production. Phycoerythrin (PE) combined with antibodies against CD14, CD19, CD34, CD45, CD73 and CD90 is used to detect the typical surface markers of hucMSC in different channels by flow cytometry. H Isolation, purification and identification of UCMSC-EV: Human umbilical cord mesenchymal stem cells (hucMSC) were cultured to a density of 80% to 90%. As mentioned earlier, the culture supernatant was collected by ultracentrifugation to separate EVs. Use BCA protein detection kit to determine the protein content of EV. The particle size distribution of hUCMSC-EV was evaluated using Nanosight LM10 system.

　　 As mentioned earlier, we used transmission electron microscopy (TEM) to identify the morphology of hUCMSC-EV. The filtered EV was resuspended in PBS, cultured and stained with 20 μL direct fluorescent antibody CD63 to detect the typical surface markers of hUCMSC-EV. The clean EV is used as NCS. The phenotype of EVS was analyzed by bd-accuri-c6 flow cytometer. Surgery and treatment of hUCMSC-EV: rats were injected intraperitoneally with 10 mg/kg xylazine and 75 mg/kg ketamine. Expose and remove the left sciatic nerve (3 mm). The contraction of the nerve creates a gap of 5 mm long. A simple 10-0 nylon direct suture was used to continuously fix the proximal and distal ends through the proximal nerve tube and silicone rubber tube. Insert the distal and proximal nerve stumps into a 1 mm deep tube. Make sure there is a gap of 5 mm. Suture the myocardium and muscle layer with 4-0 nylon sutures. Stitch the skin continuously. Twenty-four hours after the animal model was induced, 100μgh UCMSC-EV (100μl) 0.2 ml PBS or 0.2 ml PBS was injected into the tail vein of the rat. 48 male SD rats were randomly divided into hUCMSC-EVs group (rats that received hUCMSC-EVs injection, n = 24) or control group (rats that received PBS injection, n = 24). The normal control group included three rats that had not undergone surgery or treatment. Functional evaluation: According to the above scheme, walk trajectory analysis was performed before surgery and 2, 4, 6, and 8 weeks after neurogenesis. In short, soak the hind legs of rats with black ink. Next, show the mouse the standard walking path (30 x 7 cm) on the paper. Calculate the Sciatic Nerve Function Index (SFI) according to the following formula: SFI = 118.9 (ETS-NTS)/NTS-51.2 (EPL-NPL)/NPL-7.5, E and N represent experimental and normal respectively, and ETS represents experimental rats The fifth toe. TS means normal leg extension, EPL means experimental leg length for operation, and NPL means normal leg length.
Usually, 0 corresponds to a normal function? 100 means complete loss of function. Muscle weighing: The rats were euthanized 8 weeks after the operation. The gastrocnemius abdominal muscles were dissected and weighed with an electronic balance. The formula for calculating the wet weight ratio of the peroneal abdominal muscles is the operation side/normal side×100%. Hematoxylin and eosin staining: the rat neural tube and peritoneal muscle were collected, and 4% PFA was fixed in paraffin at 4°C. Prepare the longitudinal section (12μm thick) and cross section (4μm thick) of the central part of the implant, and stain with hematoxylin and eosin (H&E). Immunohistochemical analysis: antigen search was performed on nerve tissue sections 3 days after injury. Block the culture slide with monoclonal anti-IL-6 antibody diluted 1:200, monoclonal anti-IL-1β antibody diluted 1:500 or monoclonal anti-IL-10 antibody diluted 1:100. The slides were incubated with anti-mouse IgG-Western mustard peroxidase diluted 1:300. Observe the image with Nikon Ti-S microscope. Use Image-ProPlus software to determine the average density of IL-6, IL-1β, and IL-10. Transmission electron microscope: Four weeks after the operation, the midpoint of the repaired nerve tissue was excised and analyzed with a transmission electron microscope. As mentioned above, prepare tissue sections and observe under a transmission electron microscope. Calculate the number of myelin sheets and the thickness of myelin sheath in each slice. Immunofluorescence: An immunofluorescence method that measures the morphology of nerve fibers and remyelination. Rabbit anti-rat S-100 (green) (1:100 