A kit for predicting the efficacy of interferon in patients with chronic hepatitis B and its application

Abstract

The present invention relates to the field of biomedical technology, and in particular to a kit for predicting the efficacy of interferon in patients with chronic hepatitis B and its application. The kit comprises a reagent for detecting the relative expression of THBS1 mRNA. The kit predicts the efficacy of interferon treatment in patients with chronic hepatitis B by detecting the relative expression of THBS1 mRNA in the peripheral blood of the patient, thereby reducing the situation in which different groups of people have different responses to interferon due to individual differences in THBS1 expression. Compared with traditional test indicators, the relative expression of THBS1 mRNA can better reflect the human body's response to interferon.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a kit for predicting the efficacy of interferon in chronic hepatitis B patients and its application. Background Art

[0002] Interferon is the first-line drug for the clinical treatment of chronic hepatitis B. Compared with other antiviral drugs, interferon has the characteristic of enabling patients to achieve clinical cure. However, the individual differences in the efficacy of interferon are large. At present, the clinical efficacy of interferon is mainly determined by detecting indicators such as the serum hepatitis B virus genotype, HBV DNA (hepatitis B virus gene), HBsAg (hepatitis B surface antigen), and ALT (alanine aminotransferase) of patients before treatment. Specifically, the Chinese invention patent with application number 201210019389.5 discloses that the criterion for judging the efficacy of treatment can be that the status of chronic hepatitis B patients receiving treatment changes from HBeAg(+) / Anti-HBe(-) to HBeAg(-) / Anti-HBe(+), and the serum HBV DNA load of chronic hepatitis B patients drops to below 1000 copies / mL. In addition, HBV DNA before treatment is less than 2×10 8 IU / ml, ALT exceeds the upper limit of normal by 2 to 10 times, HBsAg is less than 2.5×10 4 IU / ml, HBV genotype B or C, both indicate that interferon has a good therapeutic effect.

[0003] However, the individual differences in the efficacy of interferon are very obvious, and the above indicators cannot accurately predict the efficacy of interferon. The use of the above indicators for prediction often leads to treatment failure in clinical practice, mainly manifested in high HBV DNA and persistent positivity of HBeAg (hepatitis B e antigen). Interferon is not only expensive, but also prone to cause flu-like syndrome, induce autoimmune diseases, peripheral blood cell reduction and other drug side effects. If the efficacy of interferon cannot be identified before treatment, it will not only increase medical costs, but also make patients bear unnecessary risks of drug side effects. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a kit for predicting the efficacy of interferon in patients with chronic hepatitis B and its application.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: Application of THBS1 as a marker in a kit for predicting the efficacy of interferon in patients with chronic hepatitis B

[0006] Another technical solution adopted by the present invention is: a kit for predicting the efficacy of interferon in patients with chronic hepatitis B, comprising a reagent for detecting the relative expression amount of THBS1 mRNA.

[0007] Another technical solution adopted by the present invention is: the use of the above-mentioned kit in predicting the efficacy of interferon in patients with chronic hepatitis B for non-therapeutic diagnostic purposes, comprising the following steps: taking a whole blood sample from the patient with chronic hepatitis B before treatment, extracting total RNA, performing reverse transcription PCR and real-time fluorescence quantitative PCR on the whole blood sample in sequence to obtain the Ct values ​​of THBS1 and β-actin (β-actin); using the Ct values ​​of THBS1 and β-actin to calculate the relative expression of THBS1 mRNA to determine the efficacy of interferon.

[0008] The beneficial effect of the present invention is that THBS1 is positively correlated with the expression of related genes on the interferon activation pathway, and THBS1 can be used as a marker to prepare a kit for predicting the efficacy of interferon in chronic hepatitis B patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The figure shows the ROC curve of subjects in Example 2 of the present invention using the relative expression of THBS1 to predict the efficacy of interferon;

[0010] Figure 2 The figure shows the comparison of the expression levels of related genes and antiviral expression proteins on the interferon activation pathway of HepAD38 cells before and after gene knockout in Example 4 of the present invention. DETAILED DESCRIPTION

[0011] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0012] Application of THBS1 as a marker in a kit for predicting the efficacy of interferon in patients with chronic hepatitis B.

