CN108220421A - Application of the diagnostic kit and related gene of oneself immunity hepatitis in the kit is prepared - Google Patents
Application of the diagnostic kit and related gene of oneself immunity hepatitis in the kit is prepared Download PDFInfo
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Abstract
It, can fast and effectively diagnosis of autoimmune hepatitis (AIH) using the gene marker the present invention provides a kind of gene marker and its kit for being used for diagnosis of autoimmune hepatitis (AIH).
Description
Technical field
The invention belongs to disease research fields, and in particular to the diagnosis marker of oneself immunity hepatitis (AIH) and its examination
The exploitation of agent box.
Background technology
Oneself immunity hepatitis (autoimmune hepatitis, AIH) is that one kind is mediated by abnormal autoimmune response
Chronic inflammation liver diseases, with autoantibodies are positive, transaminase level and the raising of immunoglobulin G level, tissue
Interface characteristics hepatitis is shown as on as clinical characters.In general, AIH patient is good to immunosuppressor response, however if not
Treatment can progress to hepatic sclerosis, liver failure.At present, still lack the exact data of China's AIH epidemiology.In recent years, with
It and AIH diagnosis and the continuous improvement of the level of understanding, China's AIH patient's recall rate is increased year by year.AIH has become non-viral liver
The important component of disease, is increasingly subject to the concern of people.
The cause of disease and pathogenesis of AIH not yet illustrates completely, and complication and concomitant disease are more, there is no specific treatment, course of disease warp
It crosses in chronic, fluctuation.Perspective study shows, the death rate of the serious AIH patient of untreated after diagnosis in half a year is high
Up to 40%, there is 40% to develop into hepatic sclerosis in survivor, 54% patient varices of esophagus occurs in 2 years in hepatic sclerosis, most
The patient for having 20% eventually dies of massive haemorrhage caused by Esophageal variceal rupture.Therefore diagnosis in time, and give rationally appropriate
Treatment, for delaying or reversing this disease to have very important meaning.The diagnosis of AIH acquires a certain degree of difficulty, and needs feature sex expression
Presence, easily lead to and fail to pinpoint a disease in diagnosis or mistaken diagnosis；It needs to exclude other situations, particularly virus hepatitis simultaneously.And it is diagnosed as virus
Property hepatitis also there is no lack of such people, particularly Acute onset when, be more easy to mistaken diagnosis is proved with viral hepatitis type C, increases
The uncertainty of AIH diagnosis.Hepatitis patient usually shows the feature similar to AIH.
In recent years, with the rapid development of Protocols in Molecular Biology, the morbidity of AIH is illustrated from the angle of genetic predisposition
Mechanism is increasingly becoming the hot spot of research.Human leukocyte antigen (human leukocyte antigen, HLA) be it is known most
Important AIH genetic risk factors.The correlation of HLA regions and AIH are widely studied in different regions and crowd, are mesh
The most conclusive tumor susceptibility gene of preceding evidence.
China's AIH genetics research is still in the starting stage, and there are larger gaps with western countries' research level.In China
Carry out full-length genome, large sample, the genetics research verified repeatedly in crowd to be of great significance, it is new to not only help discovery
, the AIH tumor susceptibility genes that Chinese population is special, can also be provided for the pathogenesis and personalized diagnosis and treatment for further disclosing disease according to
According to.We using high-throughput transcript profile sequencing screening and gene closely related AIH, not only contribute to research AIH generation and
The pathomechanism of development, and new diagnosis marker or new drug target can be provided for diagnose and treat AIH.
Invention content
In order to make up oneself immunity hepatitis (AIH) in the prior art lack specificity clinical manifestation and biochemical indicator,
There are the defects of certain difficulty, the present invention provides a kind of marker base available for AIH disease treatments for diagnosis in clinical position
Cause.Compared to the diagnostic and therapeutic method of traditional AIH diseases, using gene marker come diagnose and treat AIH diseases have and
Shi Xing, specificity and non-invasive.
To achieve these goals, the present invention adopts the following technical scheme that：
There is provided a kind of kit of diagnosis of autoimmune hepatitis (AIH), which is characterized in that the kit, which includes, to be passed through
The expression of KCNIP2 genes, C16orf54 genes and IL21 genes in subject's peripheral blood is detected whether to judge subject
Reagent with oneself immunity hepatitis.
Preferably, the reagent is included with RT-PCR, real-time quantitative PCR or gene chip diagnosis oneself immunity hepatitis
Reagent.
