A method for combining a human uterine smooth muscle tissue tension model and a cell tension model and application thereof

Abstract

This invention discloses a method for combining a human uterine smooth muscle tissue tension model and a cell tension model. The mechanical stimulation parameters applied in the cell tension model are determined based on the strain rate data of the tissue tension model under a specific tension load. This invention uses the strain rate value as the set value for the static stretching amplitude of human primary uterine smooth muscle cells, achieving an objective correlation between the mechanical stimulation parameters at the tissue and cell levels. Under the correlated parameters, the tissue tension model and the cell tension model are established and run to obtain tissue-level contractile function data and cell-level molecular response data, forming a cross-level data correspondence. It utilizes only human pregnant uterine smooth muscle tissue and primary cells, solving the problems of arbitrary parameter settings and disconnect between tissue and cell research in existing cell stretching models. This provides a standardized and reproducible in vitro experimental platform for studying the molecular mechanisms of mechanical tension regulating uterine smooth muscle contraction function and screening related intervention drugs.

Description

Technical Field

[0001] This invention relates to the field of experimental modeling technology, and in particular to a method for combining a human uterine smooth muscle tissue tension model and a cell tension model, and its application. Background Technology

[0002] Abnormal uterine contraction function is a core pathological link in pregnancy complications such as preterm birth and uterine atony. Mechanical tension, as a key physical signal for initiating labor, has significant clinical implications when its molecular mechanisms regulating uterine smooth muscle function are studied. However, current techniques suffer from the following limitations: 1. Cellular stretching models lack tissue physiological references. Currently widely used cellular mechanical models rely on empirically set or arbitrarily selected parameters such as stretching amplitude and frequency, lacking support from deformation data of human uterine tissue under real physiological conditions. This makes it difficult to objectively determine whether cellular-level mechanical research conclusions reflect the true in vivo situation. 2. Tissue and cell studies are disconnected. Mechanical tension signals, from the overall tissue deformation to cellular receptor activation and intracellular signal transduction, constitute a continuous, multi-level process. Under current research models, tissue and cell experiments are conducted independently, resulting in inconsistent mechanical parameters and incompatible results, making it difficult to systematically analyze the complete signaling pathways by which mechanical tension regulates uterine smooth muscle contraction. 3. Lack of parameter-correlated tissue-cell combined research methods. Currently, there is no existing experimental method that establishes an objective correlation between tissue tension models and cell tension models in terms of mechanical parameters, and allows for simultaneous tissue function testing and cellular molecular analysis under unified parameters. 4. Lack of human-derived models: Most studies use animal tissues or immortalized cell lines, which presents problems of species differences and phenotypic loss, limiting the value of research results in clinical translation.

[0003] Therefore, there is an urgent need to establish a tissue-cell combined research method based on human samples and with cellular mechanical stimulation parameters having clear tissue physiological basis. Summary of the Invention

[0004] This invention aims to overcome the shortcomings and deficiencies of existing technologies and provide a method for combining a human uterine smooth muscle tissue tension model and a cell tension model. This method has the advantages of directly deriving cell mechanical stimulation parameters from measured tissue strain rate data, allowing simultaneous analysis of tissue-level functional and cellular-level molecular responses under correlated parameters, being fully humanized, highly standardized, and highly reproducible.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for combining a human uterine smooth muscle tissue tension model and a cell tension model, wherein the mechanical stimulation parameters applied in the cell tension model are determined based on the strain rate data of the tissue tension model under a specific tension load. .

[0006] Preferably, the tissue tension model is as follows: an initial static tension of 2g is applied to isolated human uterine smooth muscle strips for 4 hours; the mechanical stimulation parameter applied in the cell tension model is 16.67% static continuous stretch for 4 hours.

[0007] Preferably, the establishment of the tissue tension model and the determination of strain rate include the following steps: (1) The isolated human uterine smooth muscle strip was suspended in the isolated perfusion system. After the strip showed regular spontaneous contraction, an initial static tension of 2g was applied to the strip and the tension was maintained for 4 hours. (2) Measure the change in length of the muscle strip before and after applying a tension of 2g, and calculate the strain rate.

