动物造模,人脐带间充质干细胞 z-bio 2021-11-16 415

　　Introduction: Peripheral nerve injury often leads to limited nerve regeneration or incomplete functional recovery. In the field of regenerative medicine, mesenchymal stem cells (mesenchymal stem cells) are considered to be cell reservoirs due to their ability to differentiate into specialized cells. MSCS can also secrete biologically active molecules to help build a regenerated microenvironment after injury. In view of these characteristics, mesenchymal stem cells are considered as a promising nerve damage repair and regeneration tool. The regenerative potential of MSC therapy is mediated by paracrine effects. Human umbilical cord mesenchymal stem cells (hucMSCs) produce more paracrine effects than bone marrow stem cells (bmcs) and adipose-derived mesenchymal stem cells. Accumulated evidence indicates that hucMSCs can promote axon regeneration after peripheral nerve injury. Guo et al. reported that hucMSCs transplantation promotes axon regeneration and functional recovery of peripheral nerve repair through paracrine function. In addition, hucMSCs can also reduce the allogeneic or xenogeneic immune response of transplanted animal models through immunosuppressive ability. Therefore, hucMSCs secrete soluble factors, enhance and assist tissue repair through paracrine mechanisms, and play a significant role in regenerative medicine. Extracellular vesicles (EVS) are one of the paracrine mechanisms of mesenchymal stem cells discovered in recent years. EVs include exosomes, microvesicles and apoptotic bodies, which are nanomembrane fragments secreted by various types of cells (such as stem cells). EVs deliver various proteins, lipids, cytokines, transcription factors and other biological materials to target cells. They are resistant to freezing and thawing processes, can avoid the problems associated with bone marrow mesenchymal stem cell transplantation, and can be used for allogeneic therapy due to their low immunogenicity. MSC-derived EVs are easy to store, have high safety, and have significant application prospects in regenerative medicine. Recent studies on animal models have shown that EVs have great potential as a cell-free therapy. MSC-EV treatment can enhance the functional recovery of ischemic stroke rats and promote axon remodeling, neurogenesis and angiogenesis. Treatment with EVs 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 EVs promoted nerve regeneration in rats with sciatic nerve crush injury. However, the effect of hUCMSC-EVs on sciatic nerve transection remains to be 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 attenuate denervated muscle atrophy. hUCMSC-EVss can accumulate in nerve defects, down-regulate pro-inflammatory cytokines (interleukin [IL]-6 and IL-1β) and up-regulate anti-inflammatory cytokines (IL-10). hUCMSC-EVs 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, weight 220-230 grams, male SD rats. Isolation and characterization of hucMSCs: Isolate and culture human umbilical cord mesenchymal stem cells (hucMSCs) according to the previously described method. The hucMSCs from the 3rd to 5th generation were used in the experiments conducted in this study. Perform Alizarin Red and Oil Red O staining as described previously. These experiments show that hucMSCs have osteogenic and adipogenic differentiation potential. Using phycoerythrin (PE) bound antibodies against CD14, CD19, CD34, CD45, CD73 and CD90, the typical surface markers of hucMSCs in different channels were detected by flow cytometry.

　　Isolation, purification and identification of hUCMSC-EVs: Human umbilical cord mesenchymal stem cells (hucMSCs) were cultured to 80%-90% confluence. As mentioned earlier, the supernatant of the culture was collected by ultracentrifugation to isolate EVs. BCA protein detection kit was used to determine the protein content of EVs. The Nanosight LM10 system was used to evaluate the particle size distribution of hUCMSC-EVs. As mentioned earlier, transmission electron microscopy (TEM) was used to identify the morphology of hUCMSC-EVs. The filtered EVs were resuspended in PBS, cultured and stained with 20μL direct fluorescent antibody CD63 to detect the typical surface markers of hUCMSC-EVs. Unstained EVs are used as NCS. The phenotype of EVS was analyzed by bd-accuri-c6 flow cytometer.

　　Surgery and hUCMSC-EVs treatment: rats were intraperitoneally injected with 10 mg/kg xylazine and 75 mg/kg ketamine. Expose and remove the left sciatic nerve (3 mm). Nerve contraction results in the formation of a 5 mm long gap. Through the epineurium and silicone rubber tube, the proximal and distal ends were continuously fixed with simple 10-0 nylon direct sutures. The distal and proximal nerve stumps are inserted into the tube 1 mm deep. Keep a gap of 5 mm long. The fascia and muscle layer were sutured with 4-0 nylon sutures. The skin is closed with continuous sutures. After the animal model was induced for 24 hours, 100μg hUCMSC-EVs (100μl) 0.2 ml PBS or 0.2 ml PBS were injected into the tail vein of the rat. 48 male SD rats were randomly assigned to the hUCMSC-EVs group (rats that received hUCMSC-EVs injection, n=24) or the control group (rats that received PBS injection, n=24). Three rats that did not undergo surgery or treatment were designated as normal control groups.

