CN109182528A - A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method - Google Patents

Abstract

Biomedicine professional domain of the invention, and in particular to a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method.The present invention depicts function of the ITGB5 gene in glioma in glioma, and is prepared into kit, for evaluating the effect of poor prognosis and chemicotherapy.

Description

A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation reagent Box and its application method

Technical field

The invention belongs to biomedical professional domains, and in particular to a kind of glioblastoma based on ITGB5 gene is auxiliary Help diagnosis, prognostic evaluation kit and its application method.

Background technique

Glioblastoma (GBM) is most important primary malignant tumor in adult human central nervous's system (CNS).Even if Using maximum operation excision, radiotherapy and auxiliary Temozolomide chemotherapy, the median survival interval of GBM patient is only 12-15 months, Only about 10% GBM patient was survived more than 5 years.In recent years, the research for tumour immunity microenvironment is more and more, is immunized and treats Method also has become a kind of new strategy for solid tumor, and revolutionizes treatment method in some certain types of cancers. For example, immunologic test point inhibitor such as nivolumab and ipilimumab have made a part with the trouble of advanced melanoma Person can long-term surviving, and former, these patients can not be cured, and median survival interval is about 8 months.Immune response is broken Bad is the main feature of GBM, especially mesenchyma hypotype.The immunosupress that tumour mediates can prevent to eradicate GBM cell and promote Into tumour progression.The cell factor such as interleukins (IL) -10 secreted by tumour cell and the relevant non-tumor cell of tumour, IL-6 and colony stimulating factor 1(CSF-1) it plays an important role in above process.

Integrin is heterodimer cell surface receptor, is made of 18 α-subunits and 8 β-subunits, they adjust cancer In multiple biological function, including proliferation, adherency, migration and invasion；It simultaneously participates in and adjusts tumour immunity microenvironment, participate in blood Pipe is formed.It participates in a variety of integrins of progression of disease and its has attraction with what the correlation of oncogene became oncotherapy The target spot of power.ITGB5 is only related to ITG α V, related with a variety of pathological conditions by multinomial research.ITGB5 passes through raw with endothelium Growth factor receptor body 2(VEGF2) cooperation is to enhance cell survival, to inhibit exogenous cells apoptosis；It is found in kinds of tumors ITGB5 participates in angiogenesis.More and more evidences support ITGB5 promoting cancer cell migration, invasion and transforming growth factor β Key effect in the epithelium-mesenchymal transformation (EMT) of (TGF-β) induction.And effect of the ITGB5 in glioma is not known still Dawn, therefore, urgent need, which probes into glioma the effect of ITGB5 and develops marker, provides auxiliary for the diagnosis and treatment of glioblastoma.

More and more studies have shown that integrin related genes, especially ITGB5 gene, can be used to instruct patient's prognosis And evaluation therapeutic effect.Currently, being had not been reported about the kit for ITGB5 gene in glioma.The present invention depicts Function of the ITGB5 gene in glioma in glioma, and it is prepared into kit, for evaluating poor prognosis and putting The effect for the treatment of.

Summary of the invention

In view of the existing technical defect, invention broadly provides a kind of glioblasts based on ITGB5 gene Tumor auxiliary diagnosis, prognostic evaluation kit and its application method find ITGB5 gene in LGG(Low grade glioma) and GBM There are significant differences for transcriptional expression level in (glioma mother cells tumor).Compared with LGG tissue, ITGB5 gene is organized in GBM The middle significant up-regulation of expression can diagnose the pernicious journey of patients with gliomas by detecting the expression of ITGB5 gene transcription level Degree.Meanwhile the expression according to ITGB5 gene, it can be estimated that patient's prognosis situation and chemicotherapy effect.

For achieving the above object, the technical solution adopted by the present invention is as follows.

A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, including amplification ITGB5 The PCR primer pair of gene, the primer pair are.

Forward primer sequence is 5 '-GGAAGTTCGGAAACAGAGGGT-3 '.

Reverse primer sequences are 5 '-CTTTCGCCAGCCAATCTTCTC-3 '.