The RT-PCR or the reagent of real-time quantitative PCR of being used for is following primer：
KCNIP2：Forward primer is 5 '-GACAGTTCGCTCCCTTCAG-3 ' (SEQ ID NO：1)；
Reverse primer is 5 '-CCCCGAAGAATCACGGACA-3 ' (SEQ ID NO：2)；
C16orf54：Forward primer is 5 '-CCAGAGCCACAAATCCTGT-3 ' (SEQ ID NO：5)；
Reverse primer is 5 '-TAGGACAACGCTCCTCTCCA-3 ' (SEQ ID NO：6)；
IL21：Forward primer is 5 '-GGACACTGGTCCACAAAT-3 ' (SEQ ID NO：7)；
Reverse primer is 5 '-GTTGGGCCTTCTGAAAGCAG-3 ' (SEQ ID NO：8).
Further, the kit also includes the amplimer of reference gene：
β-actin：Forward primer is 5 '-ATCCTGCGTCTGGACCT-3 ' (SEQ ID NO： 9)；
β-actin：Reverse primer is 5 '-ACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO：10).
The present invention also provides for detecting the reagent of the expression of KCNIP2 genes, TNFSF15 genes and IL21 genes
Application in the kit for diagnosis of autoimmune hepatitis is prepared.
The reagent includes the reagent with RT-PCR, real-time quantitative PCR or gene described in genechip detection.
The RT-PCR or the reagent of real-time quantitative PCR of being used for is SEQ ID NO：1-2, primer shown in 5-8.
The present invention by high-flux sequence screening obtain oneself immunity hepatitis (AIH) expression significantly raise or under
The gene of tune by the way that these genes are further screened and verified, therefrom determines that 3 can be used in diagnosing or predict
Whether the gene marker with AIH combines individual, and the acquisition of this combination brings great convenience for the diagnosis of AIH, has
Early diagnosis, early treatment conducive to AIH.
Description of the drawings
Fig. 1 is the electrophoretogram of the total serum IgE of extraction.
Fig. 2 is that PCR verifies that expression of the candidate gene in oneself immunity hepatitis (AIH) changes situation.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
Embodiment 1：Screening and oneself immunity hepatitis (AIH) relevant gene
1st, sample collection
Patient 25 of the collection hospital diagnosis for oneself immunity hepatitis (AIH), healthy control group (health
Control, HC) it chooses and AIH group ages, the matched healthy volunteer of gender 25.All 25 normal healthy controls ages and property
It is not matched with AIH groups, no the past hepatitis history, tumour medical history and corresponding family history.
2nd, the preparation and quality analysis of RNA sample
The preparation of 2.1RNA samples
(1) AIH patient and healthy volunteer, everyone acquires peripheric venous blood 10ml, EDTA anti-freezing.
(2) extracting peripheral blood mononuclear cells (PBMC)：Ficoll density-gradient centrifugation methods detach PBMC.
(3) total serum IgE of PBMC is detached：Trizol one-step method extracts PBMC total serum IgEs.
The quality analysis of 2.2RNA samples
NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is
1.8-2.2。
Agilent Technologies 2100Bioanalyzer detect RNA sample quality, observation 28S rRNA and 18S
RRNA master tapes are apparent, cDNA library structure is sequenced without degradation, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement
The requirement built can be used for library construction and sequencing.The RNA analysis image of sample segment is as shown in Figure 1.The RNA of such image
Belong to satisfactory RNA sample.
3rd, high-throughput transcript profile sequencing
The foundation of 3.1 sequencing libraries
After the enrichment with magnetic bead mRNA with Oligo (dT), with fragmentation buffer (Fragment buffer) by its dozen
Be broken into short-movie section, the 1st article of cDNA chain synthesized using mRNA as template with 6 base random primers, add buffer solution, RNase H,
DNTPs and DNA polymerase I synthesize the 2nd article of cDNA chain, then are purified with QiaQuiek PCR kits and add EB buffer solutions
Elution；It repairs doing end and adds poly (A) and after connecting sequence measuring joints, clip size choosing is carried out with agarose gel electrophoresis
It selects, finally carries out PCR amplification, establish sequencing library.
3.2 sequencing
Sequencing carries out the sequencing in library with Illumina HiSeq 2000, is surveyed by Shenzhen Huada Genetic Technology Co., Ltd
Sequence.
3.3 transcript abundances are assessed and the acquisition of differential gene
With composite software Trinity from the beginning transcript profile is assembled.Reads is passed through segment by SOAP denovo programs first
Overlapping is linked to be longer segment Contig；Again by Contig connect together to obtain both ends cannot extended sequence Unigene again,
De-redundancy processing, further splicing, homeodomain transcription this cluster are done to it again, obtains final Unigene.
