阅读说明：本技术 Trem-1在监测血吸虫病肝纤维化发展过程方面的应用 (Application of TREM-1 in monitoring schistosomiasis liver fibrosis development process ) 是由 朱丹丹 段义农 陈金铃 高文曦 段练 沈培 黄爱龙 于 2020-06-17 设计创作，主要内容包括：本发明提供一种TREM-1在监测血吸虫病肝纤维化发展过程方面的应用,其特征在于,采用动物模型动态监测TREM-1在血吸虫病肝脏中的表达方法。本发明还提供TREM-1在制备监测血吸虫病药物方面的应用。所述药物为药物试剂盒,所述药物试剂盒用于监测TREM-1的表达。本发明通过检测感染血吸虫的不同时间时,检测TREM-1在血吸虫病肝脏中的表达,从而监测肝纤维化发展过程,为监测肝纤维化发展过程,提供了新的监测靶点,从而提供一种新的药物试剂盒。(The invention provides an application of TREM-1 in monitoring schistosomiasis liver fibrosis development process, which is characterized in that an animal model is adopted to dynamically monitor the expression method of TREM-1 in schistosomiasis liver. The invention also provides an application of TREM-1 in preparing drugs for monitoring schistosomiasis. The drug is a drug kit used for monitoring the expression of TREM-1. The invention detects the expression of TREM-1 in the liver of schistosomiasis by detecting different time of infecting schistosomiasis so as to monitor the development process of hepatic fibrosis, and provides a new monitoring target for monitoring the development process of hepatic fibrosis, thereby providing a new medicine kit.)

1. An application of TREM-1 in monitoring the development process of schistosomiasis liver fibrosis is characterized in that an animal model is adopted to dynamically monitor the expression method of TREM-1 in schistosomiasis liver.

2. The use of a TREM-1 according to claim 1 for monitoring the development of schistosomiasis-induced liver fibrosis, comprising the steps of:

(1) establishing a mouse animal model

(1-1) animal grouping: selecting 30 female C57BL/6 mice with age of 8 weeks, and feeding in a clean space with humidity of 55% and day and night circulation of 12 h; randomly selecting 6 mice as healthy control groups, and distributing the rest mice as schistosomiasis groups;

(1-2) preparation of animal models

Taking a mouse of a schistosomiasis group, removing abdominal hair of the mouse to expose skin, fixing the mouse on a mouse frame, overflowing schistosoma japonicum cercaria from infectious oncomelania, taking the schistosoma japonicum cercaria to pick up on a cover glass, and keeping the schistosoma japonicum cercaria close to the abdominal skin of the mouse for 15min so that the cercaria fully enters the skin of the mouse to infect the mouse;

(2) RNA extraction

(2-1) respectively taking mouse liver tissues 0 week, 3 weeks, 6 weeks, 9 weeks and 12 weeks after the schistosome infected in the step (1-2), adding 1ml of Trizol into 50-100mg of the tissues, placing the tissues into a homogenizer, grinding on ice, and transferring into an EP (EP) tube; then adding chloroform according to 0.2ml chloroform/ml Trizol, gently mixing uniformly, and standing for 10min at room temperature;

(2-2) centrifuging at 12000rpm at 4 ℃ for 15 min;

(2-3) carefully sucking 0.5ml of upper-layer water phase, adding isopropanol according to the volume ratio of 1:1, gently mixing uniformly, and standing at room temperature for 10 min;

(2-4) centrifuging at 12000rpm at 4 ℃ for 20min, and removing the supernatant;

(2-5) adding 1ml of precooled 75% ethanol, shaking, uniformly mixing, and centrifuging at 4 ℃ and 12,000rpm for 5 min;

(2-6) discarding the supernatant, standing and drying RNA, and dissolving the precipitate with DEPC water;

(3) RT-qPCR detection

Reverse transcribing the RNA obtained in step (2) into cDNA using REVERTAID 1ST CDNA SYNTH KIT produced by Thermo;

the obtained cDNA was subjected to real-time quantitative PCR to detect the expression of TREM-1 using a TB Green-box Premix Ex Taq II kit generated from TAKARA.

3. The use of a TREM-1 in monitoring the development of schistosomiasis liver fibrosis according to claim 1, wherein in the step (3), the primer sequences used in the quantitative PCR assay are as follows:

mTREM-1 F: CTGCTGTGCGTGTTCTTTGTCTC；

mTREM-1 R: AGGGTTCCTTCCCGTCTGGTA；

mGAPDH F: TGGAAAGCTGTGGCGTGAT；

mGAPDH R: TGCTTCACCACCTTCTTGAT。

application of TREM-1 in preparing schistosomiasis medicine is disclosed.

5. The use of TREM-1 in the preparation of a medicament for monitoring schistosomiasis, according to claim 4, wherein the medicament is a pharmaceutical kit for monitoring the expression of TREM-1.

6. The use of TREM-1 in preparing a drug for monitoring schistosomiasis japonica according to claim 5, wherein the drug kit comprises an EP tube, chloroform, isopropanol, 75% ethanol, primers mTREM-1F, mTREM-1R, mGAPDH F and mGAPDH R for quantitative PCR detection.

7. The use of TREM-1 in the preparation of a medicament for monitoring schistosomiasis, according to claim 6, wherein the sequence of the primers used in the quantitative PCR assay is as follows:

mTREM-1 F: CTGCTGTGCGTGTTCTTTGTCTC；

mTREM-1 R: AGGGTTCCTTCCCGTCTGGTA；

mGAPDH F: TGGAAAGCTGTGGCGTGAT；

mGAPDH R: TGCTTCACCACCTTCTTGAT。

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of TREM-1 in monitoring schistosomiasis liver fibrosis development process.