Preferably, the kit further includes the PCR primer pair for expanding house-keeping gene GAPDH, and the primer pair is.

Forward primer sequence is 5 '-GGAGCGAGATCCCTCCAAAAT-3 '.

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 '.

The kit further includes SYBR Green polymerase chain reaction system, the SYBR Green polymerase chain Reaction system include PCR buffer, dNTPs, SYBR Green fluorescent dye, without enzyme water and fluorescent quantitation sample-adding plate.

The kit further includes that RNA extracts reagent, the RNA extract reagent include Trizol, chloroform, isopropanol, 75% ethyl alcohol and RNase free water.

The kit further includes the system for being cDNA by mRNA reverse transcription, 5 X RT master of the Reverse Transcription Mix is PrimeScript RTase, RNase Inhibitor, 6 mers of Random, Oligo dT Primer, dNTP Mixture, reaction Buffer, quantitative fluorescent PCR reaction system SYBR Premix Ex TaqTM.

A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method, packet Include following steps.

(1) flesh tissue that will acquire carries out RNA extracting after liquid nitrogen processing grinding.

(2) by the RNA reverse transcription extracted at corresponding cDNA.

(3) cDNA after reverse transcription is carried out to the fluorescent quantitative PCR to ITGB5 and GAPDH gene.

(4) using GAPDH as internal reference, the Ct value of each reaction is recorded, testing result is indicated with-Δ Ct, wherein Δ Ct= CtGene-CtGAPDH。

Application of the ITGB5 gene in preparation glioblastoma auxiliary diagnosis, prognostic evaluation kit.

The present invention compared with the prior art, has the following advantages and beneficial effect.

(1) it is related to glioma progression of disease that present invention firstly discloses ITGB5 genes, and ITGB5 gene is expected to become and examine The molecular marker of disconnected GBM, and new thinking is provided to study the molecule mechanism of glioma progression of disease.

(2) there are significant differences for transcriptional expression level of the ITGB5 gene in LGG and GBM, compared with LGG tissue, The significant up-regulation of expression in GBM tissue of ITGB5 gene can be diagnosed by detecting the expression of ITGB5 gene transcription level The grade malignancy of patients with gliomas, so as to prepare the glioblastoma auxiliary diagnostic box containing ITGB5 gene, benefit The method for diagnosing glioblastoma with the mode of detection gene transcription level expression is more sensitive more special, is conducive to disease The diagnosis of early stage.

(3) present invention confirms that the height that ITGB5 gene is expressed in GBM is directly related with patient's prognosis for the first time, high table Up to ITGB5 GBM patient compared with the patient of low expression ITGB5, life span substantially reduces.Water is transcribed by detection ITGB5 Flat expression can estimate the prognosis situation of GBM patient, so that glioblastoma of the preparation containing ITGB5 gene is pre- Kits for evaluation afterwards more accurate, more objective can judge patient's prognosis, while the chemicotherapy that can evaluate patient is sensitive Property.

(4) present invention also prompt can block glioma progression of disease by intervening the expression of ITGB5, possible as The specific target spot of glioma future targeted therapy, the treatment for glioma disease provide new approaches.

Detailed description of the invention

Fig. 1 is malignant phenotypes' phases such as ITGB5 gene expression same level, IDH1 wild type and interstitial type (Mesenchymal) It closes.

Fig. 2 is the diagnosis index that ITGB5 gene expression can be used as GBM and interstitial type (Mesenchymal).

Fig. 3 is that ITGB5 gene mRNA is related to the equal same level of protein expression in clinical tissue sample.

Fig. 4 is the mark that ITGB5 gene is GBM patient's poor prognosis.

Fig. 5 is that ITGB5 participates in the resistance of GBM patient's chemicotherapy.

Fig. 6 is to strike low ITGB5 expression to can inhibit glioma cell migration, invasive ability.

Specific embodiment

The present invention is described in further details below by specific embodiments and the drawings.Following embodiment is only to the present invention It is further described, should not be construed as limiting the invention.Experimental method in following embodiment unless otherwise required, For conventional method, related experiment reagent and material are conventional biochemical reagent and material unless otherwise required.

