CN114959013A

CN114959013A - Biomarker for altitude erythrocytosis and application thereof

Info

Publication number: CN114959013A
Application number: CN202210632656.XA
Authority: CN
Inventors: 骆倩倩; 丁洁; 杨万萍; 李美琦; 王国华; 朱俐
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-08-30
Anticipated expiration: 2042-06-06
Also published as: CN114959013B

Abstract

The invention discloses a biomarker of altitude erythrocytosis and application thereof, wherein the biomarker is a combined gene and consists of an HMOX1 gene, an FTL gene, an FTH1 gene, an SLC40A1 gene, a TFRC gene, an ACSL4 gene and an SLC7A11 gene. The invention can accurately, conveniently and efficiently carry out early diagnosis of the altitude erythrocytosis (HAPC), and fills the blank of the existing diagnosis technology for large-scale screening, thereby providing prediction and early warning for patients with potential HAPC morbidity risk more timely, and also providing effective treatment measures for HAPC patients and improving prognosis.

Description

Biomarker for altitude erythrocytosis and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker of altitude erythrocytosis and application thereof.

Background

The plateau is a special environment, has the characteristics of low atmospheric pressure and low oxygen partial pressure, and is easy to cause oxygen deficiency. When a plain person enters a plateau or residents on the plateau migrate to a higher altitude area, a series of altitude diseases including acute altitude diseases and chronic altitude diseases can be caused due to inadaptation to altitude increase of the plateau. Among them, High Altitude Polycythemia (HAPC) is the most common and widely-affecting chronic altitude disease. It refers to a disease that local people or immigrants living at an altitude of 2500 m or more for a long time increase Hemoglobin (HGB) concentration and Red Blood Cell (RBC) abnormally increase to increase blood viscosity and slow blood flow in order to adapt to the stimulation of high altitude hypoxia, thereby causing tissue hypoxia. The patients mainly show headache, palpitation, dyspnea, purple skin and the like, and the health of plateau people is seriously harmed. An investigation of adults living above an altitude of 3000 m has revealed that the average prevalence of HAPC is 3.78% and that the incidence of colonized populations rises from 1.41% to 15.52% with increasing altitude to 5000 m.

Currently, there are few methods for predicting HAPC in advance in clinical practice, and Erythropoietin (EPO) was considered to play a role in HAPC in the early days. Subsequent studies have shown that EPO is a major factor in erythropoiesis in acute hypoxia, but EPO does not exhibit a significant effect in chronic hypoxia. HAPC is a classical chronic altitude disease, so other factors are necessary to influence the occurrence and development of HAPC. As is well known, bone marrow is the site of RBC production, while spleen is the site of RBC recovery. Body RBC production and clearance are normally in dynamic equilibrium. In plateau exposure, a new balance of RBC production and clearance is rapidly formed in normal persons, but HAPC patients show a disruption in RBC production and clearance homeostasis, resulting in a sustained increase of RBC. Studies have confirmed that bone marrow of HAPC patients under plateau exposure has obvious splenomegaly in patients with persistent hematopoietic HAPC, suggesting that spleen function is closely related to the development of HAPC.

The development of HAPC and other more serious complications resulting therefrom are closely related to the delay of diagnosis, and since HAPC is insidious, the early symptoms are atypical, and it is easy for people to misunderstand that it is a general altitude reaction, it is very important to develop a method that can have a predictive effect before HAPC occurs. Currently, no spleen function biomarker exists clinically, so that the risk of HAPC occurrence can be predicted in advance by searching for the spleen function biomarker. The sequencing precision of the sequencing technology reaches the level of a single cell, and the further cognition of people on various diseases at the gene level is greatly promoted. A large number of researches find that a group of biomarkers obtained by screening are jointly applied to disease diagnosis or prediction, so that the defects of poor specificity and low diagnosis efficiency of a single biomarker can be overcome, and the detection sensitivity and specificity are obviously improved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the biomarker of the plateau erythrocytosis and the application thereof, the gene combination is screened out through early-stage research to predict the occurrence of HAPC, effective prediction and early warning can be timely provided for patients with potential HAPC morbidity risk, the early diagnosis and large-scale screening of HAPC can be accurately, conveniently and efficiently carried out, and the risk of the outbreak of HAPC after the people enter the plateau due to work or travel is avoided.