Background

Myeloid cell Triggering Receptors (TREM) are cell surface receptors belonging to immunoglobulin superfamily, mainly including TREM-1 and TREM-2, which play important roles in inflammation and immune regulation. The research shows that TREM-1 has a large correlation with M1 type polarization of macrophages, and the deletion of TREM-1 can promote the polarization of M1 type macrophages to M2 type macrophages. In the Schistosoma mansoni model, TREM-1 expression in spleen macrophages at the early stage of Schistosoma mansoni infection is up-regulated, and is consistent with M1 polarization trend. However, no significant change was observed in the expression of TREM-1 in liver tissue from the model of schistosoma mansoni fibrosis. Although schistosoma japonicum and schistosoma mansoni belong to the genus Schistosoma, their biological properties and mechanisms causing granuloma ovata and hepatic fibrosis are not completely the same. The expression and application of TREM-1 in schistosomiasis liver fibrosis process are not reported.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides the application of TREM-1 in monitoring the schistosomiasis liver fibrosis development process.

In order to realize the aim, the embodiment of the invention provides the application of TREM-1 in monitoring the schistosomiasis liver fibrosis development process, which is characterized in that an animal model is adopted to dynamically monitor the expression method of TREM-1 in schistosomiasis liver.

Further, an application of TREM-1 in monitoring schistosomiasis liver fibrosis development process is characterized by comprising the following steps:

(1) establishing a mouse animal model

(1-1) animal grouping: selecting 30 female C57BL/6 mice with age of 8 weeks, and feeding in a clean space with humidity of 55% and day and night circulation of 12 h; randomly selecting 6 mice as healthy control groups, and distributing the rest mice as schistosomiasis groups;

(1-2) preparation of animal models

Taking a mouse of a schistosomiasis group, removing abdominal hair of the mouse to expose skin, fixing the mouse on a mouse frame, overflowing schistosoma japonicum cercaria from infectious oncomelania, taking the schistosoma japonicum cercaria to pick up on a cover glass, and keeping the schistosoma japonicum cercaria close to the abdominal skin of the mouse for 15min so that the cercaria fully enters the skin of the mouse to infect the mouse;

(2) RNA extraction

(2-1) respectively taking mouse liver tissues 0 week, 3 weeks, 6 weeks, 9 weeks and 12 weeks after the schistosome infected in the step (1-2), adding 1ml of Trizol into 50-100mg of the tissues, placing the tissues into a homogenizer, grinding on ice, and transferring into an EP (EP) tube; then adding chloroform according to 0.2ml chloroform/ml Trizol, gently mixing uniformly, and standing for 10min at room temperature;

(2-2) centrifuging at 12000rpm at 4 ℃ for 15 min;

(2-3) carefully sucking 0.5ml of upper-layer water phase, adding isopropanol according to the volume ratio of 1:1, gently mixing uniformly, and standing at room temperature for 10 min;

(2-4) centrifuging at 12000rpm at 4 ℃ for 20min, and removing the supernatant;

(2-5) adding 1ml of precooled 75% ethanol, shaking, uniformly mixing, and centrifuging at 4 ℃ and 12,000rpm for 5 min;

(2-6) discarding the supernatant, standing and drying RNA, and dissolving the precipitate with DEPC water;

(3) RT-qPCR detection

Reverse transcribing the RNA obtained in step (2) into cDNA using REVERTAID 1ST CDNA SYNTH KIT produced by Thermo;

the obtained cDNA was subjected to real-time quantitative PCR to detect the expression of TREM-1 using a TB Green-box Premix Ex Taq II kit generated from TAKARA.

Further, in the step (3), in the quantitative PCR detection, the primer sequences used are as follows:

mTREM-1 F: CTGCTGTGCGTGTTCTTTGTCTC；

mTREM-1 R: AGGGTTCCTTCCCGTCTGGTA；

mGAPDH F: TGGAAAGCTGTGGCGTGAT；

mGAPDH R: TGCTTCACCACCTTCTTGAT。

the embodiment of the invention also provides application of TREM-1 in preparing a schistosomiasis drug.

Further, the medicament is a pharmaceutical kit for monitoring TREM-1 expression.

Further, the pharmaceutical kit comprises an EP tube, chloroform, isopropanol, 75% ethanol and primers mTREM-1F, mTREM-1R, mGAPDH F and mGAPDH R for quantitative PCR detection.

Wherein, the sequences of the primers used in the quantitative PCR detection are as follows:

mTREM-1 F: CTGCTGTGCGTGTTCTTTGTCTC；

mTREM-1 R: AGGGTTCCTTCCCGTCTGGTA；

mGAPDH F: TGGAAAGCTGTGGCGTGAT；

mGAPDH R: TGCTTCACCACCTTCTTGAT。

the technical scheme of the invention has the following beneficial effects: the invention detects the expression of TREM-1 in the liver of schistosomiasis by detecting different time of infecting schistosomiasis so as to monitor the development process of hepatic fibrosis, and provides a new monitoring target for monitoring the development process of hepatic fibrosis, thereby providing a new medicine kit. The embodiment of the invention detects TREM-1 in the liver of the schistosomiasis japonica mouse, and the result shows that the expression of TREM-1 in the liver of the schistosomiasis japonica mouse is increased, and the expression of TREM-1 in the liver tissue of the mouse is reduced after the schistosomiasis japonica mouse is infected for 12 weeks, but the TREM-1 is still maintained at a higher level.

Drawings

FIG. 1 is a graph showing the change in expression of TREM-1 in the liver tissue of schistosomiasis japonica at 0 to 12 weeks in example 1 of the present invention.

FIG. 2 is a graph showing the quantitative PCR lysis of TREM-1 in example 1 of the present invention.

FIG. 3 is a graph of the quantitative PCR dissolution curve of internal reference GAPDH in example 1 of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.