CN111413497A - Use of histone methyltransferase EZH2 in the preparation of biomarkers for the diagnosis of sepsis - Google Patents

Abstract

The present invention provides the use of histone methyltransferase EZH2 in preparing biomarkers for diagnosing the severity or prognosis of sepsis. The present invention also provides a kit for diagnosing the severity or prognosis of sepsis, which contains a reagent for detecting histone methyltransferase EZH2 in body fluid samples. The present invention found that the expression level of EZH2 in peripheral blood T cells of patients with clinical sepsis was significantly higher than that of healthy persons, and was positively correlated with the SOFA score of patients with sepsis, and it was found that the expression level of EZH2 in T cells can be determined by ROC curve analysis. As a new biomarker to predict the prognosis of sepsis, EZH2 is provided as an index for the diagnosis, prognosis and treatment monitoring of sepsis infection, and can evaluate the severity of sepsis.

Description

Histone methyltransferase EZH2 in the preparation of biomarkers for the diagnosis of sepsis use in

technical field

The invention belongs to the field of bioengineering, and relates to a biological marker, in particular to the use of histone methyltransferase EZH2 in preparing a biological marker for diagnosing sepsis.

Background technique

Sepsis is a systemic inflammatory response syndrome caused by infectious factors accompanied by acute organ dysfunction. Some septic patients are still accompanied by uncorrectable persistent hypotension after adequate fluid resuscitation, and further develop into septic shock. With the aging of the population and the increase of invasive medical methods, the incidence of sepsis continues to rise. Every year, millions of sepsis patients are added worldwide, and more than 1/4 of them die, ranking the first cause of death in intensive care units in the world. One, and the high cost of treatment (an average of more than 80,000 yuan per case in my country) has caused a heavy economic burden to patients, families and society, so the early diagnosis and treatment of sepsis and its complications is particularly important.

The etiology of sepsis is complex, and the underlying pathogenesis is not yet clear, involving complex systemic inflammatory network effects, gene polymorphisms, immune dysfunction, coagulation abnormalities, tissue damage, and abnormal host responses to different pathogenic microorganisms and their toxins. On the other hand, it is closely related to the pathophysiological changes of multiple organs and systems of the body. The clinical manifestations of sepsis are diverse and lack specificity, so many scholars are currently looking for biological markers of sepsis for the diagnosis and prognosis evaluation of sepsis. Biomarkers that have received widespread clinical attention include C-reactive protein (CRP), procalcitonin (PCT), blood lactate, IL-6, leukocyte DR antigen (HLA-DR), polysaccharide binding protein (LBP), myeloid Cell trigger receptor (TREM-1) and soluble urokinase plasminogen activator receptor (suPAR), etc., but both have certain limitations, lack of sensitivity and specificity, and cannot be used for sepsis treatment. potential target. Therefore, it is of great significance to find biomarkers with high sensitivity and specificity in order to predict disease progression and adverse outcomes early.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the use of histone methyltransferase EZH2 in the preparation of biomarkers for diagnosing sepsis, and the histone methyltransferase EZH2 is used for preparing biomarkers for diagnosing sepsis. The use in biomarkers should solve the technical problem of the lack of sensitivity and specificity of biomarkers in the prior art for diagnosing sepsis.

The present invention provides the use of histone methyltransferase EZH2 in the preparation of biomarkers for diagnosing the severity or prognosis of sepsis.

The present invention also provides a kit for diagnosing the severity or prognosis of sepsis, wherein the kit contains a reagent for detecting histone methyltransferase EZH2 in a body fluid sample.

Further, the body fluid sample is selected from serum.

Histone methyltransferase Drosophila zeste enhancer homolog 2 (EZH2) is a core component of the PcG protein family member polycombrepressive complex 2 (PRC2), with histone lysine transferase activity, Studies have found that it regulates cell proliferation and differentiation by inhibiting related target genes in the cell growth cycle. EZH2 is highly expressed in a variety of tumor tissues, and has the characteristics of promoting cell proliferation and accelerating tumor cell proliferation. It can be used as an important biomarker for the diagnosis and prognosis of related tumors, and also provides potential targets for tumor recurrence and metastasis. Treatment ideas. Unfortunately, so far, there is no study on the level of EZH2 in peripheral blood cells of sepsis patients and its correlation with abnormal function of CD4 ⁺ T and CD8 ⁺ T cells, the main acquired immune cells.

