CN107447041A - Glioblastoma united diagnostic reagent box and its application method based on CCL8, CCL26 and CXCL6 gene - Google Patents
Glioblastoma united diagnostic reagent box and its application method based on CCL8, CCL26 and CXCL6 gene Download PDFInfo
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- CN107447041A CN107447041A CN201710893865.9A CN201710893865A CN107447041A CN 107447041 A CN107447041 A CN 107447041A CN 201710893865 A CN201710893865 A CN 201710893865A CN 107447041 A CN107447041 A CN 107447041A
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
Present invention relates particularly to a kind of glioblastoma united diagnostic reagent box and its application method based on CCL8, CCL26 and CXCL6 gene, the kit includes the PCR primer pair of amplification CCL8, CCL26 and CXCL6 gene and amplification house-keeping gene GAPDH PCR primer pair, present invention firstly discovers that there is significant difference in transcriptional expression level of CCL8, CCL26 and CXCL6 gene in LGG and GBM, by the expression for detecting CCL8, CCL26 and CXCL6 gene transcription level, carry out Combining diagnosis, it can be determined that the grade malignancy of patients with gliomas.
Description
Technical field
The invention belongs to detection kit technical field, and in particular to a kind of based on CCL8, CCL26 and CXCL6 gene
Glioblastoma united diagnostic reagent box and its application method.
Background technology
Glioblastoma is grade malignancy highest, most common primary brain tumors, accounts for central nerve neuroma
50%.Although the integration scenario of joint radiation and chemotherapy turns into treatment guidelines, glioblast based on operation
The prognosis of knurl patient not be improved significantly, the mean survival time only has 12~15 months.Low grade glioma (Low
Grade glioma, LGG) the long or short time development of patient's process, it typically can all develop into High Grade Gliomas
(Glioblastoma, GBM).Currently used discriminating Low grade glioma relies primarily on histopathology with High Grade Gliomas and examined
It is disconnected, but the result of pathological diagnosis has certain subjectivity and certain unreliability.Thus, it is found that new special molecule diagnosis
Label is imperative.
Chemotactic factor (CF) is a kind of proinflammatory disease chemotaxis small cytokines, can be promoted after being combined with its acceptor immune anti-
Should, homeostasis, angiogenesis, it stick and the survival of stem cell.Chemotactic factor (CF) plays a variety of effects, bag in glioma
Control leukocyte infiltration is included to tumour, the related angiogenesis of regulation tumour, activation host to the specific immune response of tumour,
To stimulate tumor proliferation, regulation Tumor Cell Migration etc. certainly, in a manner of paracrine.The chemotactic factor (CF) during glioma progression of disease
Expression can occur different degrees of change, therefore the molecular marker that its unconventionality expression can diagnose as glioblastoma.
CCL8, CCL26 and CXCL6 are the important member of chemotactic factor (CF) family, but its expression in glioma is not yet reported.
The content of the invention
Invention broadly provides a kind of glioblastoma Combining diagnosis examination based on CCL8, CCL26 and CXCL6 gene
Agent box and its application method, it is found that transcriptional expression of CCL8, CCL26 and CXCL6 gene in LGG and GBM is horizontal and exist significantly
Difference.Compared with LGG is organized, CCL8, CCL26 and CXCL6 gene express significantly up-regulation in GBM tissues, pass through detection
The expression of CCL8, CCL26 and CXCL6 gene transcription level, the grade malignancy of patients with gliomas can be diagnosed.Its technical side
Case is as follows：
A kind of glioblastoma united diagnostic reagent box based on CCL8, CCL26 and CXCL6 gene, including amplification
The PCR primer pair of CCL8 genes, the primer pair are：
Forward primer sequence is 5 '-TGGAGAGCTACACAAGAATCACC-3 ', such as SEQ ID NO：Shown in 1；
Reverse primer sequences are 5 '-TGGTCCAGATGCTTCATGGAA-3 ', such as SEQ ID NO：Shown in 2；
PCR primer pair including expanding CCL26 genes, the primer pair are：
Forward primer sequence is 5 '-AATTGAGGCTGAGCCAAAGA-3 ', such as SEQ ID NO：Shown in 3；
Reverse primer sequences are 5 '-GGGTCCATGTAGCCTTCAGA-3 ', such as SEQ ID NO：Shown in 4；
PCR primer pair including expanding CXCL6 genes, the primer pair are：
Forward primer sequence is 5 '-AGAGCTGCGTTGCACTTGTT-3 ', such as SEQ ID NO：Shown in 5；
Reverse primer sequences are 5 '-GCAGTTTACCAATCGTTTTGGGG-3 ', such as SEQ ID NO：Shown in 6.
