A unilateral pregnancy mouse uterine mechanical tension model and a construction method and application thereof

Abstract

This invention discloses a unilateral pregnancy mouse uterine mechanical tension model, its construction method, and its application. The model construction method includes the following steps: a female mouse with unilateral tubal sterilization has its uterus on one side. After the postoperative recovery period, the female mouse is allowed to conceive naturally in the unsterilized uterus. Simultaneously, a pregnant uterus and a non-pregnant uterus are formed in the same female mouse. At specific time points during pregnancy, tissues from the pregnant and non-pregnant uterus are obtained. The model of this invention has good physiological relevance, making the research results closer to the actual pregnancy process; the variable control is rigorous, and the controls are scientific; through in vivo pairing, the pregnant and non-pregnant uteruses are used as controls, completely eliminating individual differences, and the research method is standardized; it provides an efficient and dedicated tool for studying the biology of uterine tension and screening related regulatory substances. The model construction method is simple, minimally invasive, and highly reproducible, making it easy to promote and use in basic research.

Description

Technical Field

[0001] This invention relates to the field of experimental animal model technology, and in particular to a unilateral pregnant mouse uterine mechanical tension model, its construction method and application. Background Technology

[0002] The passive mechanical tension experienced by the uterus during pregnancy, induced by fetal growth and increased amniotic fluid, is a key physical factor regulating uterine myometrial remodeling and the initiation of labor. Abnormal uterine mechanical tension is a significant contributing factor to pregnancy complications such as preterm birth and uterine atony. Therefore, elucidating the molecular mechanisms by which mechanical tension regulates uterine function has important scientific and clinical significance.

[0003] The following technical bottlenecks currently exist in this research field: First, insufficient physiological relevance of models: Existing studies often employ exogenous mechanical stretching of isolated uteruses or cultured smooth muscle cells from non-pregnant animals (such as rats and mice). The tension generated by such methods is instantaneous and non-physiological, failing to simulate the endogenous, progressively accumulating tension environment during pregnancy, resulting in discrepancies between the research results and the actual physiological state. Second, inadequate variable control and inherent flaws in control settings: In in vivo studies, when comparing different pregnant individuals (e.g., comparing pregnant uteruses of different fetal numbers or different treatment groups), unavoidable inter-individual differences exist between the experimental and control groups, including genetic background, basal hormone levels, and metabolic status. These confounding factors severely interfere with the accurate assessment of the single variable effect of "mechanical tension." Third, lack of standardized and quantifiable functional assessment systems: Different laboratories have inconsistent testing conditions for uterine contraction function (e.g., perfusion fluid composition, initial tension, stimulant concentration, etc.), leading to large differences in results and poor comparability. The lack of a widely accepted validity criterion and standardized operating procedures hinders the reliability and reproducibility of research conclusions.

[0004] In summary, current technology lacks an animal model capable of accurately isolating the effects of uterine mechanical tension in the context of physiological pregnancy and equipped with standardized functional assessment methods. This constitutes a major obstacle to further elucidating the biological mechanisms of uterine mechanical tension. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings and deficiencies of the prior art and provide a unilateral pregnant mouse uterine mechanical tension model that is simple to construct, rigorously controlled, can realistically simulate physiological uterine tension, and integrates a standardized functional testing system, as well as its construction method and application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for constructing a unilateral pregnant mouse uterine mechanical tension model, comprising the following steps: (1) By performing unilateral tubal sterilization, female mice lost the ability to conceive on one side of their uterus; (2) After the postoperative recovery period, the female mouse was allowed to conceive naturally in the uterus on the unsterilized side, and the pregnant uterus and the non-pregnant uterus were formed simultaneously in the same female mouse. (3) At a specific time point during pregnancy, the uterine tissue on the pregnant side was obtained as the tension group, and the uterine tissue on the non-pregnant side was obtained as the control group.

[0007] Preferably, the female mice in step (1) are selected from ICR, C57BL / 6J or Kunming mice, with an age of 7-12 weeks and a weight of 20-30 g.

[0008] Preferably, the unilateral tubal sterilization procedure in step (1) is selected from one of the following: thermal coagulation severance, electrocoagulation closure, suture ligation, or ultrasonic scalpel cutting.

[0009] Preferably, the unilateral tubal sterilization surgery in step (1) is performed under inhalation anesthesia or injection anesthesia; the inhalation anesthesia uses isoflurane, and the injection anesthesia uses ketamine-xylazine mixed anesthesia.

