Objective To investigate the effects of exosomes (Exo) derived from high glucose-stimulated glomerular mesangial cells (GMC) on the kidneys of C57BL/6 mice and the intervention mechanism of Tongluo Yishen Formula (TLYSF). Methods The rat GMC were divided into a normal glucose group (NG, with 5.6 mmol/L glucose) and a high glucose group (HG, with 30 mmol/L glucose). After 24 hours of culture, the supernatant was collected, and exosomes were extracted using the ultracentrifugation method. The exosomes were then identified by transmission electron microscopy and Western blot analysis. Male C57BL/6 mice were divided into three groups: NO-Exo group, NG-Exo group, and HG-Exo group. These groups were respectively administered tail vein injections of PBS buffer, exosomes derived from GMC cultured in normal glucose, and exosomes derived from GMC cultured in high glucose, three times a week for a total of 8 weeks. After 8 weeks, the mice in the HG-Exo group were randomly divided into three subgroups: the HG-Exo group [gavaged with saline], the HG-Exo+TLYSF group [gavaged with TLYSF at 34.32 g/(kg.d)], and the HG-Exo + VAL group [gavaged with valsartan suspension at 10.4 mg/(kg.d)], and the intervention lasted for 4 weeks. Urinary microalbumin (mALb), urinary N-acetyl-β-D-aminoglucosidase (NAG), glycated hemoglobin (HbA1c), serum creatinine (Scr) and urea nitrogen (BUN) were detected. Transmission electron microscopy was used to observe the ultrastructure of renal tissues. TUNEL was used to detect the DNA damage of renal tissue cells. Immunofluorescence was used to detect the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and wilms tumor 1(WT-1). RT-PCR was used to detect the mRNA levels of NLRP3, cysteinyl aspartate-specific proteinase 1 (caspase-1), interleukin-1 beta (IL-1β), miR-200c-3p and miR-148a-3p. Western Blot was employed to detect the protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 and IL-1β. Results Compared with the NG-Exo group, mice in the HG-Exo group exhibited significantly increased levels of mALb, urinary NAG, Scr and BUN. Transmission electron microscopy revealed ruptured podocyte membranes and swollen mitochondria. The positive rate of cells stained by the TUNEL increased, with elevated optical density of NLRP3 and decreased optical density of WT-1. Additionally, there was a significant increase in the level of NLRP3, caspase-1, IL-1β mRNA, as well as miR-200c-3p and miR-148a-3p. The protein expression of NLRP3, ASC, caspase-1, and IL-1β also increased. Compared with HG-Exo group, mice in the HG-Exo+TLYSF group showed decreased levels of mALb, urinary NAG, Scr, and BUN. The podocyte membranes were relatively intact, and mitochondrial damage was alleviated. The positive rate of cells stained by the TUNEL decreased, along with a reduction in the optical density of NLRP3 and an increase in the optical density of WT-1. Furthermore, the mRNA expression levels of NLRP3, caspase-1, IL-1β, miR-200c-3p, and miR-148a-3p were all downregulated to varying degrees. The protein expression levels of NLRP3, ASC, caspase-1, and IL-1β also decreased. Conclusion Exosomes derived from GMC stimulated by high glucose can damage the kidneys of mice and induce podocyte pyroptosis. TLYSF may ameliorate podocyte pyroptosis by downregulating the expression of exosomal miR-200c-3p and miR-148a-3p and inhibiting the activation of the NLRP3/ASC/caspase-1 pathway.
Animals ; Exosomes/ultrastructure* ; Glucose/pharmacology* ; Male ; Podocytes/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Mice, Inbred C57BL ; Mice ; Mesangial Cells/metabolism* ; Pyroptosis/drug effects* ; Rats ; MicroRNAs/genetics*

Stem cells are known to maintain stemness at least in part through secreted factors that promote stem-like phenotypes in resident cells. Accumulating evidence has clarified that stem cells release nano-vesicles, known as exosomes, which may serve as mediators of cell-to-cell communication and may potentially transmit stem cell phenotypes to recipient cells, facilitating stem cell maintenance, differentiation, self-renewal, and repair. It has become apparent that stem cell-derived exosomes mediate interactions among stromal elements, promote genetic instability in recipient cells, and induce malignant transformation. This review will therefore discuss the potential of stem cell-derived exosomes in the context of stromal remodeling and their ability to generate cancer-initiating cells in a tumor niche by inducing morphologic and functional differentiation of fibroblasts into tumor-initiating fibroblasts. In addition, the immunosuppressive potential of stem cell-derived exosomes in cancer immunotherapy and their prospective applications in cell-free therapies in future translational medicine is discussed.
Apoptosis ; Cell Communication ; Cell Transformation, Neoplastic ; Disease Progression ; Exosomes ; physiology ; Humans ; Immunotherapy ; methods ; Mesenchymal Stromal Cells ; physiology ; Neoplasms ; blood supply ; pathology ; therapy ; Neoplastic Stem Cells ; ultrastructure ; Neovascularization, Pathologic ; pathology ; Organelle Biogenesis ; Tumor Microenvironment

