CN107287310A - A kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit and application based on SPEXIN - Google Patents
A kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit and application based on SPEXIN Download PDFInfo
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- CN107287310A CN107287310A CN201710546255.1A CN201710546255A CN107287310A CN 107287310 A CN107287310 A CN 107287310A CN 201710546255 A CN201710546255 A CN 201710546255A CN 107287310 A CN107287310 A CN 107287310A
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Abstract
The invention discloses SPEXIN genes and its expression product can as colorectal cancer early stage auxiliary diagnosis molecular marker.The invention provides a kind of diagnostic kit for being used to detect SPEXIN gene levels in individual subjects tissue and/or blood.The present invention further discloses the concrete operation method of detection individual subjects SPEXIN gene levels.The experiment of this hair shows that SPEXIN gene expression doses have significant difference in cancerous tissue and normal structure, i.e., can tentatively judge whether the subject suffers from colorectal cancer and/or prognosis situation by detecting the level of SPEXIN genes in subject's tissue and/or blood.The inventive method is simple and convenient to operate, detect that obtained gene expression dose can provide new approaches with possibility of the auxiliary diagnosis individual test subjects with colorectal cancer and the index of colorectal cancer patients Index for diagnosis, the diagnosis and treatment of exploitation and colorectal cancer patients for colorectal cancer medicine.
Description
Technical field
The invention belongs to oncomolecularbiology technical field, and in particular to a kind of colorectal cancer auxiliary based on SPEXIN
Diagnosis and/or Index for diagnosis kit and application.
Background technology
Colorectal cancer（Colorectal cancer, CRC）It is one of malignant tumour of serious threat human life and health.
World pop disease learns investigation and shows that the incidence of disease of the colorectal cancer in all kinds of malignant tumours occupies the 3rd.With economic development and people
The change of people's growth in the living standard, life style and dietary structure, in its incidence of disease of China, to occupy malignant tumour only secondary various
The 3rd is occupied in lung cancer and breast cancer, the death rate occupies the 4th.And it was predicted that the morbidity and mortality of China's colorectal cancer exist
Will be in growing steadily trend, as China is most common, one of high incidence malignant tumour, in significant period of time from now on
Through the health as the serious threat people.
The generation of colorectal cancer and food environment heredity etc. are closely related, are the coefficient result of many factors, colon
The study on prevention of cancer has constantly caused the attention of people.Because colorectal cancer preclinical phase is asymptomatic, and the duration is longer, because
This early diagnosis and examination to colorectal cancer are simultaneously treated in time, help to reduce the incidence of disease and the death of colorectal cancer
Rate.Tumor cells mark refers to tumour cell in Carcinogenesis because increasing or decreasing for gene expression causes its content to change
Some albumen or bioactive substance become, available for diagnosing tumor, detection neoplastic process and patient evaluate more afterwards, and it is to swollen
The clinical treatment of knurl generates very big influence.So finding compared with high sensitivity and specific tumor cells mark, to knot
The diagnosis of the carcinoma of the rectum is extremely necessary.The colorectal cancer marker gene new by finding, the generation to predicting colorectal cancer
Development therapeutic effect has certain Clinical significance of MG.At present, the foundation of gene diagnosis technology and morning of colorectal cancer is developed into
Phase auxiliary diagnosis and Index for diagnosis provide approach.
SPEXIN albumen is to be based on hidden Markov model by Mirabeau team earliest（HMM）Bioinformatics search
Tactful first identified is found.People's SPEXIN genes（Ch12, orf39；~17,333 bp）Positioned at chromosome 12p12.1.
SPEXIN genes and albumen are all distributed in the central nervous system and peripheral tissues of the mankind and rodent extensively.
SPEXIN albumen participates in the regulation of the physiology such as human body energy metabolism, angiocarpy, appetite and pathologic function.However, SPEXIN genes
And/or there is not been reported so far for effect of the albumen to colorectal cancer.
The mark for being used for colorectal cancer early diagnosis clinically commonly used at present is remained in terms of specificity and sensitivity
In some shortcomings.Illustrated with cancer occurring signal Analysis in evolution, new tumor markers it is possible that.Point
Sub- biology continue to develop and technique for gene engineering continuous progress, find and utilize mark base new and that susceptibility is high
Cause, can effectively carry out the detection and diagnosis of molecular level, and then the early stage diagnosis and treatment and prevention for these diseases provide favourable bar
Part.