Embodiment.

1.ITGB5 gene significant difference in the database, is the mark of glioma malignant progression.

This research mainly includes two large database concept platforms, Chinese glioma genome (Chinese Glioma Genome Atlas, CGGA) and oncogene map plan (The Cancer Genome Atlas, TCGA).CGGA is 2012 by north Capital Tiantan Hospital initiate and build up Chinese patients with gliomas sample parsing database, China can be disclosed by being that China is distinctive The large database of people's glioma occurrence and development molecular mechanism provides a large amount of bases and clinical branch for China's glioma research It holds.In CGGA database (http://www .cgga .org .cn), amounting to includes Low grade glioma (LGG) sample 172 Example, 138 samples of GBM sample.ITGB5 gene transcription level result is tied from 2000 platform RNA-seq of Illunima Hiseq Fruit.TCGA project team is initially by American National institute of oncology (The national cancer institute, NCI) and beauty The National Human Genome Research Institute of state (The national human genome research institute, NHGRI) group At, and a very big data research platform is developed into, Low grade glioma (LGG) and glioblastoma (GBM) conduct Its one of tumour studied earliest has a large amount of comprehensive data results.It includes 625 that (RNAseq) data set, which is sequenced, in TCGA RNA Example sample, wherein 470, LGG sample, 155, GBM sample.

As shown in Figure 1A, B, in CGGA and TCGA RNA-seq database, ITGB5 gene table in GBM patient tissue (CGGA RNA-seq:P < 0.0001, TCGA RNA-seq:P is significantly risen compared with expression in LGG patient tissue up to level < 0.0001)；In addition, ITGB5 is apparently higher than classic and preceding neuron pattern GBM patient (figure in chromic fibrous GBM patient expression 1C, D) (* * * *: P < 0.0001)；Moreover, to GBM patient IDH1 mutation status the study found that ITGB5 is in IDH1 wild type Expression is higher than saltant type patient (Fig. 1 E, F) in patient.

2. based on ITGB5 gene in CGGA and TCGA RNA-seq samples of human glioma sample differential expression draw GBM and The diagnosis ROC curve of interstitial type.

Based on above-mentioned CGGA and TCGA RNA-seq samples of human glioma sample, according to the ITGB5 expression of LGG and GBM, Draw the ROC curve for diagnosing GBM.In ROC curve evaluation method, the area value AUC under ROC curve is being greater than 0.5 In the case of, closer to 1, illustrate that diagnosis effect is better.AUC has lower accuracy at 0.5~0.7, and AUC is in the .9 of 0 .7~0 When have certain accuracy, AUC has high accuracy in 0 .9 or more.As a result as shown in Fig. 2A, B, it is female thin that ITGB5 diagnoses colloid Area (AUC) is respectively CGGA RNA-seq:0.654(Fig. 2 A, P < 0.0001 under the ROC curve of born of the same parents' tumor), TCGA RNA- Seq:0.647(Fig. 2 B, P < 0.0001), it can be used as the index of diagnosis GBM.

High, the prognosis mala in view of interstitial type GBM grade malignancy, this research is further between verifying ITGB5 gene pairs GBM patient The diagnosis effect of matter type, as a result as shown in Fig. 2 C, D, area (AUC) point under the ROC curve of ITGB5 diagnosis GBM patient's interstitial type Not Wei CGGA RNA-seq:0.878(Fig. 2 C, P < 0.001), TCGA RNA-seq:0.739(Fig. 2 D, P < 0.001), I.e. diagnosis has certain accuracy.

