CN105797155A - Application of B7x in preparing anti-glioma medicine - Google Patents

Abstract

The invention belongs to biological and medical fields and the field of cancer therapy, and discloses an application of B7x as a glioma immunotherapy target. The invention provides an application of the B7x in preparing an anti-glioma medicine, and the B7x is a family member of a co-simulating molecule B7. The invention also provides a kit for detecting the B7x and an application of the kit. The B7x can be used as a drug target and a diagnostic marker for glioma. By virtue of the target, the targeted therapy of the glioma can be effectively achieved, a curative effect can be improved, and meanwhile toxic and side effects can be reduced.

Description

B7x purposes in preparing anti-glioma medicament

Technical field

The invention belongs to therapeutic field of tumor, specifically, the present invention relates to B7x purposes in preparing anti-glioma medicament.Described B7x can be used in a kind of new way of glioma immunotherapy.This approach is by blocking B7x developed by molecule thus releasing the immunosuppressive condition caused by B7x, and then activates body immune system (including tumor by local and whole body), reaches the effect of antagonism glioma.B7x can provide new target spot for the immunization therapy of patients with gliomas.

Background technology

Glioblastoma is the Primary intracranial tumor that in adult, grade malignancy is the highest, sickness rate is the highest.Clinical symptoms can have the various intracranial mass lesion effects such as headache, vomiting, papilloedema, epilepsy.One after diagnosing, about about one year life cycle of Most patients.

Current glioma tumor mainly has three big therapeutic modalities: operation, chemotherapy, radiotherapy, but the prolongation effect for the survival of patients phase is not notable.Being likely to be due to tumor cell on the one hand infiltrate, shift, there is radiation and chemotherapy resistance in Partial tumors cell on the other hand, causes that glioma can not be eliminated completely, and relapse rate is high.The research of immunotherapy is quite popular in recent years, the listing of anti-PD-1 medicine nivolumab and produce effects and also allow everybody have further confidence for tumour immunotherapy.Tumor cell, by activating body immune system, when avoiding autoimmune to occur, is effectively attacked by immunotherapy, reaches suppression tumor growth and even disappears, removes the effect of residual.Simultaneously, owing to immunocyte has relative specificity for the tumor cell being considered foreign body, in mechanism, the toxic and side effects of normal tissue to be substantially reduced compared to classic chemotherapy method, and therefore immunization therapy is expected to become the fourth-largest treatment means of glioma.

Dendritic cell is the professional antigen presenting cells in body, gains the name to stretch out many dendron samples or pseudo-Microfilament.Dendritic cell is efficiently offered antigen and activates T cells response.Dendritic cell vaccine is then a kind of extension of tumor vaccine, make use of tumor associated antigen (TAAs) to stimulate dendritic cell, and promotes that specific effector T cell is formed, the effect of its attack of the latter and dissolving tumor cell.Dendritic cell vaccine, has attempted in the clinical trial of multiple glioblastomas, but result not as everybody is willing to.In glioma immunization therapy, one of them main obstacle is that glioma self can induce immunosuppressant widely, trace it to its cause, there is scholar it is believed that the immunosuppressive agent secreted by regulatory T cells hinders producing effects of immunization therapy in being circulated by tumor cell or system, also viewpoint is had to think, in the tumor microenvironment of local, cell plays an important role in the malignant progression and treatment tolerance of tumor with intercellular connecting each other.It is reported that, tumor-infiltrated macrophage/microglia can support cell transformation to tumor, and these cells then can suppress T cell immunity.Although some excreted factor such as TGF-β, COX-2andIL-10 and some Co inhibitor have been found to play an important role in the immunomodulating of glioblastoma, but in tumor microenvironment, the accurate molecular mechanism regulating path and cell-cell interaction of these excreted factor is still in exploration.

