Immunofluorescence detection method and system for dynamically detecting mouse uterus structure development based on multiple time points

Abstract

The invention provides an immunofluorescence detection method and system for dynamically detecting mouse uterus structure development based on multiple time points, and immunofluorescence staining comprises the steps of sheet unfolding, dewaxing, repairing, cell membrane punching, sealing, primary antibody treatment and secondary antibody treatment. The system comprises a sample pretreatment control module, a multi-channel confocal microscope imaging module and a data analysis module integrating fluorescence intensity calculation, Pearson co-localization analysis and a Western blot data integration algorithm. Through a multi-time-point and multi-index immunofluorescence detection method, dynamic and systematic data support is provided for mouse uterus development mechanism research, and an application basis is provided for fundamental research of reproductive biology and construction of related disease models.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to the uterine development of newborn mice at different stages, which is suitable for studying the dynamic changes of tissue structure and the reproductive physiological mechanism during uterine development. BACKGROUND

[0002] Mammalian uterine development is a key link of reproductive system maturation, which is involved in the biological processes of menstruation formation and discharge, embryo implantation and pregnancy maintenance, and endocrine regulation. The dynamic changes of its tissue structure such as epithelial cell tight junction, extracellular matrix, and nuclear lamina structure directly affect the fertility. The development of newborn and prepubertal uterus and related abnormalities are crucial for many complex problems encountered in later life. The existing technology focuses on the uterine research of adult mice or a single development stage, and lacks systematic analysis of the continuous stages from 1-20 days after birth to prepuberty and the estrus cycle specificity. Although immunofluorescence technology is widely used for protein localization detection in tissues, there is no reported method for detecting tight junction-collagen-nuclear lamina in the uterus of newborn mice, and the pretreatment conditions such as fixation time and membrane permeabilization agent concentration of samples at different development stages need to be optimized to avoid non-specific fluorescence interference. SUMMARY

[0003] (I) Technical problem

[0004] In view of the above existing technical status, the present application mainly aims to solve the following technical problems:

[0005] 1. The purpose of the present application is to provide an immunofluorescence detection method for dynamic detection of mouse uterine structure development based on multiple time points, which synchronously detects the expression and localization of tight junction proteins, extracellular matrix and nuclear lamina related proteins by continuous sampling of mouse uterine tissue at 1, 5, 10, 15, 20 days after birth and estrus period, and solves the problems of incomplete time coverage and single index in the prior art.

[0006] 2. The immunofluorescence detection method of multiple time points and multiple indexes provides dynamic and systematic data support for the study of mouse uterine development mechanism, and provides application basis for reproductive biology basic research and related disease model construction.

[0007] (II) Technical scheme

[0008] The purpose of the present application is to overcome the deficiencies of the prior art, and to provide an immunofluorescence detection method and system for dynamic detection of mouse uterine structure development based on multiple time points.

[0009] The purpose of the present application is achieved as follows: an immunofluorescence detection method for dynamic detection of mouse uterine structure development based on multiple time points, comprising the following steps:

[0010] (1) Experimental animal preparation: Select sexually mature female mice (strains include CD1) and male mice at a ratio of 1:1, confirm pregnancy status, and use mice within 24 hours after birth as the D1 group;

[0011] Collect uterine tissue continuously at D1, D5, D10, D15, and D20 after birth;

[0012] Select 6-8 week old female mice and determine the estrous cycle stage by vaginal smear as the control group;

[0013] Estrous cycle determination:

[0014] Proestrus determination: smear with nuclear epithelial cells, occasional small amount of keratinocytes;

[0015] Estrus determination: all are anuclear keratinocytes, or with a small amount of epithelial cells, and very few leukocytes;

[0016] Metestrus determination: leukocytes, keratinocytes, and nucleated epithelial cells coexist;

[0017] Diestrus determination: large amount of leukocytes, small amount of epithelial cells and mucus.

[0018] (2) Tissue processing: After the mice are sacrificed, the uterus is separated, washed with PBS, and fixed with 4% PFA or 4°C acetone for 8-24 hours; gradient ethanol dehydration (50%, 70%, 80%, 90%, and 100% ethanol, 1 hour each); paraffin embedding;

[0019] (3) Section preparation: paraffin-embedded tissues are cut into 3-5 μm continuous sections, placed in water on a preheated water bath at 34-37°C, and attached to polylysine glass slides, then dried at 37°C overnight;

[0020] (4) Immunofluorescence staining, which includes the following steps:

[0021] (4.1) Unrolling: unroll on a 55°C unrolling table for 1 hour.