[0008] More preferably, the isolated human uterine smooth muscle strip has the following specifications: 10mm×2mm×2mm, with the muscle fibers running parallel to the long axis, and is suspended in a constant temperature water bath at 37°C and pH=7.4 filled with Krebs-Henseleit buffer, with a continuous introduction of a mixed gas of 95% O2 and 5% CO2. The Krebs-Henseleit buffer system is as follows:

[0009] Preferably, the establishment of the cell tension model includes the following steps: (1) The strain rate value is used as the stretching amplitude setting value of the cell tension model; (2) Human primary uterine smooth muscle cells were seeded on a flexible culture plate. When the cell confluence reached 90%, a cell stretching device was used to apply static continuous stretching to the cells at the stretching amplitude set in step (1) for 4 hours.

[0010] More preferably, the primary uterine smooth muscle cells of the 4th or 5th generation described in step (2) are processed at a concentration of 5 × 10⁻⁶. 5 / wells are inoculated into the well plate.

[0011] Secondly, the present invention provides the application of the above-mentioned method of combining the human uterine smooth muscle tissue tension model and the cell tension model in the study of the uterine contraction mechanical regulation mechanism for non-therapeutic diagnostic purposes; the mechanism study includes: related protein expression pathways, inflammatory signaling pathways or mechanical signal transduction pathways.

[0012] Thirdly, the present invention provides a method for studying the mechanical tension-mediated contraction function of human uterine smooth muscle for non-therapeutic diagnostic purposes, using the above-mentioned combined method and including the following steps: (1) In the tissue tension model, oxytocin was added to a final concentration of 10 nM, the contraction tension curve was recorded, and the area under the contraction curve (AUC) was calculated. (2) Cellular level molecular detection: In the cell tension model, cells or their lysates are collected to detect the expression of contraction-related proteins, gene transcriptomes, or cell contraction capacity; (3) Correlation analysis: Correlation analysis is performed between the tissue-level data obtained in step (1) and the cell-level data obtained in step (2).

[0013] Preferably, the contraction-related proteins mentioned in step (2) include, but are not limited to, oxytocin receptor (OXTR), prostaglandin intraperoxidase 2 (PTGS2), and gap junction protein 43 (GJA1).

[0014] Preferably, the gene transcription profile described in step (2) is obtained by transcriptome sequencing or single-cell transcriptome sequencing.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention combines a tissue tension model with a cell tension model. The mechanical stimulation parameters applied in the cell tension model are determined based on the strain rate data measured by an objective formula under a specific tension load in the tissue tension model. This parameter setting provides a clear tissue physiology reference and a calculable mathematical source for mechanical stimulation at the cellular level, fundamentally solving the fundamental defects of existing cell stretching models, such as arbitrary parameter settings and lack of tissue source data support.

[0016] (2) This invention establishes and runs tissue tension models and cell tension models simultaneously under associated mechanical parameters, enabling a natural correspondence between tissue-level contractile function data and cell-level molecular response data. This cross-level data correlation provides the most direct and reliable in vitro experimental method to date for studying the regulatory laws of mechanical tension signal transmission from overall tissue contraction to intracellular molecular signals.

[0017] (3) This invention uses human pregnant uterine smooth muscle tissue and primary cells, completely avoiding species differences. The research results are closer to clinical practice and have higher medical translation potential.

[0018] (4) The method parameters of the present invention are clear, the steps are complete and the source is traceable, and it has good repeatability and cross-laboratory comparability.

[0019] (5) This method is compatible with a variety of techniques such as tissue contraction function detection, cell contraction experiment, key protein expression analysis, and whole transcriptome sequencing. Under the same set of mechanical intervention protocols, it can obtain multi-level experimental data from changes in overall contraction function to changes in intracellular signaling pathways. It is an integrated research method that combines mechanism analysis and drug screening. Attached Figure Description