Summary of the invention

Present invention aim at, it is provided that B7x purposes in preparing anti-glioma medicament.The present invention is by the clinical meaning of B7x molecule, immune suppression function in research glioma and the molecular pathways regulating and controlling its expression, result shows that B7x can as glioma immunization therapy novel targets, by blocking B7x developed by molecule, body's immunity can be activated, reaching anti-glioma effect, the immunization therapy for patients with gliomas provides practicable foundation.

To achieve these goals, inventor has probed into " surgical removal tumor+dendritic cell vaccine " treatment adopted for patients with gliomas.It is known that the T cell that B7 family and receptor CD28 family thereof participate in stimulates altogether plays vital effect in regulatory T-cell response with co-suppression, thus, these paths just become very promising therapy target.The B7 family member being currently known includes B7-1 (CD80), B7-2 (CD86), B7h (CD275), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC, CD273), B7-H3 (CD276), B7x (B7-H4/B7s1) and HHLA2 (B7-H5/B7h7), and CD28 family includes CD28, CTLA-4 (CD152), ICOS (CD278), PD-1 (CD279) and CD28H (TMIGD2/IGPR-1).

I type transferring film protein B 7x (being also called B7-H4 or B7S1), is that T cell stimulates and a member in co-suppression B7 family altogether.Functionally, B7x can transmit negativity and regulate signal to T cell, effectively suppresses it to activate, breed and cause CD4+ and CD8⁺The clonal expansion of T cell.Studies have found that, the kinds of tumors tissues such as human prostate cancer, ovarian cancer, pulmonary carcinoma, breast carcinoma, cancer of pancreas, renal cell carcinoma, the esophageal carcinoma and skin carcinoma being found that, B7-H4 expresses compared with normal tissue and raises, and the poorer prognosis of the rising of this index and patient has dependency.Additionally, can detect that solubility B7-H4 in the patients serum of the thin cancer of ovarian cancer and kidney, indicate that perhaps this can become a new biomarkers.In glioma early-stage Study, inventors also found that B7-H4 can express and relevant with tumor stem cell (YaoYu, etal.JofNeuro-oncology, 2008) in glioma.

The present invention separates CD133 from samples of human glioma⁺Glioma stem cells, and therefrom extract the dendritic cell that tumor cell associated antigen (SAAs) is separated from peripheral blood for sensitization, make dendritic cell vaccine.Being expelled in patients with gliomas body by the mode of intramuscular injection by this vaccine, efficacy analysis draws, life cycle, the patient populations more than 6 months increased to some extent.Using further B7x expression in immunohistochemical analysis cerebral tissue show, it is short that B7x expresses many survival of patients phases, and B7x to express the low survival of patients phase long, it was demonstrated that there is dependency in the clinical efficacy of B7x and dendritic cell vaccine.

Then research is found that B7x is at CD133⁺Glioma stem cells and glioma are invaded all has distribution on profit macrophage/microglia, and inventor sounds out CD133⁺Glioma stem cells and glioma invade the information between profit macrophage/microglia transmits and talk with the expression for respective B7x whether have regulating and controlling effect, specifically, by finding based on the multiple-factor detection technique (FlowCytomix) of flow cytometer, neutralizing antibody, the research of Transwell Chemotaxis test, CD133⁺Cell can pass through to produce cytokine such as IL-6 and IL-10 induction gum tumor infiltrating macrophages/microglia and express B7x, and by flow cytometry (FACS), immunofluorescence and immunoblotting (westernblot) scientific discovery, glioma is invaded profit macrophage/microglia and can be induced and by IL-6 from main regulation CD133⁺The B7-H4 of glioma cell expresses；Research display, described both sides can stimulate the other side to raise the expression of B7x by secrete cytokines.