[0022] (4.2) De-waxing: de-waxing with xylene twice, then treating the sections with anhydrous ethanol, different concentrations of gradient alcohol, and distilled water; specifically, placing the sections in xylene for 10 minutes twice, anhydrous ethanol for 5 minutes twice, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, 50% alcohol for 5 minutes, and distilled water for 5 minutes.

[0023] (4.3) Repair: Microwave the slice with buffer for 8-12 minutes, the repair method is different for different antibodies; Specifically, microwave the slice in citric acid repair solution or EDTA repair solution for 10 minutes, then naturally cool at room temperature, and then rinse in 1xPBS for 3 times, 5 minutes each time; The specific repair method is different for different antibodies: use pH6.0 citric acid buffer for repair when detecting nuclear membrane-base membrane structure protein Lamin A / Laminin A5; use pH8.0 EDTA buffer for repair when detecting cell junction protein Ecadherin / ZO-1.

[0024] (4.4) Cell membrane punching: Cell membrane punching is determined according to the expression of visual signal molecules on the cell membrane and / or in the cell, and the slice is placed in a 0.1% triton x-100 cell permeabilizing agent solution for permeabilization for 8-12 minutes; The cell permeabilizing agent solution is 100 μl triton x-100 + 9.9 ml 1xPBS.

[0025] (4.5) Blocking: Prepare 10% goat serum or 5% horse serum + 1% BSA blocking solution, and block at 37°C for 45 minutes-1 hour.

[0026] (4.6) Incubation of primary and secondary antibodies: Discard the blocking solution, discard the blocking solution, and incubate the antibody combination targeting cell junction and matrix proteins at 2-4°C overnight for 16 hours; The next day, recover the primary antibody, add 1xPBS, prepare 1% BSA, dilute the secondary antibody with 1% BSA at 1:200, and the secondary antibody is a fluorescent antibody; After adding the secondary antibody to the slice and incubating at 37°C for 25-30 minutes in the dark, discard the secondary antibody, add 1xPBS, and rinse; Then use 20 μg / m of 6-diamidino-2-phenylindole dihydrochloride counterstaining, place at 37°C for 8-10 minutes, then rinse with 1xPBS, and then use an anti-fluorescence quencher to mount the slide. In this step, the antibody combination primary antibody is Ecadherin antibody (1:200), ZO-1 antibody (1:100), and MUC1 antibody (1:100), and the secondary antibody is Alexa Fluor 488 / 594. In this step, 5 μg / m of propidium iodide counterstaining can also be used, and propidium iodide is used with PI, P4170, Sigma-Aldrich.

[0027] Image analysis: Laser confocal microscope image acquisition, ImageJ analysis of fluorescence intensity and positive area ratio. The analysis area is limited to the middle of the uterine horn. The total protein amount is verified by Western blot.

[0028] Further, an immunofluorescence detection method for dynamically detecting mouse uterine structure development at multiple time points, which can be applied in quantitative evaluation of uterine development stages, and establishes the following biomarker combination threshold:

[0029] In the D10 development stage, the identification criteria are as follows: the expression intensity of E-cadherin protein is between 500-800 AU, Lamin A protein presents a discontinuous distribution pattern, and the localization rate of Collagen IV in the basement membrane is not more than 15%;

[0030] And in the estrus stage, the characteristic performance is as follows: the expression intensity of E-cadherin protein reaches 2000 AU or higher, Lamin A protein forms a complete ring-shaped continuous structure, and the localization rate of Collagen IV in the basement membrane is not less than 90%.

[0031] Further, an immunofluorescence detection method for dynamically detecting mouse uterine structure development at multiple time points, the application of the method further includes:

[0032] a) Reproductive biology research: tracking the basement membrane formation dynamics of nuclear membrane-basement membrane structure protein Laminin A5, quantifying the phase correlation (r>0.8, p<0.01) between the increase of matrix metalloproteinase activity in estrus stage and Lamin A nuclear rearrangement; in the present application, the matrix metalloproteinase activity refers to the metalloproteinase activity in the endometrial matrix. Lamin A nuclear rearrangement refers to the nuclear structure change related to Lamin A. A value of r above 0.8 indicates that there is a strong positive correlation between the two variables, that is, when the matrix metalloproteinase activity increases, the nuclear rearrangement of Lamin A will also increase accordingly. p<0.01 indicates that this correlation is statistically significant and not a random phenomenon.