[0020] Figure 1 This is a diagram of the isolated muscle strip tension measurement system in Example 1; Figure 2 This is a waveform diagram of spontaneous contraction of uterine smooth muscle tissue in Example 1; Figure 3 The results show the uterine muscle tissue contractility test and area under the curve comparison, as well as the length measurement results before and after stretching for the experimental group (Example 1) and the control group (Example 2); among them, ; Figure 4 This is a diagram of the Flexcell-6000TM cell tension stretching device from Example 2. Figure 5 This is a comparison of the cell contraction gel experiment results between the experimental group and the control group of uterine myocytes in Example 2; where, , ; Figure 6 The RNA-seq differentially expressed gene volcano plot of the tissue tension model from Example 1, as described in Example 3; Figure 7 This is a volcano plot of differentially expressed genes by RNA-seq in the cell tension model of Example 2, performed for Example 3. Figure 8 Bubble diagram of differentially expressed gene pathways in the tissue tension model of Example 1, performed as shown in Example 3; Figure 9 Bubble diagram of differentially expressed gene pathways in the RNA-seq model of cell tension in Example 2, as shown in Example 3; Figure 10 GO entries for the RNA-seq enrichment of differentially expressed upregulated genes in the tissue tension model of Example 1, as performed in Example 3; Figure 11 GO entries for the RNA-seq enrichment of differentially expressed downregulated genes in the tissue tension model of Example 1, as performed in Example 3; Figure 12 GO entries for the RNA-seq enrichment of differentially expressed upregulated genes in the cell tension model of Example 2, as performed in Example 3; Figure 13 GO entries for the RNA-seq enrichment of differentially expressed downregulated genes in the cell tension model of Example 2, as performed in Example 3; Figure 14 The figure shows the comparison of the expression levels of contraction-related proteins PTGS2, OXTR, and GJA1 in the experimental group and control group of uterine myometrium from Example 1, as shown in Example 4. , ; Figure 15 The figure shows the comparison of the expression levels of contraction-related proteins PTGS2, OXTR, and GJA1 between the experimental group and the control group of uterine myocytes in Example 2, as presented in Example 4. , . 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.

[0022] Example 1: Establishment of a human uterine smooth muscle tissue tension model and detection of its contractile function Sample Source and Ethics: With the approval of the hospital's medical ethics committee and the informed consent of the parturients, full-term, non-laboring women scheduled for elective cesarean section were selected. During the trimming of the lower uterine segment transverse incision edge during the operation, excess smooth muscle strips of approximately 1.0 cm × 1.0 cm × 0.5 cm were taken and immediately placed in pre-cooled DMEM culture medium at 4°C. The muscle strips were then transferred to the laboratory within 30 minutes.

[0023] Reagent preparation: The Krebs-Henseleit buffer used in the experiment must be freshly prepared. The method is as follows: Take 25 mL of NaCl stock solution (1.18 mol / L), 5 mL of KCl stock solution (0.47 mol / L), 5 mL of MgSO4 stock solution (0.12 mol / L), 25 mL of NaHCO3 stock solution (1.25 mol / L), 5 mL of Na2·EDTA·2H2O stock solution (0.03 mol / L), 5 mL of CaCl2 stock solution (0.28 mol / L), 5 mL of KH2PO4 stock solution (0.12 mol / L), and 5 mL of glucose stock solution (0.11 mol / L) in sequence, add them to a volumetric flask, and then make up to 500 mL with double-distilled water and mix thoroughly. Before use, a mixture of 95% O2 and 5% CO2 gas should be passed through for at least 30 minutes to stabilize the pH value at 7.4 and maintain a constant temperature of 37℃.

[0024] Tension model establishment: Completed using the isolated muscle strip tension measurement system (PanLab). Figure 1 The tension sensor was pre-calibrated and signal conversion settings were performed using a two-point method (0g and 5g weights). The uterine myometrial strip from step 1 was suspended and fixed in a solution filled with Krebs-Henseleit buffer (…). A water bath (preheated to 37℃, 10mL / batter, pH=7.4) was continuously purged with a mixture of 95% O2 and 5% CO2. The process was continued until the muscle strips exhibited regular, spontaneous contractions. Figure 2 The micro-tightening spiral was adjusted to apply an initial tension of 0 g (control group) or 2 g (experimental group) to the muscle strips, and the tension was maintained at a stable level for 4 hours. The above operation constitutes the process of establishing a tissue tension model.

