Urine protein composition for detecting helicobacter pylori related gastritis and application of urine protein composition

Abstract

The invention provides a urine protein composition for detecting helicobacter pylori related gastritis and application of the urine protein composition, and relates to the technical field of clinical gastritis detection. The urine protein composition is prepared from TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4 and RNASE6 (Ribonucleic Acid Sequence Enzyme 6). The defects in the prior art are overcome, typical difference urine proteins of healthy groups and Hp-related gastritis are screened, the proteins are adopted as markers to design detection, preliminary diagnosis can be conducted on helicobacter pylori-related gastritis noninvasively and conveniently, and a new direction is provided for clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical detection of gastritis, and particularly relates to a urine protein composition for detecting Helicobacter pylori-associated gastritis and its application. Background Art

[0002] Helicobacter pylori (Hp)-associated gastritis is a common digestive system disease that seriously affects the quality of life of patients. It is mainly caused by the infection of a class of bacteria called Helicobacter pylori. The symptoms caused by Helicobacter pylori are mainly dull pain in the upper abdomen, fullness, acid reflux, belching, nausea, etc. A small number of patients are asymptomatic (especially children), but long-term infection may lead to gastric mucosal damage and eventually form gastritis. Therefore, the early detection of Helicobacter pylori-associated gastritis is relatively important.

[0003] At present, the main detection methods for Helicobacter pylori are 13 C / 14 C urea breath test, fecal antigen detection, serological detection (IgG antibody), etc. However, these mainly detect the infection of Helicobacter pylori and cannot determine whether the patient has Helicobacter pylori-associated gastritis. If further diagnosis is required, gastroscopy is usually needed, and gastroscopy requires anesthesia and is relatively complex. Many patients without early symptoms are not willing to accept such an examination. Therefore, the early diagnosis of Helicobacter pylori-associated gastritis is often missed. And Helicobacter pylori-associated gastritis is prone to cause gastric mucosal atrophy, intestinal metaplasia, dysplasia and even gastric cancer if it gradually develops. Therefore, in order to ensure the effectiveness of treatment and prevent the deterioration of the disease, the diagnosis of Helicobacter pylori-associated gastritis is crucial.

[0004] Urine is not regulated by a homeostasis mechanism. It is a metabolite of the body, enriches various changes in the body, can be obtained in large quantities in a non-invasive manner, and is an ideal source of disease markers. It has great clinical application prospects in the early diagnosis of diseases. Based on this, it is necessary to develop urine proteins for marker diagnosis for the diagnosis of Helicobacter pylori-associated gastritis. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a urine protein composition for detecting Helicobacter pylori-associated gastritis and its application. By screening the typical differential urine proteins in the healthy group and the Hp-related gastritis group, and using these proteins as markers to design a detection method, the Helicobacter pylori-associated gastritis can be preliminarily diagnosed in a non-invasive and convenient manner, providing a new direction for clinical diagnosis.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A urine protein composition for detecting Helicobacter pylori - associated gastritis, wherein the urine protein composition is any one or more of TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, RNASE6.

[0008] Preferably, TENM4, RPL23, TFF1, RPS25, PAFAH1B2 are up - regulated in the patient group compared with the healthy group; CD300E, CTSS, MT1F, ENPP4, RNASE are down - regulated in the patient group compared with the healthy group.

[0009] Preferably, the protein composition further contains any one or more of MSMB, IGLV3 - 16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6, EMC3, IGLV1 - 40, GGH, CUL9, IGLV1 - 36, SETD2, CLPS, CGA, IGLV10 - 54, FSHB.

[0010] Preferably, MSMB, IGLV3 - 16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6 are up - regulated in the patient group compared with the healthy group; EMC3, IGLV1 - 40, GGH, CUL9, IGLV1 - 36, SETD2, CLPS, CGA, IGLV10 - 54, FSHB are down - regulated in the patient group compared with the healthy group.

[0011] Use of an identification reagent for a protein marker composition for diagnosing Helicobacter pylori - associated gastritis in the preparation of a product for diagnosing Helicobacter pylori - associated gastritis, wherein the identification reagent is a reagent for identifying the content of any one or more of TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, RNASE6 in urine.

[0012] Preferably, the identification reagent also identifies the content of any one or more of MSMB, IGLV3-16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6, EMC3, IGLV1-40, GGH, CUL9, IGLV1-36, SETD2, CLPS, CGA, IGLV10-54, FSHB in urine.

[0013] Use one or more of the above identification reagents to design a kit or chip for diagnosing Helicobacter pylori-associated gastritis.