The invention is realized by the following technical scheme:

a biomarker of high protocythemia, which is a combined gene and consists of an HMOX1 gene, an FTL gene, an FTH1 gene, an SLC40A1 gene, a TFRC gene, an ACSL4 gene and an SLC7A11 gene.

Use of a biomarker as described above in the manufacture of a product for early diagnosis or prediction of altitude erythrocytosis, said product comprising a nucleic acid capable of binding to a combinatorial gene or a substance capable of binding to a protein expressed by a combinatorial gene.

Preferably, the nucleic acid capable of binding to the combined gene is a primer for specific amplification of the combined gene used in real-time quantitative PCR;

the amplification primer of the HMOX1 gene has a sequence shown as SEQ ID No. 1-2;

the amplification primer of the FTL gene has a sequence shown as SEQ ID No. 3-4;

the amplification primer of the FTH1 gene has a sequence shown as SEQ ID No. 5-6;

the amplification primer of the SLC40A1 gene has a sequence shown as SEQ ID No. 7-8;

the amplification primer of the TFRC gene has a sequence shown as SEQ ID No. 9-10;

the amplification primer of the ACSL4 gene has a sequence shown as SEQ ID No. 11-12;

the amplification primer of the SLC7A11 gene has a sequence shown as SEQ ID No. 13-14.

Preferably, the substance capable of binding the protein expressed by the combined gene comprises a reagent for detecting the protein expressed by HMOX1 gene, the protein expressed by FTL gene, the protein expressed by FTH1 gene, the protein expressed by SLC40A1 gene, the protein expressed by TFRC gene, the protein expressed by ACSL4 gene and the protein expressed by SLC7A11 gene in a sample to be detected.

Preferably, the sample to be tested is selected from peripheral blood of a tested person.

Preferably, the absolute value of the difference value between the CT value of each gene in the combined genes and the average CT value of seven reference genes is used as the basis for judging the risk of the altitude erythrocytosis;

calculating the difference value of the CT value of each gene in the combined genes and the average CT value of seven reference genes to obtain an absolute value of delta CT, namely:

|ΔCT|＝|CT _gene -CT _{Mean of internal reference} |；

Wherein, every time the [ Delta ] CT ] exceeds 5 CTs, the risk score is 1, otherwise, the risk score is 0 if the number of the CT values is less than 5; the total score of the above 7 genes was thus divided into 3 grades: 0-2 points, and extremely low occurrence risk; 3-5 points, moderate risk; and 6-7 points, the occurrence risk is extremely high.

Preferably, the seven reference genes are respectively: ACTB gene, GAPDH gene, RNA18S gene, TBP gene, HPRT1 gene, HMBS gene, GUSB gene; wherein the content of the first and second substances,

the amplification primer of the ACTB gene has a sequence shown as SEQ ID No. 15-16;

the amplification primer of the GAPDH gene has a sequence shown as SEQ ID No. 17-18;

the amplification primer of the RNA18S gene has a sequence shown as SEQ ID No. 19-20;

the amplification primer of the TBP gene has a sequence shown as SEQ ID No. 21-22;

the amplification primer of the HPRT1 gene has a sequence shown as SEQ ID No. 23-24;

the amplification primer of the HMBS gene has a sequence shown as SEQ ID No. 25-26;

the GUSB gene amplification primer has a sequence shown as SEQ ID No. 27-28.

The application of the inhibitor/activator of the biomarker in preparing the medicine for treating the altitude erythrocytosis.

Preferably, the inhibitor is used to inhibit the SLC40a1 gene, TFRC gene, ACSL4 gene; the activator is used for activating HMOX1 gene, FTL gene, FTH1 gene, SLC7A11 gene.

Use of the above biomarker or the above inhibitor/activator for the manufacture of a product for the early diagnosis, prediction or treatment of spleen function.

