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CN104651522B - The new opplication of 1 chain of type i collagen α - Google Patents

The new opplication of 1 chain of type i collagen α Download PDF

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CN104651522B
CN104651522B CN201510100547.3A CN201510100547A CN104651522B CN 104651522 B CN104651522 B CN 104651522B CN 201510100547 A CN201510100547 A CN 201510100547A CN 104651522 B CN104651522 B CN 104651522B
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col1a1
adenocarcinoma
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CN104651522A (en
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田子强
王贵英
温士旺
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Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
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Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
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Abstract

The present invention relates to the new opplications of 1 chain of type i collagen α, and in particular to application of the antagonist of application and type i collagen α 1 chain of 1 chain of type i collagen α in diagnosing non-small cell lung cancer in preparation treatment non-small cell lung cancer drug.The COL1A1 high expression in adenocarcinoma of lung, while COL1A1 has good correlation with non-small cell lung cancer, can be used for preparing adenocarcinoma of lung assisting in diagnosis and treatment preparation, has important clinical value.

Description

The new opplication of 1 chain of type i collagen α
Technical field
The present invention relates to the new opplications of 1 chain of type i collagen α, specifically, the present invention relates to 1 chain of type i collagen α diagnose it is non- Application of the antagonist of application and 1 chain of type i collagen α in Small Cell Lung Cancer in preparation treatment non-small cell lung cancer drug.
Background technique
Type i collagen is made of 2 α l chains and 12 chain of α, the encoding gene of 2 chain of type i collagen α l chain and α be COL1A1 and COL1A2.The COL1A1 assignment of genes gene mapping is made of, full length gene 18kb in 17q21.3-q22 51 exons.Existing research table Bright, 90% or more osteogenesis imperfecta patient is mutated with type i collagen gene (COL1A1, COL1A2), especially prominent with CO L1A1 gene Become main.There are certain correlation (Hartikka, H., et in the site of type i collagen gene mutation with osteogenesis imperfecta clinical phenotypes al.,Lack of correlation between the type of COL1A1or COL1A2mutation and hearing loss in osteogenesis imperfecta patients.Human mutation,2004.24(2): p.147-154；).It shows according to another report, type i collagen, which is excessively increased, causes ECM synthesis and deposition in liver excessively, may finally lead Liver fibrosis due.In addition, it has been reported that COL1A1 in the generation, development process of breast cancer there are unconventionality expression, and may It is related to the proliferation of tumour cell and migration and the prognosis of tumor patient.
Lung cancer is one of highest malignant tumour of most common in China or even world wide and lethality, wherein about 80% Patient is non-small cell lung cancer (non-small cell lung carcinomas, NSCLC).According to statistics in annual newly-increased disease Oneself is in progressive stage when about 75% patient makes a definite diagnosis in example, and the mean survival time only 10 months.Reducing lung cancer mortality must add Strong lung cancer early diagnoses and effectively treats lung cancer, and the early diagnosis for reinforcing lung cancer has to as much as possible find and sends out with lung cancer Life develops closely related diagnosis marker.
There are many high-flux sequence types, and genetic chip, turns gene deep sequencing (genome re-sequencing) Record group deep sequencing (transcriptome re-sequencing is also known as RNA-Seq) etc., using earliest in transcript profile research And most widely be biochip technology, with the development of high throughput sequencing technologies, experimental study produces a large amount of gene core Sheet data, and the gene and signal path for influencing disease development are filtered out by gene expression chip, it has identified big Measure the gene of differential expression.But the difference for being different experiment porch and sample causes the analysis result of each genetic chip to exist Many inconsistencies, the result that Meta analysis can report the delivered correlative study of the same problem are collected, are statistical Integration, to obtain more acurrate or more result.This analysis can generate many significant difference expression genes, be avoided that list A research bring incorrectness.