[0025] Tissue contractility detection: After the tissue tension model has maintained stable tension for 4 hours, oxytocin with a final concentration of 10 nM is added to each water bath to induce enhanced contraction, and observation continues. The system continuously records its contraction tension curve, and finally uses the area under the curve (Integral or Area under curve) as an indicator to quantify the contraction intensity and changes of the muscle strip.

[0026] Quantitative calculation: The contraction wave of each specimen was measured regionally using Labchart 7.2 biosignal acquisition system software to assess contraction intensity. The contraction wave integral (Integral, g*s), i.e., the area under the contraction wave curve (AUC), reflects the contraction intensity of the muscle strip. The difference and rate of change of uterine contraction work (AUC) between T0 and T1 were compared (the rate of change of a certain index = value after treatment / value before treatment × 100%). The results showed that the contraction force of the muscle strip under 2g stretch was stronger after the addition of oxytocin than that under 0g stretch. Figure 3 Four hours after stretching, the muscle strips were immediately removed, the surface liquid was wiped dry, and the strips were flash-frozen in liquid nitrogen and then stored at -80°C for subsequent use in high-throughput transcriptome sequencing in Example 3 and molecular biological detection in Example 4.

[0027] Example 2: Establishment of a human uterine smooth muscle cell tension model and detection of contractile function Sampling and Processing: The tissue tension model described in Example 1 was obtained from the same batch of cesarean section samples from the same mother. With the approval of the hospital's medical ethics committee and the informed consent of the mother, excess smooth muscle tissue was harvested during the trimming of the lower uterine segment transverse incision edge during the cesarean section. This tissue was immediately placed in pre-cooled DMEM culture medium at 4°C and transported to the laboratory within 30 minutes. Blood stains were rinsed with pre-cooled PBS in a biosafety cabinet, and visible blood vessels were removed. A portion of the tissue was then used for primary cell separation.

[0028] Primary cell isolation: Primary cells were isolated from uterine myometrium using enzymatic digestion. Specifically, 0.25% trypsin-EDTA digestion solution was added, and the cells were gently digested at 37°C for 2 minutes. Immediately after digestion, a volume twice that of the trypsin digestion solution was added to neutralize the trypsin activity and terminate the digestion process. The mixture was repeatedly and gently pipetted using a sterile pipette to form a single-cell suspension. The cell suspension was then filtered through a cell sieve with a pore size of 70-100 μm to remove incompletely digested tissue fragments and cell clusters.

[0029] Primary cell culture: Primary cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell culture incubator.

[0030] Establishment of cell tension model: After primary cells were passaged to the 4th-5th generation, cells were divided into groups according to the cell tension model per well. Cells were seeded at a density of [number] cells per well in BioFlex™ six-well plates. When cell confluence reached approximately 90%, mechanical stretching was applied using the Flexcell-6000™ tension system (Flexcell International Corp, USA). Figure 4 This device uses a computer-controlled vacuum to deform cells at the bottom of a six-well plate coated with a flexible type I collagen, thereby simulating biomechanical strain conditions in vitro. By adjusting the vacuum intensity, the stretching amplitude of the cell attachment surface can be controlled, achieving different proportions of mechanical load (expressed as a percentage relative to the initial surface area).

[0031] Place the cell culture plate in the Flexcell-6000™ system and incubate at 37°C. Under the culture conditions, the cells were subjected to an amplitude of 16.67% (this amplitude was calculated by measuring the proportion of length change in tissue mechanical tension experiments, see...). Figure 3 The cells were subjected to static, continuous stretching for 4 hours; this group was designated as the experimental group. This process constitutes the establishment of the cell tension model. Cell samples obtained from this model were used for high-throughput transcriptome sequencing in Example 3 and molecular biological assays in Example 4. Control group cells were seeded in the same system under the same culture conditions, but without any vacuum stretching load (0% stretching). After the stretching treatment, viable cells or cell lysates from both groups were collected for subsequent functional experiments, omics analysis, and molecular biological assays.

