CN105567855B - IL-6 gene rs1524107 locus marker related to hepatitis B liver cirrhosis and application thereof - Google Patents
IL-6 gene rs1524107 locus marker related to hepatitis B liver cirrhosis and application thereof Download PDFInfo
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Abstract
The present invention relates to individual susceptibility in patients with hepatitis b cirrhosis. In particular, the invention relates to application of an rs1524107 locus marker of susceptibility gene IL-6 related to hepatitis B cirrhosis in the evaluation of patients suffering from hepatitis B cirrhosis. IL-6 gene rs1524107 locus marker related to hepatitis B liver cirrhosis, wherein the rs1524107 locus of the IL-6 gene is expressed as TT genotype. On the other hand, the invention also provides an IL-6 gene SNP rs1524107 locus marker related to the hepatitis B liver cirrhosis patient assessment, namely the IL-6 gene rs1524107 locus TT genotype and application thereof in the hepatitis B liver cirrhosis patient assessment.
Description
Technical Field
The present invention relates to individual susceptibility in patients with hepatitis b cirrhosis. In particular, the invention relates to application of an rs1524107 locus marker of susceptibility gene IL-6 related to hepatitis B cirrhosis in the evaluation of patients suffering from hepatitis B cirrhosis.
Background
The WHO updated data in 2015 showed that chronic Hepatitis B Virus (HBV) infected individuals worldwide were about 2 million 4000 tens of thousands. More than 78 tens of thousands of people die annually from hepatitis b and various complications are present worldwide, including cirrhosis and liver cancer. China is a high-incidence area of viral hepatitis, and the existing chronic HBV infected persons are about 9300 thousands of people, wherein the Chronic Hepatitis B (CHB) patients are about 2000 ten thousands of people. Generally, chronic hepatitis B is a complication caused by progressive development, but some patients are not subjected to obvious chronic hepatitis B, symptoms and signs of cirrhosis suddenly appear, and some patients are not found even in the decompensation stage. Thus, scientific early warning prediction and early diagnosis early intervention on chronic hepatitis B cirrhosis are critical for controlling disease progression and reducing mortality.
IL-6 is a classical cytokine consisting of 184 amino acids, whose gene is located on chromosome 7. IL-6 has 2 receptors: IL-6 receptor (IL-6R) and gp130.IL-6R has 2 forms, membranous IL-6R and soluble IL-6R (sIL-6R). IL-6 is mediated by membrane IL-6R by classical signaling pathways and by sIL-6R by trans signaling pathways. For healthy people, plasma levels of IL-6 are barely detectable. However, in chronic inflammatory diseases, such as chronic hepatitis b, abnormally secreted plasma IL-6 levels may be detected. There are studies showing that IL-6 levels are significantly elevated in Chronic Hepatitis B (CHB) patients than in healthy humans, and that IL-6 levels are more significantly expressed in patients with advanced liver disease (cirrhosis or liver cancer) than in CHB patients. The polymorphism of the gene may alter its expression or binding activity, thereby affecting liver injury and disease severity. Therefore, the IL-6 gene polymorphism analysis has important value for judging and early warning of the disease state of CHB patients, and has more irreplaceable significance for early warning of chronic hepatitis B progressing to liver cirrhosis.
Disclosure of Invention
In order to solve the problems, the invention provides an IL-6 gene rs1524107 locus marker related to hepatitis B liver cirrhosis, a detection reagent and a detection method thereof.
IL-6 gene rs1524107 locus marker related to hepatitis B liver cirrhosis, wherein the rs1524107 locus of the IL-6 gene is expressed as TT genotype.
A reagent for detecting rs1524107 locus of IL-6 gene.
In addition, the invention also provides a Taqman probe method for detecting the IL-6 gene SNP locus rs1524107 marker related to hepatitis B liver cirrhosis, comprising the following steps:
1) The method comprises the steps of purchasing a primer for detecting the rs1524107 locus of an IL-6 gene and a Taqman probe from an ABI company, and carrying out specific PCR amplification on sample genome DNA on a Roche LC480 II PCR instrument, wherein the Taqman probe is double-labeled by two fluorescent dyes Vic and Fam, the probes are respectively specific to different genotypes of a double-allele SNP, and only the perfectly matched probes can amplify the genotypes corresponding to the two types;
2) The reaction was carried out under the following conditions: preheating for 10min at the initial temperature of 95 ℃, then denaturing for 10s at the temperature of 95 ℃, annealing and extending for 30s at the temperature of 60 ℃, and circulating for 45 times; and scanning and reading data of the product on a Roche LC480 II type fluorescent quantitative PCR instrument, and analyzing to obtain the genotype of the research object.
Application of a reagent for detecting rs1524107 locus of IL-6 gene in preparing a diagnostic kit for hepatitis B liver cirrhosis.
