CN111265656A - Application of DIO2 in the preparation of drugs for predicting or treating intrauterine adhesions - Google Patents

Abstract

The invention discloses the application of DIO2 in the preparation of a medicament for predicting or treating intrauterine adhesions. The present invention finds that in patients with intrauterine adhesions, the expression of DIO2 in the endometrium is decreased, especially the decrease in epithelial cells is the most significant. In vitro results showed that DIO2 could effectively reverse EMT in endometrial epithelial cells by converting thyroxine T4 to T3, and inhibit epithelial cell fibrosis. Therefore, DIO2 may play an important role in the diagnosis and treatment of intrauterine adhesions, and can be used for the clinical diagnosis of intrauterine adhesions and the preparation of drugs for the treatment of intrauterine adhesions and other diseases related to uterine fibrosis.

Description

Application of DIO2 in the preparation of drugs for predicting or treating intrauterine adhesions

technical field

The invention belongs to the field of biomedicine and relates to the application of DIO2 in the preparation of a medicine for predicting or treating intrauterine adhesions.

Background technique

Intrauterine adhesion (IUA) is a serious injury to the endometrium caused by a variety of factors, resulting in the adhesion between the endometrium, the uterine cavity is completely or partially closed, and the epithelial and mesenchymal cells that constitute the endometrial are fibrotic and interstitial. Proliferative differentiation disorders are replaced by epithelium, and the endometrium becomes unresponsive to sex hormone stimulation. IUA is the most common cause of secondary infertility, accounting for up to 25-30% of infertile women. The incidence of membrane fibrosis was significantly increased. At present, the main treatment method is hysteroscopic adhesion separation surgery, which is not effective for severe and extensive endometrial injury, and the postoperative adhesion recurrence rate is as high as 62.5%. Therefore, it is urgent to find an effective method for the prediction and treatment of IUA.

DIO2, an enzyme that converts thyroxine T4 to active thyroxine T3, has significantly higher activity and expression levels in the lungs of patients with idiopathic pulmonary fibrosis than controls, and correlates with disease severity, however, Whether there is a correlation between DIO2 and intrauterine adhesions and whether it affects endometrial fibrosis has not been reported.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of DIO2 in the preparation of medicines for treating intrauterine adhesions.

Another object of the present invention is to provide the application of DIO2 in the preparation of diagnostic reagents for intrauterine adhesions.

The object of the present invention is achieved through the following technical solutions:

Application of DIO2 in the preparation of medicines for treating uterine fibrosis-related diseases.

Application of DIO2 in the preparation of medicines for treating intrauterine adhesions.

The application of DIO2 as a detection target in the preparation of intrauterine adhesion prediction or auxiliary diagnostic reagents.

According to the American Fertility Society (AFS) 1988 classification criteria, patients with intrauterine adhesions were divided into mild, moderate and severe, according to the area of the uterine cavity accounted for by endometrial fibrosis (1=<1/3, 2=1/3-2/ 3,4=>2/3), degree of adhesion (1=film-like, 2=between film and dense, 4=dense adhesion) and menstrual flow (0=normal, 2=minor menstrual, 4=amenorrhea), 1-4 is mild, 5-8 is moderate. 9-12 are classified as severe. The expression levels of DIO2 in the endometrium of patients with different degrees of intrauterine adhesions are different, so the purpose of classification of patients with intrauterine adhesions can be achieved according to the expression of DIO2.

The application of a reagent for detecting DIO2 in the preparation of an auxiliary diagnostic reagent for intrauterine adhesions.

The reagent for detecting DIO2 is preferably a reagent for detecting the expression level of DIO2.

The reagent for detecting the expression level of DIO2 is a specific primer for DIO2 gene (such as PCR primer or qPCR primer) or an antibody for detecting DIO2 protein.

The intrauterine adhesions described in the present invention refer to the formation of uterine wall adhesions characterized by endometrial fibrosis due to the damage of the endometrial basal layer and the obstacle to the regeneration and repair of the functional layer.

The diseases caused by endometrial fibrosis mentioned in the present invention refer to intrauterine adhesions or endometrial scarring and uterine infertility, recurrent miscarriage, placenta accreta and the like caused thereby.

Beneficial effects:

We found that in patients with intrauterine adhesions, the expression of DIO2 in the endometrium was significantly reduced, especially in the epithelium. Moreover, in vitro results showed that DIO2 could effectively reverse endometrial epithelial-to-mesenchymal transition (EMT) by converting thyroxine T4 to T3 and inhibit epithelial cell fibrosis. Therefore, DIO2 may play an important role in the diagnosis and treatment of intrauterine adhesions, and can be used to prepare medicines for the diagnosis and treatment of intrauterine adhesions or other diseases related to uterine fibrosis.

Description of drawings

Figure 1. A: qPCR analysis of DIO2 expression in endometrial tissue; B: immunohistochemical analysis of DIO2 expression in endometrial tissue; C: DIO2 expression in endometrial tissue of patients with normal and severe intrauterine adhesions The ROC curve of ;

Figure 2. A: The effect of DIO2 and T4 on the expression of fibrosis-related genes; B: The effect of T3 on the expression of fibrosis-related genes;

Detailed ways

Example 1 Expression of DIO2 in endometrial tissue of patients with normal and severe intrauterine adhesions

1. Materials, reagents, equipment

1.1 Source of human endometrial tissue

36 normal endometrial tissues and 36 endometrial tissues from patients with severe intrauterine adhesion syndrome were collected. When obtaining endometrial tissues, it was ensured that all the endometrial tissues were obtained in the advanced stage of proliferation. All participants signed a paper version of informed consent, which was approved by the Ethics Committee of Nanjing Drum Tower Hospital.