In order to probe into the relation of B7x and tumour immunity further, the application probes into external respectively in vivo for the function of B7x on B7x positive macrophage.By above it will be appreciated that, use CD133⁺Glioma stem cells (GSC) supernatant can make B7x positive macrophage after processing macrophage.Thus experimental design is by Healthy People macrophage, CD133⁺The macrophage of GSC supernatant process, CD133^-GSC supernatant processes macrophage and co-cultures with fluorescence E.coli granule respectively, the observation of cell phagocytosis situation to the polystyrene latex particulate of red fluorescence labelling under fluorescence microscope, find that the cell granulations of B7x positive cell phagocytosis is considerably less than other two groups, it was shown that CD133⁺Supernatant can suppress the phagocytosis of macrophage.Then, the present invention use shRNA rotaring dyeing technology carry out the expression of reticent macrophage B7x, and this macrophage and T cell are co-cultured, immunologic function by Flow cytometry T cell, it has been found that B7 silence can make T cell multiplication capacity rise, t cell proliferation reduces, lethal cytokine-expressing increases.What is more, CD133⁺The B7x that supernatant is cultivated⁺Macrophage can make T cell that the killing ability of tumor cell is decreased obviously, but after blocking B7 expression, namely this depression effect relaxes.In sum, CD133⁺Glioma stem cells can pass through to regulate the expression mediated immunity of B7x on macrophage and suppress.

The present invention has carried out experiment in vivo, adopt primary human glioma cells to set up NOD-SCID Mus intracranial and subcutaneous tumor model, Mus is divided into 5 groups: blank group (has an intravenous drip of physiological saline), treatment group 1 (defeated T cell treatment), treatment group 2 (defeated T cell+normal macrophages), treatment group 3 (defeated T cell+CD133⁺Supernatant cultivate macrophage), treatment group 4 (defeated T cell+CD133⁺Macrophage+silence the blank that supernatant is cultivated), (defeated T cell+CD133⁺Macrophage+silence B7x that supernatant is cultivated).Measuring tumor size, respectively organize the life span of tumor-bearing mice, result show, immunization therapy is worst to the effect of the mice of B7x positive macrophage group, and the tumor stasis of the macrophage group mice of silence B7x grows.

Continuing in the present invention mechanism of microglia/Expression of Macrophages B7x is studied, result display IL-6 raises B7x possibly through Stat3 path；3 example normal cerebral tissues, 13 example Low grade glioma specimen, 12 example High Grade Gliomas specimen are carried out homogenate by experiment, ELISA is adopted to detect the concentration of wherein IL-6, IL-10, result shows, IL-6 concentration in High Grade Gliomas is significantly higher than Low grade glioma, and both of which is apparently higher than normal cerebral tissue；In peripheral blood macrophage, BV-2 microglia cell line and RAW macrophage cell line, showed cell B7x albumen, phosphorylation Stat3 protein expression and IL-6 stimulate in time positive correlation and dosage positive correlation, simultaneously, the microglia cell line of experimental construction B7x gene knockout, find after Stat3 gene deregulation, B7x albumen and phosphorylation Stat3 down-regulated expression；Further by chromatin immune co-precipitation (ChIP) and the checking of luciferase plasmids reporter assay, the Stat3 of phosphorylation can pass through in conjunction with B7x gene promoter site thus regulating B7x protein expression.

Result of the test shows, B7x can express in cerebral glioma, and and CD133⁺Glioma stem cells is relevant, and small-scale DC vaccine clinical result of the test shows, B7x expression in patient's samples of human glioma is more high, and to represent the prognosis of patient more poor.Mechanism Study shows, CD133⁺Glioma stem cells and glioma invade the expression of the information transmission between profit macrophage/microglia and dialogue controllable each B7x；CD133⁺Glioma cell can raise the expression of B7x on macrophage/microglia, and then mediated immunity suppresses；And by suppressing B7x to express, then can promote that the tumor growth that T cell mediates is stagnated.Further, result shows CD133⁺Information transmission is carried out relevant with IL-6 and IL-10 with the concrete mechanism of dialogue between glioma cell with macrophage/microglia, IL-6 has raised Stat3 expression by JAK/STAT approach, the latter is attached in the promoter of coding B7x gene as transcription regulatory factor, promote B7x to express, ultimately result in immunosuppressant.Therefore, described B7x as immunotherapeutic targets, can activate immunity and cause that tumor growth stagnation is even disappeared, contributing to providing new direction for glioma immunization therapy by blocking B7x molecule.Additionally, B7x can participate in the primary dcreening operation index of immunization therapy as patients with gliomas.The present invention for patients with gliomas therapeutic scheme selection, reduce recurrence, the survival rate etc. that improves patient is all significant.

