CN113943797A - Application of hypertensive inflammation response gene as molecular marker in preparation of Xinjiang Kazakh family essential hypertension diagnostic reagent - Google Patents

Application of hypertensive inflammation response gene as molecular marker in preparation of Xinjiang Kazakh family essential hypertension diagnostic reagent Download PDF

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CN113943797A
CN113943797A CN202111501531.5A CN202111501531A CN113943797A CN 113943797 A CN113943797 A CN 113943797A CN 202111501531 A CN202111501531 A CN 202111501531A CN 113943797 A CN113943797 A CN 113943797A
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马克涛
张亮
李新芝
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Abstract

The invention provides application of a hypertensive inflammation response gene as a molecular marker in preparation of a reagent for diagnosing primary hypertension of Kazakh in Xinjiang, belonging to the technical field of molecular biological diagnosis. The gene expression spectrum and the molecular marker of the mediated hypertension inflammatory response in peripheral blood mononuclear cells of Xinjiang Kazak essential hypertension patients are obtained for the first time based on a gene chip, bioinformatics analysis and a real-time quantitative polymerase chain reaction technology, new research evidence and molecular targets are provided for screening diagnosis, disease progress and anti-inflammatory antihypertensive drug curative effect evaluation of the Xinjiang Kazak essential hypertension, and a new research idea is provided for intervention treatment of the Xinjiang Kazak essential hypertension through an immunological mechanism.

Description

Application of hypertensive inflammation response gene as molecular marker in preparation of Xinjiang Kazakh family essential hypertension diagnostic reagent
Technical Field
The invention relates to the technical field of molecular biological diagnosis, in particular to application of a hypertensive inflammation response gene as a molecular marker in preparation of a reagent for diagnosing primary hypertension of Kazakh in Xinjiang.
Background
The Xinjiang Kazakh is the second major minority of Xinjiang, and has unique living habits and dietary patterns (good drinking, high-salt and high-fat diet, and less intake of grains, vegetables, bean products and unsaturated fatty acids), and the unique living habits and dietary patterns are key pathogenic factors causing higher incidence of cardiovascular diseases, diabetes and obesity of Xinjiang Kazakh people. Essential hypertension is a common vascular disease with unknown pathogenic cause but higher than normal blood pressure, and the pathogenesis of the essential hypertension is closely related to environmental factors, life style and genetic factors. According to epidemiological investigation research, the incidence rate of primary hypertension in the Xinjiang Kazak group of 2010-2017 is as high as 40.5% -52.39%. Therefore, the reason for the high prevalence rate of the national essential hypertension is closely related to the interaction among the genetic background, the living habits and the dietary patterns.
The existing research shows that immune cell mediated low-grade systemic inflammatory reaction caused by genetic factors, bad living habits and dietary modes is an important mechanism for the occurrence and development of cardiovascular diseases such as essential hypertension, atherosclerosis, cerebral apoplexy, myocardial infarction, heart failure and the like and the damage of corresponding target organs. Peripheral blood mononuclear cells are the main cell types which play adaptive immune responses in organisms, and recent researches show that the proliferation activation, cell subsets and functional disorder of the peripheral blood mononuclear cells are closely related to the inflammatory infiltration and structural function damage of vascular endothelium, the occurrence and development of hypertension and hypertension-mediated systemic inflammatory reaction. However, the molecular mechanism of the systemic inflammatory response mediated by peripheral blood mononuclear cells in the process of cardiovascular diseases such as hypertension is still unclear, and at present, the specific markers of inflammatory response and the combination thereof suitable for predicting and evaluating the incidence and the disease progression of hypertension of Kazak in Xinjiang and the antihypertensive effect of anti-inflammatory drugs are still lacking clinically.