The invention has the following beneficial effects:

compared with the single gene expression detection in the prior art, the method has higher specificity and higher diagnostic efficiency, and can obviously improve the detection sensitivity and specificity. The invention can accurately, conveniently and efficiently carry out the early diagnosis of HAPC, and fills the blank in the prior diagnosis technology, thereby providing effective treatment measures for HAPC patients more timely and improving prognosis; the invention has good clinical application value, can solve the problem of limited early diagnosis mode of HAPC at present, is beneficial to promoting the early diagnosis and early treatment of HAPC and improving the long-term prognosis of patients so as to relieve the burden of social diseases.

Drawings

FIG. 1 is a graph of the effect of hypoxic exposure on the number of mouse blood Red Blood Cells (RBC) in example 1: a is the change of the form and the quantity of the RBC of the blood of the mouse, and the scale bar is 100 mu m; b is the reticulocyte proportion of the blood of the mouse; c is the change in RBC concentration, Hemoglobin (HGB) concentration, Hematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) of the blood of the mouse; compared with the NN (atmospheric pressure constant oxygen) group, ^* P＜0.05， ^*** p is less than 0.001; in contrast to the designated group, ^# P＜0.05， ^## P＜0.01， ^### P＜0.001；

FIG. 2 is a graph showing the changes in spleen morphology (A), volume (B) and weight (C) of mice after the exposure to high-antigen hypoxia in example 1: compared with the NN (atmospheric pressure constant oxygen) group, ^* P＜0.05， ^*** p is less than 0.001; in contrast to the designated group, ^## P＜0.01， ^### P＜0.001；

FIG. 3 is the change in expression of related proteins in the spleen of mice after 7d and 14d of high-antigen hypoxic exposure in example 1: a is a Western blot result of related proteins in the spleen of the mouse after the plateau hypoxia exposure for 7 days; b is the expression condition of related proteins in the spleen of the mouse after the plateau hypoxia exposure for 7 days; c is a Western blot result of related proteins in the spleen of the mouse after the plateau hypoxia exposure for 14 days; d is the expression condition of related proteins in the spleen of the mouse after the plateau hypoxia exposure for 14 days; compared with the NN (atmospheric pressure constant oxygen) group, ^* P＜0.05， ^** P＜0.01；

FIG. 4 is the changes in expression of ACSL4 and xCT proteins in the spleen of mice after 7d and 14d of high-antigen hypoxic exposure in example 1: a is a Western blot result and an expression condition of ACSL4 in the spleen of the mouse after 7 days of plateau hypoxia exposure; b is a Western blot result and an expression condition of ACSL4 in the spleen of the mouse after the plateau hypoxia exposure for 14 days; c is a Western blot result and an expression condition of xCT in the spleen of the mouse after the plateau hypoxia exposure for 7 d; d is a Western blot result and an expression condition of xCT in the spleen of the mouse after the plateau hypoxia exposure for 14D; compared with the NN (atmospheric pressure constant oxygen) group, ^* P＜0.05；

FIG. 5 shows the exposure of plateau in example 2Changes in expression of the relevant genes in the population of blood mononuclear cells: a is HMOX1 gene, B is FTL gene, C is FTH1 gene, D is SLC40A1 gene, E is TFRC gene, F is ACSL4 gene, G is SLC7A11 gene; compared with the Base camp (plain group), ^* P＜0.05， ^** P＜0.01， ^*** P＜0.001；

FIG. 6 is a combined gene amplification curve before plateau exposure in example 3;

FIG. 7 is a combined gene amplification curve before plateau exposure in example 3, case B.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1