The present invention completes on the basis of the above.

The invention provides B7x purposes in preparing anti-glioma medicament, described B7x is costimulatory molecules B7 family member.

People B7X is NM_001253849.1 in the accession number of NCBI, and albumen is referring to NP_001240778.1, V-setdomain-containingT-cellactivationinhibitor1isoform2.

B7x can as the diagnostic flag of glioma.

The active component of described anti-glioma medicament is the preparation blocking glioma B7x developed by molecule or releasing immunosuppressive condition caused by B7x.

Present invention also offers a kind of anti-glioma medicament, the active component of described anti-glioma medicament is the preparation blocking glioma B7x developed by molecule or releasing immunosuppressive condition caused by B7x.

In one embodiment of the invention, the active component of described anti-glioma medicament is the antibody of the antibody of IL-6 or IL-10.

Described anti-glioma medicament, by blocking glioma B7x developed by molecule, releasing the immunosuppressive condition caused by B7x, thus activating immunne response, including tumor by local response and whole body response, reaching the effect of anti-glioma.

Present invention also offers a kind of test kit detecting glioma, containing the reagent detecting B7x expression in this test kit.Utilize this test kit detection B7x expression, provide evidence for the clinical stages of glioma, glioma patient's prognosis or glioma canceration process further.

Possibly together with the standard sample of B7x or molecular weight marker in described test kit.

Such as, containing the amplimer or the Binding peptide that detect B7x in described test kit.

The present invention relates to therapeutic field of tumor, the B7x application as glioma immunotherapeutic targets has been opened in public invention.Research finds, B7x depends on JAK/STAT approach and expresses, and is immunosuppression molecule important in glioma microenvironment；And CD133⁺Between glioma stem cells and macrophage/microglia, information transmission and dialogue, having promoted the expression of respective B7x, thus having mediated immunologic escape, having become the obstacle of glioma immunization therapy.By the expression of reticent macrophage/microglia B7x, immune cell function can be strengthened in vitro, in vivo Transplanted tumor model then occur tumor growth stagnate, life span extension, it was shown that B7x can provide new target spot for glioma immunization therapy.Utilize this target spot can treat glioma by efficient targeting, improve curative effect and reduce toxic and side effects simultaneously, for carrying out the immunization therapy of glioma or even developing new individual comprehensive therapy mode, significant.

Accompanying drawing explanation

After Fig. 1: DC vaccine therapy, the relation of clinical effectiveness and patient's samples of human glioma B7x expression.

Fig. 2: CD133⁺Cell and CD133^-The kind of various cytokines and concentration in cell conditioned medium liquid.

Fig. 3: CD133⁺The relation of B7x expression and IL-6, IL-10 on the macrophage that cell conditioned medium liquid is cultivated.

Fig. 4: under In vitro culture, T cell function and the macrophage B7x relation expressed.

Fig. 5: in internal Transplanted tumor model, the relation that mouse tumor size, survival rate and macrophage B7x express.

Fig. 6: Westernblot detection macrophage B7x albumen, phosphorylation Stat3 protein expression and the IL-6 time stimulated and dosage correlation.

Fig. 7: Westernblot detection has transfected the expression of B7-H4 albumen, phosphorylation Stat3 albumen after IL-6 stimulates of the macrophage of ShRNA.