Disclosure of Invention
The invention aims to provide application of a hypertension inflammation response related gene as a molecular marker in preparation of reagents for screening and diagnosing, prognosing or detecting anti-inflammatory and antihypertensive treatment effects of Xinjiang Kazakh essential hypertension, and fills up the blank that no inflammation response specific marker suitable for predicting and evaluating the onset and progress of the Xinjiang Kazakh essential hypertension and anti-inflammatory and antihypertensive treatment effects exists in clinic.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a hypertension inflammation response related gene as a molecular marker in preparation of reagents for screening, diagnosing, prognosing or detecting anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak groups in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention also provides application of the hypertension inflammation response related gene as a molecular marker in screening anti-inflammatory antihypertensive drugs, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention also provides application of a reagent for detecting the expression level of a hypertension inflammation response related gene in preparing a reagent for screening, diagnosing, prognosing or detecting the anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak nationality in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention also provides application of a reagent for detecting the expression level of a hypertension inflammation response related gene in screening anti-inflammatory and antihypertensive medicines, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
Preferably, the Xinjiang Kazakh hypertension comprises Xinjiang Kazakh essential hypertension.
The invention also provides a primer group for detecting the expression level of the hypertensive inflammatory response related gene; the primer group comprises a CAV1 amplification primer, a PDGFA amplification primer, a VAV2 amplification primer, an IGLL1 amplification primer, a MYL2 amplification primer, a MYL9 amplification primer, an ALCAM amplification primer, an HLA-DQB1 amplification primer, an HLA-DQA1 amplification primer, an HLA-DRB1 amplification primer, an ESAM amplification primer, a CCL3 amplification primer, a CXCL5 amplification primer and a CD79B amplification primer;
the CAV1 amplification primers comprise a first primer and a second primer;
the PDGFA amplification primers comprise a third primer and a fourth primer;
the VAV2 amplification primers comprise a fifth primer and a sixth primer;
the IGLL1 amplification primers comprise a seventh primer and an eighth primer;
the MYL2 amplification primers comprise a ninth primer and a tenth primer;
the MYL9 amplification primers comprise an eleventh primer and a twelfth primer;
the ALCAM amplification primers comprise a thirteenth primer and a fourteenth primer;
the HLA-DQB1 amplification primers comprise a fifteenth primer and a sixteenth primer;
the HLA-DQA1 amplification primers comprise a seventeenth primer and an eighteenth primer;
the HLA-DRB1 amplification primers comprise a nineteenth primer and a twentieth primer;
the ESAM amplification primers comprise a twenty-first primer and a twenty-second primer;
the CCL3 amplification primers comprise a twenty-third primer and a twenty-fourth primer;
the CXCL5 amplification primers comprise a twenty-fifth primer and a twenty-sixth primer;
the CD79B amplification primers comprise a twenty-seventh primer and a twenty-eighth primer;
the nucleotide sequences of the first primer to the twenty-eighth primer are respectively shown as SEQ ID No. 1-SEQ ID No. 28.
The invention also provides a kit for detecting the expression quantity of the gene related to the hypertensive inflammatory response, which comprises the primer group and the reagent for PCR amplification in the scheme.
The invention provides application of a hypertension inflammation response related gene as a molecular marker in preparation of reagents for screening, diagnosing and prognosing essential hypertension of Kazakh in Xinjiang or detecting anti-inflammatory and antihypertensive treatment effects. The gene expression spectrum and the molecular marker of the mediated hypertension inflammatory response in peripheral blood mononuclear cells of Xinjiang Kazak essential hypertension patients are obtained for the first time based on a gene chip, bioinformatics analysis and real-time quantitative polymerase chain reaction technology, new research evidence and molecular targets are provided for the prediction diagnosis, disease progress and anti-inflammatory and antihypertensive curative effect evaluation of the Xinjiang Kazak essential hypertension, and a new research idea is provided for the intervention treatment of the Xinjiang Kazak essential hypertension.
