Application of TM9SF1 in preparation of reagent for monitoring sepsis course

Abstract

The invention provides an application of TM9SF1 in preparation of a reagent for monitoring the course of sepsis, relates to the technical field of biomedicine, and particularly provides an application of a marker in preparation of a reagent for monitoring the course of sepsis, and the marker comprises a TM9SF1 gene or an mRNA molecule corresponding to the TM9SF1 gene. According to the technical scheme, the TM9SF1 gene is adopted as a marker, a column diagram model for predicting severity of sepsis can be constructed, and through ROC curve verification, AUC reaches 0.883, sensitivity is 91.5%, and specificity is 78.4%. For predicting the death rate of patients suffering from septic shock, the C index of the column diagram model is 0.931.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of TM9SF1 in preparing a reagent for monitoring the course of sepsis. Background Art

[0002] The incidence of sepsis is increasing year by year worldwide, and the mortality rate is high, with about one-third of patients failing to survive for more than 30 days.

[0003] Currently, significant progress has been made in the treatment of sepsis, but the efficacy of the molecular markers reported so far in predicting the severity and prognosis of sepsis is still limited. There are problems such as complex detection methods, low sensitivity / specificity, and inability to effectively predict disease severity and prognosis at the same time. Summary of the invention

[0004] The main purpose of the present invention is to propose an application of TM9SF1 in the preparation of a reagent for monitoring the course of sepsis, aiming to solve the problem of disease prediction and prognosis of sepsis.

[0005] To achieve the above object, the present invention proposes the use of a marker in the preparation of a reagent for monitoring the course of sepsis, wherein the marker includes the TM9SF1 gene.

[0006] The present invention also proposes the use of a marker in preparing a reagent for monitoring the course of sepsis, wherein the marker comprises an mRNA molecule corresponding to the TM9SF1 gene.

[0007] In the technical solution of the present invention, by using the TM9SF1 gene as a marker, a nomogram model for predicting the severity of sepsis can be constructed. The AUC reaches 0.883, the sensitivity is 91.5%, and the specificity is 78.4% through ROC curve verification. For the prediction of mortality in patients with septic shock, the C index of the nomogram model is 0.931. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0009] Figure 1 This is a statistical graph of the expression level of TM9SF1 mRNA in peripheral blood mononuclear cells of different patients in Example 1 of the present invention; Figure 2This is a graph showing the results of the Spearman correlation analysis between TM9SF1 mRNA levels and cytokines in Example 2 of the present invention; Figure 3 A prediction nomogram for predicting septic shock in a patient in Example 5 of the present invention; Figure 4 The prognostic nomogram for predicting the mortality rate of patients with septic shock in Example 5 of the present invention; Figure 5 This is a graph showing the results of the mortality risk analysis of septic shock patients at different nomogram scores in Example 5 of the present invention.

[0010] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0011] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0012] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0013] The incidence of sepsis is increasing year by year worldwide, and the mortality rate is high, with about one-third of patients failing to survive for more than 30 days.

[0014] Currently, significant progress has been made in the treatment of sepsis, but the efficacy of the molecular markers reported so far in predicting the severity and prognosis of sepsis is still limited. There are problems such as complex detection methods, low sensitivity / specificity, and inability to effectively predict disease severity and prognosis at the same time.

[0015] In view of this, the present invention proposes the use of a marker in preparing a reagent for monitoring the course of sepsis, wherein the marker includes the TM9SF1 gene.

[0016] In the technical solution of the present invention, by using the TM9SF1 gene as a marker, a nomogram model for predicting the severity of sepsis can be constructed. The AUC reaches 0.883, the sensitivity is 91.5%, and the specificity is 78.4% through ROC curve verification. For the prediction of mortality in patients with septic shock, the C index of the nomogram model is 0.931.

[0017] The present invention also proposes the use of a marker in preparing a reagent for monitoring the course of sepsis, wherein the marker comprises an mRNA molecule corresponding to the TM9SF1 gene.

