| Glioma is the most common CNS neoplasms with the characteristics of high degree of malignancy, short survival and high relapse rate. The most common site of gliomas is the brain. Gliomas make up about 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors. The glioma stem cells is a group of proliferation, self-renewal and differentiation of stem like cancer cells. Many studies showed that glioma stem cells take an important role in tumorigenesis and development, metastasis and recurrence and tolerance to radiotherapy and chemotherapy. B7-H6, a new member of B7 costimulatory molecule family, is a ligand of NK cell receptor NKp30, triggers the cytotoxic activity of NK cell. B7-H6 expressed in a variety of human tumors but not in healthy adult tissues. Current research showed that B7-H6 triggers NK cell activity and enhances the killing ability against target cell in vitro, while some studies reported that B7-H6 can inhibit NK cell activity by special mechanism. Therefore the expression and function of B7-H6 in tumor cells needed further investigation.This study first isolated glioma stem-like cells from U87 cell line, and the expression of B7 family were detected. Data showed B7-H6 not only at mRNA level but also on protein level was up-regulated in U87 derived GSCs compared with initial U87 cells. siRNA was generated to interfere the expression of B7-H6, and the down regulation of B7-H6 reduced cell proliferation and enhanced cytotoxicity of NK cells. Further more, the expression and clinical significance of B7-H6 was analyzed in clinical samples. This study revealed the expression and biological function of B7-H6 in gliomas, especially in glioma stem cells, which provides theoretical basis for glioma stem cell target immunotherapy.Part 1 Isolation and identification of U87 derived GSCsObjective: To enrich and identify glioma stem-like cells from U87 glioma cell line.Method: Taking advantage of the ability to form neurospheres in vitro, GSCs were isolated from U87 cell line using subcloning. The expression of stem cell markers(Sox2, CD133, Nestin, and CXCR4) was detected by RT-PCR or flow cytometry. Chemotherapy resistance of GSCs was tested by CCK-8 reagent by treating with three kinds of drugs with different doses.Results:(1) Glioma stem-like cells were isolated from U87 cell line with the ability to form neurospheres in vitro;(2) U87 derived upregulated several stem cell markers;(3) U87 derived stem-like cells were more resistant to chemotherapy.Conclusion: Glioma stem-like cells were isolated and identified from U87 glioma cell line.Part 2 B7-H6 expression was upregulated on U87 derived stem-like cells and associated with cell proliferation and NK cell cytotoxicityObjective: To investigate the expression of B7 family molecules on U87 derived stem-like cells and discuss the meaning of upregulation of B7-H6 on it, compared to on U87 cells.Methods: Expression of B7 family molecules were analyzed by RT-PCR and flow cytometry. siRNA was generated to downregulate B7-H6. After silencing, proliferation of GSCs was detected by CCK-8 and expression of c-Myc was measured by RT-PCR. Drug resistance of GSCs were measured by CCK-8 and NK cell cytotoxicity was performed with flow cytometry detecting.Results:(1) Expression of B7-H1, B7-H3, B7-H4 and B7-H6 was detected in GSCs, cytoplasm B7-H4 and total B7-H6 expression increased in GSCs compared to initial U87 cells;(2) Inhibition of cell proliferation and decrease of c-Myc was observed after B7-H6 silencing. However no significant effect of B7-H6 on the ability to drug resistance was observed in GSCs;(3) the cytotoxicity function of NK cells were increased while co-culture with B7-H6 silenced GSCs.Conclusion: B7-H6 were up-regulated abnormally in U87 derived glioma stem-like cells, and the expression of B7-H6 had the abilities of promoting cell proliferation and inhibiting NK cell cytotoxicity was proved by B7-H6 silencing.Part 3 Expression and clinic significance of B7-H6 in glioma tissuesObjective: To investigate clinic significance of B7-H6 in glioma samplesMethod: Expression of B7-H6 and CXCR4 were detected by flow cytometry in 6 different glioma cell lines; B7-H6 protein expression was detected by immunohistochemistry in glioma paraffin section; B7-H6 mRNA expression was detected by RT-PCR and the relevance to pathology grade was analyzed.Results:(1) In 6 glioma cell lines, B7-H6 expression was detected only on UW28 which showed similar expression pattern of CXCR4;(2) in paraffin sections, normal brain tissue didn’t express B7-H6, while in glioma samples, B7-H6 expressed on cell membrane and in cytoplasm;(3) In 76 glioma samples, B7-H6 mRNA was expressed much higher in grade IV glioma samples with significant difference from normal brain tissue and low grade samples(p<0.05).Conclusion: the expression of B7-H6 had clinic significance in glioma. |
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