The inventors first screened the IL-6 gene SNP site. Firstly, screening SNP loci which are possibly related to diseases by inquiring a large number of related documents or records; then in the second generation HapMap database, rs1524107 locus is screened by taking MAF >5% as an inclusion standard. The present invention thus identifies SNP sites of IL-6 gene in patients with hepatitis B cirrhosis.
The invention also provides a Taqman probe method for detecting the IL-6 gene SNP locus rs1524107 related to the evaluation of the hepatitis B liver cirrhosis patients.
As described above, the invention adopts a method for detecting IL-6 gene SNP locus rs1524107 related to the evaluation of hepatitis B liver cirrhosis patients, and the method is a Taqman probe method, and specifically comprises the following steps:
1) For the IL-6 gene rs1524107 site, the primers and probes were purchased from ABI company and specific PCR amplification was performed on sample genomic DNA on a Roche LC480 II PCR apparatus. Taqman probes are double-labeled, which are respectively aimed at different genotypes of the double-allele SNP, and only the completely matched probes can amplify the corresponding genotypes; and the two probes are respectively marked by two fluorescent dyes Vic and Fam.
2) The reaction was carried out under the following conditions: preheating for 10min at 95 ℃ at the beginning, denaturation for 10s at 95 ℃, annealing and extension for 30s at 60 ℃ and recycling for 45 times. And scanning and reading data of the product on a Roche LC480 II type fluorescent quantitative PCR instrument, and analyzing to obtain the genotype of the research object.
On the other hand, the invention also provides an IL-6 gene SNP rs1524107 locus marker related to the hepatitis B liver cirrhosis patient assessment, namely the IL-6 gene rs1524107 locus TT genotype and application thereof in the hepatitis B liver cirrhosis patient assessment.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
Example 1
This example employs a method for detecting IL-6 gene SNP sites associated with the evaluation of patients suffering from hepatitis B liver cirrhosis.
1. Genomic DNA was extracted using Qiagen QIAamp DNA Blood Mini kit, U.S.A., and whole blood DNA was extracted according to the protocol.
Preparation before experiment: one day in advance, the blood sample was transferred to a 4 ℃ freezer and the blood was allowed to thaw. The metal bath is opened and adjusted to 56 ℃, and centrifuge tubes, masks, napkins, kitchen paper, alcohol watering cans, wastepaper baskets, waste liquid cylinders and the like are prepared. (cf. Qiagen kit)
1. 20ul of protease was added to a 1.5ml centrifuge tube; if RNA removal is desired, 4ul of RNase is added to the tube.
2. 200ul of sample was added to a 1.5ml centrifuge tube; the wall of the tube is not touched when sucking blood, so that the pipette is not polluted.
3. 200ul of buffer AL was added and mixed well upside down and shaken for 15s.
4.56 ℃ for about 1 hour.
5. 200ul of absolute ethyl alcohol is added, and the mixture is fully and uniformly mixed for 15 seconds (can not be vigorously oscillated), flocculent precipitate can possibly appear at the moment, and the mixture is centrifuged briefly to remove water drops on the inner wall of the tube cover.
6. Adding the solution obtained in the last step and flocculent precipitate into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 8000rpm for 1min, pouring out waste liquid, and placing the adsorption column into a new collecting pipe.
7. 500ul of buffer AW1 was added to the column, centrifuged at 8000rpm for 1min, the waste liquid was decanted off, and the column was placed in a new collection tube.
8. 500ul of buffer AW2 was added to the column, centrifuged at 8000rpm for 1min, the waste liquid was decanted off, and the column was placed in a new collection tube.
9. The column was placed in a fresh collection tube, centrifuged at 13000rpm for 3min, and the waste liquid was discarded. The column was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the column material.
10. Transferring the adsorption column into a clean centrifuge tube, and suspending and dripping 50ul Buffer AE into the middle part of the adsorption film. Incubating for 5min at room temperature. Centrifuging at 8000rpm for 1min.
11: step 10 is repeated. And (5) after quality inspection, storing at-80 ℃.
2. PCR amplification
The detection primer for detecting the rs1524107 locus of the IL-6 gene and the Taqman probe are purchased from ABI company, and the specific PCR amplification is carried out on the sample genome DNA on a Roche LC480 II PCR instrument, the Taqman probe is double-labeled by two fluorescent dyes Vic and Fam, the probes are respectively aimed at different genotypes of the biallelic SNP, and only the perfectly matched probes can amplify the genotypes corresponding to the probes;
the PCR reaction system was prepared as follows, and the preparation process was performed on ice.
The reaction was carried out under the following conditions:
preheating for 10min at 95 ℃ at the beginning, denaturation for 10s at 95 ℃, annealing and extension for 30s at 60 ℃ and recycling for 45 times.