At the animal level, 8-week-old male C57BL/6 mice were selected for the experiment and randomly divided by body weight into Normobaric Normoxicia (NN) group and high altitude hypoxic exposure (HH) group (comprising HH-7d and HH-14 d). The HH group of mice are placed in an animal low-pressure oxygen chamber to simulate the plateau exposure at 6000 m altitude, and an HAPC mouse model is constructed. After plateau exposure for different periods of time, mouse blood and spleen were collected separately for the following tests:

blood smear, routine blood test and flow cytometry are respectively carried out on anticoagulation blood of the mice TO observe the change of the shapes and the numbers of Red Blood Cells (RBC), the concentration of Hemoglobin (HGB), the proportion of reticulocytes (Reticulocyte), the specific volume of Hemoglobin (HCT), the mean volume of red blood cells (MCV), the mean amount of hemoglobin (MCH) of the red blood cells and the Mean Concentration of Hemoglobin (MCHC) of the red blood cells, wherein TO is Reticulocyte dye thiazole orange. The experimental results showed a significant increase in RBC, HGB, HCT in HH-3d mice compared to NN with statistical differences (P < 0.05) (fig. 1A, fig. 1C); the proportion of HH-7d reticulocytes and MCV were significantly increased, and were statistically different (P < 0.05) as HH-3d (FIG. 1B, FIG. 1C); the proportion of reticulocytes and the magnitude of MCV increase were slowed in HH exposure 14d compared to HH exposure 7d (fig. 1B, 1C), and no statistical difference was observed between MCH and MCHC at each time of HH exposure, as was observed for HH exposure 7 d. The above results suggest that HH exposure can cause a continuous increase in RBC count in the blood of mice, and successful mouse model construction.

Morphology (fig. 2A) and volume size (fig. 2B) and weight (fig. 2C) data of the spleen were recorded and photographs were taken. The volume is calculated in the following way: spleen volume is longer diameter x shorter diameter x wider diameter/6. Observation of spleen morphology in mice revealed that spleen was significantly contracted compared to NN in HH-1d group; splenomegaly was evident in the HH-3d group, and most evident in the HH-7d group; HH-14d, the splenomegaly was reduced. Spleen volume and weight were analyzed and found to be significantly reduced in weight with statistical differences (P < 0.05) in the HH-1d group; the weights and volumes of spleens of the mice in the HH-3d group are obviously increased, statistical differences exist (P is less than 0.05), and the significance is highest in the HH-7d group; spleen weight and volume were significantly reduced in the HH-14d group compared to the HH-7d group, with statistical differences (P < 0.05) (FIG. 2B, FIG. 2C). The above results suggest that HH exposure can cause splenomegaly in mice, especially for 7 days.

Western blot detection is carried out on the expression changes of protein expressed by HMOX1 gene, protein expressed by FTL gene, protein expressed by FTH1 gene, protein expressed by SLC40A1 gene, protein expressed by TFRC gene, protein expressed by ACSL4 gene and protein expressed by SLC7A11 gene in spleen, and the detection results are shown in figure 3 and figure 4. The results showed that the protein expression was found to be significantly reduced in the HH-7d and HH-14d group HO-1 (corresponding to the gene name HMOX1) compared to NN, suggesting that the decomposition ability of heme iron in spleen was reduced after HH exposure. Meanwhile, the protein expression of Ft-L (corresponding to the gene name FTL), Ft-H (corresponding to the gene name FTH1), NCOA4 and xCT (corresponding to the gene name SLC7A11) in the spleen of the HH-7d group is remarkably reduced, the protein expression changes of Fpn (corresponding to the gene name SLC40A1), TfR (corresponding to the gene name TFRC) and ACSL4 (corresponding to the gene name ACSL4) are remarkably increased, and the statistical differences (P < 0.05) exist (FIG. 3A, FIG. 3B, FIG. 4A and FIG. 4C); the expression of Fpn protein was not significantly changed when HH was exposed to 14D, and the other protein expressions were all the same as HH exposure to 7D (fig. 3C, 3D, 4B, 4D). The above results suggest that HH exposure for 7d, the iron mobilization in the spleen of mice was abnormally active, and stored iron in spleen tissue was continuously degraded by NCOA4 and exported from Fpn to blood; while the HH exposure of 14d, Fpn output to iron did not increase.