　　Functional evaluation: According to the previously described protocol, walking trajectory analysis was performed before surgery and 2, 4, 6 and 8 weeks after neuroplasty. Simply put, the rat's hind paws are dipped in black ink. Then let the rat show a standard walking trajectory on a piece of paper (30×7 cm). The sciatic nerve function index (SFI) is calculated 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; ETS represents the first to fifth toes of experimental rats. NTS means normal toe extension; EPL means experimental paw length for manipulation; NPL means normal paw length. Generally speaking, 0 corresponds to normal function, and ?100 corresponds to complete loss of function.

　　Muscle weight measurement: The rats were euthanized 8 weeks after the operation. The gastrocnemius muscle is dissected and then weighed with an electronic balance. The formula for calculating the wet weight ratio of gastrocnemius muscle is: operation side/normal side×100%. Hematoxylin and eosin staining: the nerve ducts and gastrocnemius muscles of rats were collected, fixed in 4% PFA at 4°C, and embedded in paraffin. The longitudinal section (thickness 12μm) and the cross section (thickness 4μm) of the middle part of the graft were prepared and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis: Nerve tissue sections were subjected to antigen retrieval 3 days after injury. The culture slides were blocked 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-horseradish peroxidase diluted 1:300. Observe the image using Nikon Ti-S microscope. Use Image-Pro Plus software to determine the average density of IL-6, IL-1β and IL-10. Transmission electron microscopy: 4 weeks after surgery, the midpoint of the repaired nerve tissue was removed and analyzed by transmission electron microscopy. As mentioned earlier, tissue sections are prepared and observed by transmission electron microscopy. The number of myelin sheets and the thickness of myelin sheath in each slice were calculated.

　　Immunofluorescence: immunofluorescence method to measure nerve fiber morphology and remyelination. Paraffin sections of nerve tissue with rabbit anti-rat S-100 (green) (1:100 dilution), rabbit anti-rat NF-200 (red) (1:100 dilution), rabbit anti-rat myelin basic protein (MBP) ) (Green) (1:100 dilution) or mouse monoclonal anti-Brdu (red) (1:100 dilution) for blocking and labeling. The secondary antibody uses Alexa Fluor 488 goat anti-rabbit antibody (1:400 dilution) or Cy3 goat anti-mouse antibody (1:300 dilution). Under ×400 magnification, remyelination was determined by calculating the number of myelin axons in each field of 10 randomly selected regenerated nerve tissue sections. Through confocal microscopy analysis, the directional marker EV of the distal nerve stump was determined to detect the specific Schwann cell (SC) marker S-100. The sections were blocked and labeled with rabbit anti-mouse S-100 (green) (1:100 dilution), and Alexa Fluor 488 goat anti-rabbit antibody (1:400 dilution) was used as the secondary antibody. Tracking of hUCMSC-EVs: The suspension of human umbilical cord MSC-derived extracellular vesicles (hUCMSC-EVs) was labeled with DIR, resuspended by ultracentrifugation, the solution was precipitated, and washed twice with PBS. 100 μg DiR-labeled EVs were injected intravenously into the animal model. 24 hours after injection, the rats were anesthetized and imaged in vivo. The IVIS spectral imaging system was used to detect the distribution of labeled EVs in rats.

　　Result: Typical characteristics of hUCMSCs and hUCMSC-EVs: After 10 days of initial culture, the adherent cells showed a long spindle shape, formed colonies, and reached a confluent state. As shown by Alizarin Red and Oil Red O staining, mesenchymal stem cells exhibited multi-lineage potential to differentiate into bone cells and adipocytes. Fluorescence activated cell classification showed that CD73 and CD90 cells were positive, while CD14, CD19, CD34 and CD45 cells were negative. These data indicate that we have effectively generated hUCMSCs according to the standards defined by the International Society for Cell Therapy. The isolated and purified EVs were evaluated by transmission electron microscopy, nanoparticle tracking analysis (NTA) and flow cytometry. Transmission electron microscopy showed that hUCMSC-EVs are round film particles with a typical cup-shaped shape. The diameter of hUCMSC-EVs ranges from 80 to 650 nanometers, and the average value recorded by NTA is 168 nanometers. Flow cytometry analysis showed that most human hUCMSC-EVs express the specific marker CD63, which is a representative marker of EV. As mentioned above, hucMSCs and their corresponding EVs were successfully isolated and characterized.