3. ITGB5 differential expression in clinical tissue pattern detection LGG and GBM.

It is Low grade glioma (LGG) 21, sample, glue that No. 1 Hospital Affiliated to Chinese Medical Univ, which is collected, through pathological diagnosis Matter blastoma (GBM) tissue samples 21 amounts to 42 tissue samples.RNA extraction and reverse transcription are carried out to these samples, it is right The cDNA that reverse transcription obtains carries out the fluorescent quantitative PCR of ITGB5 and GAPDH gene.As a result as shown in Figure 3A, ITGB5 base Because expression compared with expression in LGG patient tissue significantly rises (.0001 of P < 0) in GBM patient tissue.Further lead to Cross immunohistochemistry (Fig. 3 B) and western blot(Fig. 3 C) method identical tissue samples are detected, as a result and PCR result is consistent.In mature glioma cell line and glioma primary cell line by fluorescence quantitative PCR detection discovery with People's astroglia (NHA) is compared, and (Fig. 3 D), western blot are obviously increased in glioma cell line ITGB5 gene expression Result also obtained the result (Fig. 3 E) as quantitative fluorescent PCR.By the verifying of protein level, illustrate fluorescent quantitation PCR detection RNA can react the protein expression of ITGB5 well, demonstrate the degree of reliability of RNA result.

4. differential expression draws GBM and diagnoses ROC curve in clinical tissue based on ITGB5.

The rna expression of 42 samples of human glioma sample ITGB5 based on above-mentioned collection is horizontal, draws for diagnosing GBM's ROC curve.As a result as illustrated in Figure 3 F, area (AUC) is 0.943 under the ROC curve of ITGB5 diagnosis glioblastoma, that is, is examined It is disconnected that there is high accuracy.

5. ITGB5 expression height draws survivorship curve in CGGA RNAseq and TCGA RNAseq database.

In CGGA RNAseq database, for GBM patient, according to the ascending row of mRNA expression of ITGB5 Sequence, first 69 are low ITGB5 expression group, and latter 69 are high ITGB5 expression group, draw survivorship curve, and Fig. 4 A is in CGGA In RNAseq database, the survivorship curve based on ITGB5 gene mRNA height drafting.P=is analyzed to obtain by longrank test 0.0046, i.e. prognosis is statistically significant；In TCGA RNAseq database, for 155 GBM patients, according to ITGB5's The ascending sequence of mRNA expression, first 77 are low ITGB5 expression group, and latter 78 are high ITGB5 expression group, draw existence Curve, shown in Fig. 4 B, longrank test analyzes to obtain P=0.0121, i.e. prognosis is statistically significant.It further illustrates in GBM In, patient's prognosis of height expression ITGB5 is significantly worse than low expression ITGB5 patient.

6. being directed to Patients Treated by Radiotherapy situation in CGGA RNAseq and TCGA RNAseq database, expressed according to ITGB5 Height draws survivorship curve.

In CGGA RNAseq database, for GBM patient, according to the ascending row of mRNA expression of ITGB5 Sequence, first 69 are low ITGB5 expression group, and latter 69 are high ITGB5 expression group, whether receive radiotherapy according to patient, are divided into ITGB5 Low expression+radiotherapy, ITGB5 high expression+radiotherapy, ITGB5 low expression+non-radiotherapy, ITGB5 high expression+four groups of radiotherapy non-, every group of people Number is respectively 38,42,25,19, draws survivorship curve according to this four groups of patients, Fig. 5 A is in CGGA RNAseq number According in library, the patient drawn based on ITGB5 gene mRNA height receives radiotherapy situation survivorship curve.In the patient for receiving radiotherapy In, the low expression ITGB5 group survival of patients time is higher than high expression ITGB5 group, and there is system in longrank test analysis P=0.0009 Meter learns meaning；In the patient for not receiving radiotherapy, low expression ITGB5 group survival of patients time and high expression ITGB5 group difference are not Significantly, longrank test analyzes P=0.3356 and is not statistically significant.