OBJECTIVE: To provide a reference about choosing the methods of isolating exosomes derived from tumor cells including laryngocarcinima Hep-2 cells by comparing advantages and defects of two methods of isolation and extraction exosomes. METHOD: Previously, laryngocarcinoma Hep-2 cells were cultivated massively, then the cells were processed with hot shock in 42 degrees C for 1 h. Sucrose density gradient centrifugation ultrafiltration (method 1): cells culture supernatant 90 ml was gathered, the supernatant was clarified through a 3/0.8 μm small filter to remove impurities and fragments which in larger diameter. Then the filtering fluid was concentrated and purified through sucrose density gradient centrifugation and ultrafiltration, the concentrated fluid was obtained. Exosome Isolation Kit (method 2): cells culture supernatant 4 ml was gathered, the solutions of the kit were added into the supernatant in proper sequence, then filtered by the special column, the concentrated fluid was obtained. Both products are observed by high resolution transmission electron microscopy. RESULT: Both methods could isolate and extract exosomes feasibly. In single high power view of transmission electron microscopy, exosomes of method 1 disperse better, but lower density, and more impurity in background, exosomes of method 2 arrange closer, higher density, and less impurity. CONCLUSION Exosome isolation Kit require less supernatant, cost less time, process procedure briefly, harvest higher yield. It may become a new option of isolating exosomes derived from Laryngocarcinoma Hep-2 cells.
Cell Line, Tumor ; Exosomes ; ultrastructure ; Humans ; Laryngeal Neoplasms ; pathology ; Microscopy, Electron, Transmission

OBJECTIVETo isolate exosomes from rabbit aqueous humor and to investigate their immunosuppression function.

METHODSAqueous humor was collected from 40 New Zealand rabbits and exosomes were isolated by fractional separation and ultracentrifugation methods; the morphology was studied with electron microscopy. The immunosuppressive-related proteins of exosomes were detected with Western blotting; their inhibitory effect on ConA-induced proliferation of T lymphocyte was estimation with CCK-8 cells proliferation assay.

RESULTSEight milliliters of aqueous humor were collected from 40 New Zealand rabbits and 200 μg exosomes was yielded. Under electron microscope, the exosomes had typical structure of lipid bi-layer with a diameter of 50-100 nm. The results of Western blotting showed that these exosomes expressed Hsp70, CD9 and Alix but not Grp94, presenting a typical exosomes protein profile. Moreover, exosomes expressed high level of TGF-β and significantly inhibited the proliferation of T lymphocytes.

CONCLUSIONImmunosuppressive exosomes can be isolated from rabbit aqueous humor, which may be involved in immunotolerance of the eye.

Animals ; Aqueous Humor ; immunology ; Cells, Cultured ; Exosomes ; immunology ; metabolism ; ultrastructure ; Female ; Immune Tolerance ; Male ; Rabbits ; T-Lymphocytes ; immunology

OBJECTIVETo isolate and purify exosomes derived from human bladder transitional cell carcinoma T24 cells, analyze the morphology and protein composition, and investigate the antitumor effect of specific cytotoxic T lymphocytes induced by exosomes.

METHODSExosomes were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and characterized by electron microscopy and Western blot. Dendritic cells were amplified and purified from peripheral blood and pulsed with exosomes. Then they were co-cultured with T cells, and divided into 3 groups: exosome-pulsed DC group, unplused DC group and control group. Alamar-Blue assay was used to evaluate the specific cytolytic activity.

RESULTSThe exosomes were in size about 30 approximately 90 nm saucer-shaped membranous vesicles. HSP70, ICAM-1 and CK20 were detected by Western blot. The CTL induced by DC pulsed with exosomes had significant cytolytic activity (P < 0.01).

CONCLUSIONThe exosomes derived from T24 cells are loaded with immunoprotein HSP70 and ICAM-1, and DC pulsed with exosomes can promote the anti-tumor effect of CTLs in vitro.

Carcinoma, Transitional Cell ; pathology ; Cell Line, Tumor ; Coculture Techniques ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; ultrastructure ; Exosomes ; immunology ; metabolism ; ultrastructure ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Keratin-20 ; metabolism ; Lymphocyte Activation ; T-Lymphocytes ; cytology ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Urinary Bladder Neoplasms ; pathology