[0010] Preferably, the specific time point mentioned in step (3) is the second trimester, such as day 15 of pregnancy; or the third trimester, such as day 19 of pregnancy.

[0011] Preferably, the uterine tissue on the pregnant side and the uterine tissue on the non-pregnant side in step (3) are obtained in the following manner: female mice are euthanized by cervical dislocation, the uterus is removed and rinsed with pre-cooled PBS, the uterus in the tension group is cut off between each embryo, the fetal mice are separated and the placenta and decidual tissue are removed, and the uterus in the control group is removed of visible blood vessels. Both the tension group and the control group are cut transversely along the long axis of the uterus to obtain each piece of uterine tissue with a size of 5 mm × 2 mm × 2 mm.

[0012] Secondly, the present invention provides a unilateral pregnant mouse uterine mechanical tension model generated by the above-described construction method.

[0013] Thirdly, this invention provides the application of the above-mentioned unilateral pregnancy mouse uterine mechanical tension model in scientific research for non-therapeutic diagnostic purposes, including studying the biological effects of mechanical tension on uterine tissue, screening substances that regulate uterine tension response, or verifying related molecular targets.

[0014] Fourthly, the present invention provides the application of the above-mentioned unilateral pregnancy mouse uterine mechanical tension model in a testing system or screening platform, wherein the testing system or screening platform is used to evaluate the effect of substances on the uterine mechanical tension response.

[0015] Fifthly, this invention provides a method for studying the mechanical tension effect of the uterus for non-therapeutic diagnostic purposes, which is achieved using the aforementioned unilateral pregnancy mouse uterine mechanical tension model and the following steps: (1) The uterine tissue on the pregnant side and the uterine tissue on the non-pregnant side are subjected to at least one of the following analyses under in vitro conditions: muscle strip contraction function measurement, specific protein expression level detection, or transcriptomics analysis; (2) Based on the analysis results of step (1), compare the differences between the pregnant side and the non-pregnant side to obtain experimental data on the effect of mechanical tension on uterine tissue.

[0016] Preferably, the conditions for measuring the muscle strip contraction function are as follows: the obtained uterine tissues of the tension group and the control group are prepared into isolated muscle strips, and after applying 2g of initial tension equilibrium in a Krebs-Ranger buffer perfusion system at a constant temperature of 37°C and purged with 95% O2 and 5% CO2, oxytocin with a final concentration of 1 nM is used for stimulation, the contraction curve is recorded, and the contraction function is quantified by the area under the curve (AUC). The Krebs-Ranger buffer system is as follows: Glucose 11 mM.

[0017] Preferably, the specific proteins include oxytocin receptor (OXTR), prostaglandin intraperoxidase 2 (PTGS2), and gap junction protein 43 (GJA1).

[0018] Preferably, the transcriptomics analysis includes batch RNA-seq sequencing and / or single-cell RNA-seq sequencing.

[0019] Compared with the prior art, the present invention has the following beneficial effects: First, the unilateral pregnant mouse uterine mechanical tension model of this invention has good physiological relevance: it directly utilizes the endogenous and progressive mechanical tension generated by pregnancy itself, overcoming the interference of non-physiological states caused by exogenous mechanical stretching, making the research results closer to the real pregnancy process. Second, the variable control is rigorous and the control is scientific: through in vivo pairing, the pregnant and non-pregnant uteri are used as controls, completely eliminating individual differences and achieving precise research on the single variable of mechanical tension, with strong causal evidence. Third, the research method is standardized: the accompanying ex vivo muscle strip measurement system clarifies the standard conditions and quantitative indicators, solving the problems of chaotic detection methods and poor comparability of results in existing technologies. Fourth, the model is specifically designed for scientific research and in vitro screening platforms for non-therapeutic purposes, providing an efficient and dedicated tool for studying the biology of uterine tension and screening related regulatory substances. Fifth, the model construction method is simple, minimally invasive, and highly reproducible, conforming to the 3R principle of animal experimental ethics, and is easy to promote and use in basic research. Attached Figure Description