[0032] Contractile function assay: Contractile function was assayed using a cell contractile assay kit (#CBA-201, Cell Biolabs Inc. San Diego, CA, USA). The assay was performed at a concentration of [missing information - likely a reagent or dosage] per 500 μL collagen mixture (prepared according to the kit). A cell-collagen suspension was prepared at a ratio of 1:1 primary cell, mixed thoroughly, and injected into 24-well plates (500 μL / well), avoiding air bubbles. The 24-well plates were placed in a 37°C cell culture incubator for 1 hour for polymerization, followed by the addition of 1 mL of complete culture medium to each well. After 24 hours of culture, the cell-collagen gel was released from the well edges, and images were taken hourly using a ChemiDoc XRS+ (Bio-Rad) camera. The gel area was measured using Image Lab software to assess cell contractility. Results showed that the cell contractility in the stretched group was significantly enhanced compared to the unstretched group. Figure 5 ).

[0033] Cell viability assay: Cell viability was assessed using the CCK-8 Cell Counting Kit-8 Assay. After stretching treatment, cells were washed three times with PBS. 1 mL of culture medium was added to each well of a six-well plate, and after 30 minutes, 100 μL of CCK-8 solution was added to each well. Cells were incubated at 37°C for 2 hours. Then, 100 μL of culture medium from each well was transferred to a 96-well plate, and absorbance at 450 nm was measured using a microplate reader to assess cell viability. Results: There was no significant difference in cell viability between the stretching group and the control group.

[0034] Example 3: Transcriptomic analysis of a human uterine smooth muscle tissue and cell tension model Within 30 seconds of recording the contractile function of the tissue tension model described in Example 1, the uterine smooth muscle strips from the experimental group (2 g stretch) and the control group (0 g stretch) were quickly removed from the ex vivo perfusion system, wiped dry, immediately transferred to centrifuge tubes, and flash-frozen in liquid nitrogen. They were then stored at -80°C for later use. After the stretching treatment of the cell tension model described in Example 2, cell samples from the experimental group (16.67% stretch) and the control group (0% stretch) were immediately collected from BioFlex™ six-well plates, transferred to centrifuge tubes, and flash-frozen in liquid nitrogen. They were then stored at -80°C for later use.

[0035] Total RNA was extracted using the TRIzol method: magnetic beads and an appropriate amount of TRIzol reagent (15596026, Invitrogen) were added to tissue samples, and the samples were thoroughly homogenized and lysed in a low-temperature tissue homogenizer. Purification was then performed using a chloroform-ethanol-centrifugation column. The purity and concentration of RNA samples were detected using a Nanodrop 2000 microspectrophotometer, and RNA integrity was assessed using a LabChip GXTouch microfluidic capillary electrophoresis system. Total RNA samples that passed quality control (RIN value ≥ 7.0) were selected. Eukaryotic mRNA was enriched using Oligo(dT) magnetic beads, fragmented with fragmentation buffer, and then double-stranded cDNA was synthesized and purified using buffer, dNTPs, DNA polymerase I, and RNase H. The purified double-stranded cDNA underwent end repair, A-tailing, ligation of sequencing adapters, and fragment screening. cDNA fragments of approximately 350 bp were recovered to construct cDNA libraries. After passing Agilent 2100 quality control, paired-end 150 bp sequencing was performed on the Illumina NovaSeq 6000 sequencing platform. Sequencing data were aligned to the human reference genome GRCh38 / hg38 using Hisat2 software (v2.1.0, default parameters), and gene expression quantification was performed using featureCounts (v2.0.8).

[0036] Differentially expressed genes were analyzed using DESeq2 (v1.48.1) software. Genes with a fold change greater than 2 and a p-value < 0.05 were considered differentially expressed. Figure 6 ), cells with a fold change greater than 1.2 times (P < 0.05) were considered to have differentially expressed genes. Figure 7 Further gene GO function and pathway enrichment analysis was performed using the DAVID online tool, with P < 0.05 used to determine the significance of enrichment in databases such as GO, KEGG, and WIKIPATHWAY. The enriched GO and pathway results showed that differentially expressed genes in tissue and cell models were significantly enriched in biological processes such as inflammation-related pathways, signal transduction, and extracellular matrix tissue processes. Figures 8-13 This demonstrates the reliability and effectiveness of the established model.