　　hUCMSC-EVs treatment improved the functional recovery of the sciatic nerve: We constructed a rat sciatic nerve transection model to study the effect of hUCMSC-EVs on sciatic nerve defects. Figure 2a illustrates the construction of the RAT model and the collection and processing of hUCMSC-EVs. Figure 2b shows a schematic diagram of the experimental process after hUCMSC-EVs or PBS treatment. The walking trajectory analysis method was used to evaluate the recovery of rats' motor function. SFI is used to reveal the degree of improvement of hUCMSC-EVs and the control group. The walking trajectory analysis results shown in Figure 3a and b show that the PBS group showed neurological function recovery, and the hUCMSC-EVs treatment group showed improvement in function recovery. After 8 weeks of sciatic nerve transection, the walking trajectory of hUCMSC-EVs treated rats was almost similar to that of normal rats. At 4, 6, and 8 weeks after operation, the SFI score of hUCMSC-EVs group increased significantly compared with the control group. These results indicate that the application of hUCMSC-EVs treatment can improve the recovery of motor function after sciatic nerve transection.

　　Morphological analysis of nerve regeneration: We observed the morphology of the regenerated nerve in the middle segment after 8 weeks of sciatic nerve transection. The catheter was completely removed, and the two segments of the removed median nerve were connected. The results showed that both hUCMSC-EVs and the control group showed neurogenesis. Rats in the experimental group regenerated nerves larger than those 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 of the control group. S-100 is a surface marker of SCS, which wraps nerve fibers to form myelin sheath. Immunofluorescence staining showed that S-100-positive fibers appeared in hUCMSC-EVs 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. The results of Brdu staining showed that hUCMSC-EVs treatment promoted Schwann cell proliferation. The axon regeneration in the hUCMSC-EVs treatment group was better than the control group at 8 weeks postoperatively. This result indicates that hUCMSC-EVs can promote the remyelination and axon regeneration of damaged sciatic nerve.

　　Human umbilical cord MSC-derived extracellular vesicles increase the total number of myelinated fibers: by immunofluorescence analysis, the midpoint of the cross section for detecting remyelination was obtained. Figure 5a shows the midpoint of repairing nerve tissue. Eight weeks after surgery, we performed immunostaining to detect the presence of the axon marker NF-200 and the 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 the signals detected in the slices of the control group were little or no. Quantification of myelinated axons of the sciatic nerve showed that hUCMSC-EVs treatment can increase axon myelination at 8 weeks after axotomy. In addition, at 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 in rats treated with hUCMSC-EVs were higher than those in rats treated with PBS. These findings indicate that hUCMSC-EVs treatment leads to the production of a large number of axons and several SCs around individual axons, and suggests that hUCMSC-EVs may promote the myelination of regenerated axons.

　　Human umbilical cord MSC-derived extracellular vesicles reduce the degree of gastrocnemius atrophy: the gastrocnemius muscle loses weight because it is not innervated after the sciatic nerve is transected. We weighed the gastrocnemius muscle at 8 weeks to assess the recovery of muscle innervation. The wet weight of gastrocnemius in hUCMSC-EVs group was higher than that in control group. In addition, the gastrocnemius wet weight ratio in the hUCMSC-EVs group was higher than that in the control group. These results indicate that hUCMSC-EVs treatment leads to extensive innervation of the gastrocnemius muscle. H&E staining showed that gastrocnemius muscle fiber atrophy was weakened in hUCMSC-EVs group. The fiber morphology of gastrocnemius muscle in hUCMSC-EVs group was similar to that of normal muscle. However, the fiber area of the gastrocnemius in the control group decreased, suggesting severe gastrocnemius atrophy.

　　Human umbilical cord MSC-derived extracellular vesicles accumulate in rat nerve defects and regulate its inflammatory response: Mesenchymal stem cells (MSCs) can survive and graft in damaged tissues. Therefore, we investigated whether the MSC from the EV has a similar homing function. We used the IVIS lumina II system to evaluate the biodistribution of EVS in rats by fluorescence imaging. 24 hours after injection into the tail vein of rats, DiR-labeled EVs from HUCMSC gathered in the nerve defect. The rats were killed immediately, and the nerves at the distal end of the nerve defect were collected for tissue slices. The results of immunofluorescence staining of longitudinal sections showed that DiR-labeled EV reached the distal end of the nerve defect. There is evidence that MSC-EVS limits post-ischemic inflammation that leads to ischemic brain injury. We also investigated whether the immune response induced by nerve injury is regulated by hUCMSC-EVs. Histochemical staining of the distal nerve stump 3 days after surgery