Preferably, the kit also includes amplification house-keeping gene GAPDH PCR primer pair, and the primer pair is：
Forward primer sequence is 5 '-GCACCGTCAAGGCTGAGAAC-3 ', such as SEQ ID NO：Shown in 7；
Reverse primer sequences are 5 '-TGGTGAAGACGCCAGTGGA-3 ', such as SEQ ID NO：Shown in 8.
Preferably, the kit also includes SYBR Green PCR systems, and the SYBR Green gather
Polymerase chain reaction system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes, is loaded without enzyme water and fluorescent quantitation
Plate.
Preferably, the kit also includes RNA extracts reagents, and the RNA extracts reagents include Trizol, chloroform, different
Propyl alcohol, 75% ethanol and without enzyme water.
Preferably, the kit also includes the system that mRNA reverse transcriptions are cDNA, the reverse transcription system including gDNA
Eraser, gDNA Eraser Buffer, T repeat oligonucleotides Oligo (dT), inverse transcription reaction liquid, reverse transcriptase and
dNTPs。
A kind of application method of the glioblastoma united diagnostic reagent box based on CCL8, CCL26 and CXCL6 gene,
Comprise the following steps：
(1) flesh tissue of acquisition is subjected to RNA extractings after liquid nitrogen processing grinding；
(2) by the RNA reverse transcriptions extracted into corresponding cDNA；
(3) cDNA after reverse transcription is subjected to the quantitative fluorescent PCR expansion to CCL8, CCL26, CXCL6 and GAPDH gene
Increase；
(4) using GAPDH as internal reference, the Ct values each reacted are recorded, testing result is represented with Δ Ct, wherein Δ Ct=
CtGene-CtGAPDH。
Application of CCL8, CCL26 and CXCL6 gene in glioblastoma united diagnostic reagent box is prepared.
Using such scheme, the present invention has advantages below：
(1) it is related to glioma progression of disease present invention firstly discloses CCL8, CCL26 and CXCL6 gene, CCL8,
CCL26 and CXCL6 genes are expected to turn into diagnosis GBM molecular marker, and are the molecule mechanism of research glioma progression of disease
New thinking is provided；
(2) there is significant difference in transcriptional expression level of CCL8, CCL26 and CXCL6 gene in LGG and GBM, with LGG
Tissue is compared, CCL8, CCL26 and CXCL6 gene GBM tissue in express significantly up-regulation, by detect CCL8, CCL26 and
The expression of CXCL6 gene transcription levels, the grade malignancy of patients with gliomas can be diagnosed, so as to prepare containing
The glioblastoma united diagnostic reagent box of CCL8, CCL26 and CXCL6 gene, using detect gene transcription level expression
The method that mode diagnoses glioblastoma is more sensitive more special, is advantageous to the diagnosis of disease early stage；
(3) present invention also prompting can block glioma progression of disease by intervening CCL8, CCL26 and CXCL6 expression,
, possible as the specific target spot of glioma future targeted therapy, the treatment for glioma disease provides new approaches for it.