Fig. 8: chromatin immune coprecipitation (ChIP) detects the relation of B7x promoter sequence, Stat3 albumen and IL-6.

The expression of Fig. 9: B7x depends on JAK/STAT approach schematic diagram.

Detailed description of the invention

According to the research of tumour immunity and the present invention early-stage Study to Treatment for Glioma, the immunization therapy of glioma has wide potentiality equally.Therefore B7x molecule is expected to the target spot as glioma immunization therapy, for immunotherapy of patients scheme selection so that include operation, chemotherapy, radiotherapy and immunization therapy individual comprehensive therapy provide practicable foundation, meanwhile, this is also significant for glioma Basic Research Results is transformed into clinical practice.

In the present invention, block glioma B7x developed by molecule, release the immunosuppressive condition caused by B7x, it is possible to activate immunne response, including tumor by local response and whole body response, reach the effect of anti-glioma.

And CD133⁺Glioma stem cells and glioma invade the expression of the information transmission between profit macrophage/microglia and dialogue controllable each B7x.CD133⁺Glioma cell can pass through to secrete IL-6 and raise the expression of B7x on macrophage/microglia, and then mediated immunity suppresses.

Further study showed that, the expression of B7x depends on IL-6/STAT3 approach, and IL-6 can pass through JAK/STAT approach and raise Stat3 expression, and Stat3 is as transcription regulatory factor, it is attached in the promoter of coding B7x gene, promotes B7x to express, ultimately result in immunosuppressant.

The present invention does with respective drawings in conjunction with the embodiments and explains explanation further, and following example are for illustration purposes only, and are not used in the restriction scope of the invention.

After embodiment 1DC vaccine therapy, there is relation in the clinical effectiveness of patient and samples of human glioma B7x expression

The inclusion criteria of DC vaccine therapy: the recurrent malignant patients with gliomas that histopathology confirms；Postoperative Contrast-enhanced MRI confirms that tumor excises >=80% in maximum safety range；Karnofsky marks >=60 points；Good organ dysfunction, as follows: sufficient marrow function deposit: leukocyte >=3.0 × 10⁹/ L, platelet >=100 × 10⁹/L；Hemoglobin>=9g/dL, liver: aspartate transaminase (AST) and alanine aminotransferase (ALT)<2 times of upper limits of normal, kidney: the serum creatinine of normal range；

The preparation of DC vaccine: separate CD133 from fresh samples of human glioma⁺Glioma stem cells, forms and extracts tumor associated antigen (SAAs) through x-irradiation (6Gy), for the dendritic cell that sensitization is separated from peripheral blood, makes specific dendritic shape cell vaccine (DC).In surrounding after recurrent glioma operation in patients, this DC is expelled in patients with gliomas body by the mode that triangular muscle is injected.Inject weekly once.

Immunohistochemical staining step is as follows:

Dewaxing and aquation: cerebral tissue paraffin section is placed in 30min in dimethylbenzene I, 20min in dimethylbenzene II, 10min in dimethylbenzene III, dehydrated alcohol soaks 5mins, 95% ethanol soaks 5min, 80% ethanol soaks 5mins, soaking 5min in 60% ethanol, single steaming soaks 5min in water.

Antigen retrieval: taking a certain amount of CB buffer in a high-temperature resistant container, put in microwave oven, power10 × 2min, so as to seethe with excitement.Then section is carefully placed into wherein, more slowly puts into heating, power3 × 5min in microwave oven.Finally, room temperature natural cooling 30-40min it is placed on.

Dyeing: take out section, TBS rinses 4-5 time, and drying is cleaned.Adding 30ulH2O2, close 30min in the wet box of room temperature, drying is cleaned.Dropping mouse-anti people B7-H4 primary antibodie (1:50, AbDSerotec) 40ul, 4 DEG C are overnight, TBST rinses 4-5 time, drips biotinylation two anti-20ul, incubated at room 1h, TBS rinses 4-5 time, drips enzyme mark Avidin 20ul, incubated at room 30min, TBS rinses 4-5 time, rinses well rapidly, drip 1 hematoxylin after dropping 30ul substrate is painted, 5-10min, dehydration, mounting, microscopy.