[0020] Figure 1 Schematic diagram illustrating the construction of an animal model for the regulation of uterine contractions by pregnancy tension; Figure 2 This is a graph showing the muscle strip contraction detection and area under the contraction curve comparison of uterine tissue from the experimental group and the control group in Example 1; where A is the tension curve and B is the quantitative analysis result graph; n=5. ; Figure 3 This image shows the protein expression levels of PTGS2, OXTR (oxytocin receptor), and GJA1 in the uterine tissues of the experimental and control groups in Example 1; n=4. ; Figure 4 The diagram shows the results of differentially expressed genes and signaling pathways in the uterine tissues of the experimental group and the control group in Example 1; where A is a volcano diagram of all differentially expressed genes between the two groups, and B is a bubble diagram of significantly enriched pathways of differentially expressed genes between the two groups. Figure 5 This is an example of the biological function enrichment analysis of differentially expressed genes in uterine tissues of the experimental group and the control group in Example 1; where A is a bar chart of biological function of significantly enriched upregulated genes in the experimental group; and B is a bar chart of biological function of significantly enriched downregulated genes in the experimental group. Figure 6 This is a single-cell sequencing cell type UMAP diagram of the unilateral pregnancy mouse uterine mechanical tension model from Example 1; Figure 7 This is a graph showing the percentage of cell types in the experimental and control groups of the unilateral pregnant mouse uterine mechanical tension model in Example 1. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless specifically indicated or separately defined, the scientific and technical terms used in this application have the same unambiguous meaning as commonly known to those skilled in the art to which this invention pertains. Example 1

[0022] Experimental animals: SPF-grade CD-1 (ICR) female mice (8-10 weeks old, weighing 25±3 g) were purchased from Guangdong Provincial Medical Laboratory Animal Center and were acclimatized in a standard environment (temperature 22℃, humidity 50%, 12h / 12h light-dark cycle).

[0023] Unilateral sterilization surgery ( Figure 1 Mice were anesthetized with isoflurane (3% induction, 2% maintenance). The skin was prepared in a 2×2 area beside the right lumbar spine. After disinfection with 75% alcohol, the skin, fascia, muscle, and peritoneum were sequentially incised 1 cm lateral to the lumbar spine and at the level of the right costal arch using tissue scissors. A white adipose tissue was exposed in the abdominal cavity. This tissue was gently removed with toothless forceps, fully exposing the right ovary, fallopian tube, and uterus attached to the adipose tissue. Another toothless forceps was heated red-hot and used to thermally coagulate and sever the right fallopian tube. The uterus and ovary were then returned to the abdominal cavity. The peritoneum, muscle layer, fascia, and skin were intermittently sutured layer by layer using 4-0 Vicryl sutures. The mice were disinfected with 75% alcohol and revived on a 38°C warming pad. They were then fed and recovered for two weeks using standard feeding methods.

[0024] Induction of unilateral pregnancy: Recovered mice were housed with male mice at a female:male ratio of 3:1. The day a vaginal plug was detected was defined as day 0.5 of pregnancy (P<0.5). Subsequent procedures were performed on day 19 of pregnancy (P19).

[0025] Sampling: Pregnant mice were euthanized by cervical dislocation, and their intact uteruses were quickly removed by laparotomy.

[0026] Tension group: The uterus was taken from the left side of the uterus at conception. The uterus was cut at each embryonic interval, and the fetal rat, placenta and decidual tissue were separated and removed, while the myometrium was preserved.

[0027] Control group: The uterus was taken from the right side of a ligated, non-pregnant woman. Visible blood vessels were removed.

[0028] After rinsing the two groups of uterine tissue with pre-cooled PBS, the tissue was transversely cut perpendicular to the long axis of the uterus to prepare uterine tissue with a size of 5 mm × 2 mm × 2 mm.

[0029] Example 2: Measurement of Muscle Strip Contraction Function Experimental Procedure: The experiment was conducted using an isolated muscle strip tension measurement system (PowerLab 8 / 35 eight-channel research high-speed recording host, eight-channel bridge amplifier, tension sensor, eight-chamber isolated tissue perfusion device, etc., ADInstruments). The experiment was carried out in a constant-temperature perfusion bath, which maintained Krebs-Ranger buffer at 37°C (10 mL / chamber; Krebs-Ranger buffer system as follows:) The environment was prepared by adjusting the pH to 7.4 and continuously introducing a mixed gas containing 95% O2 and 5% CO2 to maintain physiological acidity and provide oxygen. Before the experiment, the Labchart 7.2 software was used to reset the signal to zero. All channel electrical signals were set to 10mV, and the unit conversion was set to "mV to g". The tension sensor was pre-calibrated and the signal conversion settings were configured using a two-point method (0g and 5g weights). The uterine tissue prepared in the example was vertically suspended in a water bath and given an initial tension of 2g. After a continuous and stable spontaneous contraction wave was observed, the system was allowed to equilibrate for 1 hour (marked as T0). Oxytocin with a final concentration of 1 nM was added to the water bath to induce enhanced contraction. The system continuously recorded the muscle strip contraction tension curve 1 hour after drug administration (marked as T1).