Brief description of the drawings
Fig. 1 is that CCL8, CCL26 and CXCL6 gene mRNA are suffered from 530 LGG patients, 167 GBM in TCGA databases
Expression in person's tissue；
Fig. 2 is the independent GBM drawn based on CCL8, CCL26 and CXCL6 gene mRNA expression in TCGA databases
Diagnose ROC curve；
Fig. 3 be based in TCGA databases CCL8, CCL26 and CXCL6 gene mRNA expression draw CCL8,
CCL26 and CXCL6 joint GBM diagnosis ROC curves；
Fig. 4 is that CCL8, CCL26 and CXCL6 gene mRNA are suffered from 181 LGG patients, 144 GBM in CGGA databases
Expression in person's tissue；
Fig. 5 is that CCL8, CCL26 and CXCL6 gene mRNA in clinical samples source are suffered from 41 LGG patients, 41 GBM
Expression in person's tissue；
Fig. 6 is the independent GBM diagnosis that CCL8, CCL26 and CXCL6 gene expression dose based on clinical samples source are drawn
ROC curve；
Fig. 7 is the joint GBM diagnosis that CCL8, CCL26 and CXCL6 gene expression dose based on clinical samples source are drawn
ROC curve.
Embodiment
Specific embodiment
Experimental method in following examples is conventional method unless otherwise required, involved experiment reagent and material
Material is routine biochemistry reagent and material unless otherwise required.
First, CCL8, CCL26 and CXCL6 differential expression in TCGA databases contrast LGG and GBM
The plan of oncogene collection of illustrative plates (The Cancer Genome Atlas, TCGA) project team is initially swollen by American National
Knurl research institute (The national cancer institute, NCI) and American National human genome research institute (The
National human genome research institute, NHGRI) composition, and develop into a very big data
Research platform.Low grade glioma (LGG) and glioblastoma (GBM) have big as one of its tumour studied earliest
Measure comprehensive data result.LGG and GBM data sources are in (https in TCGA://genome-cancer.ucsc.edu), its
Middle LGG has included 530 tumor samples, and GBM has included 167 samples.CCL8 gene transcription levels result comes from Illumina
The RNA-seq results of HiSeq microarray datasets.Fig. 1 is CCL8, CCL26 and CXCL6 gene mRNA in TCGA databases 530
Expression in example LGG patient, 167 GBM patient tissues.As shown in figure 1, CCL8, CCL26 and CXCL6 gene are suffered from GBM
Expression significantly rises (P compared with expression in LGG patient tissues in person's tissue<0.0001).
2nd, based on CCL8, CCL26 and CXCL6, differential expression draws independent GBM diagnosis ROC curve in TCGA
Based on above-mentioned TCGA samples of human glioma sample LGG and GBM CCL8, CCL26 and CXCL6 expression, draw and use
In diagnosis GBM ROC curve.In ROC curve evaluation method, the area value AUC under ROC curve in the case of more than 0.5,
Closer to 1, illustrate that diagnosis effect is better.AUC has relatively low accuracy at 0.5~0.7, and AUC has necessarily at 0.7~0.9
Accuracy, there is high accuracy when AUC is more than 0.9.As a result as shown in Fig. 2 the ROC curve of CCL8 diagnosis glioblastomas
Lower area (AUC) be 0.775, CCL26 diagnosis glioblastoma ROC curve under area (AUC) be that 0.758, CXCL6 is examined
Area (AUC) is 0.708 under the ROC curve of disconnected glioblastoma, i.e. independent diagnostics have certain accuracy.
3rd, based on CCL8, CCL26 and CXCL6, differential expression draws joint GBM diagnosis ROC curves in TCGA
Based on above-mentioned TCGA samples of human glioma sample LGG and GBM CCL8, CCL26 and CXCL6 expression, draw and use
In Combining diagnosis GBM ROC curve.As a result such as Fig. 3, Combining diagnosis AUC are 0.820, and diagnosis is with certain accuracy and equal height
In independent diagnostics AUC.
4th, CGGA databases obtain CCL8, CCL26 and CXCL6 differential expression in LGG and GBM
Chinese glioma genome (Chinese Glioma Genome Atlas, CGGA) is 2012 by Beijing day
The database for the Chinese patients with gliomas sample parsing that altar hospital initiates and built up, Chinese's glue can be disclosed by being that China is distinctive
The large database of matter knurl occurrence and development molecular mechanism, a large amount of bases and clinical support are provided for China's glioma research.