This research screening recurrent glioma patient accepts " tumor+DC vaccine is won in operation " treatment, hereafter observes 8 patients, and wherein 3 survival of patients phases were more than 6 months, and 5 survival of patients phases were less than 6 months.Consider that the median survival interval of recurrent gliomas patient was lower than 6 months, therefore life cycle is represented respectively less than 6 months with more than 6 months short life cycle and long life cycle.The samples of human glioma of these patients with recurrents is made immunohistochemical staining, tissue B7-H4 is expressed and the analysis that takes statistics life cycle.

With reference to Fig. 1, brown color dyeing prompting B7x positive cell, contrast statistics draws, after dendritic cell vaccine is treated, it is short that B7x expresses many survival of patients phases, and B7x to express the low survival of patients phase long.

Embodiment 2 detects CD133 based on the multiple-factor detection technique (FlowCytomix) of flow cytometer⁺The cytokine kind of supernatant and concentration.

Coated 96 orifice plates of antibody add degree CD133⁺,CD133^-Cell conditioned medium liquid, in duplicate, after the specific antibody and supernatant cytokine that are combined with fluorescence polystyrene microsphere have been hatched, adds biotin and streptavidin-PE.Sample is excited by flow cytometer 690nm light subsequently, and uses FlowCytomixPro2.4 software analysis, and the cytokine detected includes MIP-1 α, MCP-1, MIP-1 β, IL-8, RANTES, MIG, IL-6, IL-10, GM-CSFandIL-4.

With reference to Fig. 2, CD133⁺Cell and CD133^-The concentration of various cytokines in cell conditioned medium liquid, wherein IL-6, IL-10, MIP-1 α andMCP-1 concentration is higher.

Embodiment 3CD133⁺The relation of B7x expression and IL-6, IL-10 on the macrophage that cell conditioned medium liquid is cultivated

Macrophage is cultivated: macrophage is placed in following 4 groups of culture fluid and cultivates 72 hours: group 1 (CD133⁺GSC supernatant), group 2 (CD133⁺GSC supernatant+anti-IL-6 monoclonal antibody), group 3 (CD133⁺GSC supernatant+anti-IL-10 monoclonal antibody), group 4 (CD133⁺GSC supernatant+anti-IL-6 monoclonal antibody+anti-IL-10 monoclonal antibody), wherein monoclonal antibody is anti-IL-6:clone6708；500ngml-1)andIL-10(anti–IL-10,clone23738；50ngml-1)

Flow cytometry: 100 μ lPBS re-suspended cells, adds the 5 μ lAPC anti-human CD11b antibody combined and the anti-human B7-H4 antibody (1:5, eBioscience) of 5 μ lPE combination, hatch 15min for 4 DEG C, PBS washes 2 times, 300 μ l paraformaldehyde re-suspended cells, upper machine testing.

With reference to Fig. 3, being simultaneously introduced anti-IL-6 and anti-IL-10 antibody can make B7-H4 down-regulated expression on macrophage, lower modulation is more than independent anti-IL-6 or IL-10, it is known that, CD133⁺In cell conditioned medium liquid, IL-6 or IL-10 can raise macrophage B7x expression.

Under embodiment 4 In vitro culture, the relation that T cell function and macrophage B7x express.