[0037] Example 4: Molecular biological detection of a human primary uterine smooth muscle cell tension model The expression levels of contraction-related proteins PTGS2 (prostaglandin intraperoxide synthase 2), OXTR (oxytocin receptor), and GJA1 (gap junction protein 43) in the tissue tension model of Example 1 and the cell tension model of Example 2 were detected by Western blotting to verify the consistency of the contractile force response of the two models to the same physiological tension stimulus at the protein level. The specific steps are as follows: Total protein (n=3) was extracted from the tissue tension model and the cell tension model using RIPA lysis buffer (R0278, Sigma) containing a mixture of protease inhibitors (P8340-1 ml, Sigma), and the 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. Using GAPDH as an internal control, the protein expression levels of PTGS2, OXTR, and GJA1 in the uterine tissue of the pregnant and non-pregnant sides were detected. The results show that ( Figure 14 , Figure 15 At the tissue and cellular levels, compared with the non-stretching group, the expression of the aforementioned contraction-related proteins in the uterine tissue of the stretching group was significantly upregulated. The antibodies used included 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. These results are consistent with the functional performance of the model, confirming that stretching stimulation can significantly upregulate the expression of contraction-related factors at the protein level, further validating the reliability and effectiveness of the established model in elucidating the molecular mechanisms of uterine contraction function.

[0038] 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 combining a human uterine smooth muscle tissue tension model and a cell tension model, characterized in that, The mechanical stimulation parameters applied in the cell tension model are determined based on the strain rate data of the tissue tension model under a certain tension load, .

2. The method of using according to claim 1, wherein, The tissue tension model was as follows: an initial static tension of 2g was applied to isolated human uterine smooth muscle strips for 4 hours; the mechanical stimulation parameter applied in the cell tension model was 16.67% static continuous stretch for 4 hours.

3. The combined method according to claim 1, characterized in that, The establishment of the tissue tension model and the determination of strain rate include the following steps: (1) The isolated human uterine smooth muscle strip was suspended in the isolated perfusion system. After the strip showed regular spontaneous contraction, an initial static tension of 2g was applied to the strip and the tension was maintained for 4 hours. (2) Measure the change in length of the muscle strip before and after applying a tension of 2g, and calculate the strain rate; The establishment of the cell tension model includes the following steps: (1) The strain rate value is used as the stretching amplitude setting value of the cell tension model; (2) Human primary uterine smooth muscle cells were seeded on a flexible culture plate. When the cell confluence reached 90%, a cell stretching device was used to apply static continuous stretching to the cells at the stretching amplitude set in step (1) for 4 hours.

4. The method of using according to claim 1, wherein, The isolated human uterine smooth muscle strips were 10mm × 2mm × 2mm in size, with the muscle fibers running parallel to the long axis. They were suspended in a constant temperature water bath at 37°C and pH 7.4 filled with Krebs-Henseleit buffer, and a mixture of 95% O2 and 5% CO2 gas was continuously introduced.

5. The method of using according to claim 1, wherein, The fourth or fifth passage of the primary uterine smooth muscle cells described in step (2) were seeded at 5 x 10 5 cells per well in well plates.

6. The application of the method of combining the human uterine smooth muscle tissue tension model and the cell tension model as described in any one of claims 1-5 in the study of the uterine contraction mechanical regulation mechanism for non-therapeutic diagnostic purposes.

7. Use according to claim 6, characterized in that, The mechanism studies include: related protein expression pathways, inflammatory signaling pathways, or mechanical signal transduction pathways.

8. A method for studying the mechanical tension-mediated contractile function of human uterine smooth muscle, not for therapeutic or diagnostic purposes, characterized in that, The combined method according to any one of claims 1-5 includes the following steps: (1) In the tissue tension model, oxytocin was added to a final concentration of 10 nM, the contraction tension curve was recorded, and the AUC was calculated; (2) Cellular level molecular detection: In the cell tension model, cells or their lysates are collected to detect the expression of contraction-related proteins, gene transcriptomes, or cell contraction capacity; (3) Correlation analysis: Correlation analysis is performed between the tissue-level data obtained in step (1) and the cell-level data obtained in step (2).

9. The method according to claim 8, characterized in that, The contraction-related proteins mentioned in step (2) include, but are not limited to, OXTR, PTGS2, and GJA1.

10. The method according to claim 8, characterized in that, The gene transcription profile described in step (2) is obtained by transcriptome sequencing or single-cell transcriptome sequencing.

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