(http in CGGA databases://www.cgga.org.cn), altogether comprising LGG 181,144 samples of GBM.CCL8、
CCL26 and CXCL6 gene transcription levels result comes from the platform RNA-seq results of Illunima Hiseq 2000.Fig. 2 is CGGA
Expression feelings of CCL8, CCL26 and CXCL6 gene mRNA in 181 LGG patients, 144 GBM patient tissues in database
Condition.As shown in figure 4, CCL8, CCL26 and CXCL6 gene in GBM patient tissues expression compared with being expressed in LGG patient tissues
It is horizontal significantly to rise (P<0.05).
5th, CCL8, CCL26 and CXCL6 differential expression in clinical tissue pattern detection LGG and GBM
It is Low grade glioma (LGG) 41 and high-level glue that affiliated hospital of Zhengzhou University first, which is collected, through pathological diagnosis
Matter knurl (GBM) tissue samples 41, altogether 82 tissue samples.RNA extractions and reverse transcription are carried out to these samples, to reverse transcription
Obtained cDNA carries out the fluorescent quantitative PCR of CCL8, CCL26, CXCL6 and GAPDH gene.As a result it is as shown in Figure 5.By
Fig. 5 understands that CCL8, CCL26 and CXCL6 gene expression in GBM patient tissues show compared with expression in LGG patient tissues
Write and rise (P<0.01).
6th, the ROC that independent diagnostics GBM is drawn based on CCL8, CCL26 and CXCL6 differential expression in clinical tissue sample is bent
Line
82 samples of human glioma sample CCL8, CCL26 and CXCL6 expressions based on above-mentioned collection, draw for examining
Disconnected GBM ROC curve.As a result as shown in fig. 6, area (AUC) is 0.880 under the ROC curve of CCL8 diagnosis glioblastomas,
Area (AUC) is the ROC songs that 0.827, CXCL6 diagnoses glioblastoma under the ROC curve of CCL26 diagnosis glioblastomas
Area (AUC) is 0.684 under line, i.e. CCL8 and CCL26 have the certain accuracy for diagnosing GBM, and CXCL6 individually has diagnosis
GBM relatively low accuracy.
7th, the ROC that Combining diagnosis GBM is drawn based on CCL8, CCL26 and CXCL6 differential expression in clinical tissue sample is bent
Line
82 samples of human glioma sample CCL8, CCL26 and CXCL6 expressions based on above-mentioned collection, draw for joining
Close diagnosis GBM ROC curve.As a result as shown in fig. 7, Combining diagnosis TG-AUC is 0.946, diagnosis has high accuracy
And independent diagnostics AUC is above, Combining diagnosis significant effect.
8th, the preparation and use of the glioblastoma united diagnostic reagent box based on CCL8, CCL26 and CXCL6 gene
1. glioblastoma united diagnostic reagent box includes the PCR primer of amplification CCL8, CCL26 and CXCL6 gene
To, amplification house-keeping gene GAPDH PCR primer to, SYBR Green PCRs system, RNA extracts reagents,
MRNA reverse transcriptions are cDNA system.
Wherein expand CCL8 genes PCR primer to for：
Forward primer sequence is 5 '-TGGAGAGCTACACAAGAATCACC-3 ', such as SEQ ID NO：Shown in 1；
Reverse primer sequences are 5 '-TGGTCCAGATGCTTCATGGAA-3 ', such as SEQ ID NO：Shown in 2.
Expand CCL26 genes PCR primer to for：
Forward primer sequence is 5 '-AATTGAGGCTGAGCCAAAGA-3 ', such as SEQ ID NO：Shown in 3；
Reverse primer sequences are 5 '-GGGTCCATGTAGCCTTCAGA-3 ', such as SEQ ID NO：Shown in 4.
Expand CXCL6 genes PCR primer to for：
Forward primer sequence is 5 '-AGAGCTGCGTTGCACTTGTT-3 ', such as SEQ ID NO：Shown in 5；
Reverse primer sequences are 5 '-GCAGTTTACCAATCGTTTTGGGG-3 ', such as SEQ ID NO：Shown in 6.
