Application of KLRG1 gene as marker in preparation of SLE secondary HLH detection preparation

Abstract

The invention discloses application of a killer cell lectin-like receptor G1 (KLRG1) gene as a marker in preparation of a detection preparation for systemic lupus erythematosus (SLE) secondary hemophagic lymphocytosis (HLH). The preparation is used for detecting the expression level of the KLRG1 gene in a biological sample. Researches find that the expression level of the KLRG1 gene in an SLE secondary HLH patient is obviously lower than that of a healthy control group and an SLE patient, and the KLRG1 gene has good diagnosis sensitivity and specificity. The invention further discloses a preparation for detecting the expression level of the KLRG1 gene in a biological sample of a patient, the preparation contains a primer pair for detecting the mRNA level expressed by the KLRG1 gene or an antibody for detecting the protein level expressed by the KLRG1 gene, whether the SLE patient has secondary HLH or not can be detected only by drawing blood and extracting peripheral blood mononuclear cells (PBMC), the operation is simple and rapid, and the result is accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of disease diagnosis, and in particular to the application of KLRG1 gene as a marker in the preparation of a SLE secondary HLH detection preparation. Background Art

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening complication of systemic lupus erythematosus (SLE). Currently, its diagnosis is based on the HLH-2004 criteria proposed by the International Histiocyte Society. There is a lack of biomarkers for early diagnosis of HLH. HLH includes eight specific diagnostic criteria: (1) fever: body temperature >38.5°C, duration >7 days; (2) splenomegaly; (3) pancytopenia (involving two or three peripheral blood lineages); (4) hypertriglyceridemia and / or hypofibrinogenemia; (5) hemophagocytic cells found in the bone marrow, spleen, liver or lymph nodes; (6) ferritin increased ≥500 μg / L; (7) decreased or absent NK cell activity; (8) elevated sCD25. HLH can be diagnosed if five of the above eight criteria are met. Although the diagnosis of HLH based on this standard has high specificity, there are many criteria, including fever, pancytopenia and other criteria that overlap with the clinical manifestations of SLE. Hemophagocytic cells require invasive procedures to be identified, and sCD25 and NK cell activity are not available in medical institutions and need to be sent to an external company for testing. As a result, the diagnosis of HLH secondary to SLE is delayed and the missed diagnosis rate is high, leading to poor prognosis. Summary of the invention

[0003] The purpose of the present invention is to address the problems of cumbersome diagnosis and high missed diagnosis rate of HLH-2004 standard in the prior art, and to provide the use of KLRG1 gene as a marker in the preparation of SLE secondary HLH detection preparations, which can quickly and accurately diagnose.

[0004] The technical solution of the present invention is described in detail as follows:

[0005] In a first aspect, the present invention provides the use of KLRG1 gene as a marker in the preparation of a preparation for detecting HLH secondary to SLE, wherein the preparation is used to detect the expression level of KLRG1 gene in a biological sample.

[0006] KLRG1 is killer cell lectin-like receptor G1. Studies have found that the expression level of KLRG1 in patients with secondary HLH in SLE is significantly lower than that in HC (healthy control) and SLE groups, and has good sensitivity and specificity for diagnosing secondary HLH in SLE. The expression level of the KLRG1 gene can be either the mRNA expression level or the protein expression level.

[0007] Optionally or preferably, the preparation is used to detect the mRNA expression level of the KLRG1 gene in a biological sample.

[0008] Compared with normal healthy controls (HC) and SLE patients, the mRNA expression level of KLRG1 gene in biological samples of SLE secondary HLH patients was significantly decreased.

[0009] Optionally or preferably, the biological sample is peripheral blood mononuclear cells (PBMC).

[0010] In a second aspect, the present invention provides a preparation for detecting the expression level of KLRG1 gene in a patient's biological sample, comprising a primer pair for detecting the mRNA level of KLRG1 gene expression, or an antibody for detecting the protein level of KLRG1 gene expression.

[0011] Optionally or preferably, the preparation contains a primer pair for detecting the mRNA level of KLRG1 gene expression, and the nucleotide sequence of the primer pair is as follows:

[0012] Sense strand: 5'-AACCCAAGCCCAGAATGACT-3' (SEQ ID NO: 1),

[0013] Antisense strand: 5'-CAGCTGGCACAAGTGGAGTA-3' (SEQ ID NO: 2).