[0030] Quantitative Analysis: To quantitatively assess the contractile function of uterine tissue, this application used LabChart 7.2 software to perform region analysis on the contraction wave, calculating the area under the contraction curve (AUC) as a quantitative indicator of the work done by uterine contractions, and presenting the results as the integral of contractions (%). The results are as follows: Figure 2 As shown: Figure 2 A tension curve showed that after stimulation with oxytocin (OXT) (Post-OXT), the amplitude and frequency of contractions of the fetalized uterus were significantly enhanced compared with those before stimulation (Pre-OXT), while the changes in the non-fertilized uterus were not obvious. Figure 2 Quantitative analysis of B further confirmed that the contraction score of the pregnant uterus after oxytocin stimulation was significantly higher than that before stimulation, and the pregnant uterus was more responsive to oxytocin compared with the non-pregnant uterus.

[0031] Example 3: Detection of contraction-related protein expression (Western Blot) To further validate the reliability of the model, we examined the expression levels of contraction-related proteins: Western blotting detected the contraction-related proteins PTGS2 (prostaglandin intraperoxide synthase 2), OXTR (oxytocin receptor), and GJA1 (gap junction protein 43) in pregnant and non-pregnant uterine tissues. First, total protein (n=4) from the pregnant and non-pregnant uterine tissues prepared in Example 1 was extracted using RIPA lysis buffer (R0278, Sigma) containing a protease inhibitor mixture (P8340-1ml, P8340), and protein concentration was determined using a BCA protein quantification kit (#23227, Thermo Scientific). After separation by SDS-PAGE gel electrophoresis, the protein samples were transferred to a PVDF membrane (IPVH00010, Millipore) and visualized using a ChemiDoc XRS+ chemiluminescence imaging system. The expression levels of contraction-related proteins were detected using GAPDH as an internal control. Western blot analysis was performed on uterine tissues from the pregnant and non-pregnant sides (antibodies used were OXTR antibody (1:2000, T58142, Abmart), connexin43 / GJA1 antibody (1:2000, Ab097313, Aladdin), PTGS2 antibody (1:2000, ab179800, Abcam), and GAPDH antibody (1:20000, GTX100118, GeneTEX). All experiments were independently repeated three times). Results are as follows: Figure 3 As shown, compared with the non-fertilized uterus, the protein expression levels of contraction-related proteins PTGS2, OXTR, and GJA1 were significantly increased in the fertilized uterus. This result is consistent with the model's functional performance, confirming the significant upregulation of contraction-related proteins in the pregnant uterus and demonstrating the effectiveness of this model in detecting the molecular mechanisms of uterine contraction function. Example 4

[0032] 1. Transcriptome sequencing (RNA-seq) To further investigate the transcriptomic differences between the pregnant and non-pregnant uterine tissues, the applicant performed RNA-seq sequencing analysis on two groups of uterine tissues (n=3) prepared in Example 1. First, total RNA was extracted: Under ice-bath pre-cooling conditions, uterine tissue blocks were transferred to dedicated centrifuge tubes pre-filled with magnetic beads and an appropriate amount of Trizol reagent (15596026, Invitrogen) and thoroughly homogenized in a low-temperature tissue homogenizer. Direct lysis was performed, and all samples were purified using chloroform-ethanol-centrifugation column. Sample purity and concentration were detected using a Nanodrop 2000 microspectrophotometer, and RNA sample integrity was detected using a Labchip GX touch microfluidic capillary electrophoresis system. Total RNA samples that passed quality control (RNA integrity index RIN value ≥ 7.0) were selected. Eukaryotic mRNA was enriched using Oligo(dT) magnetic beads. Fragmentation buffer was added to fragment the mRNA, followed by the addition of buffer, dNTPs, DNA polymerase I, and RNase H to synthesize double-stranded cDNA. The cDNA was then purified, and the purified double-stranded cDNA underwent end repair, A-tailing, and sequencing adapter ligation. Fragment selection was performed, and approximately 350 bp of cDNA was recovered to obtain a cDNA library. After passing Agilent 2100 quality control, paired-end 150 bp sequencing was performed on an Illumina NovaSeq 6000 sequencing platform. Hisat2 with default parameters was used to map the RNA-seq sequencing data to the mouse genome. Differentially expressed genes were statistically analyzed using DESeq2, with P < 0.05 considered statistically significant. Enrichment analysis was performed using the DAVID tool to assess pathway enrichment in the gene list, with P < 0.05 considered statistically significant. Biological information pathway analysis was performed using the GO, KEGG, and WIKIPATNWAY databases.