1.2 Main reagents

TRNzol (Tiangen), Reverse Transcription Reagent (Yase), SYBR Ggreen (Yase), chloroform, isopropanol, xylene, absolute ethanol, 95%, 80%, 75% ethanol, 3% hydrogen peroxide, lemon Acid repair solution, DIO2 primer sequence (Forward primer 5'-3' ACTCGGTCATTCTGCTCAAG (SEQ ID NO. 1) Reverse primer 5'-3' ATTGCCACTGTTGTCACCTC (SEQ ID NO. 2)), DIO2 antibody (abcam), DAB, hematoxylin.

1.3 Main instruments

qPCR machine (Roche), PCR machine (ABI), oven, microscope (Leica).

1.4 Main methods

1.4.1 Tissue RNA extraction and real-time quantitative PCR

Total RNA was extracted by Trizol method. 1ug RNA was reverse transcribed to obtain cDNA, diluted with an appropriate volume of RNase-free water, and the SYBR Green method was used for fluorescence quantitative PCR detection. The expression of different target genes was calculated using 18S as the internal reference standard.

1.4.2 Endometrial tissue immunohistochemistry

1. Bake the slices in a constant temperature oven at 1.60°C for 60 minutes, treat with xylene for 3 times, each time for 5 minutes, after treatment with gradient alcohol (100% ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 75% ethanol for 5 minutes), rinse with water for an appropriate time, 3 Soak in %H2O2 for 15min (remove endogenous peroxidase), rinse with water for an appropriate time; after citric acid repair, the primary antibody is incubated at 37°C for 2h, rinsed 3 times with PBST, 5min each time, incubated with the secondary antibody for 8min, rinsed 3 times with PBST, 5min each time. DAB coloration, hematoxylin staining of nuclei, microscope observation and photographing after mounting.

2. Results

In the endometrial tissue of patients with severe intrauterine adhesions, the expression of DIO2 was significantly reduced, with the most significant decrease in epithelial cells (Figure 1).

Example 2 The effect of DIO2 on endometrial epithelial cell fibrosis

1. Materials, reagents, equipment

1.1 Main reagents

Collagenase Type I (Sigma), Hyaluronidase (Sigma), DNAase (Roche), Epithelial Cell Medium (Gibco), DMEM/F12 Medium (Wisent Inc), Serum (Gibco), Trizol (Invitrogen). TGFβ1 (PeproTech), Thyroxine T4/T3 (MCE), FN/N-cad/E-cad/a-SMA/DIO2 antibody (abcam), electrophoresis fluid, transfer fluid, skim milk (bio-rad), TBST

1.2 Main instruments

Cell incubator, shaker, PVDF nitrocellulose membrane, double vertical electrophoresis tank, gel imaging analysis system

1.3 Main methods

1.3.1 Isolation and culture of primary endometrial epithelial cells

Place the endometrial tissue in a new 60mm petri dish, chop the tissue until there are no lumps visible to the naked eye, add the prepared digestive solution, mix by pipetting, and place it in a 37°C incubator for 3 min; take out the petri dish and observe the tissue under a microscope Digestion situation; pipetting the digested tissue, adding DMEM/F12 to stop the digestion, and dropping the tissue suspension into a 40 μm sieve. The remaining tissue in the sieve was transferred to a clean 60mm petri dish, DMEM/F12 medium was added, and the suspension was added dropwise to a 100μm sieve after pipetting and mixing to obtain epithelial cells, and the digestion was repeated 3-5 times. After 24 hours, the cells were replaced. When the cells grew to a suitable density, they were treated with TGFβ1 (10ng/ml) for 24h, then added with thyroxine T4 (100nM)/transfected with DIO2/transfected with DIO2+T4/added with thyroxine T3 (20nM/100nM) and treated for 48h, and then the protein was extracted. .

1.3.2WB

Discard the culture medium, wash 3-4 times with pre-cooled PBS, add an appropriate amount of cell lysis buffer, put it on ice for lysis for 30 minutes, aspirate into an EP tube and centrifuge for 15 minutes to take the supernatant, measure the protein concentration, and calculate according to the loading amount of 30 μg volume, add loading buffer, heat in a metal water bath at 99°C for 10-15min, run the 10% gel to the bottom of bromophenol blue, transfer the membrane for 1-2h, block with 5% milk for 1h; incubate the primary antibody at 4°C overnight, wash with TBST for 5 min, and wash for 3 The secondary antibody was incubated at room temperature for 1 h, washed with TBST for 5 min, and washed 3 times; exposure.

2. Results

After TGF-β treatment for 24h, the expression of E-cad decreased, and the expression of N-cad, FN, a-SMA increased. After T3 alone or DIO2 and T4 co-treatment, the expression of E-cad increased, and the expression of N-cad, FN, a-SMA decreased, indicating that DIO2 can convert thyroxine T4 to T3, reverse EMT of endometrial epithelial cells and inhibit the process of fibrosis.

sequence listing

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Claims (6)

1. The application of DIO2 as a detection target in preparing a reagent for predicting intrauterine adhesion or assisting diagnosis.
2. Use of DIO2 in the manufacture of a medicament for the treatment of a disease caused by uterine fibrosis.
3. 3. The use according to claim 2, wherein DIO2 is used in the preparation of a medicament for the treatment of intrauterine adhesions.
4. 4. Application of a reagent for detecting DIO2 in preparing a reagent for predicting intrauterine adhesion or assisting diagnosis.
5. 5. The use of claim 4, wherein the reagent for detecting DIO2 is a reagent for detecting the expression level of DIO 2.
6. 6. The use of claim 5, wherein the reagent for detecting the expression level of DIO2 is a specific primer of DIO2 gene or an antibody for detecting DIO2 protein.

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