| Object:Osteosarcoma is the most common primary bone malignant tumor. Currently, the essential treatment, in clinic, is surgical operation, supplemented by radiotherapy and chemotherapy. However, the curative effect is far from ideal. The cancer stem cell theory proposed that the root of recurrence and refractory of tumors are the existance of cancer stem cells(CSCs) in tumors. At present, the majority of scholars consider CD 133 and CD44 as osteosarcoma stem cell surface marker. Studies showed that on the cell surface of lung adenocarcinoma cancer stem cells, the abnormal expression of β1,6 branch of N-sugar chain increased. While on breast cancer, the lack of (31,6 branch of N-sugar chain could induce macrophage M1 phenotype conversion, which played a role in anti-tumor effect. However, the role of β1,6 branch of N- sugar chain in osteosarcoma has seldom been reported. Based on this, the study intends to detect the expression of N glucosides on the cell surface of osteosarcoma cancer stem cells, and discuss its effect on the macrophage phenotype conversion, so as to further illuminate the tumor immune escape mechanism of osteosarcoma, and lay the foundation for osteosarcoma immune therapy.Method:1. Culture mouse osteosarcoma cell line LM8 by serum free culture method, and then obtain the preliminary osteosarcoma cell suspension ball. Get the CD 133 and CD44 double positive LM8 cells after two positive sorting by MACS method. Detect the expression of CD 133 and CD44 molecules on these cell surface after the cell sorting by flow cytometry.2. Use different doses of N glycosylation inhibitor -swainsonine to treat these osteosarcoma cancer stem cells, which are gotten from the cell corting. Detect the expression level of beta-1,6 branch of N- sugar chains on cell surface by lectins binding experiment.3. Obtain mice bone marrow cells, and then GM-CSF is added to induce monocytes to differentiate into macrophages. Eight days later, detect the expression level of specific F4/80 molecular on macrophage cell surface by flow cytometry.4. Co-culture osteosarcoma like tumor stem cells treated by the different doses of swainsonine with bone marrow-derived macrophages. Detect the expression level of the iNOS and Arg-1 RNA in macrophages by real-time fluorescence quantitative PCR method. Detect the arginase activity in the macrophage culture supernatant by arginase assay kit. Detect TNF-a and IL-10 content in the macrophage culture supernatant by ELISA.Result:1. Flow cytometry detection result showed that the proportion of the CD 133 and CD44 double positive LM8 cells in these cells which were cultured in the serum free culture mediun and then preliminary separated, and finally, cell sorted by the MACS method, is 96.5+1.2%. The result shows that we successfully isolated osteosarcoma like tumor stem cells2.. Lectin binding experiments showed the binding rate of osteosarcoma like tumor stem cells and L-PHA is higher, up to 90.3+2.1%, combined with the binding rate of LM8 cells and L-PHA (54.3+3.1%). The difference is statistically significant(P<0.05). Treated with 1 mg/mL swainsonine, the positive expression rate of beta-1,6 branch of N-sugar chain on the surface of osteosarcoma like cancer stem cells is 90%. While treated with 5mg/mL swainsonine, the positive expression rate is 21%. The above results show that with the increase of swainsonine doses, the positive expression rate of beta-1,6 branch of N-sugar chain on the cell surface of osteosarcoma like tumor stem cells decrease.3. Flow cytometry examination showed that after inducing cultured for 8-days, the F4/80 positive cell rate in bone marrow-derived macrophages, is 95.4%, which proves bone marrow-derived macrophages were cultured successfully.4.48 hours later, compared with macrophages alone, the expression of Arg-lmRNA in bone marrow macrophages co-cultured with osteosarcoma like tumor stem cells increased(40+2.6%v. s.4+2.6%, P<0.05), while the expression of iNOSmRNA had no significant difference (12+1.3%+4.6%v. s.8, P> 0.05); the content of IL-10 in the co-culture supernatant increased (150+6.8+3.8 pg/mL v. s.50 pg/mL, P<0.05), while the content of TNF-a had no significant difference(50+3.3+4.8pg/mLv. s.55pg/mL, P>0.05). The results show that co-cultured with osteosarcoma like tumor stem cells, the expression of M2 type biomarkers Arg-1 and IL-10 in macrophages increased, the expression of Ml type biomarkers iNOS and TNF-a had no significant difference, which suggests osteosarcoma like tumor stem cells can induce macrophages to differentiate into M2 type. Compared with untreated osteosarcoma like tumor stem cells> when we co-cultured osteosarcoma like tumor stem cells treated with swainsonine (lmg/mL) with bone marrow macrophages. the expression of M2 type biomarkers Arg-l(12+ 3. 1%v. s.40+2.6%, P<0.05) and IL-10 (90+4.4pg/mLv. s.150+6.8 pg/mL, P<0.05) in macrophages decreased, while the expression of M1 type biomarkers iNOS(50+2.1%v.s.12+1.3%, P<0.05) and TNF-a (240+8.1pg/mL v. s.50+3.3pg/mL, P<0.05) in macrophages increased, and with the increase of the swainsonine concentration, these changes were further enhanced.Conclusuon:Osteosarcoma like tumor stem cells highly express β1,6 branch of N-sugar chain, and swainsonine could reduce the expression of β1,6 branch of N-sugar chain on cell surface. Osteosarcoma like tumor stem cells can induce bone marrow-drived macrophages to differentiate into M2 type, when reducing the expression of N glycoside on cell surface of osteosarcoma like tumor stem cells with swainsonine, its ability of inducing macrophages to differentiate into M2 type decreased. Various inhibitors, aiming at inhibiting β1,6 branch of N-sugar chain synthesis, such as swainsonine, are expected to become a new assistant drug for the treatment of osteosarcoma. |
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