The content of the invention
It is an object of the invention to provide a kind of colorectal cancer auxiliary diagnosis based on SPEXIN and/or Index for diagnosis reagent
Box, the expression that the kit energy specific detection goes out SPEXIN genes whether there is downward, so as to be used as the diagnosis basis of colorectal cancer.Should
Kit detection sensitivity is high, high specificity.
The purpose of the present invention is achieved through the following technical solutions：A kind of colorectal cancer auxiliary diagnosis based on SPEXIN and/
Or Index for diagnosis kit, the kit include primer pair I and primer pair II；The positive sequence such as SEQ of the primer pair I
ID NO：Shown in 1（cacagagcct cgcctttgc）, reverse sequence such as SEQ ID NO：Shown in 2（ccaccatcac
gccctgg ）；The positive sequence of the primer pair II such as SEQ ID NO：Shown in 3（tccgcagaga ctgttggaga）, reversely
Sequence such as SEQ ID NO：Shown in 4（gttgctgcct ccggaatagt）.
Described colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit based on SPEXIN, in addition to reagent I, examination
Agent II and solution group, solution group include solution a-i, and the composition of reagent I, reagent II and solution a-i is as follows：
。
The application of described colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit based on SPEXIN, its feature exists
In including following operating procedure：
（1）Total serum IgE is extracted from the blood or colorectal carcinoma of subject；
（2）Detect the expression of SPEXIN in sample and control；
（3）The level to SPEXIN in sample and control carries out analysis and evaluation respectively.
The step（1）Middle colorectal carcinoma is Colorectal Carcinoma or cancer beside organism.
The step（2）For：By step（1）The total serum IgE reverse transcription of extraction into cDNA as template, be with primer pair I
The testing result of template as reference gene expression quantity, using primer pair II be template when testing result be used as target gene
SPEXIN expression quantity.
The step（2）Middle SPEXIN expression refer to SPEXIN genes, SPEXIN polypeptides, SPEXIN protein,
The level of the structural derivative of SPEXIN polypeptides or the functional analogue of SPEXIN albumen.
The SPEXIN polypeptides or SPEXIN protein contain such as SEQ ID NO：Amino acid sequence shown in 5
（MKGLRSLAATTLALFLVFVFLGNSSCAPQRLLERRNWTPQAMLYLKGAQGRRFISDQSRRKDLSDRPLPERRSPNP
QLLTIPEAATILLASLQKSPEDEEKNFDQTRFLEDSLLNW）.
Advantages of the present invention and beneficial effect are：（1）It is related to colorectal cancer present invention firstly discloses SPEXIN
Property, foundation can be provided for the preparation of the diagnostic kit based on SPEXIN；（2）The diagnostic kit that the present invention is provided can be with
Contain the possibility with colorectal cancer by detecting gene expression situation to assess subject, make detection more using the kit
Plus it is sensitive and convenient, be conducive to early diagnosis and the Index for diagnosis of colorectal cancer.
Brief description of the drawings
Fig. 1 is colorectal cancer group and the statistical results chart of normal group SPXIN gene expression amounts；
Fig. 2 is colorectal cancer patients SPXIN gene expression amounts and sex and the graph of a relation at age.
Embodiment
The present invention will further the present invention will be further described in the following embodiments and illustrates, but the present invention is not
It is confined to these embodiments.Involved experimental method is conventional method unless otherwise specified in the examples below, institute
Reagent and material for being related to etc., unless otherwise specified, can be obtained by commercial sources.
The preparation of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit based on SPEXIN
, can be by detecting the expression water of SPEXIN genes according to the gene expression abundance of SPEXIN genes and the correlation of colorectal cancer
The flat auxiliary characteristics as diagnosis of colorectal carcinoma.Based on this, the invention provides a kind of colorectal cancer auxiliary based on SPEXIN
Diagnosis and/or Index for diagnosis kit, the key component of the diagnostic kit include：Expand SPEXIN genes and beta-actin
Gene（Probe）Primer pair, SYBR Green PCR reaction systems, semiquantitive PCR system.Amplification beta-actin genes draw
Thing pair：Forward primer sequence, such as SEQ NO：Shown in 1；Reverse primer sequences, such as SEQ NO：Shown in 2；The length of amplified production is
434bp.Expand SPEXIN gene primers pair：Forward primer sequence, such as SEQ NO：Shown in 3；Reverse primer sequences, such as SEQ NO：
Shown in 4；The length of amplified production is 306 bp.Also include solution group（Solution a to i）With reagent I, reagent II, this kit institute
Comprising reagent and its content it is as shown in table 1.
Colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit forms of the table 1. based on SPEXIN
。
Embodiment 1
Semiquantitive PCR method detects Colorectal Carcinoma SPEXIN mRNA expressions
（1）The collection of tissue specimen：Collect Patients with Colorectal Cancer sample, cancer between section of Huaihe River hospital of He'nan University 2013-2016
Tissue and cancer beside organism's number pair, are respectively placed in 1.5 mL RNase/DNase-free centrifuge tubes, and -80 are transferred to through liquid nitrogen bucket
DEG C low temperature refrigerator is preserved.
（2）Colorectal cancer patients tissue samples mRNA is extracted using conventional method.[Sambrook J, Pehanorm Brooker,
Russell DW, et al., Molecular Cloning:A Laboratory guide 2002:Science Press], specifically：
1. the tissue block of soybean grain size is taken, is put into corresponding EP pipes, notes operating on ice, often pipe adds 1.0 mL
TRI Reagent, historrhexis is carried out with Ultrasonic Cell Disruptor, and room temperature places 5 min；
2. according to 10:Often pipe adds 100 μ L BCP to 1 ratio, 15 s of mixing that are acutely vortexed after EP pipe lids is covered, in ventilation
Room temperature places 8 min in cupboard；
③ 4℃、12000 g15 min are centrifuged, the μ L of upper strata about 500 aqueous phase supernatant is transferred to new mark by centrifugation after terminating
In clear 1.5ml RNase-Free centrifuge tube；
4. isometric isopropanol is added in each EP pipes, mixing more than 10 times of being turned upside down after lid is covered, under normal temperature condition
10 min are stood, then 4 DEG C, 12000g10 min are centrifuged, centrifugation abandons supernatant after terminating；
5. added in EP pipes and be cooled to 4 DEG C of the mL of 75% ethanol 1.0 in advance in advance, featheriness suspends, fully 4 DEG C after washing, 7500g
5 min are centrifuged, are repeated once after absorbing supernatant.The mRNA that EP ttom of pipe white depositions are proposed for us；
6. place and 20 min dried in super-clean bench, added after fully drying appropriate about 20 μ L without EP pipes are put after RNase water
Enter and be preheated to the min of water-bath 10 in 55 DEG C of water-bath in advance, fully dissolving after, flick tube wall, put be stored in from after -80 DEG C surpass
It is standby in low temperature refrigerator；
7. NANODROP 2000 spectrophotometers are used, wherein OD260/OD280 ratios detect protein in RNA sample
Pollution condition, makes OD260/280 ratios between 1.8-2.0, and determine RNA concentration.
（3）Reverse transcription PCR obtains cDNA：2 μ g total serum IgEs are taken as template and carry out reverse transcription synthesis cDNA, reaction system
For 25 μ L, following component is added in PCR pipe：
Above-mentioned mixed liquor is placed in PCR instrument, 42 DEG C of 1.5 h of extension, 95 DEG C, 5 min inactivations, under the conditions of obtaining cDNA, -20 DEG C
Storage is standby.
（4）Pcr amplification reaction
200 μ L PCR pipes are taken, following reagent is sequentially added by following system：
Outer ginseng gene/SPEXIN Gene response systems
Gently mix, centrifuge.Set PCR programs：94℃ 2 min；By 30 circulations（94℃ 30 sec；56℃ 30
sec；72℃ 60 sec）；72 DEG C of 5 min of extension after amplification.
（5）Gel electrophoresis, gel imaging and quantitative analysis
Take the μ L of pcr amplification product 10 and 10 μ L solution f and 4 μ L solution h to mix, draw 7 μ L to 1% reagent I agar
Sugared gel electrophoresis, puts upper 5 μ L reagent IIs, to determine the size of amplified production fragment.The min of 80 V electrophoresis 15, it is public with SynGene
Department's imaging system images are taken pictures, and gained band carries out the half-quantitative detection of gray value with Image J softwares, with SPEXIN/ACTIN
Ratio and is analyzed data as the final result of SPEXIN gene mRNA expression amounts.
Embodiment 2
Quantitative real-time PCR detects Colorectal Carcinoma SPEXIN mRNA expressions
（1）Sample collection and Total RNAs extraction, with reference to embodiment 1；
（2）Prepared by cDNA, with reference to embodiment 1；
（3）Real-time quantitative PCR：Detection reaction is carried out on the quantitative real time PCR Instruments of ABI 7500.Using 20 μ L reaction systems：According to
5 μ L cDNA samples of secondary addition；10 μ L solutions is；1 μ L primer pairs I（Outer ginseng gene）Or primer pair II（SPEXIN genes）；4
μ L solution e.To ensure the reliability of data, each sample sets 3 multiple holes, it is ensured that all steps are all operated on ice.