dilution), rabbit anti-rat NF-200 (red) (1:100 dilution), nerve tissue containing rabbit anti-rat myelin basic protein (MBP) Paraffin section) (green) (1:100 dilution) or mouse monoclonal anti-Brdu (red) for blocking and labeling (1:100 dilution). For the secondary antibody, please use Alexa Fluor488 goat anti-rabbit antibody (1:400 dilution) or Cy3 goat anti-mouse antibody (1:300 dilution). The myelin sheath remodeling was determined by calculating the number of myelin sheath axons in each field of 10 randomly selected regenerated nerve tissue sections at 400x magnification. Through confocal microscopy analysis, the distal nerve stump direction marker EV was determined to detect the specific Schwan cell (SC) marker S-100. The sections were blocked and labeled with rabbit anti-mouse S-100 (green) (1:100 dilution), and Alexa Fluor488 goat anti-rabbit antibody (1:400 dilution) was used as the secondary antibody. Follow-up of hUCMSC-EV: DIR-labeled the suspension of extracellular vesicles derived from human umbilical cord MSC (hUCMSC-EV), resuspended by ultracentrifugation, precipitated the solution and washed twice with PBS. The animal model was injected intravenously with 100 μg DiR-labeled EV. Twenty-four hours after the injection, the rats were anesthetized and imaged in vivo. The IVIS spectral imaging system is used to detect the distribution of labeled EVs in rats. Results: Typical characteristics of hucMSC and hUCMSC-EV: After 10 days of initial culture, the attached cells became long fusiform, settled and reached a fusion state. As shown by Alizarin Red and Oil Red O staining, mesenchymal stem cells show the potential of different lineages to differentiate into bone and adipocytes. In the fluorescence-activated cell classification, CD73 and CD90 cells were positive, while CD14, CD19, CD34 and CD45 cells were negative. These data indicate that hUCMSC is effectively produced according to the standards defined by the International Society for Cell Therapy. The separated and purified electric vehicles were evaluated by transmission electron microscopy, nanoparticle tracking analysis (NTA) and flow cytometry. Transmission electron microscopy has shown that hUCMSC-EV is a typical cup-shaped round film particle. The diameter of hUCMSC-EV is 80-650 nm, and the average value recorded by NTA is 168 nm. Flow cytometry analysis showed that most human hUCMSC-EVs express specific marker CD63, which is a representative marker of EV. As mentioned above, hucMSC and its corresponding EV have been successfully isolated and characterized.

　　 HUCMSC-EVs treatment can improve the recovery of sciatic nerve function: establish a rat sciatic nerve cross-section model to study the effect of hUCMSC-EVs on sciatic nerve defects. Figure 2a shows the construction of the RAT model and the collection and processing of hUCMSC-EV. Figure 2b shows a schematic diagram of the experimental process after hUCMSC-EV or PBS treatment. The walking trajectory analysis method was used to evaluate the recovery of rat's motor function. SFI is used to determine the degree of improvement of hUCMSC-EV and the control group. The gait trajectory analysis results shown in Figures 3a and 3b showed that the PBS group showed a recovery of neurological function, while the hUCMSC-EVs treatment group showed an improvement in function recovery. Eight weeks after sciatic nerve transection, the gait trajectory of rats treated with hUCMSC-EVs was similar to that of normal rats. Four, six and eight weeks after the operation, the SFI score of the hUCMSC-EVs group was significantly improved compared with the control group. These results indicate that the application of hUCMSC-EVs can improve the recovery of motor function after sciatic nerve transection. Morphological analysis of nerve regeneration: Eight weeks after the diaphragm muscle was transected, the morphology of the regeneration nerve was observed in the middle of the sciatic nerve. The catheter has been completely removed, and the two segments of the removed median nerve are connected. The results showed that both hUCMSC-EV and the control group showed neurogenicity. Rats in the experimental group regenerated larger nerves than rats in the control group.