Beyond blank group, the macrophage of pretreatment is longitudinally divided into 5 groups: group 1 (Healthy People macrophage), group 2 (CD133⁺GSC supernatant cultivate macrophage), group 3 (CD133^-GSC supernatant cultivate macrophage), group 4 (CD133⁺The macrophage+silence that GSC supernatant is cultivated is blank), organize 5 (CD133⁺Macrophage+silence B7x that GSC supernatant is cultivated), often group macrophage and the CTL of tumour-specific are by the mixing of following proportioning, namely laterally it is divided into 4 groups again: 0:1,0.1:1,0.2:1,0.5:1, the U87MG glioma cell of these 20 groups of cells Yu CSFE labelling is co-cultured (10:1), after 72 hours, annexin V/PI is adopted to detect death or the apoptosis percentage ratio of U87MG glioma cell.

With reference to Fig. 4: at CD133⁺The B7x cultivated⁺Macrophage can suppress antineoplastic T cell function, and after retardance B7x, this suppression is eased, and tumor mortality and apoptosis rate rise.

In embodiment 5 body in Transplanted tumor model, the relation that mouse tumor size, life cycle and macrophage B7x express

Primary people's U87MG glioma cell is adopted to set up NOD-SCID Mus intracranial and subcutaneous tumor model.Mus is divided into 5 groups: blank group (has an intravenous drip of physiological saline), treatment group 1 (defeated T cell treatment), treatment group 2 (defeated T cell+normal macrophages), treatment group 3 (defeated T cell+CD133⁺Supernatant cultivate macrophage), treatment group 4 (defeated T cell+CD133⁺Macrophage+silence the blank that supernatant is cultivated), treatment group 5 (defeated T cell+CD133⁺Macrophage+silence B7x that supernatant is cultivated).Wherein, macrophage uses after different disposal 12 days.Measure tumor size, respectively organize the life span of tumor-bearing mice.With reference to Fig. 5, the mice tumors grew having injected normal macrophages and reticent B7x macrophage is stagnated, and the survival time of mice having injected reticent B7x macrophage significantly extends.

Embodiment 6Westernblot detects macrophage B7-H4 albumen, phosphorylation stat3 protein expression and the IL-6 time stimulated and dosage correlation

BV-2 microglia is divided into process in 12 hours and processes two big groups in 24 hours, is sub-divided into three groups in group, uses IL-6 to stimulate cell, and concentration is 0ng/ml, 12.5ng/ml, 24ng/ml respectively.Two groups of treated 12 hours and 24 h before harvest cells, detect the expression of cell protein with Westernblot.

Prepared by protein example: cerebral tissue adds homogenate buffer, machinery or ultrasound wave room temperature homogenate 0.5-1min after shredding.Then 4 DEG C, 12,000g centrifugal 15min.Take supernatant as sample.

Electrophoresis: preparation SDS-PAGE running gel, adds sample-loading buffer, after being cooled to room temperature, protein sample loading to gel is carried out electrophoresis in sample.Transferring film: adhesive tape is cut to suitable size after terminating by electrophoresis, balances with transferring film buffer, 5min × 3 time.Cut out in advance and an equal amount of filter paper of adhesive tape and NC film, immerse 10min in transferring film buffer.Membrane-transferring device from bottom to up successively by carbon anode plate, 24 metafiltration paper, NC film, gel, 24 metafiltration paper, negative electrode carbon plate order put well, filter paper, gel, NC film Accurate align, each step remove bubble, upper ballast, liquid unnecessary on carbon plate is blotted.Switch on power, constant current 300mA, shifts 1.5h.After transfer terminates, deenergization takes the film out.

Antibody incubation: cut film bar to be measured, wash film, 5min × 3 time with 0.01MPBS.Add confining liquid, steadily shake, room temperature 2h.Abandon confining liquid, wash film, 5min × 3 time with 0.01MPBS.Add the anti-human B7-H4 primary antibodie (1:5000, Epitomics) of rabbit, place more than 12h for 4 DEG C.Negative control, replaces primary antibodie with 1%BSA, and all the other steps are identical with experimental group.Abandon primary antibodie and 1%BSA, wash film, 5min × 4 time with 0.01MPBS respectively.Add the two of horseradish peroxidase to resist, steadily shake, room temperature 2h.Abandon two to resist, wash film, 5min × 4 time with 0.01MPBS.