[0014] The present invention provides a urine protein composition for detecting Helicobacter pylori-associated gastritis and its application. Compared with the prior art, the advantages are as follows:

[0015] Through experimental research, the present invention found that compared with the urine of the healthy group, there are 29 up-regulated and 15 down-regulated differential proteins in the urine of Hp-associated gastritis patients. The top five proteins with the most prominent up-regulation and down-regulation are TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, RNASE6 respectively. By detecting the up-regulated and down-regulated proteins in urine, the risk of patients suffering from Hp-associated gastritis can be effectively detected. Designing detection reagents, kits, chips and other products for these proteins can conveniently and non-invasively achieve early screening of Hp-associated gastritis, providing a good application prospect for the clinical detection of Hp-associated gastritis. Brief Description of the Drawings

[0016] Figure 1 It is a statistical bar chart of DIA identification results in the embodiment of the present invention, where identified Peptides represents the total number of identified peptide segments; Identified Proteins represents Protein groups, the total number of identified proteins; different colors represent different groups, and the dotted line in the figure represents the number of proteins / peptide segments identified in 50% of the samples;

[0017] Figure 2 It is a Venn diagram of inter-group samples in the embodiment of the present invention;

[0018] Figure 3 It is a 2D PCA distribution diagram of patient group and healthy group samples in the embodiment of the present invention;

[0019] Figure 4It is the 3D PCA distribution map of the samples of the patient group and the healthy group in the embodiments of the present invention;

[0020] Figure 5 It is the PCC analysis diagram of the proteins identified from the samples of the patient group and the healthy group in the embodiments of the present invention;

[0021] Figure 6 It is the RSD analysis diagram of the proteins identified between the sample groups in the embodiments of the present invention;

[0022] Figure 7 It is the scatter plot of the protein abundance distribution of the patient group and the healthy group in the embodiments of the present invention, where the abscissa is the ranking of the protein expression level and the ordinate is the intensity value of the protein (log10 transformed);

[0023] Figure 8 It is the bar chart of the quantitative difference results of the proteins of the patient group and the healthy group in the embodiments of the present invention;

[0024] Figure 9 It is the volcano plot of the protein differences between the patient group and the healthy group in the embodiments of the present invention. Detailed implementation manners

[0025] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0026] Embodiment:

[0027] Determination of differential proteins related to Hp-associated gastritis:

[0028] 1. Sample acquisition:

[0029] Twenty healthy individuals and twenty individuals diagnosed with Hp-related gastritis were selected as volunteers. The urine of the volunteers was collected and divided into the patient group 1-20 (Pos100, Pos104, Pos17, Pos18, Pos19, Pos20, Pos21, Pos25, Pos27, Pos30, Pos41, Pos42, Pos45, Pos55, Pos62, Pos63, Pos76, Pos84, Pos88, Pos99) and the healthy group 1-20 (Neg14, Neg16, Neg22, Neg33, Neg36, Neg37, Neg48, Neg57, Neg58, Neg69, Neg70, Neg73, Neg77, Neg78, Neg79, Neg80, Neg82, Neg85, Neg86, Neg87) for testing;

[0030] 2. Sample pretreatment:

[0031] Each sample was analyzed by LC-MS / MS (DIA mode, Astral mass spectrometer), and qualitative and quantitative analysis of peptides and proteins was performed; after independent sample preparation and protein enzymatic digestion of each sample, DIA (data-independent acquisition) analysis was performed on the instrument respectively. The obtained DIA raw files were imported into DIA-NN for analysis;

[0032] The mass spectrometry experiment analysis process mainly includes protein extraction, peptide enzymatic digestion, liquid chromatography-tandem mass spectrometry (LC-MS / MS) DIA data acquisition, database retrieval, qualitative and quantitative result analysis, and bioinformatics analysis.

[0033] 3. Identification quantity analysis:

[0034] 3.1 Identification quantity analysis

[0035] Among them, the statistics of identification and quantification results are as Figure 1 , and the proteins identified in each group are displayed in the form of a Venn diagram. See specifically Figure 2 .

[0036] 2D and 3D PCA analysis was performed on all samples, and the results are as Figure 3 and Figure 4 shown. In Figure 3 and Figure 4 , PC1 represents the first principal component, PC2 represents the second principal component, PC3 represents the third principal component, each point represents a sample, and different colors represent different groups respectively.

[0037] A heatmap is plotted by calculating the Pearson correlation coefficient between all pairs of samples. This coefficient is a value that measures the degree of linear correlation between two sets of data: when the Pearson coefficient is closer to -1, it indicates a negative correlation; when it is closer to 1, it indicates a positive correlation; when it is closer to 0, it indicates no correlation. The results are as shown in Figure 5 as follows.

[0038] The smaller the relative standard deviation (RSD) of the protein quantification values between samples, the better the quantitative repeatability of proteomics. The RSD analysis of the proteins identified between sample groups is as shown in Figure 6 as follows.