[0013] From the above description, it can be seen that the beneficial effect of the present invention is that the THBS1 gene is located on the 14th band of human chromosome 15, and has a total of 22 exons. The inventors found through research that THBS1 is positively correlated with the expression of related genes in the interferon activation pathway, which is specifically described as follows:

[0014] THBS1 inhibits the transport of FFA (Free Fatty Acid) by cells by binding to the receptor CD36, downregulates PPARA (Peroxisome Proliferator Activated Receptor Alpha) signaling, inhibits fatty acid oxidation and the production of unsaturated fatty acids, and promotes the polarization of macrophages to the M1 type. On the one hand, M1 macrophages secrete cytokines such as IL-1β (catabolite), IL-6 (interleukin 6) and TNF-α (Tumor Necrosis Factor α), which synergize with interferon to enhance the body's innate immunity and play a role in clearing HBV. On the other hand, by upregulating ISGF3 (Interferon-stimulated gene factor 3), M1 macrophages enhance the expression of antiviral proteins downstream of the interferon signaling pathway, including ADAR1 (Adenosine Deaminase), PKR (double-stranded RNA-dependent protein kinase), OAS (Oligoadenylate synthetase) and Mx protein, thereby playing an anti-HBV role.

[0015] Another technical solution adopted by the present invention is: a kit for predicting the efficacy of interferon in patients with chronic hepatitis B, comprising a reagent for detecting the relative expression amount of THBS1 mRNA.

[0016] From the above description, it can be seen that due to individual differences in THBS1 expression, different populations have different responses to interferon. The kit of the present invention predicts the efficacy of interferon treatment for chronic hepatitis B patients by detecting the relative expression of THBS1 mRNA in the patient's peripheral blood. Compared with traditional test indicators, the relative expression of THBS1 mRNA can better reflect the human body's response to interferon.

[0017] Furthermore, the kit includes a real-time fluorescence quantitative PCR reaction system, and the real-time fluorescence quantitative PCR reaction system includes a THBS1 primer set.

[0018] Furthermore, the THBS1 primer set consists of two primers shown in SEQ ID NO: 1 and SEQ ID NO: 2, see Table 1.

[0019] Furthermore, the real-time fluorescence quantitative PCR reaction system also includes a β-actin primer set, which consists of two primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, as shown in Table 1.

[0020] Table 1

[0021]

[0022] From the above description, it can be seen that the β-actin primer set is used as an internal reference.

[0023] Furthermore, the kit also includes a reverse transcription PCR reaction system.

[0024] Furthermore, the reverse transcription PCR reaction system includes polythymidine (polythymidine), buffer, dNTP (deoxyribonucleoside triphosphate), reverse transcriptase and RNase inhibitor.

[0025] Another technical solution adopted by the present invention is: the use of the above-mentioned kit in predicting the efficacy of interferon in patients with chronic hepatitis B for non-therapeutic diagnostic purposes, that is, using the above-mentioned kit to predict the efficacy of interferon on patients with chronic hepatitis B, comprising the following steps: taking a whole blood sample from the chronic hepatitis B patient before treatment, extracting total RNA, performing reverse transcription PCR and real-time fluorescence quantitative PCR on the whole blood sample in sequence to obtain the Ct values ​​of THBS1 and β-actin; using the Ct values ​​of THBS1 and β-actin to calculate the relative expression of THBS1 mRNA to determine the efficacy of interferon.

[0026] Furthermore, the specific steps of reverse transcription PCR are: adding the sample obtained after total RNA extraction to polythymidine and mixing to perform the first step PCR reaction; after the first step PCR reaction is completed, adding buffer, dNTP, reverse transcriptase and RNase inhibitor to perform the second step PCR reaction.

[0027] From the above description, it can be seen that in the process of reverse transcription PCR, the first step of PCR reaction is carried out to specifically bind polythymine and the polyA tail of mRNA. In this process, the role of polythymine is equivalent to the primer of reverse transcription PCR. After the reaction is completed, buffer, dNTP, reverse transcriptase and RNase inhibitor are added to carry out the second step of PCR reaction.

[0028] Furthermore, a relative expression level of THBS1 mRNA ≥8.5 indicates that the patient has a good therapeutic effect of interferon; a relative expression level of THBS1 mRNA <8.5 indicates that the therapeutic effect of interferon is poor.