In TCGA RNAseq database, for GBM patient, according to the ascending row of mRNA expression of ITGB5 Sequence, first 77 are low ITGB5 expression group, and latter 78 are high ITGB5 expression group, whether receive radiotherapy according to patient, are divided into ITGB5 Low expression+radiotherapy, ITGB5 high expression+radiotherapy, ITGB5 low expression+non-radiotherapy, ITGB5 high expression+four groups of radiotherapy non-, every group of people Number is respectively 64,63,9,12, draws survivorship curve according to this four groups of patients, Fig. 5 B is in TCGA RNAseq data In library, the patient drawn based on ITGB5 gene mRNA height receives radiotherapy situation survivorship curve.In the patient for receiving radiotherapy, The low expression ITGB5 group survival of patients time is higher than high expression ITGB5 group, and longrank test analyzes P=0.0171, there is system Meter learns meaning；In the patient for not receiving radiotherapy, low expression ITGB5 group survival of patients time and high expression ITGB5 group difference are not Significantly, longrank test analyzes P=0.4501, is not statistically significant.Pass through CGGA RNAseq and TCGA RNAseq Height survivorship curve is expressed for the ITGB5 that Patients Treated by Radiotherapy situation is drawn in database, illustrates that ITGB5 takes part in radiotherapy of glioma Resistance process can predict GBM Patients Treated by Radiotherapy sensibility according to ITGB5 expression quantity.

7. being directed to Chemotherapy in Patients situation in CGGA RNAseq and TCGA RNAseq database, expressed according to ITGB5 Height draws survivorship curve.

In CGGA RNAseq database, for GBM patient, according to the ascending row of mRNA expression of ITGB5 Sequence, first 69 are low ITGB5 expression group, and latter 69 are high ITGB5 expression group, whether receive chemotherapy according to patient, are divided into ITGB5 Low expression+chemotherapy, ITGB5 high expression+chemotherapy, ITGB5 low expression+non-chemotherapy, ITGB5 high expression+four groups of chemotherapy non-, every group of people Number is respectively 45,38,18,23, draws survivorship curve according to this four groups of patients, Fig. 5 C is in CGGA RNAseq number According in library, the patient drawn based on ITGB5 gene mRNA height receives chemotherapy situation survivorship curve.In patient of chemotherapy In, the low expression ITGB5 group survival of patients time is higher than high expression ITGB5 group, and there is system in longrank test analysis P=0.0058 Meter learns meaning；In non-patient of chemotherapy, low expression ITGB5 group survival of patients time and high expression ITGB5 group difference are not Significantly, longrank test analyzes P=0.8661 and is not statistically significant.In TCGA RNAseq database, for GBM Patient, according to the ascending sequence of mRNA expression of ITGB5, first 77 are low ITGB5 expression group, and latter 78 are height Whether ITGB5 expression group receives chemotherapy according to patient, is divided into ITGB5 low expression+chemotherapy, ITGB5 high expression+chemotherapy, ITGB5 Low expression+non-chemotherapy, ITGB5 high expression+four groups of chemotherapy non-, each group of people is respectively 56, and 55,11,18, according to this Four groups of patients draw survivorship curve, and Fig. 5 D is to be drawn in TCGA RNAseq database based on ITGB5 gene mRNA height Patient receives chemotherapy situation survivorship curve.In patient of chemotherapy, the low expression ITGB5 group survival of patients time is higher than high table Up to ITGB5 group, longrank test analyzes P=0.0072, statistically significant；In non-patient of chemotherapy, low table Not significant up to ITGB5 group survival of patients time and height expression ITGB5 group difference, the longrank test analysis .5297 of P=0 does not have It is statistically significant.The ITGB5 drawn by being directed to Chemotherapy in Patients situation in CGGA RNAseq and TCGA RNAseq database Height survivorship curve is expressed, illustrates that ITGB5 takes part in glioma chemoresistance process, GBM can be predicted according to ITGB5 expression quantity Chemotherapy in Patients sensibility.

8. the Cox multinomial logistic regression based on CGGA RNAseq and TCGA RNAseq database GBM.

Whether age, IDH1 mutation status receive radiotherapy, whether receive chemotherapy and the expression of ITGB5 is included into Cox Multinomial logistic regression model analyzes the relationship of these factors Yu GBM prognosis.The multifactor COX analysis of analysis result such as table 1.(mentions Show that the model can be used as CGGA RNAseq, independent prognostic factor in GBM database), table 2.(multifactor COX analysis prompt should Model can be used as CGGA RNAseq, independent prognostic factor in GBM database).