Expand house-keeping gene GAPDH PCR primer to for：
Forward primer sequence is 5 '-GCACCGTCAAGGCTGAGAAC-3 ', such as SEQ ID NO：Shown in 7；
Reverse primer sequences are 5 '-TGGTGAAGACGCCAGTGGA-3 ', such as SEQ ID NO：Shown in 8.
The SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescence dye
Expect, without enzyme water and fluorescent quantitation sample-adding plate.
The RNA extracts reagents include Trizol, chloroform, isopropanol, 75% ethanol and without enzyme water.
The system that the reverse transcription is cDNA includes gDNA Eraser, gDNA Eraser Buffer, T and repeats few nucleosides
Sour Oligo (dT), inverse transcription reaction liquid, reverse transcriptase and dNTPs.
2. the detection process of diagnostic kit is as follows：
1) flesh tissue to be measured is ground under liquid nitrogen effect, 1mL Trizol is added in the tissue after fragmentation, are made
Blown and beaten repeatedly with 1mL liquid-transfering gun, be incubated at room temperature 5min, be sufficiently separated nucleoprotein complex；
2) often pipe adds 500 μ L chloroforms, mixing 10 times of turning upside down.After solution is fully emulsified, 5min is stored at room temperature.4
DEG C centrifugation, 12000rpm, 15min；
3) centrifuge tube is carefully taken out from centrifuge, now homogenate is divided into three layers, i.e.,：It is colourless aqueous phase, middle white
Chromoprotein layer and with coloured lower floor's organic phase.Extract water phase transfer is into new centrifuge tube；
4) isometric isopropanol is added into aqueous phase, after the centrifuge tube that turns upside down fully mixes, is stored at room temperature 10min；4
DEG C, 12 000rpm centrifugation 10min, after centrifugation, precipitation occurs in EP bottom of the tube；
5) careful supernatant discarding, the ethanol (being sure not to touch precipitation) of lmL precoolings 75% slowly is added along centrifugation tube wall,
12000rpm, 4 DEG C of centrifugation 5min, supernatant discarding；
6) drying at room temperature precipitation 2-5min, appropriate RNase-free water dissolving precipitation is added, liquid-transfering gun can be used if necessary
Gently piping and druming precipitation；
7) Nanodrop detections RNA concentration and purity are utilized.OD260/OD280 ratios are between 1.80-2.0, explanation
RNA purity meets requirement of experiment；
8) reverse transcription：5 × gDNA Eraser Buffer (2 μ L), gDNA Eraser (1 μ L), RNA (1 μ) are configured first
With RNase-free water mixed systems, totally 10 μ L, 42 DEG C, 2min；By 5 × PrimeScript buffer 2 (4 μ L),
PrimeScript RT Enzyme Mix (1 μ L), RT Primer Mix (1 μ L), RNase-free water (4 μ L) and above-mentioned mixed
Close liquid (10 μ L) to be mixed, PCR instrument, reaction condition are put into after of short duration centrifugation：37 DEG C of reaction 15min, 85 DEG C are reacted 5 seconds, are obtained
Obtain CDNA；
9) it is loaded：Fluorescent quantitation plate is placed on ice, each sample sets 2 multiple holes, by cDNA (4~5 times of dilution, dilution
Afterwards plus 2 μ L), SYBER Green and RCR buffer solutions be mixed every μ L of hole 12, it is laggard to have configured total system per the μ L of hole 8 for primer
Row packing, reduces error；
10) after sample-adding terminates, using PE gloves carefully by fluorescent quantitation plate membrane cover upper sealing panel, centrifugation, bubble is avoided to produce
It is raw；
11) program is set：95℃10min；95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 10s, 40 circulation+solubility curves；
12) analysis of experimental data：Using GAPDH as internal reference, the Ct values each reacted are recorded, Ct values are each reaction tube
Interior fluorescence signal reaches the period undergone during the threshold value of setting.Δ Ct=CtGene-CtGAPDH, Δ Ct is smaller to represent that it rises
Beginning copy number is more, and expression is higher.