PCR reaction mixtures are added in 200 μ L PCR reaction tubes with liquid-transfering gun according to above-mentioned 20 μ L reaction systems,
It is gently quick to mix, centrifuge after capping, it is put into quantitative real time PCR Instrument and expands, PCR amplification programs is set as follows（From
Step 2 to step 4 continues 40 circulations）：
。
（4）Data analysis：Using SBYR Green as fluorescent marker, relative quantification is carried out with Δ Δ CT methods.Purpose base
Because the SPEXIN CT values expanded are corrected with the CT values of outer ginseng gene beta-actin amplifications.Gene relative expression quantity=2- Δ Δ CT。Δ
Δ CT=(CT1-T2)-CT3-T4), CT1 refers to the CT values of Colorectal Carcinoma sample SPEXIN genes；CT2 refers to Colon and rectum
The CT values of cancer tissue samples beta-actin genes；CT3 refers to the CT values of cancer beside organism/normal tissue sample SPEXIN genes；CT4
Refer to by cancer/normal tissue sample beta-actin CT values.
CT3 refers to the CT values of cancer beside organism/normal tissue sample SPEXIN genes；CT4 refers to by cancer/normal tissue sample
Beta-actin CT values.
The real-time fluorescence quantitative PCR of table 2 detection Colorectal Carcinoma SPEXIN mRNA expression（Some cases）
。
Embodiment 2 test result indicates that, compared with normal group, colorectal cancer group SPXIN gene relative expression quantities are obvious
Lower, expression is only the 22% of normal structure.Statistical analysis shows that difference has statistical significance, P<0.05, referring to Fig. 1.
In addition, the expression quantity of colorectal cancer patients SPEXIN genes does not have significant correlation with sex or age, referring to Fig. 2.Knot is straight
The ratio of patients with bowel cancer cancerous tissue SPEXIN mrna expression amounts and normal structure SPEXIN mrna expression amounts is as shown in table 2.Through
Clinical further checking shows that these cases suffer from colorectal cancer really, illustrates, the testing result of kit prepared by the present invention
It is straightforward with one with clinical detection result.Inferring this kit testing result accordingly can distinguish whether patient suffers from colorectal cancer
And the prognosis situation of colorectal cancer, can as clinical diagnosis index, and next step clinical treatment provide reference frame.Together
When, SPEXIN can also as treatment of colorectal cancer potential target site.Morbidity of the present invention to further research colorectal cancer
Risk assessment, early diagnosis and the Index for diagnosis of mechanism and colorectal cancer are extremely important.
The embodiment and specific embodiment that the present invention is provided are only used for explaining the present invention, but this can not limit this hair
Bright protection domain.It should be pointed out that research or technical staff for this area, are not departing from spirit of the invention and principle
On the premise of and/or some improvement or modification that are carried out on the basis of the present invention, also in rights protection of the presently claimed invention
Within the scope of.
SEQUENCE LISTING
<110>He'nan University
<120>A kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit and application based on SPEXIN
<130> 2017
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
cacagagcct cgcctttgc 19
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
ccaccatcac gccctgg 17
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tccgcagaga ctgttggaga 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gttgctgcct ccggaatagt 20
<210> 5
<211> 116
<212> PRT
<213>People（Homo sapiens）
<400> 5
Met Lys Gly Leu Arg Ser Leu Ala Ala Thr Thr Leu Ala Leu Phe Leu
1 5 10 15
Val Phe Val Phe Leu Gly Asn Ser Ser Cys Ala Pro Gln Arg Leu Leu
20 25 30
Glu Arg Arg Asn Trp Thr Pro Gln Ala Met Leu Tyr Leu Lys Gly Ala
35 40 45
Gln Gly Arg Arg Phe Ile Ser Asp Gln Ser Arg Arg Lys Asp Leu Ser
50 55 60
Asp Arg Pro Leu Pro Glu Arg Arg Ser Pro Asn Pro Gln Leu Leu Thr
65 70 75 80
Ile Pro Glu Ala Ala Thr Ile Leu Leu Ala Ser Leu Gln Lys Ser Pro
85 90 95
Glu Asp Glu Glu Lys Asn Phe Asp Gln Thr Arg Phe Leu Glu Asp Ser
100 105 110
Leu Leu Asn Trp
115
Claims (7)
1. a kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit based on SPEXIN, it is characterised in that：The reagent
Box includes primer pair I and primer pair II；The positive sequence of the primer pair I such as SEQ ID NO：Shown in 1, reverse sequence such as SEQ
ID NO：Shown in 2；The positive sequence of the primer pair II such as SEQ ID NO：Shown in 3, reverse sequence such as SEQ ID NO：4 institutes
Show.
2. colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit as claimed in claim 2 based on SPEXIN, its feature
It is：Also include reagent I, reagent II and solution group, solution group includes solution a-i, the composition of reagent I, reagent II and solution a-i
It is as follows：
3. colorectal cancer auxiliary diagnosis as claimed in claim 1 based on SPEXIN and/or the application of Index for diagnosis kit,
It is characterized in that including following operating procedure：
(1) total serum IgE is extracted from the blood or colorectal carcinoma of subject；
(2) expression of detection sample and SPEXIN in control；
(3) level respectively to SPEXIN in sample and control carries out analysis and evaluation.
4. application as claimed in claim 3, it is characterised in that：Colorectal carcinoma is Colorectal Carcinoma in the step (1)
Or cancer beside organism.
5. application as claimed in claim 3, it is characterised in that the step (2) is：The total serum IgE reversion that step (1) is extracted
The cDNA recorded for the testing result of template as reference gene expression quantity, with primer pair II is mould using primer pair I as template
Testing result during plate is used as target gene SPEXIN expression quantity.
6. application as claimed in claim 3, it is characterised in that SPEXIN expression refers to SPEXIN in the step (2)
Gene, SPEXIN polypeptides, SPEXIN protein, the functional analogue of the structural derivative of SPEXIN polypeptides or SPEXIN albumen
Level.
7. application as claimed in claim 3, it is characterised in that the SPEXIN polypeptides or SPEXIN protein contain such as SEQ
ID NO：Amino acid sequence shown in 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710546255.1A CN107287310A (en) | 2017-07-06 | 2017-07-06 | A kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit and application based on SPEXIN |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710546255.1A CN107287310A (en) | 2017-07-06 | 2017-07-06 | A kind of colorectal cancer auxiliary diagnosis and/or Index for diagnosis kit and application based on SPEXIN |
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|---|---|
| CN107287310A true CN107287310A (en) | 2017-10-24 |
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ID=60100951
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580948A (en) * | 2018-11-30 | 2019-04-05 | 中国科学院上海有机化学研究所 | Application of the combination based on dihydrothymine metabolin in diagnosis of colorectal carcinoma and prognosis prediction |
| CN109957621A (en) * | 2019-04-01 | 2019-07-02 | 中国人民解放军海军军医大学国家肝癌科学中心 | It is a kind of for measuring the method and its kit of people's circulating excretion body circular rna |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103391784A (en) * | 2010-10-15 | 2013-11-13 | 纽约市哥伦比亚大学理事会 | Obesity-related genes and their proteins and uses thereof |
| CN104878121A (en) * | 2015-06-29 | 2015-09-02 | 黄文林 | Kit for colorectal cancer auxiliary diagnosis and/or prognosis |
-
2017
- 2017-07-06 CN CN201710546255.1A patent/CN107287310A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103391784A (en) * | 2010-10-15 | 2013-11-13 | 纽约市哥伦比亚大学理事会 | Obesity-related genes and their proteins and uses thereof |
| CN104878121A (en) * | 2015-06-29 | 2015-09-02 | 黄文林 | Kit for colorectal cancer auxiliary diagnosis and/or prognosis |
Non-Patent Citations (1)
| Title |
|---|
| CHENG-YUAN LIN,ET AL: "Spexin Enhances Bowel Movement through Activating L-type Voltagedependent Calcium Channel via Galanin Receptor 2 in Mice", 《SCIENTIFIC REPORTS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580948A (en) * | 2018-11-30 | 2019-04-05 | 中国科学院上海有机化学研究所 | Application of the combination based on dihydrothymine metabolin in diagnosis of colorectal carcinoma and prognosis prediction |
| CN109957621A (en) * | 2019-04-01 | 2019-07-02 | 中国人民解放军海军军医大学国家肝癌科学中心 | It is a kind of for measuring the method and its kit of people's circulating excretion body circular rna |
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Application publication date: 20171024 |