ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) derived exosomes (MSCs-exosomes) in tissue repair. MethodsThe literature about MSCs-exosomes in tissue repair was reviewed and analyzed. ResultsExosomes are biologically active microvesicles released from MSCs which are loaded with functional proteins, RNA, and microRNA. Exosomes can inhibit apoptosis, stimulate proliferation, alter cell phenotype in tissue repair of several diseases through cell-to-cell communication. ConclusionMSCs-exosomes is a novel source for the treatment of tissue repair. Further research of MSCs-exosomes biofunction, paracellular transport, and treatment mechanism will help the transform to clinical application.
ObjectiveTo observe the expression of S100A8 in plasma exosomes, microvesicles (MV), plasma and vitreous in patients with diabetic retinopathy (DR), and verify it in a diabetic rat model, and to preliminarily explore its role in the occurrence and development of DR.MethodsA case-control study. From September 2018 to December 2019, a total of 73 patients with type 2 diabetes, hospitalized patients undergoing vitrectomy, and healthy physical examinations in the Tianjin Medical University Eye Hospital were included in the study. Among them, plasma were collected from 32 patients and vitreous fluid were collected from 41 patients, which were divided into plasma sample research cohort and vitreous sample research cohort. The subjects were divided into simple diabetes group (DM group), non-proliferative DR group (NPDR group) and proliferative DR group (PDR group) without fundus changes; healthy subjects were regarded as normal control group (NC group). In the study cohort of vitreous samples, the control group was the vitreous humor of patients with epimacular membrane or macular hole. Plasma exosomes and microvesicles (MVs) were separated using ultracentrifugation. Transmission electron microscopy, nanometer particle size analyzer and Western blot (WB) were used to characterize exosomes and MVs. The mass concentration of S100A8 was determined by enzyme-linked immunosorbent assay. Eighteen healthy male Brown Norway rats were divided into normal control group and diabetic group with 9 rats in each group by random number table method. The rats of diabetes group were induced by streptozotocin to establish diabetic model. Five months after modeling, immunohistochemical staining and WB were used to detect the expression of S100A8 in the retina of rats in the normal control group and the diabetes group. t test was used for the comparison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data.parison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data.ResultsExosomes and MVs with their own characteristics were successfully separated from plasma. The concentrations of plasma exosomes and vitreous S100A8 in the PDR group were higher than those in the NPDR group, DM group, NC group, and the difference was statistically significant (P=0.039, 0.020, 0.002, 0.002, P＜0.000,＜0.000). In the plasma sample cohort study, It was not statistically significant that the overall comparison of the S100A8 mass concentrations of plasma and plasma MV in the four groups of subjects (F=0.283, 0.015; P=0.836, 0.996). Immunohistochemical staining showed that retinal ganglion cells, bipolar cells, cone rod cells and vascular endothelial cells in the diabetic group all expressed S100A8 protein. Compared with the normal control group, the expression level of S100A8 in the retina of the diabetic group increased, and the difference was statistically significant (t=8.028, P=0.001).ConclusionsThe level of S100A8 protein in circulating exosomes increases significantly with the severity of DR in patients with type 2 diabetes. S100A8 may be an influential factor in the inflammatory environment of DR and a potential anti-inflammatory therapeutic target.