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FIG. 1 is a cluster analysis graph and volcano plot of differentially expressed mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients; wherein A is mRNA clustering heat map of differential expression in peripheral blood mononuclear cells of 6 Kazakh normal controls and 6 Kazakh essential hypertension patients, red bars in the heat map represent up-regulated mRNA, and green bars represent down-regulated mRNA; b is mRNA volcano graph differentially expressed in peripheral blood mononuclear cells of the essential hypertension patient and a normal control, the abscissa and the ordinate in the volcano graph are expression multiple change and P value size of the differentially expressed mRNA respectively, red dots represent up-regulated mRNA, blue dots represent down-regulated mRNA, and green dots represent non-differentially expressed mRNA;
FIG. 2 shows the results of GSEA analysis of mRNA up-regulated in peripheral blood mononuclear cells of patients with primary hypertension of the Kazakh family; 6 gene clusters which are expressed in peripheral blood mononuclear cells of Kazakh essential hypertension patients and up-regulate mRNA enrichment and participate in inflammatory response are respectively as follows: local adhesion (a), chemokine signaling pathway (B), leukocyte transendothelial migration (C), B cell receptor signaling pathway (D), cytokine and its receptor interaction signaling pathway (E), T cell receptor signaling pathway (F); the peak value in the line graph is the enrichment fraction of the gene cluster, and the gene before the peak value is the core gene in the gene cluster; rank in ordered dataset represents the position of gene ordering, and the upper vertical line represents the ordering position of each gene in a gene cluster; the Ranked list metric represents a value of the gene order quantity;
FIG. 3 is a GO functional biological process analysis of upregulated expressed mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients, with the abscissa representing the enrichment fraction of the biological process predicted to be involved by the upregulated expressed mRNA;
FIG. 4 is a GO functional cell fraction analysis of upregulated expressed mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients, with the abscissa representing the fraction of cell fraction enrichment predicted to be involved in upregulated expressed mRNA;
FIG. 5 is a GO functional molecular function analysis of upregulated expressed mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients, with the abscissa representing the fraction of molecular function enrichment predicted to be involved by upregulated expressed mRNA;
FIG. 6 is a KEGG signal pathway enrichment analysis of upregulated expressed mRNA in peripheral blood mononuclear cells of Kazakh essential hypertension patients, with the abscissa representing the fraction of signal pathway enrichment predicted to be involved by upregulated expressed mRNA;
FIG. 7 is a network diagram of the interaction relationship of KEGG signal pathways involved in upregulating mRNA expressed in peripheral blood mononuclear cells of patients with primary hypertension in Kazakh, wherein the size of a circle in the network diagram represents the degree of the signal pathway in the network, i.e., the number of neighbors interacting with other signal pathways, and light gray and dark gray represent that the two signal pathways are in negative correlation and positive correlation respectively;
FIG. 8 shows QRT-PCR validation of inflammatory response-associated genes in peripheral blood mononuclear cells of Kazak essential hypertension patients (n-30); histogram is the relative expression levels of 14 inflammation response-associated mrnas in peripheral blood mononuclear cells of patients with primary hypertension of the kazakh family; the measurements are expressed as Mean ± standard deviation (Mean ± SD); p <0.01 compared to control; KHS: a Kazakh normal control; KHP: kazakh hypertension patients.
Detailed Description
The invention provides application of a hypertension inflammation response related gene as a molecular marker in preparation of reagents for screening, diagnosing, prognosing or detecting anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak groups in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention provides application of a hypertension inflammation response related gene serving as a molecular marker in screening anti-inflammatory antihypertensive drugs, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention provides application of a reagent for detecting the expression level of a hypertension inflammation response related gene in preparing a reagent for screening, diagnosing, prognosing or detecting anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
The invention provides application of a reagent for detecting the expression level of a hypertension inflammation response related gene in screening anti-inflammatory and antihypertensive medicines, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B. .
The invention utilizes gene chip, bioinformatics and real-time quantitative polymerase chain reaction technology to screen mRNA related to the hypertensive inflammatory response in peripheral blood mononuclear cells of Xinjiang Kazak essential hypertension patients, and provides theoretical basis and specific molecular markers for immunological mechanism research, clinical diagnosis and accurate prevention and treatment of national essential hypertension.
In the present invention, the Sinkiang Kazakh hypertension includes Sinkiang Kazakh essential hypertension.
