| Glioma is the most common intracranial malignant disease. The prognosis and outcome are poor because of tumor infiltrating brain parenchyma and lacking effective treatment. The median survival is about 10-15 months. Seeking biological therapy methods is important apart from the traditional surgical operation, chemotherapy, and radiotherapy. Now brain is not thought as immunologic privileged. Several lymphocyte circulation routes are clarified and brain blood barrier is often compromised of glioma. Glioma cells could express MHC molecule and antigen presenting cell (APC) exist in glioma tissue, therefore making it possible that immunology reactions happen in brain.Many immunotherapy research about glioma are in progress. Among those, depending on effective T cells is the most promising means as it could target tumor cells. The outcome is not optimistic till now owing to the difficulty of finding tumor special antigen (TSA) and the immunosuppressive mechanism existing in glioma patients. Immunosuppressive cells and cytokines secreted by glioma could hamper the effective immunotherapy. Many immunosuppressive cytokines such as TGF-??IL-6, IL-10, PGE2 etc. exist in peripheral blood and tumor tissue of glioma. In addition, there is an unbalance of T helper subset. It shows low Thl type IFN-y and high Th2 type IL-6, Tregs etc.Th17 is still unknown in glioma till now. Th17 emerges as a new arm of adaptive immunology. It was discovered from research about autoimmune disease. It is a new member of CD4+ family co-expressing CD4 and IL-17 and Naive CD4+ cells differentiate into Th17 by activating transcription factor RORC under influence of TGF-?and IL-6. Other cytokines such as IL-23, IL-1?were concerned with Th17 differentiation and function. Thl7 has been investigated deeply in autoimmune disease. It can defense infection and induce sustained inflammation through IL-17. Th17 is now been paid attention of its function in tumor. Th17 could promote tumor at least through:(1) promotion of angiogenesis through the upregulation of VEGF, CD31, etc.; (2) promotion of tumorigenesis through the IL-6-STAT3 signaling pathway; (3) downregulation of IL-12R?2 then impairing Thl function; and (4) co-expression with CD8, thereby making CTLs lose their cytotoxic effect. Cytokines of IL-6, TGF-?, IL-23, IL-1?existing in tumor microenvironment could pilot Th17 differentiation and other cytokines such as RANTES, MCP-1 could recruit Th17 thereby make Th17 aggregated in tumor site and in turn may promote tumor progress. Therefore clarifying expression, differentiation mechanism and possible function of Thl7 in glioma is essential to understanding the T helper subset status, especially immunosuppressive part of glioma. It is indispensable to search effective immunotherapy. This study wants to answer the questions brought up above. Reliable results was obtained and listed below.(1). Expression pattern of Th17 in gliomaThis part wants to know expressions of Th17 and its related cytokines in glioma. Thl7 volume and serum concentrations of IL-17, IL-23, IL-6, TGF-?, IFN-?were measured from 35 glioma patients and 20 healthy donors. Meanwhile,24 glioma tissues and 5 trauma tissues were measured of mRNA of IL-17, RORC, IL-23, IL-6, TGF-?, IFN-y, IL-1?. Results showed that volume of IL-17+CD4+/CD4+ cells, serum concentrations of IL-17, IL-23, IL-6, TGF-?, IFN-y were not with statistical significances between glioma and healthy donors, nor in grades of glioma. IL-17 and RORC mRNA were high positive ratios in glioma tissues with 19/24 and 18/24 respectively. However, these two cytokines were negative in 5 trauma tissues. IL-23 was negative in all samples but IL-1?was nearly all positive in all samples. IL-6, IFN-y were positive with different degrees and have no statistical significance between groups. TGF-P was higher in trauma and grade II compared to grade IV glioma (p<0.05), though no statistical difference between the former two groups. Further,15 mRNA IL-17 positive glioma samples were positive of intercellular or intracellular immunohistochemical staining for IL-17.