[0018] TM9SF1 mRNA expression levels are significantly upregulated in patients with sepsis and are closely related to shock and death. At the same time, the predictive ability of TM9SF1 in terms of sepsis severity and mortality risk is significantly higher than that of common clinical detection indicators. Considering that the detection method of TM9SF1 mRNA expression level is simple and can simultaneously predict the severity of sepsis and the prognosis, TM9SF1 can effectively solve the current problems of complex detection methods and low predictive efficiency in the clinical diagnosis and treatment of sepsis.

[0019] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0020] Example 1 Expression level of TM9SF1 mRNA in sepsis patients Blood samples were collected from 45 patients with sepsis at admission and during follow-up. Peripheral blood mononuclear cells (PBMCs) of different populations were extracted from the blood samples, and the expression level of TM9SF1 mRNA in PBMCs was detected.

[0021] The expression level of TM9SF1 mRNA in PBMCs of different populations is as follows Figure 1 As shown in the figure, Figure A shows the expression level of TM9SF1 mRNA in patients with sepsis and non-sepsis, Figure B shows the expression level of TM9SF1 mRNA in patients with sepsis and septic shock, Figure C shows the paired samples of sepsis patients and septic shock group, Figure D shows the expression level of TM9SF1 mRNA in the survival group and the death group in patients with sepsis, and Figure E shows the expression level of TM9SF1 mRNA in the survival group and the death group in patients with septic shock.

[0022] The results showed that the TM9SF1 mRNA level in sepsis patients was significantly higher than that in normal controls (0.25±0.02vs.0.10±0.02, P<0.001; Figure 1A). The difference in TM9SF1 expression between the septic shock group and the sepsis group was also detected ( Figure 1 B), TM9SF1 mRNA level in septic shock patients was significantly higher than that in sepsis patients (0.29±0.02vs. 0.16±0.01, P<0.001). Paired t test to compare the expression of TM9SF1 in shock group and non-shock patients showed that ( Figure 1 C), As the condition of sepsis patients improved from severe to non-severe, TM9SF1 mRNA levels decreased significantly (0.25±0.04 vs. 0.13±0.02, P<0.001).

[0023] In addition, to evaluate the difference in TM9SF1 expression between patients who progressed and those who died, we performed an independent sample t-test ( Figure 1 D and Figure 1 E). The results showed that the TM9SF1 mRNA levels in patients with sepsis (0.30±0.03 vs. 0.19±0.02, P<0.001) and patients with septic shock (0.37±0.04 vs. 0.23±0.02, P = 0.005) were significantly higher in patients who died than in those who survived.

[0024] Example 2 Correlation between TM9SF1 expression and cytokines and clinical parameters in patients with sepsis Excessive production of cytokines is one of the important characteristics of sepsis and a key factor leading to related tissue damage. In order to explore the correlation between TM9SF1 mRNA levels and cytokines in patients with sepsis, this example uses Spearman correlation analysis, and the expression levels of TM9SF1 and cytokines are detected by RT-PCR technology. The primer sequences are shown in Table 1, where GAPDH is used as an internal reference.

[0025] Table 1 Primer sequences for RT-PCR experiments

[0026] The results of Spearman correlation analysis are as follows Figure 2 The results showed that in all patients with sepsis, the expression level of TM9SF1 mRNA was significantly positively correlated with a variety of key cytokines, including IFN-γ (r = 0.788, P<0.001), TNF-α (r = 0.610, P<0.001), IL-6 (r = 0.646, P<0.001), IL-17A (r = 0.818, P<0.001) and Forkhead Box P3 (FOXP3) (r = 0.607, P<0.001).