[0014] Optionally or preferably, the preparation further contains a qPCR reaction solution.

[0015] Optionally or preferably, the preparation further contains a primer pair for detecting the expression level of the internal reference gene GAPDH, and the nucleotide sequence of the primer pair is as follows:

[0016] Sense strand: 5'-TTGCCCTCAACGACCACTTT-3' (SEQ ID NO: 3),

[0017] Antisense strand: 5'-TGGTCCAGGGGTCTTACTCC-3' (SEQ ID NO: 4).

[0018] Optionally or preferably, the preparation further contains a PBMC separation and extraction reagent, a total RNA extraction reagent and a reverse transcription reagent.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a new diagnostic marker KLRG1 for SLE secondary HLH. The gene expression level in SLE secondary HLH patients is significantly lower than that in HC and SLE patients, and has good diagnostic sensitivity and specificity.

[0021] The present invention provides a preferred biological sample peripheral blood mononuclear cells (PBMC), which has the following advantages: ① The sample is easy to obtain: PBMC is a mixture of mononuclear cells contained in peripheral blood (i.e., blood other than bone marrow), and 2 ml of EDTA anticoagulated whole blood can meet the experimental needs, and can be separated and obtained by Percoll non-continuous density gradient centrifugation. ② The sample is closely related to the occurrence and development of the disease: PBMC includes NK cells, T cells, and B cells, which are the location of the body's immune cells and are a type of cell commonly used to study the pathogenesis of immune diseases. Therefore, paying attention to PBMC is a key link in understanding the occurrence and development of SLE secondary HLH. ③ High value of clinical differential diagnosis: The expression level of KLRG1 in PBMC in patients with SLE secondary HLH is significantly lower than that in the HC and SLE groups, and has good sensitivity and specificity for the diagnosis of SLE secondary HLH. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 In order to screen the differentially expressed mRNAs between SLE and SLE secondary HLH groups in the embodiment, a volcano plot was drawn after the differentially expressed mRNA data were standardized;

[0023] Figure 2 In order to screen the differentially expressed mRNAs between SLE and SLE secondary HLH groups in the embodiment, a heat map was drawn after the differentially expressed mRNA data were standardized;

[0024] Figure 3 Statistical results of the mRNA expression level of KLRG1 in different groups of PBMCs in the example (left) and ROC curve analysis (right). DETAILED DESCRIPTION

[0025] In order to make the technical personnel in this field better understand the present application scheme, the present application will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only embodiments of a part of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should belong to the scope of protection of this application. The instruments and reagents used in the embodiments are all derived from commercial channels unless otherwise specified.

[0026] Example 1 Screening of differentially expressed genes in patients with SLE secondary to HLH

[0027] There were three groups in the experiment, namely HC (healthy control) group, SLE (systemic lupus erythematosus) group, and SLE secondary HLH (systemic lupus erythematosus secondary hemophagocytic syndrome) group.

[0028] 1. Isolation of human PBMC

[0029] 2 ml of peripheral EDTA anticoagulated whole blood was collected, and the Percoll non-continuous density gradient centrifugation method was used to obtain peripheral blood PBMCs from healthy subjects and disease groups. The specific operation method is as follows:

[0030] 2. Transcriptome sequencing to screen differentially expressed mRNAs in HC group, SLE group and SLE secondary HLH group

[0031] PBMC samples from 3 HC (healthy controls), 3 moderately active SLE (systemic lupus erythematosus) patients, and 3 SLE secondary HLH patients were sent to Novogene for transcriptome sequencing. The differentially expressed mRNAs were obtained according to Fold change ≥ 0 and P-value < 0.05. At the mRNA level, there were 601 differentially expressed genes in the SLE secondary HLH group, of which 365 genes were upregulated and 236 genes were downregulated. Figure 1 and Figure 2 shown.