[0029] From the above description, it can be seen that the relative expression level can be used to predict the efficacy of interferon, and the judgment standard can be digitized, which is more convenient and quick.

[0030] Embodiment 1 of the present invention is: a kit for predicting the efficacy of interferon in patients with chronic hepatitis B. The reagents contained are shown in Table 2, and the specific sequences of the primers in Table 2 are shown in Table 1.

[0031] Table 2

[0032]

[0033]

[0034] Embodiment 2 of the present invention is: using the kit of embodiment 1 to construct an interferon efficacy judgment standard.

[0035] S1. Select samples:

[0036] Thirty patients with chronic hepatitis B who responded to interferon (average age 27.86±4.50, male to female ratio 21 / 9) and 35 patients with chronic hepatitis B who did not respond to interferon (average age 27.8571±6.0979, male to female ratio 26 / 9) were selected. The specific sample grouping criteria are as follows:

[0037] Patients with chronic hepatitis B who are responsive to interferon and receive regular (24 to 72 weeks) conventional interferon or pegylated interferon treatment all meet the following conditions:

[0038] 1) Virological response: serum HBV DNA is undetectable (PCR method) or below the detection limit or decreased by ≥2 log IU / mL compared with baseline;

[0039] 2) Serological response: serum HBeAg negative or HBeAg seroconversion, or HBsAg negative or HBsAg seroconversion;

[0040] 3) Biochemical response: Serum ALT and AST returned to normal.

[0041] Patients with chronic hepatitis B who are non-responsive to interferon, after regular treatment (24 to 72 weeks) with conventional interferon or pegylated interferon, still fail to meet the requirements of the response group after 48 weeks of follow-up.

[0042] S2. Specimen pretreatment:

[0043] For each sample, 500 μL of whole blood specimen was taken, 1000 μL of red blood cell lysis solution was added, and the mixture was kept in the dark for 10 min, centrifuged at 10,000 rpm for 5 min, and then the supernatant was discarded and the total RNA extraction reagent-TRIzol was added and mixed.

[0044] S3. Total RNA extraction:

[0045] Add 150 μL of chloroform to the treated specimen, shake and mix, let stand for 10 minutes, and centrifuge at 10,000 rpm and 4°C for 10 minutes. Pipette the supernatant into a new tube, add 200 μL of isopropanol and mix, and centrifuge at 10,000 rpm and 4°C for 12 minutes. Discard the supernatant, add 75% ethanol to wash twice, dry naturally, add 20 μL of DEPC (diethyl pyrocarbonate) and mix to obtain the RNA sample;

[0046] S4, PCR:

[0047] Take two RNA samples and use THBS1 primer set and β-actin primer set for PCR reaction respectively. The reaction process is as follows:

[0048] S41, reverse transcription PCR:

[0049] Take 2μL of the above RNA sample, add 1μL Oligo(dT) and 9μL ddH2O and mix thoroughly. Perform the first step of PCR reaction at 65℃, 5min. After amplification, add 4μL buffer, 2μL dNTP, 1μL reverse transcriptase and 1μL RNase inhibitor. The second step of PCR reaction conditions are set at 42℃ for 60min and 70℃ for 5min.

[0050] S42, Real-time fluorescence quantitative PCR:

[0051] Take 1 μL of reverse transcription PCR product, 5 μL SYBR Green PCR master mix, 0.15 μL of primer set mixture and 3.85 μL ddH2O and mix thoroughly for real-time fluorescence quantitative PCR. The PCR conditions are ① 90℃ for 1 min, ② (90℃ for 5 s, 60℃ for 34 s, read the fluorescence value) × 40 cycles.

[0052] S5. Data processing and analysis:

[0053] Record the Ct values ​​of THBS1 and β-actin respectively, and define △Ct = Ct THBS1 –Ct β-actin , the relative expression of THBS1 was 2 -△Ct t. The relative expression of THBS1 in each chronic hepatitis B patient was calculated, and the ROC curve (receiver operating characteristic curve) of the relative expression of THBS1 was drawn using SPSS 26.0 software. Figure 1 , and calculate the Youden index. Figure 1In the figure, the gray curve represents the diagnostic value of THBS1 for predicting the efficacy of interferon, the dark diagonal line is used to judge the predictive value, and the gray curve above the dark diagonal line indicates that it has diagnostic value. The larger the area under the gray curve, the higher the diagnostic value. Figure 1 The relative expression of THBS1 corresponding to the maximum Youden index was found to be 8.5, which was the optimal cutoff value. Therefore, the relative expression of THBS1 of 8.5 was used to divide the prediction of poor interferon efficacy and the prediction of good interferon efficacy.