　　 Specifically, H&E staining showed that the diameter of regenerated nerve fibers in the hUCMSC-EVs group was significantly larger than that in the control group. S-100 is the surface marker of SCS, which wraps nerve fibers to form myelin sheath. Immunofluorescence staining showed that S-100-positive fibers appeared in hUCMSC-EV and the control group. However, the immunofluorescence density of s-100-positive fibers in the hUCMSC-EVs group was higher than that in the control group. Brdu staining showed that hUCMSC-EVs treatment can promote Schwan cell proliferation. At 8 weeks postoperatively, the axial regeneration of the hUCMSC-EVs treatment group was better than that of the control group. This result indicates that hUCMSC-EV can promote the remyelination and axon regeneration of the damaged sciatic nerve. Extracellular vesicles derived from human umbilical cord MSC increase the total number of myelin fibers. Immunofluorescence analysis provides the midpoint of the cross-section used to detect remyelination. Figure 5a shows the midpoint of nerve tissue repair. Eight weeks after surgery, immunostaining was performed to detect the presence of axon markers NF-200 and SC marker MBP in the cross-section of the regenerated sciatic nerve. As shown in Figure 5b, the fluorescence signals of NF-200 and MBP were mainly detected in the slices of the hUCMSC-EVs group, while little or no signal was detected in the slices of the control group. The quantification of the myelin axis in the sciatic nerve showed that hUCMSC-EVs treatment can increase the axon myelin formation 8 weeks after axon amputation. In addition, 4 weeks after surgery, the expression of MBP in the hUCMSC-EVs treatment group was up-regulated compared with the PBS control group. Transmission electron microscopy showed that the number of myelin fibers and the thickness of myelin sheath of rats treated with hUCMSC-EV were higher than those of rats treated with PBS. These findings indicate that treatment with hUCMSC-EVs resulted in multiple axons and several SCs around a single axon, while hUCMSC-EVs promoted the myelination of regenerated axons. Suggest possible. The extracellular vesicles derived from human umbilical cord MSC reduced the degree of peroneal abdominal muscle atrophy. When the sciatic nerve is cut, the peroneal abdominal muscles are not innervated and lose weight. To assess the recovery of muscle innervation, we weighed the peroneal abdominal muscles at week 8. The wet weight of the peroneal abdominal muscles in the hUCMSC-EV group was higher than that in the control group. In addition, the wet weight ratio of gastrocnemius muscle in the hUCMSC-EVs group was higher than that in the control group. These results indicate that hUCMSC-EVs treatment resulted in extensive innervation of the common peroneal abdominal muscle. H&E staining showed that the atrophy of peroneal muscle fibers in the hUCMSC-EVs group was weakened. The fiber morphology of the peroneal abdominal muscle in the hUCMSC-EVs group was similar to that of normal muscle. However, the fibrous area of the fibular muscle in the control group was reduced, suggesting severe peritoneal muscle atrophy. Extracellular vesicles derived from human umbilical cord MSC accumulate in rat nerve defects and regulate its inflammatory response: mesenchymal stem cells (MSCs) can survive and can be transplanted into damaged tissues.

　　 Therefore, we studied whether the MSC of electric vehicles has a similar homing function. The IVISlumina II system was used to evaluate the biodistribution of EVS in rats by fluorescence imaging. Twenty-four hours after injection into the tail vein of the rat, HUCMSC DiR-labeled electric cars gathered in the nerve defect. The rats were killed immediately, and the nerves distal to the nerve defect were collected for tissue sectioning. Longitudinal immunofluorescence staining results showed that DiR-labeled EV reached the distal end of the nerve defect. There is evidence that MSC-EVS can limit post-ischemic inflammation that leads to ischemic brain injury, and we also investigated whether hUCMSC-EV modulates the immune response caused by nerve injury. Histochemical staining was performed on the distal nerve stump 3 days after surgery. Compared with the control group, the hUCMSC-EVs treatment group promoted the down-regulation of inflammatory cytokines (IL-6 and IL-1β) and the up-regulation of anti-inflammatory cytokines (IL-10). These results indicate that hUCMSC-EV can implant nerve defects and modulate inflammation. This effect may promote the regeneration of defective nerves. Conclusion: hUCMSC-EV has been proven to effectively promote the functional recovery and nerve regeneration in rat models of sciatic nerve defect. Our research provides potential for clinical peripheral nerve repair. The clinical application of hUCMSC-EV is more advantageous than stem cell administration.