[0033] The results showed that, compared with the non-pregnant side, a total of 2123 differentially expressed genes were identified in the uterine tissue of the pregnant side, of which 1344 genes were upregulated and 779 genes were downregulated. Figure 4 A). Pathway enrichment analysis showed that differentially expressed genes were significantly enriched in biological processes such as muscle contraction, inflammatory response, GPCR signal transduction, and extracellular matrix tissue formation. Figure 4 B). Further GO enrichment analysis revealed that contraction-related genes, such as adrenergic receptors (ADRB2, ADRA1A, etc.), showed significant enrichment in biological processes (BP), cellular components (CC), and molecular functions (MF). Figure 5 These results confirm at the transcriptomic level that the pregnant uterus activates multiple signaling pathways related to contraction regulation under mechanical stretching, demonstrating the effectiveness and reliability of this model in studying the molecular mechanisms of uterine contraction.

[0034] 2. Single-cell transcriptome sequencing (scRNA-seq) The myometrial tissues (n=3) from the pregnant and non-pregnant sides prepared in Example 1 were washed with pre-cooled physiological saline, finely minced using a scalpel in a biosafety cabinet, and digested in a digestion buffer containing 2 mg / mL collagenase II, 10 mg / mL Dispase II, and 50,000 U / mL DNase I on a shaker at 37°C for 30 minutes, with the digestion process observed under a microscope every 5 minutes until a single-cell suspension was formed. The suspension was then filtered through a 40 μm cell filter and centrifuged at 250 g, 4°C for 4 minutes. AO / PI double staining was used, requiring cell viability >80% and cell clumping rate <10% for library construction.

[0035] Using the 10X Genomics Chromium Controller system, single-cell suspensions were added to the Chromium microfluidic chip to generate single-cell gel bead emulsions. After gel bead-in-EMulsion (GEMs) generation and reverse transcription, full-length cDNA with barcodes was amplified by PCR using an amplification reaction system containing cDNA primers. The PCR reaction volume was 100 μL, and the thermal cycling program was: 98°C pre-denaturation for 3 min; 98°C denaturation for 15 s, 63°C annealing for 20 s, 72°C extension for 1 min, for a total of 11-12 cycles; final extension at 72°C for 1 min, and final hold at 4°C. The amplified products were purified using SPRIselect magnetic beads and used for subsequent library construction. The library quality was evaluated using an Agilent 2100, and paired-end 150 bp sequencing was performed on the Illumina NovaSeq 6000 sequencing platform. Raw sequencing data underwent sample demultiplexing, barcoding, and gene quantification using the CellRanger workflow, and the sequencing data was aligned to a mouse reference genome. CellRanger output was imported into Seurat for dimensionality reduction and subsequent analysis. To exclude low-quality cells, filtering criteria were set: cells with fewer than 2000 or more than 30000 genes, and cells with more than 6000 detected genes were removed; cells with mitochondrial genes accounting for more than 20% were also excluded. Batch effects were corrected using the Harmony method, and marker genes for each cluster were identified using the "FindAllMarkers" function. Cell types were annotated based on specific gene expression profiles.

[0036] We successfully constructed cellular atlases of uterine tissue from the pregnant and non-pregnant sides using single-cell transcriptome sequencing. Figure 6As shown, UMAP dimensionality reduction clustering clearly divided the cells into 13 major populations, including smooth muscle cells (SMCs), fibroblasts, endothelial cells, macrophages, and T cells, demonstrating the cellular heterogeneity of uterine tissue. Further analysis of the changes in the proportion of cell types in different samples... Figure 7 The study found that, compared with the control groups (dpc15.5_Control and dpc19.5_Control), the proportions of smooth muscle cells, fibroblasts, and some immune cells (such as macrophages and T cells) were significantly altered in the model groups (dpc15.5_Model and dpc19.5_Model), suggesting that mechanical stretching may regulate uterine contractions by modulating the abundance and function of these cell populations. These results validated the model's effectiveness at the single-cell level and provide an important cellular basis for further investigation into the molecular mechanisms of uterine contractions under mechanical stretching.