Objective To explore a method of loading exosomes onto absorbable stents. MethodsBy building a stent-(3-aminopropyl) triethoxysilane-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 5000]-exosomes connection, the exosomes were loaded onto absorbable stents to obtained the exosome-eluting absorbable stents. The surface conditions of the stents and absorption of exosomes were observed by scanning electron microscope and identified through the time-of-flight mass spectrometry; the roughness of the stents’ surfaces was observed by atomic force microscope; the appearances and sizes of the stents were observed by stereomicroscope; and the radial force was tested by tensile test machine. The absorbable stents were used as control. Results The scanning electron microscope observation showed that the exosome-eluting absorbable stents had some small irregular cracks on the surface where many exosomes could be seen. The atomic force microscopy observation showed that within the range of 5 μm2, the surface roughness of the absorbable stents was ±20 nm, while the surface roughness of the exosome-eluting absorbable stents was ±70 nm. In the results of time-of-flight mass spectrometry, both the exosome-eluting absorbable stents and exosomes had a peak at the mass charge ratio of 81 (m/z 81), while the absorbable stents did not have this peak. The peak of exosome-eluting absorbable stents at m/z 73 showed a significant decrease compared to the absorbable stents. The stereomicroscope observation showed that the sizes of exosome-eluting absorbable stents met standards and the surfaces had no cracks, burrs, or depressions. The radial force results of the exosome-eluting absorbable stents met the strength standards of the original absorbable stent. Conclusion By applying the chemical connection method, the exosomes successfully loaded onto the absorbable stents. And the sizes and radial forces of this exosome-eluting absorbable stents meet the standards of the original absorbable stents.
Exosomes are nanovesicles actively secreted by cells, which selectively encapsulate biologically active molecules such as proteins, RNA, and cytokines. They play an important role in intercellular communication, immune regulation, and maintenance of homeostasis, which can also be used as carriers for targeted drug delivery. Retinal ischemia-reperfusion injury (RIRI) is a retinopathy that seriously threatens human vision. At present, the clinical treatment of these diseases are symptomatic treatments, and some patients have poor efficacy or even blindness. As extracellular vesicles rich in functional proteins and RNAs, exosomes can not only be used as drugs for the treatment of RIRI, but also be used as carriers for drug delivery to play synergistic therapeutic effects. In the future, with the deepening of the research on the molecular structure, contents and biological functions of exosomes, as well as the continuous development of ophthalmic biology and genetic engineering technology, exosomes are expected to exert their great potential as therapeutic drugs and carriers, and become an important means of treating RIRI.
ObjectiveTo observe the effects of exosomes derived from rat mesenchymal stem cells (MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model.MethodsTwelve Lewis rats were randomly divided into experimental group and control group by random number table, with 6 rats in each group. Rats in the experimental group were established with EAU model, 100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer. At different time points after modeling, the retinal structure was observed by hematoxylin and eosin (HE) staining, and the clinical and pathological manifestations were evaluated. T cells from the two groups were analyzed by flow cytometry. Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68. The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays. And flow cytometry was used to detect Th1, Th17 and regulatory T cells Variety. Electroretinogram (ERG) was used to evaluate the retinal function. Data were compared between the two groups using the t test.ResultsHE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling. Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group. On the 15th day after modeling, the retinal pathological score of the experimental group was lower than that of the control group. On the 9th to 13th day after modeling, compared to the control group, the average clinical scores of the retina in the experimental group were lower, and the difference was statistically significant (t=3.665, 3.21, 3.181, 4.121, 3.227; P＜0.01). The results of T cell proliferation assay showed that exosomes (1.0, 10.0 μg/ml) inhibited the proliferation of T cells under different concentrations of R16 (1, 10, 30 μg/ml), and the difference was statistically significant (F=11.630, 4.188, 6.011; P＜0.05). The results of flow cytometry showed that the number of Th1, Th17 and Treg cell subsets in the experimental group was decreased compared with the control group, and the difference was statistically significant (t=7.374, 4.525, 6.910; P＜0.01). There was no difference in the proportion of cells in the T cells and lymph nodes (t=1.126, 0.493, 0.178; P=0.286, 0.632, 0.862). The results of ERG showed that, compared with the control group, the amplitudes of 0.01, 3.0 cd/m2 a wave and b wave of the experiment group were all increased on the 15th day after modeling, and the differences were statistically significant (t=3.604, 4.178, 4.551, 2.566, P＜0.05).ConclusionsMSC-exosomes can reduce the clinical and pathological manifestations of EAU, protect retinal function, reduce ocular macrophage infiltration, down-regulate the proportion of inflammatory cells in the eye, and inhibit T cell proliferation.