[0032] 3. Screening of differentially expressed mRNA molecules in patients with secondary HLH due to SLE

[0033] Using Fold Change>2 and P value<0.05 as the criteria for differential gene screening, it was found that the expression of KLRG1 in the HC group, SLE group and SLE secondary HLH group showed a progressive downward trend. See Table 1 for details.

[0034] Table 1 KLRG1 mRNA expression in HC, SLE and SLE-HLH

[0035]

[0036] The screening results showed that the expression level of KLRG1 in the SLE secondary HLH group was significantly lower than that in the healthy control group and the disease control group (SLE group).

[0037] KLRG1 gene sequence (NM_001329099 1401 bp, SEQ ID NO: 5):

[0038]

[0039] The present invention also sets the internal reference gene GAPDH.

[0040] GAPDH gene sequence (NM_001256799 1386 bp, SEQ ID NO: 6):

[0041]

[0042] Example 2 Clinical verification of the diagnostic value of the mRNA molecule KLRG1 in PBMC in SLE secondary to HLH

[0043] Fourteen HC and 50 active SLE patients were collected. The SLE patients were divided into SLE group (36 cases) and SLE secondary HLH group (14 cases) according to the presence or absence of secondary HLH.

[0044] 1. Isolation of PBMC

[0045] Take 2ml EDTA anticoagulated whole blood, centrifuge at 3000rpm for 10min, carefully aspirate the cells in the buffy coat layer, and resuspend them with 2ml sterile 1×PBS for later use; prepare 2ml of 75% and 60% percoll cell separation solution dilutions with sterile 1×PBS for later use; prepare a clean 15ml centrifuge tube, add 75% percoll cell separation solution, 60% percoll cell separation solution and the resuspended buffy coat layer at once; centrifuge at 2600rpm / min for 20min to obtain the PBMC layer, mix it with 10ml 1×PBS, and centrifuge it at 1800rpm for 5min, discard the supernatant, add an appropriate amount of red blood cell lysis solution, lyse the mixed red blood cells, and collect the cell precipitate as PBMC.

[0046] 2. Total RNA extraction (Tiangen's total RNA extraction kit: D419)

[0047] Take 1 ml TRIzol (total RNA extraction reagent) and blow the above cell pellet, let it stand at room temperature for 10 minutes; add 200 μl chloroform, shake vigorously for 1 minute, let it stand at room temperature for 3 minutes, and centrifuge at 12000g at 4°C for 10 minutes; take 500 μl of the upper aqueous phase and add 250 μl of anhydrous ethanol, blow and mix well, transfer to the RNA extraction column, and centrifuge at 12000g at 4°C for 1 minute; discard the waste liquid, add 500 μl of deproteinization solution to the column, and centrifuge at 12000g at 4°C. 1min; discard the waste liquid, add 500μl washing solution to the column, let it stand at room temperature for 2min, centrifuge at 12000g at 4℃ for 1min; repeat the previous step; discard the supernatant, centrifuge at 12000g at 4℃ for 2min; let it stand at room temperature for 10min until the ethanol is completely evaporated; add 30μl enzyme-free water in the center of the column, let it stand at room temperature for 2min; centrifuge at 12000g at 4℃ for 2min to obtain total RNA, and detect the RNA concentration and absorbance A260, A280 and A230.

[0048] 3. Reverse transcription (Yisheng Company: 11141ES60)

[0049] 1) First remove residual genomic DNA

[0050] Prepare the mixture shown in Table 2 in an RNase free centrifuge tube and mix it by gently pipetting. Incubate at 42°C for 2 min.

[0051] Table 2 Mixture for removing residual genomic DNA

[0052] Components Usage <![CDATA[RNase free ddH2O]]> Up to 30 μL 5×gDNA digester 6μL Total RNA 4 μg

[0053] 2) Preparation of reverse transcription reaction system

[0054] Directly add 2×HifairTMⅡSuperMix plus as shown in Table 3 to the reaction tube in step 1) and mix by gently pipetting.

[0055] Table 3 Reverse transcription reaction system

[0056]

[0057] 3) Reverse transcription program settings, as shown in Table 4:

[0058] Table 4 Standard reverse transcription procedures

[0059] temperature time 25℃ 5min 42℃ 30min 85℃ 5min

[0060] To obtain template cDNA, add 80 μL RNase free ddH2O to each sample for later use.