The study of the embodiment finds that when the mouse is exposed to the high altitude hypoxia environment, the expression changes of the HMOX1 gene, the FTL gene, the FTH1 gene, the SLC40A1 gene, the TFRC gene, the ACSL4 gene and the SLC7A11 gene in the spleen of the mouse are consistent with the changes of impaired spleen function (splenomegaly), and the impaired spleen function is closely related to the development of HAPC.

Example 2

At the human level, GSE46480 in the GEO database was analyzed and the data set was divided into two groups, one group being the plains group (Base camp) and the other group being the highlands group (Altitude), with a sample size of 98 per group. The experimental method comprises the following steps: the same person collected blood at McMurdo Station (48 m), immediately transferred to the Armonton-Scott South Pole Station (Amundsen-Scott South Pole Station, 2835m), and collected blood on the third day to isolate mononuclear cells for testing. After extracting RNA from the two groups of mononuclear cell samples, carrying out high-throughput detection by adopting Affymetrix HG U133 Plus 2array, selecting the concerned genes and analyzing the change trend of the concerned genes in the population before and after plateau exposure. As shown in FIG. 5, compared with the gene before the plateau (Base camp), the expression levels of HMOX1, FTL, FTH1 and SLC7A11 genes were significantly reduced, and the expression levels of RNA of SLC40A1, TFRC and ACSL4 were significantly increased. The result is consistent with the expression change of the corresponding gene in the spleen sample of the plateau exposed mouse, which shows that the expression change of the related gene in the blood mononuclear cell sample of the population can reflect the protein expression change of the same gene in the spleen of the HAPC mouse.

Example 3

The case A and the case B are healthy men, and the people enter the plateau (the elevation is about 5300 meters) in 2013 for 5 months, extract peripheral blood, separate white blood cells, extract RNA and perform reverse transcription and PCR amplification. The gene amplification primer sequences are shown in Table 1. The method comprises the following specific steps:

1. peripheral blood mononuclear cell isolation

Collecting human anticoagulated whole blood, transferring 10mL of whole blood into a 50mL centrifuge tube, adding 10mL of PBS solution for dilution, and gently mixing uniformly; two 15mL centrifuge tubes were taken and 5mL of ficoll solution was added first. Then, slightly adding the diluted blood to the upper layers of the ficoll of the two centrifuge tubes, wherein the diluted blood must be gentle to avoid mixing the two solutions together, and 10mL of the diluted blood is added to each centrifuge tube; 2000rpm for 20min, the second white layer was peripheral blood mononuclear cells, which were pipetted into another clean 15mL centrifuge tube. After washing once with PBS, resuspend until needed.

2. Mononuclear cell RNA extraction

Adding chloroform according to the volume ratio of 1/5, swirling for 10s, standing for 10min, centrifuging at 12000g at 4 ℃ for 15min, taking the supernatant, adding isopropanol in equal proportion, slightly inverting, and standing for 10 min. Centrifuging at 12000g at 4 deg.C for 10min, collecting precipitate, washing with 75% anhydrous ethanol (prepared with DEPC water) for 2 times, centrifuging at 12000g at 4 deg.C for 5min, collecting precipitate, adding appropriate amount of DEPC water, dissolving, and standing by, all steps being carried out on ice.

3. RNA concentration determination and reverse transcription

(1) mu.L of mRNA was taken on a Nanodrop 2000 Spectrophotpmeter and the concentration of mRNA and A were determined ₂₆₀ /A ₂₈₀ Relative absorbance, data were recorded.

(2) Reversing the amount of 2000ng mRNA according to a 20 uL system, sequentially adding a corresponding amount of mRNA, non-enzyme water and 4 uL 4 XgDNAwiper Mix to form a total volume of 16 uL, heating at 42 ℃ for 2min, adding 4 uL 5 XHiscopt III Supermix, mixing uniformly, and setting a reverse transcription program by using a PCR instrument to: 15min at 50 ℃; 5s at 85 ℃. After completion, the cDNA was stored at-20 ℃.