[0037] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a unilateral pregnant mouse uterine mechanical tension model, characterized in that, Includes the following steps: (1) By performing unilateral tubal sterilization, female mice lost the ability to conceive on one side of their uterus; (2) After the postoperative recovery period, the female mouse was allowed to conceive naturally in the uterus on the unsterilized side, and the pregnant uterus and the non-pregnant uterus were formed simultaneously in the same female mouse. (3) At a specific time point during pregnancy, the uterine tissue on the pregnant side was obtained as the tension group, and the uterine tissue on the non-pregnant side was obtained as the control group.

2. The construction method according to claim 1, characterized in that, Includes at least one of the following (a)-(d): (a) The female mice described in step (1) are selected from ICR, C57BL / 6J or Kunming mice, with an age of 7-12 weeks and a weight of 20-30 g; (b) The unilateral tubal sterilization procedure described in step (1) is selected from one of the following: thermal coagulation transection, electrocoagulation closure, suture ligation, or ultrasonic scalpel transection; (c) The unilateral tubal sterilization procedure described in step (1) is performed under inhalation anesthesia or injection anesthesia; (d) The specific time point mentioned in step (3) is the second trimester, for example, day 15 of pregnancy; or the third trimester, for example, day 19 of pregnancy.

3. The construction method according to claim 1, characterized in that, The uterine tissue on the pregnant side and the uterine tissue on the non-pregnant side mentioned in step (3) were obtained as follows: female mice were euthanized by cervical dislocation, the uterus was removed and rinsed with pre-cooled PBS, the uterus in the tension group was cut off between each embryo, the fetal mice were separated and the placenta and decidual tissue were removed, and the visible blood vessels in the uterus of the control group were removed. Both the tension group and the control group were cut transversely perpendicular to the long axis of the uterus to obtain each piece of uterine tissue with a size of 5 mm × 2 mm × 2 mm.

4. A unilateral pregnancy mouse uterine mechanical tension model, characterized in that, Produced by the construction method described in any one of claims 1-3.

5. The application of the unilateral pregnancy mouse uterine mechanical tension model of claim 4 in scientific research for non-therapeutic diagnostic purposes, wherein the scientific research includes studying the biological effects of mechanical tension on uterine tissue, screening substances that regulate uterine tension response, or verifying relevant molecular targets.

6. The application of the unilateral pregnancy mouse uterine mechanical tension model of claim 4 in a testing system or screening platform, wherein the testing system or screening platform is used to evaluate the effect of substances on the uterine mechanical tension response.

7. A method for studying the mechanical tension effect of the uterus for non-therapeutic diagnostic purposes, characterized in that, This is achieved using the unilateral pregnancy mouse uterine mechanical tension model as described in claim 4, and through the following steps: (1) The uterine tissue on the pregnant side and the uterine tissue on the non-pregnant side are subjected to at least one of the following analyses under in vitro conditions: muscle strip contraction function measurement, specific protein expression level detection, or transcriptomics analysis; (2) Based on the analysis results of step (1), the differences between the pregnant side and the non-pregnant side samples are compared to obtain experimental data on the effect of mechanical tension on uterine tissue.

8. The method for studying the mechanical tension effect of the uterus for non-therapeutic diagnostic purposes according to claim 7, characterized in that, The conditions for measuring the contractile function of the muscle strips were as follows: the obtained uterine tissues from the tension group and the control group were prepared into isolated muscle strips. After applying 2g of initial tension equilibration in a Krebs-Ranger buffer perfusion system at a constant temperature of 37℃ and purged with 95% O2 and 5% CO2, oxytocin with a final concentration of 1 nM was used for stimulation, the contraction curve was recorded, and the contractile function was quantified by the area under the curve.

9. The method for studying the mechanical tension effect of the uterus for non-therapeutic diagnostic purposes according to claim 7, characterized in that, The specific proteins include OXTR, PTGS2, and GJA1.

10. The method for studying the mechanical tension effect of the uterus for non-therapeutic diagnostic purposes according to claim 7, characterized in that, The transcriptomics analysis includes batch RNA-seq sequencing and / or single-cell RNA-seq sequencing.

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