ObjectiveTo investigate whether exosomes derived from atorvastatin (ATV)-pretreated human umbilical cord mesenchymal stem cells (ATV-MSC-EXO) alleviate high glucose-induced injury in human retinal vascular endothelial cells (HREC) via the protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathway. MethodsThe optimal pretreatment concentration of ATV was determined using the cell counting Kit-8 (CCK-8) assay. Exosomes derived from mesenchymal stem cells (MSC-EXO) and ATV-pretreated MSC (ATV-MSC-EXO) were isolated and extracted, and their morphology and surface markers were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting (WB). The uptake capacity of exosomes by human retinal vascular endothelial cells (HREC) was evaluated using a fluorescence labeling assay. In vitro cultured HREC were divided into the following groups: normal control group (NC group), high glucose group (HG group), high glucose+MSC-EXO group (MSC-EXO group), high glucose+ATV-MSC-EXO group (ATV-MSC-EXO group), high glucose+ATV-MSC-EXO+AKT inhibitor group (ATV-MSC-EXO-MK-2206-2HCL group), and high glucose+ATV-MSC-EXO+eNOS inhibitor group (ATV-MSC-EXO-L-NAME group). Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The protein expression levels of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated protein (Bax), and Caspase-3 were measured by WB. In addition, the regulatory effects of ATV-MSC-EXO on the AKT/eNOS signaling pathway and its downstream functional molecules were analyzed by detecting the phosphorylation levels of AKT (P-AKT/AKT) and eNOS (P-eNOS/eNOS) via WB, the mRNA expression levels of AKT and eNOS by quantitative real-time polymerase chain reaction, and the concentrations of nitric oxide (NO) and endothelin-1 (ET-1) using commercial NO and ET-1 assay kits. ResultsThe optimal pretreatment concentration of ATV was 1 μmol/L. ATV-MSC-EXO exhibited similar morphology and particle size to MSC-EXO and were efficiently taken up by HREC. Under high glucose conditions, ATV-MSC-EXO significantly enhanced the viability of HREC (F=83.24, P＜0.000 1) and inhibited apoptosis (F=77.39, P＜0.000 1). WB analysis further confirmed that ATV-MSC-EXO upregulated the expression of the anti-apoptotic protein Bcl-2 (F=53.17), while downregulating the pro-apoptotic proteins Bax (F=36.49) and Caspase-3 (F=60.75) (P＜0.001). In addition, ATV-MSC-EXO markedly increased the protein levels of P-AKT/AKT (F=107.60) and P-eNOS/eNOS (F=38.59), as well as the relative mRNA expression of AKT, eNOS (F=203.60, 315.00; P＜0.000 1). Furthermore, ATV-MSC-EXO promoted NO production (F=407.40) and suppressed the relative expression of ET-1 (F=49.76) (P＜0.000 1). ConclusionATV-MSC-EXO enhances the viability and inhibits apoptosis of HREC under high glucose conditions by activating the AKT/eNOS signaling pathway.
This research aims to investigate the encapsulation and controlled release effect of the newly developed self-assembling peptide R-LIFE-1 on exosomes. The gelling ability and morphological structure of the chiral self-assembling peptide (CSAP) hydrogel were examined using advanced imaging techniques, including atomic force microscopy, transmission electron microscopy, and cryo-scanning electron microscopy. The biocompatibility of the CSAP hydrogel was assessed through optical microscopy and fluorescent staining. Exosomes were isolated via ultrafiltration, and their quality was evaluated using Western blot analysis, nanoparticle tracking analysis, and transmission electron microscopy. The controlled release effect of the CSAP hydrogel on exosomes was quantitatively analyzed using laser confocal microscopy and a BCA assay kit. The results revealed that the self-assembling peptide R-LIFE-1 exhibited spontaneous assembly in the presence of various ions, leading to the formation of nanofibers. These nanofibers were cross-linked, giving rise to a robust nanofiber network structure, which further underwent cross-linking to generate a laminated membrane structure. The nanofibers possessed a large surface area, allowing them to encapsulate a substantial number of water molecules, thereby forming a hydrogel material with high water content. This hydrogel served as a stable spatial scaffold and loading matrix for the three-dimensional culture of cells, as well as the encapsulation and controlled release of exosomes. Importantly, R-LIFE-1 demonstrated excellent biocompatibility, preserving the growth of cells and the biological activity of exosomes. It rapidly formed a three-dimensional network scaffold, enabling the stable loading of cells and exosomes, while exhibiting favorable biocompatibility and reduced cytotoxicity. In conclusion, the findings of this study support the notion that R-LIFE-1 holds significant promise as an ideal tissue engineering material for tissue repair applications.