Example 2
This example uses the method of example 1 to analyze genotypes and alleles at locus rs1524107 of the IL-6 gene for 191 chronic hepatitis B patients, 177 hepatitis B cirrhosis patients, 48 hepatitis B liver failure patients, 93 carriers negative for both HBsAg and HBeAg, and 112 healthy individuals in the International health care center.
1. Test subjects
During the period from 11 months 2013 to 11 months 2015, 93 cases of carriers with negative HBsAg and HBeAg, 191 cases of chronic hepatitis B patients, 177 cases of hepatitis B cirrhosis patients, 48 cases of hepatitis B liver failure patients and 112 cases of health physical examination persons in the international health physical examination center, which are in hospital or clinic at the university of Zhejiang medical institute, were collected, and 621 cases were taken.
Patient enrollment criteria
Case diagnosis is performed according to the case judgment standard specified in the code 2015 "guidelines for prevention and treatment of chronic hepatitis b":
1. the carrier: serum HBsAg positive, with more than 2 consecutive follow-up times within 1 year, showed serum ALT and AST in the normal range, with no or minimal lesions in the liver histological examination.
2. Chronic hepatitis b: in the past, the history of hepatitis B or HBsAg positive is more than 6 months, and the HBsAg and/or HBV DNA are still positive, and ALT is continuously or repeatedly abnormal or has hepatitis lesions in liver histology examination.
3. Hepatitis b liver cirrhosis: (1) histological or clinical evidence suggests the presence of cirrhosis; (2) evidence of HBV infection with clear etiology. Other common causes of cirrhosis such as HCV infection, alcohol and drugs are identified or excluded by medical history or corresponding examination.
Case diagnosis is performed according to case judgment criteria specified in 2012 edition of diagnosis and treatment guidelines for liver failure:
4. hepatitis b liver failure: (1) the jaundice is deepened rapidly, and the serum TBIL is 10 times larger than the upper limit of the normal value or rises more than or equal to 17.1 mu mol/L per day; (2) bleeding tendency, PTA.ltoreq.40/o (or INR.gtoreq.1.5), and others.
Patient exclusion criteria
1) Excluding patients with other liver diseases such as other hepatitis virus infection, drug-induced liver disease, autoimmune liver disease, alcoholic liver disease, etc.;
2) Patients who have recently been excluded from liver damage due to other factors, such as poisoning and bacterial infection;
3) Excluding history of long-term drinking, typically over 5 years;
4) Excluding patients with severe organic lesions of heart, kidney, brain, lung, etc.;
5) Patients with primary or metastatic biliary tract tumors and gastrointestinal tract tumors are excluded.
Healthy control inclusion criteria
1) HBsAg, HBeAg, HBV-DNA and other markers negative;
2) Normal liver function.
Standard of exclusion for healthy controls
HBsAg, HBeAg or HBV-DNA marker positive, and exclude other physical health people with viral hepatitis, autoimmune liver disease, alcoholic liver disease, kidney, heart, brain, etc.
2. Hardy-Weinberg genetic balance test
IL-6 gene rs1524107 locus is tested by Hardy-Weinberg genetic balance law, accords with Hardy-Weinberg genetic balance and has population representativeness (table 1).
Table 1 IL-6 Gene rs1524107 locus genotype genetic balance test
3. Correlation analysis of IL-6 Gene polymorphism and hepatitis B liver cirrhosis
At the rs1524107 locus of the IL-6 gene, the genotype frequency comparison difference of the hepatitis B liver cirrhosis group and the control group has significance (χ) 2 =6.52, p=0.04) (table 2). The comparisons between the remaining groups were not statistically significant.
TABLE 2 distribution of IL-6 Gene rs1524107 locus genotypes and alleles in two groups
Therefore, the method provided by the invention is used for analyzing the relationship between the rs1524107 locus of the IL-6 gene and the hepatitis B liver cirrhosis.
It will be appreciated that after reading the above description of the invention, those skilled in the art may make various changes or modifications to the invention, but equivalents of the changes or modifications are also within the scope defined in the claims of the present application.
Claims (1)
1. The application of a reagent for detecting the TT genotype of the rs1524107 locus of the IL-6 gene in preparing a diagnosis kit for hepatitis B liver cirrhosis.
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Non-Patent Citations (3)
| Title |
|---|
| Analysis of IL-6, STAT3 and HSPA1L Gene Polymorphisms in Anti-Tuberculosis Drug-Induced Hepatitis in a Nested Case-Control Study;Jing Wang等;《PLOS ONE》;20150319;第10卷(第3期);e0118862 * |
| 基因变异及心血管危险因素与单核细胞分泌白细胞介素6和10的关系;谢高强等;《北京大学学报（医学版）》;20140831;第46卷(第4期);589-595 * |
| 社区人群中白细胞介素-6基因单核苷酸多态性与其血清水平的关系;谢高强等;《中国分子心脏病学杂志》;20141231;第14卷(第5期);1055-1059 * |
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