The invention also provides a primer group for detecting the expression level of the hypertensive inflammatory response related gene; the primer group comprises a CAV1 amplification primer, a PDGFA amplification primer, a VAV2 amplification primer, an IGLL1 amplification primer, a MYL2 amplification primer, a MYL9 amplification primer, an ALCAM amplification primer, an HLA-DQB1 amplification primer, an HLA-DQA1 amplification primer, an HLA-DRB1 amplification primer, an ESAM amplification primer, a CCL3 amplification primer, a CXCL5 amplification primer and a CD79B amplification primer;
the CAV1 amplification primers comprise a first primer and a second primer;
the PDGFA amplification primers comprise a third primer and a fourth primer;
the VAV2 amplification primers comprise a fifth primer and a sixth primer;
the IGLL1 amplification primers comprise a seventh primer and an eighth primer;
the MYL2 amplification primers comprise a ninth primer and a tenth primer;
the MYL9 amplification primers comprise an eleventh primer and a twelfth primer;
the ALCAM amplification primers comprise a thirteenth primer and a fourteenth primer;
the HLA-DQB1 amplification primers comprise a fifteenth primer and a sixteenth primer;
the HLA-DQA1 amplification primers comprise a seventeenth primer and an eighteenth primer;
the HLA-DRB1 amplification primers comprise a nineteenth primer and a twentieth primer;
the ESAM amplification primers comprise a twenty-first primer and a twenty-second primer;
the CCL3 amplification primers comprise a twenty-third primer and a twenty-fourth primer;
the CXCL5 amplification primers comprise a twenty-fifth primer and a twenty-sixth primer;
the CD79B amplification primers comprise a twenty-seventh primer and a twenty-eighth primer;
the nucleotide sequences of the first primer to the twenty-eighth primer are respectively shown as SEQ ID No. 1-SEQ ID No. 28.
The invention also provides a kit for detecting the expression quantity of the gene related to the hypertensive inflammatory response, which comprises the primer group and the reagent for PCR amplification in the scheme.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Peripheral blood mononuclear cells of primary hypertension patients of the Xinjiang Kazak family are taken as research objects, mRNA chips, bioinformatics analysis and real-time quantitative polymerase chain reaction technology are adopted to preliminarily explore the effect of inflammatory reaction mediated by peripheral blood immune cells in the pathogenesis process of the Xinjiang Kazak family primary hypertension, mRNA related to the inflammatory response of the hypertension in the peripheral blood mononuclear cells of the primary hypertension patients of the Xinjiang Kazak family is screened, and specific molecular markers and gene/drug intervention targets are provided for clinical diagnosis and accurate prevention and treatment of the hypertension.
1 object and method
1.1 sample Collection
60 Kazak hypertensive patients (15 male patients and 15 female patients) and normal physical examination crowd (20 male patients and 10 female patients) which are sourced from the department of cardiology and the department of residence and outpatient clinic at the first subsidiary hospital of Xinjiang stone river university medical college from 2018 to 2019 and 5 are selected, standardized inquiry and physical examination are carried out on the Kazak hypertensive patients, and the selected subjects are collected according to the following inclusion and exclusion standards:
essential hypertension and normal healthy controls were included as standards: A. the age is more than or equal to 38 years, and subjects have no relationship with blood; B. according to the diagnosis standards of hypertension published by the world health organization in 2010, the international union for hypertension and the Chinese guideline for hypertension control in 2010, the systolic pressure is more than or equal to 140mmHg and/or the diastolic pressure is more than or equal to 90mmHg, and all the patients are the patients who have been diagnosed as primary hypertension for the first time or have been taken for more than or equal to 2 weeks after the inquiry of medical history before admission; C. the outpatient with 3 blood pressure measurements on different days, the average systolic pressure is less than 140mmHg, and the average diastolic pressure is less than 90mmHg is the normal blood pressure contrast.
Exclusion criteria: A. patients with secondary hypertension and liver and kidney insufficiency; B. has serious cardiovascular and cerebrovascular diseases, history of excessive drinking, rheumatic diseases, inflammatory bowel disease, chronic obstructive pulmonary disease, recent operation or trauma history or patients with multiple organ function failure and malignant tumor, and patients taking contraceptive or other drug-induced diseases which may affect blood pressure. C. People with white coat hypertension, mental diseases and cognitive dysfunction. The study was reviewed by the ethical committee of the first hospital affiliated of the medical college of the university of rock river, Xinjiang, the content of the study was in accordance with the declaration of Helsinki, and all participants signed informed consent to voluntarily participate in the study.