Table 1. and table 2 analyze result are as follows: the result explanation obtained in CGGA RNAseq and TCGA RNAseq database ITGB5 expression has the value (CGGA:P=0.0039, TCGA:P=0.0132) of independent judgment prognosis to GBM.

9. detecting ITGB5 in glioma cell line and glioma primary cell line to the shadow of glioma invasion, migration It rings.

ITGB5 siRNA is constructed, in glioma cell line LN229 and glioma primary cell line DGC1228 respectively That verifies two couples of siRNA strikes inefficient fruit, as shown in FIG, siITGB5-368, siITGB5-1218 two to siRNA with Control group is compared, and the mRNA level in-site of ITGB5 in LN229 and DGC1228 cell line has significantly been lowered, and Fig. 6 B further passes through Western blot method demonstrates siITGB5-368 in protein level, and siITGB5-1218 two is to siRNA and control group It compares, has significantly lowered the expression of ITGB5 in LN229 and DGC1228 cell line.As shown in Figure 6 C, Transwell experimental result It was found that siITGB5-368 compared with the control group, siITGB5-1218 lower ITGB5 expression can significantly inhibit LN229 and The migration of DGC1228 and invasive ability, invasion and transfer ability directly react the recurrence of GBM patient, ITGB5 expression and glue Matter tumor invades transfer ability correlation, can predict the GBM Patients on Recurrence time by ITGB5 expression quantity.

10. the preparation of the glioblastoma auxiliary diagnostic box based on ITGB5 gene.

Glioblastoma auxiliary diagnostic box include expand ITGB5 gene PCR primer to, amplification house-keeping gene The PCR primer of GAPDH to, SYBR Green polymerase chain reaction system, RNA extract reagent, mRNA reverse transcription is cDNA System.

Wherein expand ITGB5 gene PCR primer to for.

A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, including amplification ITGB5 The PCR primer pair of gene, the primer pair are.

Forward primer sequence is 5 '-GGAAGTTCGGAAACAGAGGGT-3 '.

Reverse primer sequences are 5 '-CTTTCGCCAGCCAATCTTCTC-3 '.

The kit further includes the PCR primer pair for expanding house-keeping gene GAPDH, and the primer pair is.

Forward primer sequence is 5 '-GGAGCGAGATCCCTCCAAAAT-3 '.

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 '.

The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green fluorescence dye Material, RNase free water and fluorescent quantitation are loaded plate.

It includes Trizol, chloroform, isopropanol, 75% ethyl alcohol and RNase-free water that the RNA, which extracts reagent,.

The system that the reverse transcription is cDNA is mix: PrimeScript RTase, RNase of 5 X RT master 6 mers of Inhibitor, Random, Oligo dT Primer, dNTP Mixture, reaction Buffer.

The application method of glioblastoma auxiliary diagnostic box of 11, based on ITGB5 gene.

Specific step is as follows for detection process.

1) flesh tissue to be measured is ground under liquid nitrogen effect, 1mL Trizol is added in the tissue after fragmentation, makes It is blown and beaten repeatedly with the liquid-transfering gun of 1mL, is incubated at room temperature 5min, is sufficiently separated nucleoprotein complex.

2) 200ul chloroform is added in every pipe, turns upside down 10 times, is placed at room temperature for 3min, with 12000rpm revolving speed in 4 DEG C from Heart 15min is shifted in upper strata aqueous phase to new EP pipe (about 500ul).

3) addition and the isometric isopropanol of supernatant (about 500ul, 4 DEG C of pre-coolings), are mixed by inversion, are stored at room temperature 10- 20min (average 15min), with 12000rpm revolving speed in 4 DEG C of centrifugation 10min, abandons supernatant, it is seen that a little white precipitate.

4) 75% ethyl alcohol (RNase-free water dilutes dehydrated alcohol) of 1ml pre-cooling is added, is slowly added along tube wall, on Lower mild reverse concussion, with 7500rpm revolving speed in 4 DEG C of centrifugation 5min.