(2). Effect of IL-17 in U87MG xenograft tumorigenesisWhen results showed IL-17 was higher in glioma, then this study wants to explore what exact function of IL-17 exert on tumor formation by using glioma cell line xenograft in nude mice. First, eukaryotic expression vector pEGFP-N1-IL-17 was constructed and transferred into U87MG cell lines, with vector pEGFP-N1 as a parallel contrast. Monoclone of positive transferred cells was shifted by limiting dilution and afterwards augmented cultured, then IL-17 expression was verified by fluorescence detection, Real time PCR and ELISA. Cells of pEGFP-N1-IL-17-U87MG?pEGFP-N1-U87MG?U87MG were adjusted to 5×105 then inoculated s.c. to 6 weeks nude mice (10 mice each group). Tumor volumes were measured on 32D,37D, and 39D. Results showed that only 3/5 mice were burdened with tumor of pEGFP-N1-U87MG group, and tumor volume less than the other 2 groups at all 3 times (p<0.01). All mice of groups pEGFP-N1-IL-17-U87MG and U87MG were burdened with tumor. Tumor volumes of pEGFP-N1-IL-17-U87MG group were larger than U87MG at all 3 the times but only has statistical significance at 32D (p<0.05). At 39D, the volume of spleen changed similarly to tumor volume between the 3 groups. Spleen volume of pEGFP-N1-IL-17-U87MG, U87MG were larger than pEGFP-N1-U87MG (p<0.05). CD31 mRNA of pEGFP-N1-IL-17-U87MG group is higher than pEGFP-N1-U87MG and U87MG groups (p<0.01) but no difference between the latter two groups. Similar result is observed of microvascular density by immunohistochemistry staining (p<0.01). Then molecular changes were analyzed between the 3 cell lines. Chemokines of CXCL1, CXCL5, CXCL8, CXCL10, CXCL11, MCP-1(CCL2), RANTES(CCL5), CCL20, CCR4, and CCR6, immunology regulation factors of?2-MG, PD-L1, PGE2, TGF-?, IL-6, and STAT3, intercellucar adhesion molecules of MMP3, ICAM-1, VEGF were measured between pEGFP-N1-IL-17-U87MG, pEGFP-Nl-U87MG, and U87MG cell lines by Real time PCR. pEGFP-N1-IL-17-U87MG and pEGFP-N1-U87MG were higher than U87MG of CXCL10 and CCL20, however, pEGFP-Nl-U87MG was higher pEGFP-N1-IL-17-U87MG of MCP-1, and no difference between pEGFP-N1-IL-17-U87MG and U87MG. Differences of varying degrees were observed between the 3 cell lines of immunology regulation factors except IL-6. As for intercellucar adhesion molecules, pEGFP-N1-U87MG was higher of MMP3, ICAM-1 than the other 2 cell lines.(3). Differentiation effect on Naive CD4+cells exerted by culture supernatant of U87MG.Tumor cells and immunology system influence mutually. Our previous result confirmed IL-17 expression in glioma and further observed that IL-17 can promote U87MG xenograft tumorigenesis on nude mice. IL-17 mainly secreted by Th17 cells. Here the following work wants to know if culture supernatant of U87MG has any effect on differentiation of Naive CD4+ cells. PBMC (1×108) was bought from Shanghai Blood Center. PBMC was negative selected by immunomagnetic beads and afterwards Naive CD4+ cells counted about (1×106). U87MG culture supernatant was added to the Naive CD4+ culture medium as 1/5 volume. Naive CD4+ cells was then cultured on pre-coated anti-hCD3 plate and anti-hCD28 and IL-2 were added to the culture system. Culture medium was changed with same ingredient 3 days later and harvested cells were stained for Th1, Th2, Th17, and Treg 7 days later. Results showed that Th1, Th2 subset was lower of U87MG-Naive CD4+ than Naive CD4+ but only significant difference with Th2. Th17 and Treg cells were with no differences between the 2 groups.Our study showed that IL-17, RORC were with higher positive ratios in glioma tissues compared to trauma tissues. In addition, TGF-p was higher of trauma and grade II glioma than grade IV. Over expression of IL-17 can accelerate tumor formation of U87MG in nude mice. U87MG secreted molecules alone can't render Naive CD4+ cells differentiate towards Th17 cells. |
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