1.2 clinical information Collection
A unified questionnaire is formulated, and clinical information of all enrolled subjects is collected, which mainly comprises: age, sex, nationality, past medical history, history of hypertension and contraceptive, smoking and drinking.
1.3 blood pressure measurement
The subject is rested quietly for at least 15min before blood pressure measurement, and is not allowed to smoke, drink alcohol or drink coffee and the like within 30 min. The subject takes a sitting position and keeps the upper arm and the heart at the same level, the mercury sphygmomanometer is used for continuously measuring the blood pressure three times, the interval of each time is not less than 5min, and the average value of the blood pressure of 3 times is taken as the measured value of the blood pressure.
1.4 physical examination and blood routine Biochemical index detection
The physical examination of the testee mainly comprises height, weight, body mass index and conventional biochemical index detection. Before the conventional biochemical test of blood generation, a subject is ordered to fast for 12h before blood sampling, 5mL of elbow median venous blood of the subject is extracted in the morning on an empty stomach, and after serum is separated, a BeckmanAU-5800 full-automatic biochemical analyzer is adopted to measure various biochemical indexes including Fasting Blood Glucose (FBG), glycosylated hemoglobin (GHb), serum Total Cholesterol (TC), Triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and serum creatinine (Cr) in various groups of patients and normal people according to a standard method.
1.5 Primary reagents
Human peripheral blood mononuclear cell separation liquid, Beijing Soilebao science and technology Co., Ltd; the total RNA extraction reagent Trizol was purchased from Invitrogen, USA; RNA purification Kit (Qiagen RNAeasy Mini Kit) purchased from Qiagen biotechnology (shanghai) ltd; human LncRNA-The mRNAV4.0 gene chip is purchased from Agilent technologies, Inc.; cDNA reverse transcription kit (PrimeScript)TMRT reagent Kit) and real-time quantitative polymerase chain reaction Kit (TB)
Figure BDA0003401832930000081
Premix Ex TaqTMII) purchased from Baori physician technology (Beijing) Ltd.
1.6 peripheral blood mononuclear cell separation and Total RNA extraction
Randomly drawing 5ml of venous blood of hypertension patients meeting the inclusion standard and normal controls thereof, separating peripheral blood mononuclear cells by adopting a Ficoll-Hypaque density gradient centrifugation method, detecting cell activity by a trypan blue staining method, adding an RNA extraction reagent Trizol into the peripheral blood mononuclear cells, and storing the peripheral blood mononuclear cells in a refrigerator at-80 ℃ for later use. After total RNA in each set of peripheral blood mononuclear cells was extracted by Trizol reagent, total RNA concentration, purity and integrity were examined using Thermo Nanodrop ND-2000C nucleic acid/protein analyzer (Thermo Scientific Co.), formaldehyde denaturing agarose gel electrophoresis and Agilent 2100 bioanalyzer (Agilent Technologies Co.).
1.7mRNA expression chip hybridization assay
In each group of subjects, 6 total RNA samples qualified for quality inspection were selected and used
Figure BDA0003401832930000091
Purifying total RNA by using an RNA Clean-up Kit, synthesizing double-stranded cDNA by using the purified RNA (200- & 500ng) as a template, synthesizing cRNA by using the cDNA as the template, purifying the cRNA by using a Qiagen RNAeasy Mini Kit, synthesizing cDNA containing Cyanine 3-dCTP fluorescent markers again by taking 5-10 mu G of the purified cRNA, and hybridizing the fragmented cNDA in an Agilent G2545A gene chip hybridization furnace (45 ℃, 20rpm) by using an Agilent human LncRNA/mRNA V4.0 gene chip for 16 h; after hybridization, the chip was washed and immediately scanned by the Agilent G2565CA chip scanner on the machine to obtain the original image. And finally, processing the original image by adopting Agilent Feature Extraction (version 11.0.1.1) software, and collecting the fluorescence signal value in the chip probe.