The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to matching spy in every 1,000,000 sequencing segment
Determine the segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated values is calculated by Bayesian inference method.
The screening of differential gene is first with FC (Fold-change, multiple variation) and statistic mixed-state P value primary election, Ran Houyong
FDR (False discovery ratio) methods of calibration carry out false positive inspection to P values.As FDR≤0.001, log2
(Ratio) (Ratio is the ratio of Unigene FPKM in AIH patient Unigene FPKM and normal healthy controls during absolute value >=1
Value), the Unigene differential expressions are notable.
3.4 result
The RNA-seq sequencing results of early period filter out the gene of 103 difference expression genes, wherein expression up-regulation altogether
45, the gene 62 that expression is lowered.
Embodiment 2PCR verifies that expression of the candidate gene in oneself immunity hepatitis (AIH) patient changes situation
According to high throughput transcript profile deep sequencing early period as a result, according to the size of P value, the size of FC and text
Offer investigation selection KCNIP2 (its expression is lowered in AIH patient), TNFSF15 (its expression is raised in AIH patient),
C16orf54 (its expression is lowered in AIH patient), IL21 (its expression is raised in AIH patient) gene are verified.It collects
Hospital diagnosis be oneself immunity hepatitis (AIH) patient 25, healthy control group (health control, HC) choose with
AIH group ages, the matched healthy volunteer of gender 25 carry out classical molecular biology experiment using PCR and verify, specific to grasp
It is as follows to make step：
1st, RNA is extracted：Referring to embodiment 1.
2nd, reverse transcription：Reverse transcription synthesis cDNA is carried out to 1 μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems,
Each sample takes 1 μ g total serum IgEs to be separately added into following components in PCR pipes as template ribonucleic acid：DEPC water, 5 × reverse transcription buffering
Liquid, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid. 42
DEG C be incubated 1h, 72 DEG C of 10min, of short duration centrifugation.
3rd, PCR amplification：
KCNIP2：Forward primer is 5 '-GACAGTTCGCTCCCTTCAG-3 ' (SEQ ID NO：1)；
Reverse primer is 5 '-CCCCGAAGAATCACGGACA-3 ' (SEQ ID NO：2)；
TNFSF15：Forward primer is 5 '-GATCTGGGACTGAGCTTTG-3 ' (SEQ ID NO：3)；
Reverse primer is 5 '-TCTCCCGACTCTGGGATCA-3 ' (SEQ ID NO：4)；
C16orf54：Forward primer is 5 '-CCAGAGCCACAAATCCTGT-3 ' (SEQ ID NO：5)；
Reverse primer is 5 '-TAGGACAACGCTCCTCTCCA-3 ' (SEQ ID NO：6)；
IL21：Forward primer is 5 '-GGACACTGGTCCACAAAT-3 ' (SEQ ID NO：7)；
Reverse primer is 5 '-GTTGGGCCTTCTGAAAGCAG-3 ' (SEQ ID NO：8)；
β-actin：Forward primer is 5 '-ATCCTGCGTCTGGACCT-3 ' (SEQ ID NO： 9)；
β-actin：Reverse primer is 5 '-ACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO： 10).
Each ingredient in reaction mixture：Forward primer, reverse primer, 10 × PCR buffer solutions, MgCl2、dNTP、Taq
Archaeal dna polymerase, cDNA templates amount for be respectively 0.2,0.2,0.4,0.4,1.0,1.0,0.2,0.1 and 5 μ L, finally supplement
Distilled water makes reaction system be 10 μ L.The reaction condition of PCR is as follows：94 DEG C, 5min pre-degenerations；94 DEG C, 30s denaturation；56 DEG C,
30s anneals；72 DEG C, 30s extensions；Totally 36 cycles, electrophoresis detection pcr amplification product.DNA is weighed with the brightness of DNA bands
Amount, band is brighter, and representation DNA amount is more.Internal reference control is made with β-actin, by the brightness of the band of each gene amplification product
Homogenization processing is carried out, compares the ratio of the brightness of each gene amplification product in normal sample and AIH patient's samples.As a result such as
Shown in Fig. 1, compared with normal control, the expression of KCNIP2 is lowered in AIH patient, and the expression of C16orf54 is in AIH patient
It lowers, the expression of IL21 is raised in AIH patient, consistent with RNA-sep results.The expression of TNFSF15 in AIH patient with
Normal control is inconsistent with RNA-seq results without significant difference.