[0027] Further subgroup analysis of patients with septic shock showed that the strong correlation was still significant in this subgroup, especially for IFN-γ (r = 0.834, P<0.001), IL-6 (r = 0.784, P<0.001) and TNF-α (r = 0.709, P<0.001). These results indicate that there is a close correlation between TM9SF1 expression levels and plasma cytokine levels, suggesting that it may play a regulatory role in the harsh microenvironment of sepsis.

[0028] Example 3: Upregulation of TM9SF1 expression increases the severity of sepsis and the risk of death in patients The correlation between TM9SF1 expression level and sepsis severity was evaluated by univariate and multivariate logistic regression analysis (Figure 2 and Table 3).

[0029] Table 2 TM9SF1 Correlation analysis between expression level and severity of sepsis

[0030] *** P <0.001; a Univariate analysis; b Adjustment variables: age, gender, smoking history, drinking history, and disease history.

[0031] Table 3 Correlation analysis between TM9SF1 expression level and prognosis of patients with septic shock

[0032] *** P <0.001; a Univariate analysis; b Adjustment variables: age, gender, smoking history, drinking history, and disease history.

[0033] The results showed that after adjusting for age, sex, smoking status, drinking status, and medical history, high levels of TM9SF1 were significantly associated with increased severity of sepsis (OR = 47.14, 95% CI = 8.06-275.65, P<0.001). After patients were divided into high-risk and low-risk groups according to the median TM9SF1 level (0.14), the high-risk group was 2.94 times more likely to develop severe sepsis than the low-risk group (OR = 3.29, 95% CI = 1.88-5.78, P<0.001).

[0034] In order to further explore the relationship between TM9SF1 expression and the risk of death in patients, this example uses Cox regression analysis and Kaplan-Meier survival curve analysis. After adjusting for age, gender, smoking status, drinking status and disease history, the risk of death in patients increased with the increase of TM9SF1 levels (HR = 37.21, 95% CI = 12.80-108.21, P<0.001). In addition, patients with higher TM9SF1 expression levels had significantly shorter survival times than patients with lower expression levels (HR = 11.12, 95% CI = 4.35-28.45, P<0.001).

[0035] Example 4 TM9SF1 has better predictive ability in sepsis severity and mortality risk than traditional clinical indicators ROC curve analysis was used to evaluate the predictive ability of TM9SF1 and various clinical indicators for the severity of sepsis and the risk of death. The results are shown in Table 4.

[0036] Table 4 Predictive ability of each index for the severity of sepsis

[0037] This example simultaneously analyzes the effect of TM9SF1 and clinical indicators such as WBC, LYM%, MON%, NEU%, NLR, PLR, MLR, CRP, ESR, D-dimer, SaO2, OI, and lactate in predicting the severity of sepsis. The results show that among all indicators, TM9SF1 has the largest area under the ROC curve (AUC = 0.822, 95% CI = 0.766 - 0.878). The AUCs for ESR and OI severity prediction are 0.768 (95% CI = 0.705-0.832) and 0.615 (95% CI = 0.541-0.688), respectively. It suggests that TM9SF1 is the best marker for distinguishing patients with sepsis from septic shock, with a sensitivity of 89.4% and a specificity of 72.9%.

[0038] The correlation between TM9SF1 expression level and important clinical indicators (WBC, PLT, CRP, ESR, NEU%, LYM%, MON%, NLR, MLR, PLR, D-dimer and Lactate) in patients with sepsis was analyzed. The analysis results are shown in Table 5.

[0039] Table 5 Correlation analysis between TM9SF1 expression levels and important clinical indicators in patients with sepsis

[0040] Time-dependent ROC analysis was used to evaluate the predictive ability of TM9SF1 on the risk of death in patients and compared with other independent variables. The results are shown in Table 6.