5) supernatant is abandoned, drying at room temperature precipitates 1-2min, and RNase-free water 50-100ul is added, can use liquid relief when necessary Rifle gently blows and beats precipitating.

6) concentration and purity of Nanodrop detection RNA are utilized.OD260/OD280 ratio is between 1.80-2.0, explanation RNA purity meets requirement of experiment.

7) reverse transcription system.

1ugRNA volume: X ul.

5X PrimeScript RT Master Mix: 4ul。

RNase free water: 16-Xul.

Total system: 20ul.

Reverse transcription condition are as follows: 37 DEG C of 15min, 85 DEG C of 5S, 4 DEG C of 1min.

8) quantitative fluorescent PCR is loaded: fluorescent quantitation plate being placed on ice, each sample sets 3 multiple holes, by cDNA (dilution 4~5 times, 2 μ L are added after dilution).

Quantitative fluorescent PCR system is.

SYBR Premix Ex TaqTM: 10ul。

Upstream primer (10umol): 1ul.

Downstream primer (10umol): 1ul.

CDNA template: 2ul.

RNase free water: 6ul.

Total system: 20ul.

9) after being loaded, using PE gloves carefully by fluorescent quantitation plate membrane cover upper sealing panel, centrifugation avoids bubble from producing It is raw.

10) program is arranged: 95 DEG C of 10min95 DEG C of 10min, 95 DEG C of 5S, 60 DEG C of 10S, 72 DEG C of 10S carry out 40 altogether and follow Ring circulation+solubility curve.

11) analysis of experimental data: using GAPDH as internal reference, the Ct value of each reaction is recorded, Ct value is in each reaction tube Fluorescence signal recurring number experienced when reaching the threshold value of setting.Δ Ct=CtGene-CtGAPDH, Δ Ct are smaller to indicate it Starting copy number is more, express it is higher, using-Δ Ct as ITGB5 expression quantity progress evaluation index.

It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention Within.

Claims (7)

1. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that including Expand the PCR primer pair of ITGB5 gene, the primer pair are as follows:

Forward primer sequence is 5 '-GGAAGTTCGGAAACAGAGGGT-3 '

Reverse primer sequences are 5 '-CTTTCGCCAGCCAATCTTCTC-3 '.

2. a kind of interferon related kit predicted for prognostic evaluation in glioma and chemotherapy effect, which is characterized in that excellent Choosing, the kit further includes the PCR primer pair for expanding house-keeping gene GAPDH, the primer pair are as follows:

Forward primer sequence is 5 '-GGAGCGAGATCCCTCCAAAAT-3 '

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 '.

3. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that including SYBR Green polymerase chain reaction system, the SYBR Green polymerase chain reaction system include PCR buffer, DNTPs, SYBR Green fluorescent dye are loaded plate without enzyme water and fluorescent quantitation.

4. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that described Kit includes that RNA extracts reagent, and it includes Trizol, chloroform, isopropanol, 75% ethyl alcohol and RNase that the RNA, which extracts reagent, Free water.

5. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that described Kit includes the system for being cDNA by mRNA reverse transcription, and 5 X RT master mix of the Reverse Transcription is PrimeScript RTase、RNase Inhibitor、Random 6 mers、Oligo dT Primer 、dNTP Mixture, reaction Buffer, quantitative fluorescent PCR reaction system SYBR Premix Ex TaqTM.

6. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that reagent Box application method, comprising the following steps:

(1) flesh tissue that will acquire carries out RNA extracting after liquid nitrogen processing grinding；

(2) by the RNA reverse transcription extracted at corresponding cDNA；

(3) cDNA after reverse transcription is carried out to the fluorescent quantitative PCR to ITGB5 and GAPDH gene；

(4) using GAPDH as internal reference, the Ct value of each reaction is recorded, testing result is indicated with-Δ Ct, wherein Δ Ct= CtGene-CtGAPDH。

7. a kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit, which is characterized in that ITGB5 Application of the gene in preparation glioblastoma auxiliary diagnosis, prognostic evaluation kit.