T- is carried out after inventor is normalized by the result to 15 sets of adenocarcinoma of lung genetic chips in the present invention Test screens difference expression gene, filters out 1063 difference expression genes altogether, wherein gene 464 of expression up-regulation, Gene 599 of expression downward.In order to preferably study the function of difference expression gene, we are by DAVID to difference Expressing gene carries out the enrichment of GO function and the enrichment of KEGG access.KEGG access enrichment 37 genes can be in KEGG as the result is shown It is sifted out in library, concentrates on this signal path of talin, it may be related to the transfer of adenocarcinoma of lung.Based on our research direction, We select COL1A1 and carry out classical molecular biology experiment verifying as candidate gene.By to the further of candidate gene Molecular biology verifying, it was confirmed that COL1A1 high expression in adenocarcinoma of lung, while COL1A1 and non-small cell lung cancer are with fine Correlation, can be used for preparing adenocarcinoma of lung assisting in diagnosis and treatment preparation, have important clinical value.
Summary of the invention
It is an object of the present invention to provide new application of the COL1A1 in preparation adenocarcinoma of lung diagnostic preparation.
Inventor carries out Meta analysis to the result for the 15 sets of adenocarcinoma of lung genetic chips delivered, and obtains 1063 difference tables Up to gene, wherein gene 464 of expression up-regulation, lower gene 599 of expression, and utilize KEGG, GO, OMIM Functional analysis is carried out to difference expression gene etc. analysis tools such as various biological information databases and MATLAB, and then is filtered out Influence adenocarcinoma of lung occurrence and development key gene and important signal path, be additionally based on data mining analysis result and we Research direction, filter out candidate gene COL1A1.In turn, the present invention is by fluorescence quantifying PCR method in 5 adenocarcinoma of lung illness The cancerous tissue of patient and 5 normal tissues are that the adenocarcinoma of lung Disease-causing gene COL1A1 that sample verifying screens is sick in adenocarcinoma of lung illness The cancerous tissue of people and the expression of normal tissue, COL1A1 is expressed in the cancerous tissue of adenocarcinoma of lung diseased patient as the result is shown It significantly rises.
It is an object of the present invention to provide a kind of adenocarcinoma of lung detection kit, the detection kit detects COL1A1 egg It is white.Further, the kit further includes other detection reagents.
Further, the detection kit is to detect the ELISA detection kit of COL1A1 albumen.In the kit Commercially available COL1A1 monoclonal antibody can be used in antibody.Further, the kit includes: coating COL1A1 monoclonal antibody Solid phase carrier, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid Deng the IgG antibody of preferably HRP label, horseradish peroxidase.
Further, the detection kit is to detect the gold-immunochromatographyreagent reagent for assay box of COL1A1 albumen, and the antibody can Using commercially available COL1A1 monoclonal antibody.Further, the gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod skill Art or colloidal gold percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has Anti- COL1A1 monoclonal antibody, quality control region (C) specking have goat anti-mouse immunoglobulin IgG.
It is an object of the present invention to provide a kind of adenocarcinoma of lung detection kit, the detection kit detects COL1A1 base Cause.Further, the kit further includes other detection reagents.
Further, the detection kit is to detect the PCR kit for fluorescence quantitative of above-mentioned COL1A1 expression.It is excellent Choosing uses primer pair for SEQ ID NO.1 and SEQ ID NO.2.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal control primer, quantitative fluorescent PCR Reaction solution.Wherein the specific primer includes upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.1, Downstream primer sequence is SEQ ID NO.2.The internal control primer is β-actin internal control primer, and upstream primer sequence is SEQ ID NO.3, downstream primer sequence are SEQ ID NO.4.
The kit also includes RNA extraction agent.It is preferred thatReagent carries out sample rna extraction.
The present invention also has detected this kit sensitivity, this kit detection range is 10 as the result is shown6-102copies/μ L, minimum concentrations are 100copies/ μ l.
The answering in the diagnosis product of preparation adenocarcinoma of lung it is an object of the present invention to provide COL1A1 gene and/or COL1A1 albumen With.