Through a series of explorations in the present invention, it is found that EZH2 is involved in the occurrence and development of sepsis, and can be used as a good biomarker for the diagnosis, prognosis and treatment monitoring of sepsis.

The present invention firstly detects the expression levels of EZH2 in peripheral blood CD4+ and CD8+ T cells of 48 sepsis patients and 40 healthy people (healthy control group) patients by flow cytometry. The positive rate of EZH2 in CD4+ T cells, the average fluorescence intensity of EZH2 in CD4+ T cells, the positive rate of EZH2 in CD8+ T cells, and the average fluorescence intensity of EZH2 in CD8+ T cells in the sepsis patient group were significantly higher than those in the healthy control group. Patients with sepsis have exacerbated disease and increased EZH2 expression. The mean fluorescence intensity of EZH2 in CD8+ T cells on day 1 of sepsis patients was an independent risk factor for predicting the death of patients on day 28. ROC curve analysis: the area under the curve of the mean fluorescence intensity of CD8+ T cells EZH2 is 0.768 (0.619-0.917), which can predict the prognosis at 28 days, and the mean fluorescence intensity of CD8+ T cells EZH2 predicts the sensitivity and specificity of death at 28 days The specificity was 75% and 82.14%, respectively, and the specificity was better than the SOFA score. The Spearman correlation between the mean fluorescence intensity of CD8 ⁺ T lymphocyte EZH2 ⁺ and the disease severity, survival time, and hospitalization time in the sepsis group showed that: The APACHEII score on the 3rd day and the 7th day was positively correlated, and negatively correlated with the survival time. These results suggest that the expression level of EZH2 in T lymphocytes can be used as an indicator for sepsis diagnosis, prognostic assessment and treatment monitoring.

Compared with the prior art, the present invention has significant technical progress. The invention provides a histone methyltransferase EZH2 as a candidate biomarker for sepsis diagnosis and prognosis judgment, which is used to solve the sensitivity of the biomarkers currently used for sepsis diagnosis, prognosis evaluation and treatment monitoring. and lack of specificity.

Description of drawings

Figure 1 shows the comparison of EZH2 expression on peripheral blood T lymphocytes of healthy control group, sepsis patients on day 1, sepsis patients on day 3, and sepsis patients on day 7. Among them, A: Comparison of EZH2 positive rate and mean fluorescence intensity on peripheral blood CD4 ⁺ T lymphocytes of healthy control group and sepsis patients; B: EZH2 positive rate of peripheral blood CD8 ⁺ T lymphocytes of healthy control group and sepsis patients Compared with the mean fluorescence intensity, *P<0.05, **P<0.01.

Figure 2.1 shows the comparison of EZH2 expression on peripheral blood T cells on day 1 between the survival group and the death group of sepsis patients. A: Comparison of the positive rate and mean fluorescence intensity of EZH2 on peripheral blood CD4 ⁺ T lymphocytes between the death group and the survival group on the first day after the diagnosis of sepsis patients; B: The death group and the survival group on the first day after the diagnosis of sepsis patients Comparison of EZH2 positive rate and mean fluorescence intensity on peripheral blood CD8 ⁺ T lymphocytes, *P<0.05, **P<0.01.

Figure 2.2 shows the comparison of EZH2 expression on peripheral blood T cells on day 3 between the survival group and the death group of sepsis patients. Among them, A: comparison of the positive rate and mean fluorescence intensity of EZH2 on peripheral blood CD4 ⁺ T lymphocytes between the death group and the survival group on the 3rd day after the diagnosis of sepsis patients; B: the death group and the survival group on the 3rd day after the diagnosis of sepsis patients Comparison of the positive rate and mean fluorescence intensity of EZH2 on peripheral blood CD8 ⁺ T lymphocytes in the survival group, *P<0.05, **P<0.01.

Figure 2.3 shows the comparison of EZH2 expression on peripheral blood T cells on day 7 between the survival group and the death group of sepsis patients. Among them, A: comparison of the positive rate and mean fluorescence intensity of EZH2 on peripheral blood CD4 ⁺ T lymphocytes between the death group and the survival group on the 7th day after the diagnosis of sepsis patients; B: the death group and the survival group on the 7th day after the diagnosis of sepsis patients Comparison of the positive rate and mean fluorescence intensity of EZH2 on peripheral blood CD8 ⁺ T lymphocytes in the survival group, *P<0.05, **P<0.01.