3 difference expression gene of embodiment is used to predict the compliance test result whether unknown sample suffers from AIH diseases
The other sample being different from Examples 1 and 2 is collected, suffers from AIH diseases, sample to be diagnosed including not knowing whether
This 20, it is known that 10, the sample of healthy control group (health control, HC).
The mononuclearcell (PBMC) of sample peripheral blood is extracted, detaches total serum IgE.After reverse transcription, using shown in embodiment 2
Primer respectively to 20 unknown samples and 10 3 gene KCNIP2, C16orf54 of healthy samples (as control),
IL21 and reference gene β-actin carries out PCR amplification.Each ingredient in reaction mixture：Forward primer, reverse primer, 10 ×
PCR buffer solutions, MgCl2, dNTP, Taq archaeal dna polymerase, cDNA templates amount for be respectively 0.2,0.2,0.4,0.4,1.0,
1.0th, 0.2,0.1 and 5 μ L, finally supplementing distilled water makes reaction system be 10 μ L.The reaction condition of PCR is as follows：94 DEG C, 5min is pre-
Denaturation；94 DEG C, 30s denaturation；56 DEG C, 30s annealing；72 DEG C, 30s extensions；Totally 36 cycles, electrophoresis detection pcr amplification product.
Amount of DNA is weighed with the brightness of DNA bands, band is brighter, representation DNA amount is more.Internal reference control is made with β-actin, it will be each
The brightness of the band of gene amplification product carries out homogenization processing.The homogenization of each gene amplification product in 10 healthy samples
The average value of numerical value afterwards compares the homogenization numerical value of each gene and the standard value of each gene in unknown sample as standard value
Between ratio.It is as shown in table 1 to embody situation, the results showed that, (P is significantly lowered in KCNIP2, C16orf54 expression<0.05)
And IL21 expression significantly up-regulation (P<0.05) unknown sample totally 3 (No. 2, No. 6, No. 20), thus it is speculated that be AIH patient.
Multiple (unknown sample/normal healthy controls sample) is lowered in 120 unknown samples of table in the expression of each gene
Hermes etc. accurately diagnoses AIH for the ease of simple in clinical position, and Cultivation has been formulated in 2008
Standard.4 independent variables are included in the standard whether to distinguish patient with AIH：Liver histological, autoantibody titer,
IgG and exclusion virus hepatitis.It specifically diagnoses as shown in the table：
Oneself immunity hepatitis simplifies ranking criterion (2008)
Clinical diagnosis is carried out according to the standard, it is thus identified that No. 2, No. 6, No. 20 are really AIH patient.And remaining not by
Be speculated as in 15 individuals of AIH, 3 be primary biliary cholangitis (PBC), 1 be hepatitis B, 11 for health
Individual is not admitted to AIH diseases after diagnosing.Therefore, the AIH patient speculated using the upper downward of these three gene markers
Conclusion be accurate.
It can be seen that the present invention provides can genetic test level speculate individual whether be AIH patient mark
Object can be realized using genetic test to AIH diseases promptness, specificity and non-invasive diagnosis.
Although the embodiments of the present invention have been disclosed as above, but its be not restricted in specification and embodiment it is listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, it is of the invention and unlimited
In specific details and legend shown and described herein.
Sequence table
<110>Li Xuan
<120>Application of the diagnostic kit and related gene of oneself immunity hepatitis in the kit is prepared
<160>10
<170>PatentIn version 3.5
<210>1
<211>19
<212>DNA
<213>Artificial sequence
<400>1
GACAGTTCGC TCCCTTCAG
<210>2
<211>19
<212>DNA
<213>Artificial sequence
<400>2
CCCCGAAGAA TCACGGACA
<210>3
<211>19
<212>DNA
<213>Artificial sequence
<400>3
GATCTGGGAC TGAGCTTTG
<210>4
<211>19
<212>DNA
<213>Artificial sequence
<400>4
TCTCCCGACT CTGGGATCA
<210>5
<211>19
<212>DNA
<213>Artificial sequence
<400>5
CCAGAGCCAC AAATCCTGT
<210>6
<211>20
<212>DNA
<213>Artificial sequence
<400>6
TAGGACAACG CTCCTCTCCA
<210>7
<211>18
<212>DNA
<213>Artificial sequence
<400>7
GGACACTGGT CCACAAAT
<210>8
<211>20
<212>DNA
<213>Artificial sequence
<400>8
GTTGGGCCTT CTGAAAGCAG
<210>9
<211>17
<212>DNA
<213>Artificial sequence
<400>9
ATCCTGCGTC TGGACCT
<210>10
<211>18
<212>DNA
<213>Artificial sequence
<400>10
ACTTGCGCTC AGGAGGAG 。
Claims (8)
1. a kind of kit of diagnosis of autoimmune hepatitis (AIH), which is characterized in that the kit include by detection by
In examination person's peripheral blood the expression of KCNIP2 genes, C16orf54 genes and IL21 genes come judge subject whether suffer from from
The reagent of body autoallergic.