The present invention is extracted the cancerous tissue and 5 normal tissues of 5 adenocarcinoma of lung diseased patients respectively first, to its total serum IgE Extract, 2 primer SEQ ID NO.1 and SEQ ID NO.2 for expanding COL1A1 gene are devised, for expanding 2 primer SEQ ID NO.3 and SEQ ID NO.4 of reference gene β-actin.It is prepared for the mark containing COL1A1 gene order Quasi- DNA profiling, and carried out sensitivity experiments.In turn, COL1A1 gene is compared in pulmonary adenocarcinoma using the method for qRT-PCR With the expression in normal tissue.The result shows that: qRT-PCR stable amplification result, wherein COL1A1 is in the normal tissue Expression is apparently higher than adenocarcinoma of lung cancerous tissue, experiment show bioinformatics the selection result.Further, inventor exists COL1A1 expression conditions are detected in the cancerous tissue of 134 adenocarcinoma of lung diseased patients and cancer beside organism, as the result is shown 133 lungs COL1A1 gene expression is higher than the expression in cancer beside organism in the cancerous tissue of gland cancer diseased patient, detects accuracy rate up to 99%, i.e., COL1A1 gene has good correlation with adenocarcinoma of lung, can be used for preparing adenocarcinoma of lung auxiliary diagnosis or treatment preparation.
Detailed description of the invention
Fig. 1 COL1A1 gene by fluorescence quantitative amplification curve diagram
Fig. 2 COL1A1 gene by fluorescence quantitative solubility curve figure
Fig. 3 COL1A1 gene relative expression's spirogram in cancerous tissue and normal tissue
Specific embodiment
Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer Part examinations.
1 adenocarcinoma of lung high throughput transcript profile data Meta of embodiment analysis
We carry out t-test after the result of reported 15 sets of adenocarcinoma of lung genetic chips is normalized, screening Difference expression gene filters out 1063 difference expression genes altogether, wherein gene 464 of expression up-regulation, expression The gene of downward 599.In order to preferably study the function of difference expression gene, we are by DAVID to difference expression gene Carry out the enrichment of GO function and the enrichment of KEGG access.37 genes as the result is shown of KEGG access enrichment can sift out in the library KEGG (being shown in Table 1) concentrates on this signal path of talin, may be related to the transfer of adenocarcinoma of lung.Based on our research direction, choose COL1A1 is taken to carry out classical molecular biology experiment verifying as candidate gene.
The signal path of 1 difference expression gene of table enrichment
2 pulmonary adenocarcinoma of embodiment and normal tissue COL1A1 expression conditions
One material and method
1, material
Pulmonary adenocarcinoma and normal tissue are taken from -2010 years 2005 inpatients, have taken 5 respectively.
2, method
The extraction of 2.1 pulmonary adenocarcinomas and normal tissue total serum IgE
The RNA that tissue is extracted by Trizol kit specification is proved the integrality of RNA by gel electrophoresis, uses nucleic acid The concentration and purity of albumen instrument measurement RNA.It is extracted using total serum IgE extraction agent box.Main operational steps are as follows:
(1) histocyte is cracked with cellular lysate liquid BL, centrifuging and taking upper cell is added lml's in upper cell Trizol is lashed lysate 10 times with the disposable syringe with syringe needle；
(2) it stands after five minutes, the chloroform of 200 μ l is added, after firmly overturning centrifuge tube mixing, be stored at room temperature and be allowed to be layered, 12000g is centrifuged 5 minutes, carefully pipettes water phase into the centrifuge tube of 1.5ml；
(3) isometric isopropanol is added, after thoroughly mixing, takes out 750 μ l and moves into adsorption column, be centrifuged 30 seconds, outwell receipts Liquid in collector moves into adsorption column in same collecting pipe, will all enter remaining in adsorption column, is centrifuged 30 seconds, outwells Liquid in collecting pipe moves into adsorption column in the same collecting pipe；
(4) 500 μ L RP liquid are added, are centrifuged 30 seconds.The liquid in collecting pipe is outwelled, adsorption column is moved into same collecting pipe In；
(5) by the W3 liquid of 500 μ l, 1 minute is stood, is centrifuged 15 seconds；
(6) adsorption column is moved into a clean collecting pipe, 500 μ l W3 liquid is added, be centrifuged 15 seconds；
(7) liquid in collecting pipe is outwelled, then adsorption column is moved into same collecting pipe, is centrifuged 1 minute；
(8) adsorption column is put into the centrifuge tube of another clean 1.5ml, 50 μ l pure water is added in adsorbed film center, After being stored at room temperature l/min, it is centrifuged 1 minute, RNA is stored in -70 DEG C；
(9) total serum IgE integrality is identified: being taken 2 μ l RNA samples at 1.5% agarose gel electrophoresis (80v, 15min), is separated After zone, EB is dyed, and Zone electophoresis band is observed under ultraviolet lamp；
(10) with the concentration and purity of nucleic acid-protein instrument measurement RNA.