1.8 mRNA functional annotation and KEGG pathway enrichment analysis of differential expression
The mRNA with up-regulated expression in peripheral blood mononuclear cells was functionally annotated and analyzed for KEGG signaling pathway enrichment using the "Bioconductor" software package in the R language, the Cluster Profiler R software package, the Gene Set Enrichment Analysis (GSEA) software, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the Gene Ontology (GO) (http:// www.geneongoloty.org /) database, the biomolecule function annotation System software (http//: biooinfo. capitalbio. com/MAS) and the DAVID software. Firstly, mapping an input gene set with up-regulated expression to a database gene, counting the number of target genes included in each GO item, carrying out FDR correction by using a QVALUE and Benjamini-Hochberg multiple hypothesis test method, calculating the significance of target gene enrichment in each GO item, and finally selecting a biological function with the P being less than or equal to 0.05 for significant enrichment. In addition, a KEGG database is used for carrying out signal path enrichment analysis on the differentially expressed genes, the significance of gene enrichment in each signal path item is measured by the statistical test method, the signal path with the significant enrichment P less than or equal to 0.05 is selected, and finally, an R language software package is used for drawing a histogram of function and path enrichment.
1.9 Quantitative real-time polymerase chain reaction (QRT-PCR)
Extracting the total RNA of the peripheral blood mononuclear cells of each group by adopting a Trizol reagent, and detecting the concentration, purity and integrity of the total RNA; taking 500 ng-1 mu g of total RNA, adopting PrimeScriptTM1st Strand cDNA Synthesis Kit cDNA template was synthesized. Taking each group of cDNA samples obtained by reverse transcription as a template, adopting Oligo 7.0 and Primer 5.0 software to design specific primers (table 1) of a gene to be detected and an internal reference gene GAPDH synthesized by Shanghai biological engineering Co., Ltd, and adopting a QRT-PCR kit to carry out QRT-PCR in a CFX96 real-time PCR instrument of Berle company under the reaction condition of 95 ℃ for 5 min; 10s at 95 ℃, 15s at 60 ℃, 20s at 72 ℃ and 40 cycles. 2 parallel wells were set for detection of all genes of interest per sample. Finally according to
Figure BDA0003401832930000102
The formula calculates the relative expression level of the gene of interest.
TABLE 1 QRT-PCR primer sequences
Figure BDA0003401832930000101
Figure BDA0003401832930000111
1.10 statistical treatment
For chip data processing, raw data is firstly normalized by using a "Percentile 75" method in Agilent GeneSpring GX v13.0 software, and then chip scanning quality is evaluated through a box plot. And screening the differential expression genes of which the up-regulation or down-regulation multiple change value FC is more than or equal to 1.5 and P is less than or equal to 0.05 by adopting the difference Fold Change (FC) values of the difference significance P value and the standardized signal value obtained by the correction of t test and the Benjamini Hochberg method. Hierarchical clustering analysis was then performed on differentially expressed genes using CLUSTER 3.0 software, and functional analysis was performed on up-regulated expressed mRNA using GSEA software, Go (http:// www.geneongoloty.org /) database and biomolecule functional annotation system software (http//: biolnfo. capitalbio. com/MAS /). Other data were statistically analyzed using GraphPad Prism 8.3.0 software, data statistics are expressed as Mean. + -. standard deviation (Mean. + -. SD), and comparisons of data between groups were tested using Student's t with P <0.05 as the difference being statistically significant.
2 results
2.1 general clinical indices of Kazakh essential hypertension patients and Kazakh Normal controls
Clinical indicators of Kazakh essential hypertension patients and their normal controls are shown in Table 2. Wherein gender, mean age, Body Mass Index (BMI), history of smoking, history of drinking, fasting plasma glucose, glycated hemoglobin, total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) levels were not significantly different between the Kazakh hypertensive group and the Kazakh normal control group (P >0.05, Table 2). The systolic and diastolic blood pressure of Kazakh hypertensive patients were significantly higher than that of the normal control group (P <0.01, Table 2).
TABLE 2 clinical data of Kazakh essential hypertension patients and their normal controls (n ═ 30)
Figure BDA0003401832930000121
Figure BDA0003401832930000131
Note: measure data to
Figure BDA0003401832930000132
And (4) showing. BMI is body mass index; FBG, fasting blood glucose; GHb is glycosylated hemoglobin; TC is total cholesterol; TG is triglyceride; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.