[0041] Table 6 Predictive ability of each indicator for sepsis mortality risk based on time-dependent ROC analysis

[0042] The results in Table 6 show that TM9SF1 has a high predictive accuracy, with a Harrell's C index of 0.853 (95% CI = 0.782-0.924), a sensitivity of 91.8%, and a specificity of 78.3%. In contrast, the Harrell's C index for mortality prediction of ESR was 0.680 (95% CI = 0.584-0.775), NLR was 0.668 (95% CI = 0.567-0.769), and lactate was 0.749 (95% CI = 0.654-0.874). These results suggest that TM9SF1 has a high predictive ability in assessing the severity of sepsis and the risk of death. Therefore, TM9SF1 may be a new biomarker with great potential for predicting the prognosis of sepsis.

[0043] Example 5 Construction of a prediction model for sepsis severity and mortality risk based on TM9SF1 In order to construct a prediction model for the severity of sepsis, this example first performed a multivariate logistic regression analysis on the variables with P<0.05 in the univariate logistic regression analysis. After adjusting other covariates, only the variables with P<0.05 were included in the final model.

[0044] The results showed that TM9SF1, D-dimer, ESR and CRP were independent risk factors for predicting the severity of sepsis, with OR values ​​of 47.81 (95% CI = 28.33-74.41, P<0.001), 1.20 (95% CI = 1.10-1.30, P<0.001), 1.04 (95% CI = 1.02-1.05, P<0.001) and 1.13 (95% CI = 1.02-1.47, P =0.040), respectively.

[0045] The mortality risk of patients with sepsis was further evaluated by multivariate COX proportional hazards regression analysis. The results showed that the mortality risk of patients with septic shock was significantly associated with higher TM9SF1 levels (HR = 28.69, 95%CI = 17.19-47.48, P<0.001), CRP (HR = 1.14, 95%CI = 1.03-3.07, P = 0.027), ESR (HR = 1.05, 95%CI = 1.01-1.37, P = 0.046), lactate (HR = 8.69, 95%CI = 5.01-11.13, P<0.001), and oxygenation index (HR = 0.08, 95%CI = 0.04-0.26, P = 0.047).

[0046] Based on the above multivariate logistic regression analysis results, this embodiment constructs a new nomogram prediction model, such as Figure 3 As shown in the figure, it is used to quantitatively predict the probability of septic shock in patients. The nomogram model combines TM9SF1, D-dimer, ESR and CRP levels to calculate the probability of septic shock in each patient by adding up the scores of these four variables. Similarly, the significant independent predictors based on COX regression analysis include TM9SF1, ESR, CRP, lactate and OI, and a prognostic nomogram for predicting mortality in patients with septic shock is constructed, as shown in the figure. Figure 4 By summing the predictors in the nomogram, the probability of death in patients with septic shock can be calculated.

[0047] The nomogram model for predicting the severity of sepsis was validated by the ROC curve, with an AUC of 0.883 (95% CI = 0.839-0.927), a sensitivity of 91.5%, and a specificity of 78.4% (Table 4). For the prediction of mortality in patients with septic shock, the C index of the nomogram model was 0.931 (95% CI = 0.884-0.978, Table 6).

[0048] In addition, this embodiment divides patients into high-risk group and low-risk group according to the total score of the nomogram prediction model. The optimal total score cutoff value for risk classification is 44.30. The Kaplan-Meier survival curve is used to analyze the mortality risk of patients with septic shock at different nomogram scores. The analysis results are as follows: Figure 5As shown, low level refers to patients with septic shock whose TM9SF1 expression level is lower than 0.22, and high risk refers to patients with septic shock whose TM9SF1 expression level is not lower than 0.22. The results showed that the possibility of severe sepsis in the high-risk group was 8.39 times that in the low-risk group (HR = 8.39, 95% CI = 3.74-18.80, P<0.001).

[0049] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. Use of a marker in the preparation of a reagent for monitoring the course of sepsis, characterized in that: The markers include the TM9SF1 gene.

2. Use of a marker in the preparation of a reagent for monitoring the course of sepsis, characterized in that: The markers include mRNA molecules corresponding to the TM9SF1 gene.

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