2.2COL1A1 genetic test design of primers and synthesis
According to PCR primer design principle, using the OligoArchitect of Premier 5.0 and enhanced editionTMSoftware carries out Design of primers.
The upstream and downstream primer sequence of COL1A1 is respectively as follows:
Upstream primer: 5'-TGATGCCAATGTGGTTCGTG-3'；SEQ ID NO.1
Downstream primer: 5'-TTGGTTGGGGTCAATCCAGTA-3'；SEQ ID NO.2
Product length is 184bp.
The upstream and downstream primer sequence of reference gene ACTIN is respectively
Upstream primer: 5'-ACTTAGTTGCGTTACACCCTT-3'；SEQ ID NO.3
Downstream primer: 5'-GTCACCTTCACCGTTCCA-3'；SEQ ID NO.4
Product length is 156bp.
The foundation of 2.3 quantitation curves
The preparation of standard DNA template
To specifications, it from pulmonary adenocarcinoma and normal tissue, is extracted using Invitrogen RTIZOL kit total RNA, and further useRNA clean-up kit (MACHEREY-NAGEL, Germany) is to total serum IgE Column purification was carried out, reverse transcription reaction is then carried out:
The first step removes genomic DNA reaction, and reaction system and condition are as follows:
Reagent Usage amount
5×gDNA Eraser Buffer 2.0μl
gDNA Eraser 1.0μl
Total RNA 1μg
RNase Free dH2O Add water to 10 μ l
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows:
Reagent Usage amount
The reaction solution of step 1 5.0μl
PrimeScript RT Enzyme Mix I 0.5μl
RT Primer Mix 0.5μl
5×PrimeScript Buffer 2 2μl
RNase Free dH2O 2.0μl
Total volume 10μl
By above-mentioned component 37 DEG C of incubation 15min after mixing, then 85 DEG C of inactivation 5sec are to get arriving cDNA.It will reverse The cDNA that record reaction obtains carries out Standard PCR, and reaction system and condition are as follows: 10 × Ex Taq buffer 5 μ L, dNTP Mixture (each 2.5mmol/L) 2 μ L, 2 μ L of upstream primer (10pmol), downstream primer (10pmol) 2 μ L, cDNA (0.1-2 μ g) 2.5 μ L, Ex Taq archaeal dna polymerase, 0.25 μ L, distilled water polishing to 50uL.Reaction condition is 94 DEG C of initial denaturation 5min；94 DEG C of changes Property 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 50s, 30cycles；Last 72 DEG C of extensions 10min.
5 μ L are sampled, agarose gel electrophoresis detection is carried out to the product of PCR amplification, gel extraction is carried out and purifies (recycling Use kit: EZ-10Spin Column DNA Gel Extraction Kit), purified product is connected to pGM-T clone Carrier is then transformed into DH5 α competent cell.Drawn by the specificity that sequence is SEQ ID NO.1 and SEQ ID NO.2 Object screening positive clone.Plasmid DNA is extracted after positive colony amplification, Plasmid DNA uses NanoDrop ND-1000 nucleic acid quantification Instrument quantifies (NanoDrop Technologies, Wilmington, Delaware) and does 10 times and is serially diluted as standard items use In the preparation of standard curve, (standard DNA template concentration range is 108-102copies/μl)。
2.4 sensitivity experiments
Recombinant plasmid is taken to be diluted to 10 in proportion8、107、106、105、104、103、102A copy/μ L carries out fluorescent quantitation PCR, using the minimum concentration of test positive as the detection sensitivity of this method.This research institute establish method detection range be 108-102Copies/ μ L, minimum concentrations are 100copies/ μ L.