2. kit according to claim 1, which is characterized in that the reagent include with RT-PCR, real-time quantitative PCR or
The reagent of gene chip diagnosis oneself immunity hepatitis.
3. kit according to claim 2, which is characterized in that the reagent for being used for RT-PCR or real-time quantitative PCR
For following primer：
KCNIP2：Forward primer is 5 '-GACAGTTCGCTCCCTTCAG-3 ' (SEQ ID NO：1)；
Reverse primer is 5 '-CCCCGAAGAATCACGGACA-3 ' (SEQ ID NO：2)；
C16orf54：Forward primer is 5 '-CCAGAGCCACAAATCCTGT-3 ' (SEQ ID NO：5)；
Reverse primer is 5 '-TAGGACAACGCTCCTCTCCA-3 ' (SEQ ID NO：6)；
IL21：Forward primer is 5 '-GGACACTGGTCCACAAAT-3 ' (SEQ ID NO：7)；
Reverse primer is 5 '-GTTGGGCCTTCTGAAAGCAG-3 ' (SEQ ID NO：8).
4. kit according to any one of claim 1-3, which is characterized in that the kit also includes reference gene
Amplimer：
β-actin：Forward primer is 5 '-ATCCTGCGTCTGGACCT-3 ' (SEQ ID NO：9)；
β-actin：Reverse primer is 5 '-ACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO：10).
5. the reagent for detecting the expression of KCNIP2 genes, C16orf54 genes and IL21 genes is being prepared for diagnosing
Application in the kit of oneself immunity hepatitis.
6. application according to claim 5, which is characterized in that the reagent is included with RT-PCR, real-time quantitative PCR or base
Because chip detects the reagent of the gene.
7. application according to claim 6, which is characterized in that described to be for the reagent of RT-PCR or real-time quantitative PCR
Following primer：
KCNIP2：Forward primer is 5 '-GACAGTTCGCTCCCTTCAG-3 ' (SEQ ID NO：1)；
Reverse primer is 5 '-CCCCGAAGAATCACGGACA-3 ' (SEQ ID NO：2)；
C16orf54：Forward primer is 5 '-CCAGAGCCACAAATCCTGT-3 ' (SEQ ID NO：5)；
Reverse primer is 5 '-TAGGACAACGCTCCTCTCCA-3 ' (SEQ ID NO：6)；
IL21：Forward primer is 5 '-GGACACTGGTCCACAAAT-3 ' (SEQ ID NO：7)；
Reverse primer is 5 '-GTTGGGCCTTCTGAAAGCAG-3 ' (SEQ ID NO：8).
8. according to the application described in any one of claim 5-7, which is characterized in that the kit also includes reference gene
Amplimer：
β-actin：Forward primer is 5 '-ATCCTGCGTCTGGACCT-3 ' (SEQ ID NO：9)；
β-actin：Reverse primer is 5 '-ACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO：10).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680549A (en) * | 2020-12-31 | 2021-04-20 | 郑州大学第一附属医院 | Oral microbial gene marker for distinguishing autoimmune hepatitis from healthy people and application thereof |
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|---|---|---|---|---|
| JP2007143504A (en) * | 2005-11-29 | 2007-06-14 | Ehime Univ | Gene marker of hypertension |
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|---|---|---|---|---|
| JP2007143504A (en) * | 2005-11-29 | 2007-06-14 | Ehime Univ | Gene marker of hypertension |
| EP2210942A1 (en) * | 2007-10-30 | 2010-07-28 | The University of Tokyo | Gene associated with liver cancer, and method for determination of the risk of acquiring liver cancer |
| CN101998964A (en) * | 2008-02-15 | 2011-03-30 | 太平洋艾瑞有限公司 | Blocking the metastasis of cancer cells and the uses of new compounds thereof |
Non-Patent Citations (1)
| Title |
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| 周威等: "肝纤维化相关基因C16orf54的克隆及表达", 《胃肠病学和肝病学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112680549A (en) * | 2020-12-31 | 2021-04-20 | 郑州大学第一附属医院 | Oral microbial gene marker for distinguishing autoimmune hepatitis from healthy people and application thereof |
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