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@#[Abstract] Objective: To investigate the effect of exosomes derived from osteosarcoma on the differentiation of tumor-related macrophages and its mechanism. Methods: From March 2018 to October 2019, tumor tissues and corresponding normal tissues from 18 patients with primary osteosarcoma who underwent osteosarcoma resection and pathological diagnosis in the Departments of Orthopedics and Pediatric Surgery of the Affiliated Hospital of North Sichuan Medical College were collected. The expression level of Tim-3 was detected by Western blotting; Exosomes of osteosarcoma MG63 cells (MG63-Exo) were isolated and identified by transmission electron microscopy and nanoparticle size analysis, and its phagocytosis by macrophages was verified by Dual fluorescent staining; The effects of MG63-Exo on macrophage differentiation and the expression levels of IL-10, TGF-β and VEGF were detected by qPCR; The effects of MG63-Exo induced macrophages on the migration and invasion of MG63 cells and the expression of EMT related proteins were detected by Transwell invasion and migration assay and Western blotting; CRISPR/cas9 was used to knock out Tim-3 in MG63 cells, and its knockout efficiency was verified by Western blotting, and then qPCR, transwell assay and Western blotting were used to detect the effect of MG63-Exo with Tim-3 knock-out on macrophage differentiation, as well as migration, invasion and expression of EMT related proteins in MG63 cells; Finally, the mouse model of osteosarcoma lung metastasis was used to verify the effect of exosomes from different sources on the lung metastasis of osteosarcoma. Results: Transmission electron microscopy and nanoparticle size assay confirmed that MG63-Exo were successfully isolated, and Confocal fluorescence results confirmed that it could be phagocytized by macrophages; qPCR results showed that MG63-Exo significantly promoted M2 differentiation of macrophages compared with PBS (P<0.05); Compared with PBS control group, M2 macrophages induced by MG63-Exo significantly promoted the migration, invasion and EMT of osteosarcoma cells (all P<0.05); The mRNA and protein expressions of Tim-3 in the MG63 cells knocked out by CRISPR/cas9 (Tim-3-KO) were significantly reduced (all P<0.05), and Tim-3 could be transferred into macrophages in the form of exosomes; Compared with MG63-Exo co-cultured macrophages, the M2 type differentiation of macrophages treated with Tim-3-KO-exo was significantly decreased (P<0.05); Compared with the MG63 cells co-cultured with macrophages induced by MG63-Exo, the migration, invasion and EMT were significantly reduced while the lung metastasis was significantly promoted in MG63 cells co-cultured with macrophages induced by Tim-3-KO-Exo (all P<0.05). Conclusion: Exosomes derived from osteosarcoma can induce M2 polarization of macrophages through Tim-3 and promote the invasion and metastasis of tumor.
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A highly skilled surgical team relies on a standardized training program. This article presents a comprehensive description on the current status, problems, and strategies in Hepato-bilio-pancreatic surgery education reform in Zhejiang University School of Medicine (ZJU), China. A multiple-discipline joint training program was set up by integrating the basic laboratory and clinical research as well as routine clinical practice. Reform includes resident training in the Royal College of Surgeons of Edinburgh, curriculum resetting, surgery textbook rewriting, clinical teaching and training bases construction, teaching methods improvement, English reading, writing and reporting skills training and a research assignment. ZJU establishes rigorous processes for managing teaching quality and ensuring graduates promote evidence-based surgical practice. ZJU has built its leading position in the Hepato-Bilio-Pancreatic surgery in China. In 2011, 6 post-doctoral researchers, 52 Ph.D. students and 83 master students were enrolled in the 7year MD program and 8year Ph.D. MD/Ph.D. program. The admission scores have been listed as the top three of the 264 master degrees programs and 181 doctoral degrees programs in Zhejiang University, which ranks among the top three in all Chinese Universities. Historical experience and the advanced experience of Western countries are adopted as a reference to build up a surgical education system with Chinese characteristics to achieve a continuous, sound, and rapid development of surgical education in ZJU, China. However, there are differences relating to the healthcare systems of China and Western countries. These differences present major challenges for the internationalization of surgical education. This article may facilitate the process of surgical training development in a global context.
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Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.