Objective To observe the effects of co-transfection of Nogo extracellular peptide residues 1-40 (NEP1-40) and neurotrophin 3 (NT-3) genes with Schwann cell-derived exosomes (SCDEs) on the survival and differentiation of neural stem cells (NSCs), and lay the foundation for the in vivo experiments of SCDE and NSC co-transplantation. Methods The NEP1-40 and NT-3 genes were transfected into Schwann cells by lentiviral vector, and SCDEs were collected for identification. The NSCs that have been passaged for 3 times were selected and inoculated into the inoculation plate, and they were divided into conventional culture group, simple exosome culture group (adding empty vector plasmid to modify SCDE for culture) and two genes exosome culture group (adding two genes modified SCDE for culture). The activity of cells in each group was detected. The survival and differentiation of NSCs were evaluated by immunofluorescence detection of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and galactosylceramidase (GALC) positive cells. Results After transfection of these two genes, the fluorescence intensity was higher and the cell state was better. The relative expression levels of messenger RNA and protein of NEP1-40 and NT-3 in the two gene groups were higher than those in the empty plasmid group (P<0.05). The relative expression levels of NEP1-40 and NT-3 proteins in SCDE of the two gene groups were higher than those of the empty vector group (P<0.05). There was no significant difference in the relative expression level of CD63 protein in SCDE between the two groups (P>0.05). In terms of cell activity, the cell activity of the two genes exosome culture group was the strongest, followed by the simple exosome culture group, and the conventional culture group was the weakest. The differences between any two groups were statistically significant (1.28±0.04 vs. 0.72±0.09 vs. 0.41±0.04, P<0.05). In terms of cell survival, NeuN-positive cells (5.23±0.22 vs. 2.36±0.09 vs. 1.00±0.01) and GALC-positive cells (2.29±0.06 vs. 1.75±0.02 vs. 1.00±0.04) of the two genes exosome culture group were the best, followed by the simple exosome culture group, and the conventional culture group were the weakest. The differences between any two groups were statistically significant (P<0.05). In terms of cell differentiation, NeuN-positive cells (0.44±0.02 vs. 0.29±0.01 vs. 0.16±0.01) and GALC-positive cells (0.38±0.07 vs. 0.23±0.02 vs. 0.12±0.01) of the two genes exosome culture group were the best, followed by the simple exosome culture group, and the conventional culture group were the weakest. The differences between any two groups were statistically significant (P<0.05). The differentiation of GFAP-positive cells in the conventional culture group was the best, followed by the simple exosome culture group, and the two genes exosome culture group was the worst (0.52±0.05 vs. 0.42±0.03 vs. 0.30±0.09). The differences between any two groups were statistically significant (P<0.05). Conclusion NEP1-40 and NT-3 genes can be successfully transfected into Schwann cells by lentiviral vector, which can effectively increase the content of related proteins in SCDE, and the exosomes can effectively promote the survival and differentiation of NSCs in vitro.
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.
Bone malignancies exhibit the characteristics of high incidence, poor prognosis, and strong chemoresistance. Exosomal microRNAs can regulate the proliferation of bone malignant cells, improve chemoresistance, influence cell communication and the microenvironment, and have significant potential in the diagnosis and treatment of bone malignancies. Due to their stability, exosomal microRNAs can serve as non-invasive biomarkers for diagnosis and prognosis. However, their widespread application in clinical settings requires standardized research. This review summarizes the progress of exosomal microRNA research in various bone malignancies including osteosarcoma, chondrosarcoma, Ewing sarcoma, and fibrosarcoma, to provide new theoretical foundations and perspectives for the field.