Colour developing: add nitrite ion, prepare film in darkroom.

With reference to Fig. 6, according to shade it can be seen that cell B7x albumen, phosphorylation stat3 protein expression stimulate in time positive correlation and dosage positive correlation with IL-6.

Embodiment 7Westernblot detection has transfected the expression of B7x albumen, phosphorylation Stat3 albumen after IL-6 stimulates of the macrophage/microglia of ShRNA.

The little hair fastener shRNA sequence designed for Stat3 and mock sequence are inserted in PLKO carrier, and PLKO is packaged into lentiviral particle, by slow virus carrier, shRNA is transfected in BV-2 microglia, stably by STAT3 Knockdown.After giving suitable IL-6 stimulation, Westernblot is used to detect the expression of B7x albumen, Stat3 albumen, phosphorylation Stat3 in cell.

With reference to accompanying drawing: although by the stimulation of IL-6, but ShRNA still significantly suppress the expression of macrophage B7x albumen, phosphorylation Stat3 albumen

Embodiment 8 chromatin immune coprecipitation (ChIP) detects the relation between B7x promoter sequence, phosphorylation Stat3 albumen and IL-6

Chromatin immune co-precipitation test is for analyzing the phosphorylation Stat3 albumen combined with B7x promoter sequence.Cell DNA, after formaldehyde treated, is decomposed into the small fragment of 200-1000bp by BV-2 microglia through Sonication.DNA fragmentation and IgG and the hatching of anti-Stat3 antibody, precipitation, collect after, carry out PCR detection.PCR forms according to the design data of UCSC.

With reference to Fig. 8: BV-2 microglia through IL-6 stimulate after, with B7x promoter sequence directly in conjunction with phosphorylation Stat3 albumen dramatically increase.

Expression with reference to Fig. 9: B7x depends on JAK/STAT approach schematic diagram.

Above content described in this specification is only illustration made for the present invention.Described specific embodiment can be done various amendment or supplements or adopt similar mode to substitute by those skilled in the art; without departing from the content of description of the present invention or surmount the scope that present claims book is defined, protection scope of the present invention all should be belonged to.

Claims (10)

1.B7x purposes in preparing anti-glioma medicament, it is characterised in that: described B7x is costimulatory molecules B7 family member.

2. purposes as claimed in claim 1, it is characterised in that: B7x is the diagnostic flag of glioma.

3. purposes as claimed in claim 1, it is characterised in that: the active component of described anti-glioma medicament is the preparation blocking glioma B7x developed by molecule or releasing immunosuppressive condition caused by B7x.

4. an anti-glioma medicament, it is characterised in that the active component of described anti-glioma medicament is the preparation blocking glioma B7x developed by molecule or releasing immunosuppressive condition caused by B7x.

5. anti-glioma medicament as claimed in claim 4, it is characterised in that: the active component of described anti-glioma medicament is the antibody of the antibody of IL-6 or IL-10.

6. anti-glioma medicament described in claim 4, it is characterized in that: described anti-glioma medicament, by blocking glioma B7x developed by molecule, releases the immunosuppressive condition caused by B7x, thus activating immunne response, including tumor by local response and whole body response, reach the effect of anti-glioma.

7. the test kit detecting glioma, it is characterised in that containing the reagent detecting B7x expression in described test kit.

8. test kit as claimed in claim 7, it is characterised in that possibly together with the standard sample of B7x or molecular weight marker in described test kit.

9. test kit as claimed in claim 7, it is characterised in that containing the amplimer or the Binding peptide that detect B7x in described test kit.

10. the test kit described in claim 7 is being used for detecting B7x expression, and measures the purposes in the clinical stages of glioma, glioma patient's prognosis or glioma canceration process.