Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

Preliminary study of bone marrow mesenchymal stem cell-derived exosomes in repair of spinal cord injury in rats

AIM:To study the influence of bone marrow mesenchymol stem cell-drived exosomes (BMSC-exosomes) on hindlimb activity, and the numbers of reactive astrocytes and residual neurons in spinal cord injury (SCI) rats. METHODS:BMSCs were cultured using the whole bone marrow adherent culture method and surface markers CD90 and CD34 were verified by flow cytometry. Exosomes were isolated by ultracentrifugation and the morphology of exosomes was observed under transmission electron microscope. The protein markers CD63 and CD9 were verified by Western blot. After exosomes were applied to SCI rats, the Basso, Beattie and Bresnahan locomotor rating scale score, the Nissl staining of the lesion site, and the numbers of reactive astrocytes and residual neurons were assessed at various time points. RESULTS:Transmission electron microscopic observation revealed the presence of saucer-shaped vesicles. BMSC-exosomes were found to express high levels of CD63 and CD9. Compared with injury group, significant improvement of hindlimb activity scores from day 14 after injury in treatment group was observed (P<0.05), and less reactive astrocytes and more residual neurons from day 7 after injury were also observed (P<0.05). CONCLUSION:BMSC-exosomes inhibit reactive astrocytes and death of neurons, and improve hindlimb activity in the rats after SCI.     

Effect of interleukin 18 gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes

AIM: To study the effect of interleukin 18( IL-8 ) gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes.METHODS: Exosomes isolated from the supernatants of IL-18 gene-modified NCI-H460 lung cancer cells (IL-18/H460), pcDNA3.1⁺ vector-modified cancer cells (DNA3.1/H460) and non-modified NCI-H460 lung cancer cells (NCI-H460) were observed under transmission electron microscope.The expression of heat-shock protein 70(HSP70),human leukocyte antigen(HLA) and IL-18 were determined by Western blotting.T lymphocytes were activated by exosomes or exosome-pulsed dendritic cells(DCs).The activity of T cells for killing lung cancer cells were detected by lactate dehydrogenase (LDH) method.The killing rates were calculated and compared.RESULTS: Exosomes showed typical morphous under transmission electron microscope.The protein levels of HSP70 and HLA were detected in the exosomes of all 3 groups, and IL-18 protein was only observed in IL-18/H460 group.The killing rates of exosome-activated T cells in IL-18/H460 group with the ratio of effector cell to target cell at 25∶ 1, 10∶ 1 and 5∶ 1 were (38.45±5.42)%, (25.17±3.94)% and (11.75±3.22)%, respectively.The killing rates of exosome-pulsed DC-activated T cells in this group were (89.05±4.06)%, (64.97±6.02)% and (40.16±4.98)%, respectively.The killing rates in IL-18/H460 group were higher than those in DNA3.1/H460 group and NCI-H460 group.The anti-tumor efficacy of exosome-pulsed DC－activated T cells was stronger than that of exosome-activated T cells.CONCLUSION: IL-18 gene modification enhances the anti-tumor activity induced by NCI-H460 lung cancer cell-derived exosomes.     

外泌体miRNA在配子发育和受精中的调控作用

外泌体(Exosomes,EXs)是一类膜状结构的纳米级细胞外囊泡,携带大量的生物活性调节分子进行细胞间信息转导。EXs携带的miRNA在配子发生、发育和受精过程中发挥重要的调控作用。本文主要对EXs中的miRNA在卵母细胞和精子成熟以及受精过程中的调控作用做一综述。     

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages， macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

外泌体调控卵泡中母细胞发育的研究进展

为了探究外泌体在卵泡中调控卵母细胞生长发育以及后续受精中的调控作用及机制，归纳了最近外泌体泡的文献。首先综述了外泌体的组成和功能，其次在此基础上选择重点阐述外泌体在卵泡中如何调控卵母细胞与卵泡体细胞的互作机制，然后进一步分析了外泌体介导精卵结合的调控机制。从而得出了外泌体在卵泡中通过其中包含的蛋白和RNA等物质调控卵母细胞生长发育的结论，建议在研究卵母细胞生长发育调控的研究方面应该重视对于外泌体的研究。最终，首次统性论述了外泌体对于卵母细胞的发育的影响，期望对于研究同行有一定的意义。     

Decidual macrophages modulate trophoblast cells by transferring exosomes in unexplained recurrent spontaneous abortion

AIM: To investigate whether decidual macrophages have ability to regulate trophoblast cells by secreting exosomes in unexplained recurrent spontaneous abortion (URSA). METHODS: The decidual macrophages were isolated from deciduas of URSA and normal early pregnant induced abortion women. Transmission electron microscopy revealed the morphology and size of the macrophages-derived exosomes. Besides, the characterization of the exosomes was confirmed by the presence of known exosomal marker CD63 by Western blot. The HTR8/Snveo cells were treated with exosomes extracted from the decidual macrophages of URSA or normal early pregnanty induced abortion women. Confocal microscopy, MTS assay and Transwell assay were used to explore the effect of URSA macrophages-derived exosomes on viabi-lity and migration ability of trophoblasts cells. RESULTS: Electron microscopy showed exosomes secreted by macrophages displayed a round-shaped appearance, ranging 40~80 nm in diameter. The results of Western blot indicated that exosomes exacted from the macrophages expressed CD63, a member of tetraspanin family protein. Confocal microscopy revealed the exosomes were taken up by the HTR8/Snveo cells. MTS assay indicated the viability of HTR8/Snveo cells was declined when incubated with the URSA macrophages-derived exosomes (P<0.05). In URSA macrophages-derived exosomes group, the migration ability of the HTR8/Snveo cells was decreased significantly as compared with normal early pregnant induced abortion macrophages-derived exosome (P<0.05). CONCLUSION: Decidual macrophages of URSA inhibits the viability and migratory properties of trophoblast cells by exosomes, which participates in regulating maternal-fetal immune in URSA.     

Effect of inhibiting release of exosomes on biological characteristics of bone marrow mesenchymal stem cells

AIM:To investigate the effect of inhibiting the release of exosomes on the biological characteristics of bone marrow mesenchymal stem cells (BM-MSCs) and the underlying mechanisms. METHODS:The exosome releasing-deficient mouse model was constructed by knockout of Rab27a using TALEN technique. The BM-MSCs were isolated and cultured. The exosomes were extracted from the culture medium using total exosome isolation kit and quantified by nanoparticle tracking analysis (NTA). The size and morphology of the exosomes were observed under transmission electron microscope. To evaluate the proliferation ability of BM-MSCs, the BM-MSCs were labeled with 5-ethynyl-2'-deoxyuridine (EdU) and the expression level of proliferating cell nuclear antigen (PCNA) was determined by Western blot. Moreover, hypoxia tolerance of BM-MSCs in vitro was evaluated via TUNEL staining and MTS assay. RESULTS:The count of exosomes released by BM-MSCs isolated from Rab27a knockout mice was significantly reduced. Inhibition of exosome release resulted in decreases in the viability of the BM-MSCs and their resistance to hypoxia. CONCLUSION:Inhibition of exosome release from the BM-MSCs results in significantly decreased proliferation ability and resistance to hypoxia.