Figure 3 shows the ROC curve analysis (day 1 post-diagnosis indicator) predicting 28-day mortality in sepsis patients. Note: AUC: area under the curve CI: confidence interval MFI: mean fluorescence intensity; EZH2: Drosophila zeste gene enhancer homolog 2.

Figure 4 shows the linear correlation between APACHEII and EZH2 ⁺ /CD4 ⁺ Tcells (%).

Figure 5 shows the linear correlation between APACHEII and MFIofEZH2onCD8 ⁺ Tcell.

Figure 6 shows the linear correlation between SOFA and MFIofEZH2onCD8 ⁺ Tcell.

Detailed ways

The experiments involved in the following examples are described as follows: Sepsis patients admitted to the emergency ICU and central ICU of Dongfang Hospital Affiliated to Tongji University from January 2019 to February 2020, as well as healthy volunteers in the health examination center, were divided into two groups. For the sepsis group and the healthy control group. Inclusion criteria: Diagnosed according to the guidelines of the Rescue Sepsis Movement released in 2016, that is, the infection focus + SOFA score > 2 points can be diagnosed. The exclusion criteria included (1) patients under the age of 18; (2) malignant tumors; (3) autoimmune deficiency diseases; (4) hematological diseases; (5) pregnant and lactating women. Subjects All studies were approved by the Science and Ethics Committee of Dongfang Hospital Affiliated to Tongji University. Extract 6ml of peripheral blood from patients with clinically diagnosed sepsis on the same day, on the 3rd day, on the 7th day and normal people in the physical examination center in the same period of time, put them in K2EDTA anticoagulation tubes, store them in a 4 ℃ refrigerator, and process them immediately on the same day. The expression of EZH2 on human peripheral blood T lymphocytes was continuously detected by cytometry. SPSS 20.0 was used for data analysis.

Example 1 Comparison of EZH2 expression changes on T lymphocytes in sepsis and healthy control groups

Compared with healthy controls, the positive rate and mean fluorescence intensity of EZH2 expression in lymphocytes in sepsis patients were significantly increased (Figure 1). Compared with the healthy control group, the positive rate of EZH2 in peripheral blood CD4 ⁺ lymphocytes of patients on day 1, 3 and 7 was significantly increased (P<0.001); the expression intensity was also significantly increased (P<0.001).

We further detected and analyzed the expression of EZH2 on CD8 ⁺ lymphocytes in patients with sepsis, and found that both the positive rate and the mean fluorescence intensity were significantly higher than those of the control group. For further analysis on the 1st, 3rd, and 7th days in the sepsis patient group, the positive rate of EZH2 on CD4+ T lymphocytes on the 7th day was significantly higher than that on the 1st day (P<0.01) and the 3rd day (P<0.01). P<0.01); the mean fluorescence intensity of EZH2 on CD4+ T lymphocytes on day 7 was higher than that on day 3 (P<0.05); the positive rate of EZH2 on CD8+ T lymphocytes on day 7 was significantly higher than that on day 1 day (P<0.05) and day 3 (P<0.01). Example 2 Comparison of EZH2 expression on T cells of sepsis patients with different prognosis

It can be seen from Figures 2.1, 2.2 and 2.3 that the EZH2 positive rate and expression intensity (MF) of CD4 ⁺ T cells in the death group at 1, 3 and 7 days were significantly higher than those in the survival group, and the difference was statistically significant (P<0.05). The positive rate and expression intensity (MF) of EZH2 in CD8 ⁺ T cells were significantly higher than those in the survival group, and the difference was statistically significant (P<0.05).