2.2 mRNA differentially expressed in peripheral blood mononuclear cells of Kazakh essential hypertension patients
The results of the gene chip analysis showed that 2549 mRNAs were detected in total. Under the conditions that the screening threshold value is FC (fiber channel) is more than or equal to 1.5 and P is less than or equal to 0.05, compared with the normal control of Kazakh, 522 mRNAs with significant differential expression are totally contained in peripheral blood mononuclear cells of a Xinjiang Kazakh hypertensive patient, wherein 411 genes with up-regulated expression and 111 mRNAs with down-regulated expression are contained in the peripheral blood mononuclear cells (figure 1); the first 15 mrnas with the highest fold up expression were shown in table 3.
TABLE 3 mRNA with upregulated expression in peripheral blood mononuclear cells of Primary hypertension patients of the Kazakh family
Figure BDA0003401832930000133
2.3 GSEA analysis of upregulated expression mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients
According to the standard that P is less than or equal to 0.05, the present invention firstly carries out GSEA analysis on mRNA with up-regulated expression, and the result is shown in Table 4 and figure 2, and the result shows that 14 gene clusters (Table 4) are obtained, wherein 6 gene clusters (local adhesion, chemokine signal path, leukocyte transendothelial migration, B cell receptor signal path, cytokine and receptor interaction signal path, T cell receptor signal path) (figure 2) are possibly related to the hypertensive inflammatory response.
TABLE 4 GSEA analysis of upregulated expression mRNA in peripheral blood mononuclear cells of Kazak essential hypertension patients
Figure BDA0003401832930000141
2.4 GO function and KEGG signal channel enrichment analysis of upregulated and expressed mRNA in peripheral blood mononuclear cells of Kazakh essential hypertension patients
Performing GO function and KEGG enrichment analysis on the up-regulated mRNA through R software, and displaying that biological processes related to inflammatory response in the up-regulated mRNA mainly relate to platelet activation, immune response regulation, immune response activation and signal transduction thereof through results of the up-regulated mRNA; the cellular components mainly relate to immunoglobulin complexes, local adhesion, major histocompatibility class II protein complexes, and the like; the molecular functions mainly relate to antigen binding, immunoglobulin receptor binding, polypeptide antigen binding, and the like, as shown in fig. 3 to 5. The results of KEGG enrichment analysis show that mRNA up-regulated and expressed in peripheral blood mononuclear cells of Kazak essential hypertension patients mainly participates in signal pathways related to inflammatory responses such as local adhesion, cell adhesion molecules, intestinal immune signal pathways for synthesizing IgA, chemokine signal pathways, leukocyte transendothelial migration, interaction of cytokines and receptors thereof, B cell receptor signal pathways and the like (figure 6), and some enrichment pathways are consistent with the results of GSEA analysis (figure 2).
2.5 Co-expression network construction of Primary hypertension inflammatory response-related Signal pathways of Kazakh
For further analyzing the relevant genes of Kazakh essential hypertension inflammatory responseParticipating in the possible interaction relationship among signal paths, and drawing a network diagram of the signal paths related to the Kazakh essential hypertension inflammatory response in the KEGG enrichment analysis result. The results show that there are interactions between T cell receptor signaling pathways and cell adhesion molecules, between cell adhesion molecules and leukocyte transendothelial migration, between local adhesion and extracellular matrix receptors during the inflammatory response to primary hypertension in the kazakh family (fig. 7). And the T cell receptor signal pathway can be related to multiple inflammatory response related signal pathways (NF-kB, PI3K, MAPK, Ca)2+Signal path) to produce an interaction (fig. 7).
2.6 screening of molecular markers associated with inflammatory response to essential hypertension of Kazakh
Induction, sorting and intersection analysis are carried out on the signal paths related to the inflammatory response and the up-regulated expression genes thereof in the KEGG signal paths and the GSEA analysis results, after the genes related to the inflammatory response which are not reported in documents at present are removed, 14 candidate target difference genes which are involved in a plurality of signal paths related to the inflammatory response are finally selected from the intersection results of the two bioinformatics analyses, and the results are shown in Table 5.