[0061] 4. qPCR (Yisheng Company: 11184ES08)

[0062] 1) Prepare the qPCR mixed solution (see Table 5 below) in a 96-well plate, set up 3 replicate wells for each sample, seal the plate and centrifuge to mix.

[0063] Table 5 qPCR Mixture

[0064]

[0065]

[0066] (Note: The nucleotide sequence of the upstream primer is the same as that of the aforementioned sense strand, and the nucleotide sequence of the downstream primer is the same as that of the aforementioned antisense strand)

[0067] 2) qPCR program settings

[0068] Table 6 qPCR program

[0069]

[0070] 3) Result calculation

[0071] GAPDH was used as the internal reference gene, and the minimum cycle number required to reach the threshold set by the instrument was used as the CT value. –ΔΔCT Calculate expression levels.

[0072] 5. Statistical results

[0073] The results of statistical analysis showed that the mRNA levels of KLRG1 in PBMCs were significantly different among the HC group, SLE group, and SLE secondary HLH group, and the expression level in the SLE secondary HLH group was significantly lower than that in the HC group and SLE group, see Table 7 and Figure 3 (Left).

[0074] Table 7 Comparison of expression levels of candidate mRNA molecules in three groups of PBMCs

[0075]

[0076] Further ROC curve analysis was performed to detect the efficacy of KLRG1 in the classification and diagnosis of secondary HLH in SLE. The area under the ROC curve (AUC) was 0.927, the specificity was 0.929, and the sensitivity was 0.889, as shown in Table 8 and Figure 3 (Right) It is superior to the currently used clinical indicators such as ferritin (Fer), triglyceride (TG) and fibrinogen (FIB) for the diagnosis of HLH secondary to SLE.

[0077] Table 8 Diagnostic value of KLRG1 mRNA expression level in PBMC in HLH secondary to SLE

[0078]

[0079] In summary, the present invention has a high diagnostic value for diagnosing secondary HLH in SLE patients, with a sensitivity of 0.889 and a specificity of 0.929. It is not only simple and feasible, but also can effectively improve the early diagnosis rate of secondary HLH, provide effective treatment in time, reduce HLH-related complications, and improve HLH-related adverse prognosis.

[0080] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. Use of the KLRG1 gene as a marker in the preparation of a preparation for detecting HLH secondary to SLE, wherein the preparation is used to detect the expression level of the KLRG1 gene in a biological sample.

2. The use according to claim 1, characterized in that: The preparation is used for detecting the mRNA expression level of the KLRG1 gene in a biological sample.

3. The use according to claim 1 or 2, characterized in that: Compared with normal healthy people and SLE patients, the mRNA expression level in biological samples of SLE secondary HLH patients was significantly reduced.

4. The use according to claim 1 or 2, characterized in that: The biological sample is peripheral blood mononuclear cells (PBMC).

5. A preparation for detecting the expression level of KLRG1 gene in a patient's biological sample, characterized in that: The kit contains a primer pair for detecting the mRNA level of KLRG1 gene expression, or contains an antibody for detecting the protein level of KLRG1 gene expression.

6. The preparation according to claim 5, characterized in that Contains a primer pair for detecting the mRNA level of KLRG1 gene expression, and the nucleotide sequence of the primer pair is as follows: Sense strand: 5'-AACCCAAGCCCAGAATGACT-3' (SEQ ID NO: 1), Antisense strand: 5'-CAGCTGGCACAAGTGGAGTA-3' (SEQ ID NO: 2).

7. The preparation according to claim 6, characterized in that Also contains qPCR reaction solution.

8. The preparation according to claim 6, characterized in that It also contains a primer pair for detecting the expression level of the internal reference gene GAPDH, and the nucleotide sequence of the primer pair is as follows: Sense strand: 5'-TTGCCCTCAACGACCACTTT-3' (SEQ ID NO: 3), Antisense strand: 5'-TGGTCCAGGGGTCTTACTCC-3' (SEQ ID NO: 4).

9. The preparation according to claim 6, characterized in that It also contains PBMC isolation and extraction reagents, total RNA extraction reagents and reverse transcription reagents.

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