Example 328 Independent Risk Factor Analysis of Death

Univariate logistic regression analysis was performed according to the indicators with statistical differences between the death group and the survival group in the sepsis group, and the multivariate logistic regression analysis was performed on the indicators with statistical significance in a single factor. index. Combined with the test results, the independent risk factors for 28-day death were found to be the use of ventilator (OR: 4.107 (1.168-14.436). p=0.028), APACHEII (OR: 1.440 (1.159-1.791) among the indicators of sepsis patients on the first day ), p=0.001), SOFA (OR: 1.495 (1.168-1.915), p=0.001), percentage of CD8 ⁺ T lymphocytes (%) (OR: 1.114 (1.008-1.231), p=0.034), CD8 ⁺ Mean fluorescence intensity of EZH2 ⁺ in T lymphocytes (OR: 1.022 (1.002-1.043), p=0.032); (as shown in Table 3.1), the indicators were included in the ROC curve analysis to evaluate the predictive power related to 28-day death.

Table 3.1: Multivariate analysis of independent risk factors for death at 28 days (indicators on day 1 after diagnosis)

Note: Age: age; Ventilator: ventilator; Lac: lactic acid; IL-6: interleukin-6; MFI: mean fluorescence intensity; EZH2: Drosophila zeste gene enhancer homolog 2; p<0.05 has a statistical difference .

As shown in Table 3.2 ^and Figure 3, after multivariate logistic ^regression analysis, the independent risk factors for death at 28 days were obtained ^. Fluorescence intensity, and the area under the ROC curve of these two indicators were analyzed to evaluate the prognostic predictability. The areas under the ROC curves of the two were 0.696 (0.536-0.857) and 0.768 (0.619-0.917), respectively, and the sensitivity was 64.29% and 75%, respectively. The specificity of the mean fluorescence intensity of CD8 ⁺ T lymphocyte EZH2 ⁺ in predicting 28-day death was better than that of SOFA score, which had certain clinical significance.

Table 3.2 The percentage of CD8 ⁺ T lymphocytes and the mean fluorescence intensity of CD8 ⁺ T lymphocytes EZH2 ⁺ on day 1 in patients with sepsis in predicting death at 28 days

Note: AUC: area under the curve CI: confidence interval MFI: mean fluorescence intensity; EZH2: Drosophila zeste gene enhancer homolog 2; Example 4 Correlation analysis of EZH2 expression on T cells and sepsis score

(1) To analyze the correlation between the mean fluorescence intensity of ^{CD8 +} T lymphocyte EZH2 ⁺ and the severity of disease, survival time, and hospitalization time in patients with sepsis ^. APACHEII score on day 3 (Spearman'sr=0.474, p=0.002) and APACHEII score on day 7 (Spearman'sr=0.365, p=0.043) were positively correlated with survival time (Spearman'sr=-0.304, p=0.045) It was negatively correlated, and had no correlation with SOFA on day 1, day 3, and day 7. The mean fluorescence intensity of EZH2 ⁺ of CD8 ⁺ T lymphocytes on the 3rd day and the APACHEII score on the 1st day (Spearman'sr=0.717, p=0), the APACHEII score on the 3rd day (Spearman'sr=0.505, p=0.002), The 7-day APACHEII score (Spearman'sr=0.424, p=0.027) was positively correlated with survival time (Spearman'sr=-0.665, p=0) and hospital days (Spearman'sr=-0.362, p=0.032) It was positively correlated with SOFA score on day 1 (Spearman'sr=0.457, p=0.006) and SOFA score on day 3 (Spearman'sr=0.505, p=0.036), but had no correlation with SOFA score on day 7.

(2) The correlation between the positive rate of EZH2 on CD4 ⁺ T lymphocytes and the severity of the disease in patients with sepsis was analyzed, and it was positively correlated with the SOFA score on day 1 (Spearman'sr=0.342, p=0.023) (Figure 4, 5, 6).

In summary, the present invention found that the expression of EZH2 in peripheral blood T cells of clinical sepsis patients was significantly higher than that of healthy subjects, and was positively correlated with the SOFA score of sepsis patients. The expression level of cells can be used as a novel biomarker to predict the prognosis of sepsis, providing EZH2 as an indicator for the diagnosis, prognosis and treatment monitoring of sepsis infection, and can assess the severity of sepsis.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (3)

1. Use of histone methyltransferase EZH2 in the preparation of biomarkers for diagnosing the severity or prognosis of sepsis. 2. A kit for diagnosing the severity or prognosis of sepsis, characterized in that: the kit contains a reagent for detecting histone methyltransferase EZH2 in a body fluid sample. 3. The kit for diagnosing the severity or prognosis of sepsis according to claim 1, wherein the body fluid sample is selected from serum.

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