2.5qRT-PCR detects COL1A1 gene expression amount
The total serum IgE for taking above-mentioned 5 pulmonary adenocarcinomas and normal tissue to extract, carries out reverse transcription reaction:
The first step removes genomic DNA reaction, and reaction system and condition are as follows:
Reagent Usage amount
5×gDNA Eraser Buffer 2.0μl
gDNA Eraser 1.0μl
Total RNA 1μg
RNase Free dH2O Add water to 10 μ l
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows:
Reagent Usage amount
The reaction solution of step 1 5.0μl
PrimeScript RT Enzyme Mix I 0.5μl
RT Primer Mix 0.5μl
5×PrimeScript Buffer 2 2μl
RNase Free dH2O 2.0μl
Total volume 10μl
By above-mentioned component 37 DEG C of incubation 15min after mixing, then 85 DEG C of inactivation 5sec are to get arriving cDNA.Using TAKARA SYBR Premix Ex TaqTMII (TIi RNaseH Plus) fluorescence quantitative kit (article No. RR820A).50μL QRT-PCR reaction system includes: upstream primer (10 μm of ol/L) 2 μ L；Downstream primer (10 μm of ol/L) 2 μ L；4 μ L of sample cDNA； 50×ROX Reference Dye 1μL；2 × SYBR Premex Ex Taq II (TIi RNaseH Plus) 25 μ L, add ion Water is to 50 μ L.Quantitative fluorescent PCR program: 95 DEG C of 30s initial denaturations meet 40 circulations: 95 DEG C of 5s, 60 DEG C of 30s.
Two experimental results
11.5 software of SPSS For Windows used to qRT-PCR reaction result, related data using chi-square criterion and Fisher exact propability is handled, and P < 0.05 is statistically significant；QRT-PCR reaction is statisticallyd analyze soft using MedCalc Part calculates.
Real-time quantitative PCR amplification curve (see Fig. 1) inflection point understands that amplification curve entirety collimation is good, shows each reaction tube Amplification efficiency it is close, the limit it is flat and without raising up now, exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample expands Volume increase object solubility curve (see Fig. 2) be it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the phase of qRT-PCR To quantitative equation: 2- Δ Ct × 100% compares expression of the COL1A1 gene in pulmonary adenocarcinoma and normal tissue.Knot Fruit shows: qRT-PCR stable amplification result, and wherein COL1A1 is apparently higher than normal tissue in the expression of pulmonary adenocarcinoma, These results suggest that COL1A1 high expression in pulmonary adenocarcinoma.
3 non-small cell type cancerous lung tissue of embodiment and cancer beside organism's COL1A1 expression conditions
One material and method
1, material
Non-small cell type lung cancer is taken from -2010 years 2005 inpatients, takes group by 134 adenocarcinoma of lung cancerous tissues and cancer It knits, it is numbered respectively.
2, method
Experimental method in reference implementation example 2.
Two results
In 134 non-small cell type lung cancer samples, expression of the COL1A1 in cancerous tissue is higher than cancer in 133 samples Side is organized, and expression of the COL1A1 in cancerous tissue is not distinguished clearly with cancer beside organism in only 1 sample, and accuracy rate is 99%, show that COL1A1 gene has good correlation with adenocarcinoma of lung, can be used for preparing adenocarcinoma of lung auxiliary diagnosis or prognosis Preparation.
A kind of kit and application method for detecting COL1A1 gene of embodiment 4
RNA extracts reagent: Invitrogen RTIZOL reagent
Reverse Transcription: TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time)
Fluorescent quantitation reagent:

Claims (2)

  1. Application of the 1.COL1A1 gene in the diagnostic preparation of preparation adenocarcinoma of lung, which is characterized in that the diagnosis system of the adenocarcinoma of lung Agent is to detect COL1A1 mrna expression with fluorescence quantifying PCR method, method for gene chip, and COL1A1 gene mRNA exists Expression quantity is high in patients with lung adenocarcinoma tissue, the product with COL1A1 gene in fluorescence quantifying PCR method detection adenocarcinoma of lung Primer containing a pair of of specific amplification COL1A1 gene；The genetic chip includes miscellaneous with the nucleic acid sequence of COL1A1 gene The probe of friendship, the primer upstream primer sequence of the pair of specific amplification COL1A1 gene are SEQ ID NO.1, downstream primer Sequence is SEQ ID NO.2.
  2. 2. a kind of application of PCR kit for fluorescence quantitative for detecting adenocarcinoma of lung in the diagnostic preparation of preparation adenocarcinoma of lung, feature It is, the kit detects gene C OL1A1 mRNA expression, using special upstream primer and downstream primer, upstream Primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
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