[0054] Example 3 of the present invention is: Application of a kit for predicting the efficacy of interferon in patients with chronic hepatitis B

[0055] Whole blood samples from 85 chronic hepatitis B patients who were undergoing interferon treatment were selected as experimental samples for the validation set. The chronic hepatitis B patients in the validation set were receiving conventional interferon or pegylated interferon treatment when sampling occurred, and the efficacy was unknown.

[0056] The relative expression of THBS1 of the patient was calculated by methods S2 to S6 in Example 2, and the patients were divided into patients with predicted good interferon efficacy (relative expression of THBS1>8.5) and patients with predicted poor interferon efficacy (relative expression ≤8.5) according to the relative expression of THBS1.

[0057] The number of patients predicted to have good interferon efficacy was 32, and the number of patients predicted to have poor interferon efficacy was 53. After the patients finished interferon treatment, HBeAg in serum was tested. If serum HBeAg turned negative or HBeAg serological conversion occurred, it was considered to have poor efficacy. The efficacy was compared with the predicted efficacy, and the accuracy rate was 82.3%.

[0058] Example 4 of the present invention is: Detecting the relationship between THBS1 and related genes in the interferon activation pathway

[0059] The THBS1 gene of HepAD38 cells was knocked out and the cells were stimulated with interferon. The expression levels of each gene after stimulation were shown in Figure 2 ( Figure 2Figure A is a comparison of the expression levels of genes related to the interferon activation pathway in HepAD38 cells before and after the THBS1 gene was knocked out; Figure B is a statistical chart of the expression levels of antiviral expression proteins in HepAD38 cells under interferon stimulation before and after the THBS1 gene was knocked out. After interferon stimulation, the phosphorylation level of interferon-stimulated gene factor 3 (ISGF3) in HepAD38 cells with the THBS1 gene knocked out was downregulated. Not only that, the gene expression levels of antiviral proteins such as Myxoma Resistance Protein (Mx), 2',5'-Oligoadenylate Synthase 1 (2'-5'-Oligoadenylate Synthase 1, OAS1) and Adenosine Deaminase (Adenosine Deaminase ADAR1) were also significantly downregulated after the THBS1 was knocked out. It can be seen that THBS1 is positively correlated with the expression of genes related to the interferon activation pathway.

[0060] In summary, the present invention adopts THBS1 as a marker to predict the efficacy of interferon in patients with chronic hepatitis B, and prepares a related kit. The kit and application for predicting the efficacy of interferon in patients with chronic hepatitis B provided by the present invention predict the efficacy of interferon treatment in patients with chronic hepatitis B by detecting the relative expression of THBS1 mRNA in the peripheral blood of patients. Compared with traditional test indicators, it can better reflect the response of the human body to interferon, is easy to sample and detect, and is suitable for promotion and use in conventional medical processes.

[0061] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Application of reagents for quantitative detection of THBS1 in the preparation of a kit for predicting the efficacy of interferon in patients with chronic hepatitis B.

2. The use according to claim 1, characterized in that: The kit comprises a real-time fluorescence quantitative PCR reaction system for detecting the relative expression of THBS1 mRNA to reflect the response of the human body to interferon, and the real-time fluorescence quantitative PCR reaction system comprises a THBS1 primer set and a β-actin primer set; the THBS1 primer set consists of two primers shown in SEQ ID NO:1 and SEQ ID NO:2, and the β-actin primer set consists of two primers shown in SEQ ID NO:3 and SEQ ID NO:

4.

3. The use according to claim 2, characterized in that: The kit also includes a reverse transcription PCR reaction system.

4. The use according to claim 3, characterized in that: The reverse transcription PCR reaction system comprises polythymidine, buffer, dNTP, reverse transcriptase and RNase inhibitor.

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