[0039] Scatter plot analysis is performed on the protein abundances identified in the samples of all groups. The specific results are as shown in Figure 7 as follows.

[0040] 3.2. Differential expression analysis

[0041] Statistics of the number of differential results: In the screening of significantly differential proteins, with the expression fold change (FC) > 1.5 times (upregulation greater than 1.5 times or downregulation less than 0.67 times) and P value < 0.05 (T-test or others) as the criteria, the numbers of upregulated and downregulated proteins between comparison groups are obtained, that is, 29 significantly upregulated proteins and 15 significantly downregulated proteins are obtained, as shown in Figure 7 .

[0042] The 29 significantly upregulated proteins are: MSMB, IGLV3-16, RPS13, ERMAP, IDH2, HYDIN, RPL23, PSMC3, DLD, TFR2, RPS16, RPS25, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, PAFAH1B2, TRIM47, TFPI2, EEF1D, TFF1, PCDHB13, LAMA1, TENM4, GALNT6

[0043] The 15 significantly downregulated proteins are: ENPP4, EMC3, CD300E, MT1F, IGLV1-40, GGH, CUL9, IGLV1-36, SETD2, RNASE6, CTSS, CLPS, CGA, IGLV10-54, FSHB.

[0044] To show the significant differences in proteins between the comparison groups, volcano plots were drawn for the proteins in the comparison groups based on two factors: fold change in expression and P value (T-test). Proteins with significant downregulation were marked in blue (FC < 0.67 and p < 0.05), proteins with significant upregulation were marked in red (FC > 1.5 and p < 0.05), and proteins with no difference were in gray. The top 5 proteins with the most significant differences in up- and downregulation were marked. The results are as shown in Figure 9 which shows the 5 most significantly upregulated and downregulated proteins. The upregulated proteins are NPNT, TFAP2D, TMPRSS13, JSRP1, and SORCS2; the downregulated proteins are ERMAP, NKX2-2, HLA-DMB, KIF5B, and CRYAA.

[0045] And the average values of the protein profiles of the corresponding differential proteins in each group and the ratio of the patient group / healthy group were detected. The specific results are shown in the following table:

[0046]

[0047]

[0048] That is, there are significant differences in the 29 upregulated proteins and 15 downregulated proteins in the diseased group and the healthy group, and TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, and RNASE6 have the most significant differences.

[0049] By designing corresponding urine protein detection reagents based on the above significant differences, patients who may have Hp-related gastritis can be effectively and preliminarily screened out, providing a good direction for actual clinical detection.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A urine protein composition for detecting Helicobacter pylori - associated gastritis, characterized in that, The urine protein composition is any one or more of TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, RNASE6.

2. The urine protein composition according to claim 1, wherein: TENM4, RPL23, TFF1, RPS25, PAFAH1B2 are up-regulated in the patient group compared with the healthy group; CD300E, CTSS, MT1F, ENPP4, RNASE are down-regulated in the patient group compared with the healthy group.

3. The urinary protein composition according to claim 1, wherein: The protein composition further contains any one or more of MSMB, IGLV3-16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6, EMC3, IGLV1-40, GGH, CUL9, IGLV1-36, SETD2, CLPS, CGA, IGLV10-54, FSHB.

4. The urine protein composition according to claim 3, wherein: MSMB, IGLV3-16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6 are up-regulated in the patient group compared with the healthy group; EMC3, IGLV1-40, GGH, CUL9, IGLV1-36, SETD2, CLPS, CGA, IGLV10-54, FSHB are down-regulated in the patient group compared with the healthy group.

5. Use of an identification reagent for a protein marker composition for diagnosing Helicobacter pylori-associated gastritis in the preparation of a product for diagnosing Helicobacter pylori-associated gastritis, characterized in that: The identification reagent is a reagent for identifying the content of any one or more of TENM4, RPL23, TFF1, RPS25, PAFAH1B2, CD300E, CTSS, MT1F, ENPP4, RNASE6 in urine.

6. The urine protein composition according to claim 5, characterized in that: The identification reagent also identifies the content of any one or more of MSMB, IGLV3-16, RPS13, ERMAP, IDH2, HYDIN, PSMC3, DLD, TFR2, RPS16, ISLR2, HBM, PSMD4, CSRP1, RPS18, YOD1, HMGB1, DNAJB2, TRIM47, TFPI2, EEF1D, PCDHB13, LAMA1, GALNT6, EMC3, IGLV1-40, GGH, CUL9, IGLV1-36, SETD2, CLPS, CGA, IGLV10-54, FSHB in urine.

7. A kit or chip for diagnosing Helicobacter pylori - associated gastritis, characterized in that: The kit or chip contains the identification reagent according to any one of claims 5-6.

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