4. Real-time quantitative PCR

Prepare 10 μ L reaction system: SYBR Green 5. mu.L, forward primer (2. mu.M) 0.2. mu.L, reverse primer (2. mu.M) 0.2. mu.L, H ₂ O3.6. mu.L, and 1. mu.L of template cDNA. Setting a program: 5min at 95 ℃; 95 ℃ 30s, 60 ℃ 30s, 72 ℃ 30s (40 cycles); 72 ℃ to 95 ℃ (dissolution curve).

TABLE 1 primer sequence Listing

The results are shown in fig. 6 (case a) and fig. 7 (case B), and gene amplification CT values of case a and case B were obtained, respectively. Calculating the difference between the CT value of each gene (HMOX1 gene, FTL gene, FTH1 gene, SLC40A1 gene, TFRC gene, ACSL4 gene and SLC7A11 gene) in the combined genes and the average CT value of seven reference genes (ACTB, GAPDH, RNA18S, TBP, HPRT1, HMBS and GUSB) to obtain the absolute value of delta CT:

|ΔCT|＝|CT _gene -CT _{Mean of internal reference} |

If the < | delta > CT | exceeds 5 CTs, the risk score is 1, otherwise, if the < 5 > CT value is less, the risk score is 0; the total score of the above 7 genes was thus divided into 3 grades: 0-2 points, and extremely low occurrence risk; 3-5 points, moderate risk; and 6-7 points, the occurrence risk is extremely high.

The combined gene results are shown in tables 2 and 3, and the case A confirmed diagnosis of HAPC and the case B no confirmed diagnosis of HAPC after one year of stay in plateau, which confirms that the prediction and evaluation system of the invention is completely applicable.

TABLE 2 case A combination Gene detection prediction HAPC incidence Risk assessment Table

TABLE 3 case B combinatorial gene testing and prediction HAPC morbidity risk assessment table

The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Sequence listing

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Claims

1. The biomarker of the plateau erythrocytosis is a combined gene and consists of an HMOX1 gene, an FTL gene, an FTH1 gene, an SLC40A1 gene, a TFRC gene, an ACSL4 gene and an SLC7A11 gene.

2. Use of a biomarker according to claim 1 in the manufacture of a product for early diagnosis or prediction of hypercytosis, wherein the product comprises a substance capable of binding to a nucleic acid of a combinatorial gene or to a protein expressed by a combinatorial gene.

3. The use according to claim 2, wherein the nucleic acid capable of binding to the combinatorial gene is a primer for the specific amplification of the combinatorial gene used in real-time quantitative PCR;

the amplification primer of the SLC7A11 gene has a sequence shown in SEQ ID No. 13-14.

4. The use according to claim 2, wherein the substance capable of binding to the protein expressed by the combination gene comprises a reagent for detecting the protein expressed by HMOX1 gene, the protein expressed by FTL gene, the protein expressed by FTH1 gene, the protein expressed by SLC40A1 gene, the protein expressed by TFRC gene, the protein expressed by ACSL4 gene, the protein expressed by SLC7A11 gene in the sample to be tested.

5. The use of claim 4, wherein the sample is selected from peripheral blood of a subject.

6. The use of claim 2, wherein the absolute value of the difference between the CT value of each gene in the combined genes and the average CT value of seven reference genes is used as the basis for determining the risk of altitude polycythemia;

|ΔCT|＝|CT _gene -CT _{Internal referenceAverage} |；

7. The use of claim 6, wherein the seven reference genes are respectively: ACTB gene, GAPDH gene, RNA18S gene, TBP gene, HPRT1 gene, HMBS gene, GUSB gene; wherein the content of the first and second substances,

the GUSB gene amplification primer has a sequence shown as SEQ ID No. 27-28.

8. Use of the biomarker inhibitor/activator of claim 1 in the manufacture of a medicament for the treatment of elevated erythrocytosis.

9. The use of claim 8, wherein the inhibitor is for inhibiting the SLC40a1 gene, TFRC gene, ACSL4 gene; the activator is used for activating HMOX1 gene, FTL gene, FTH1 gene, SLC7A11 gene.

10. Use of the biomarker of claim 1 or the inhibitor/activator of claim 8 for the preparation of a product for the early diagnosis, prognosis or treatment of spleen function.