TABLE 5 expression upregulation genes in the Primary hypertensive inflammatory response-related signalling pathway of the Kazakh family
Figure BDA0003401832930000151
Figure BDA0003401832930000161
2.7 verification of expression level of molecular marker related to inflammatory response of Primary hypertension of Kazakh family
In order to confirm the reliability of mRNA chip expression data and the effect of the screened molecular marker related to the hypertensive inflammatory response in the onset of the essential hypertension of the Kazakh family, QRT-PCR was adopted to verify the screened candidate molecular marker in 15 cases of patients with the essential hypertension of the Kazakh family. The results showed that the expression of 14 inflammation response-associated genes (CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5, CD79B) involved in local adhesion, cell adhesion molecules, IgA-synthesizing intestinal immune signaling pathway, chemokine signaling pathway, leukocyte transendothelial migration, cytokine interaction with its receptor, and B-cell receptor signaling pathway in peripheral blood mononuclear cells of the primary hypertensive patient of the kazakh group were significantly up-regulated compared to the normal control of the kazak group (fig. 8, P <0.01), which was consistent with mRNA chip expression data.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (7)

1. The application of the hypertension inflammation response related gene as a molecular marker in preparing reagents for screening, diagnosing, prognosing or detecting anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak nationality in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
2. The application of the hypertension inflammation response related gene as a molecular marker in screening anti-inflammatory antihypertensive drugs, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
3. The application of a reagent for detecting the expression level of a hypertension inflammation response related gene in preparing a reagent for screening, diagnosing, prognosing or detecting the anti-inflammatory and antihypertensive treatment effects of primary hypertension of Kazak in Xinjiang, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
4. The application of the reagent for detecting the expression level of the hypertension inflammation response related gene in screening anti-inflammatory and antihypertensive medicines, wherein the hypertension inflammation response related gene comprises one or more of CAV1, PDGFA, VAV2, IGLL1, MYL2, MYL9, ALCAM, HLA-DQB1, HLA-DQA1, HLA-DRB1, ESAM, CCL3, CXCL5 and CD 79B.
5. The use of any one of claims 1 to 4, wherein the hypertension of the Kazakh, Xinjiang comprises essential hypertension of the Kazakh, Xinjiang.
6. A primer group for detecting the expression level of a gene related to hypertensive inflammatory response; the primer group comprises a CAV1 amplification primer, a PDGFA amplification primer, a VAV2 amplification primer, an IGLL1 amplification primer, a MYL2 amplification primer, a MYL9 amplification primer, an ALCAM amplification primer, an HLA-DQB1 amplification primer, an HLA-DQA1 amplification primer, an HLA-DRB1 amplification primer, an ESAM amplification primer, a CCL3 amplification primer, a CXCL5 amplification primer and a CD79B amplification primer;
the CAV1 amplification primers comprise a first primer and a second primer;
the PDGFA amplification primers comprise a third primer and a fourth primer;
the VAV2 amplification primers comprise a fifth primer and a sixth primer;
the IGLL1 amplification primers comprise a seventh primer and an eighth primer;
the MYL2 amplification primers comprise a ninth primer and a tenth primer;
the MYL9 amplification primers comprise an eleventh primer and a twelfth primer;
the ALCAM amplification primers comprise a thirteenth primer and a fourteenth primer;
the HLA-DQB1 amplification primers comprise a fifteenth primer and a sixteenth primer;
the HLA-DQA1 amplification primers comprise a seventeenth primer and an eighteenth primer;
the HLA-DRB1 amplification primers comprise a nineteenth primer and a twentieth primer;
the ESAM amplification primers comprise a twenty-first primer and a twenty-second primer;
the CCL3 amplification primers comprise a twenty-third primer and a twenty-fourth primer;
the CXCL5 amplification primers comprise a twenty-fifth primer and a twenty-sixth primer;
the CD79B amplification primers comprise a twenty-seventh primer and a twenty-eighth primer;
the nucleotide sequences of the first primer to the twenty-eighth primer are respectively shown as SEQ ID No. 1-SEQ ID No. 28.
7. A kit for detecting an expression level of a gene involved in a hypertensive inflammatory response, comprising the primer set according to claim 6 and a reagent for PCR amplification.
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