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Sequence table
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<120>Glioblastoma united diagnostic reagent box and its application method based on CCL8, CCL26 and CXCL6 gene
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Claims (7)
- A kind of 1. glioblastoma united diagnostic reagent box based on CCL8, CCL26 and CXCL6 gene, it is characterised in that：Institute Stating kit includes the PCR primer pair of amplification CCL8 genes, and the primer pair is：Forward primer sequence is 5 '-TGGAGAGCTACACAAGAATCACC-3 ', such as SEQ ID NO：Shown in 1；Reverse primer sequences are 5 '-TGGTCCAGATGCTTCATGGAA-3 ', such as SEQ ID NO：Shown in 2；PCR primer pair including expanding CCL26 genes, the primer pair are：Forward primer sequence is 5 '-AATTGAGGCTGAGCCAAAGA-3 ', such as SEQ ID NO：Shown in 3；Reverse primer sequences are 5 '-GGGTCCATGTAGCCTTCAGA-3 ', such as SEQ ID NO：Shown in 4；PCR primer pair including expanding CXCL6 genes, the primer pair are：Forward primer sequence is 5 '-AGAGCTGCGTTGCACTTGTT-3 ', such as SEQ ID NO：Shown in 5；Reverse primer sequences are 5 '-GCAGTTTACCAATCGTTTTGGGG-3 ', such as SEQ ID NO：Shown in 6.
- 2. the glioblastoma united diagnostic reagent according to claim 1 based on CCL8, CCL26 and CXCL6 gene Box, it is characterised in that：The kit also includes amplification house-keeping gene GAPDH PCR primer pair, and the primer pair is：Forward primer sequence is 5 '-GCACCGTCAAGGCTGAGAAC-3 ', such as SEQ ID NO：Shown in 7；Reverse primer sequences are 5 '-TGGTGAAGACGCCAGTGGA-3 ', such as SEQ ID NO：Shown in 8.
- 3. the glioblastoma united diagnostic reagent according to claim 1 based on CCL8, CCL26 and CXCL6 gene Box, it is characterised in that：The kit also includes SYBR Green PCR systems, the SYBR Green polymerizations PCR system include PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes, without enzyme water and fluorescent quantitation sample-adding plate.
- 4. the glioblastoma united diagnostic reagent according to claim 1 based on CCL8, CCL26 and CXCL6 gene Box, it is characterised in that：The kit also includes RNA extracts reagents, and the RNA extracts reagents include Trizol, chloroform, isopropyl Alcohol, 75% ethanol and without enzyme water.
- 5. the glioblastoma united diagnostic reagent according to claim 1 based on CCL8, CCL26 and CXCL6 gene Box, it is characterised in that：The kit also includes the system that mRNA reverse transcriptions are cDNA, the reverse transcription system including gDNA Eraser, gDNA Eraser Buffer, T repeat oligonucleotides Oligo (dT), inverse transcription reaction liquid, reverse transcriptase and dNTPs。
- 6. a kind of application method of the glioblastoma united diagnostic reagent box based on CCL8, CCL26 and CXCL6 gene, its It is characterised by：Comprise the following steps：(1) flesh tissue of acquisition is subjected to RNA extractings after liquid nitrogen processing grinding；(2) by the RNA reverse transcriptions extracted into corresponding cDNA；(3) cDNA after reverse transcription is subjected to the fluorescent quantitative PCR to CCL8, CCL26, CXCL6 and GAPDH gene；(4) using GAPDH as internal reference, the Ct values each reacted are recorded, testing result is represented with Δ Ct, wherein Δ Ct=CtGene- CtGAPDH。
- Application of 7.CCL8, CCL26 and CXCL6 gene in glioblastoma united diagnostic reagent box is prepared.
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| CN110452980A (en) * | 2019-09-23 | 2019-11-15 | 武汉儿童医院 | A kind of mitochondrial encephalomyopathy diagnostic kit and application |
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Application publication date: 20171208 |