[0033] b) Disease model construction: construction of adenomyosis pathological characteristics of the uterus.

[0034] c) Drug evaluation: Lamin A nuclear membrane repair rate (Δ continuity index≥0.4) and MUC1 regeneration speed (48-hour positive rate increase≥30%) are used as key potency indicators of endometrial repair drugs.

[0035] Further, an immunofluorescence detection method for dynamically detecting mouse uterine structure development at multiple time points, which evaluates the following in the pathological characteristics of adenomyosis of the uterus:

[0036] Abnormal deposition of Collagen IV interstitium: the amount of Collagen IV deposition exceeds 200% of the normal value, and presents diffuse rather than localized distribution;

[0037] Basement membrane disorganization: Collagen IV deposition increased by >50% compared to normal group and diffused distribution;

[0038] Nuclear instability: Lamin A appeared to be broken or fragmented distribution;

[0039] E-cadherin polarity loss: mislocalization with cell polarity markers was determined by Pearson's colocalization coefficient <0.4.

[0040] The matrix metalloproteinase activity during estrus had a significant correlation with Lamin A nuclear rearrangement (r>0.8, p<0.01). In addition, Collagen IV deposition, cadherin diffusion, and polarity loss (Pearson's coefficient <0.4) were found in the model, indicating that these factors collectively led to abnormal changes in uterine structure, providing a precise criterion for disease characteristics.

[0041] Based on the above three, which together constitute the matrix-cell-nucleus cascade damage marker, the index of Lamin A repair rate ≥80% and E-cadherin polarity recovery synchronization after treatment response drug intervention was designed to verify the functional coupling mechanism of the three.

[0042] A mouse uterine development detection system comprises:

[0043] Sample pretreatment control module: integrated dewaxing, repair, punching, sealing, antibody incubation time and temperature control, supporting 96 samples / batch processing;

[0044] Imaging module: multi-channel confocal microscope (FITC / TRITC / DAPI synchronous acquisition); realize protein subcellular localization visualization, such as Lamin A in the annular continuous structure of nuclear membrane (estrus marker), Collagen IV basement membrane localization rate ≥90% quantitative determination, analysis area limited to the middle segment of uterine horn, avoiding bias caused by anatomical position difference.

[0045] Data analysis module: integrated fluorescence intensity calculation, Pearson's colocalization analysis and Western blot data integration algorithm, the data analysis module is built-in ResNet-50 model, the ResNet-50 model inputs image and outputs uterine development stage score. ResNet-50 model combined with Western blot data integration algorithm and Pearson's colocalization analysis (such as Ecadherin and Lamin A colocalization rate ≥0.7 as polarity maturity marker), improves the repeatability and efficiency of the results.

[0046] Further, the professional terms involved in the method are as follows: Ecaherin is E-cadherin; ZO1 is the abbreviation of zonula occludens 1, which represents tight junction protein 1; Muc1 is the abbreviation of Mucin1, which represents mucin 1; College IV is collagen 4; Laminin is laminin; Laminin A5 is laminin A5; Lamin A is a nuclear skeleton protein, PI is propidium iodide, which represents iodinated propyl iodide: a fluorescent dye.

[0047] (Three) beneficial effects

[0048] 1. Existing research focuses on adult mice or a single development stage, while the present scheme covers continuous sampling from D1 to D20 after birth and specific comparison in estrus period, filling the gap of dynamic data of uterine development from newborn to pre-sexual maturity.

[0049] 2. The present application synchronously detects tight junction protein E-cadherin, basement membrane component Collagen IV, and nuclear lamina protein Lamin A, breaking through the limitation of traditional immunofluorescence single index and establishing the spatiotemporal expression correlation of the three for the first time. Not only does it reveal the three-level regulation mechanism of "extracellular matrix-cell junction-nucleus" in uterine remodeling, but also establishes a precise disease typing standard for multi-index combination, providing a new tool for regenerative medicine and targeted therapy.

[0050] 3. Non-specific fluorescence is easily produced in newborn mouse tissues, and the fixation and membrane permeation conditions need to be optimized. The present scheme adjusts the membrane permeation agent concentration for different ages, and uses vaginal smear combined with serum E2 / P4 hormone detection for estrus determination, improving the accuracy of stage-specific sampling.

[0051] 4. The present application quantifies the phase correlation (r>0.8, p<0.01) between matrix metalloproteinase activity and LaminA nuclear rearrangement in estrus period, revealing the molecular mechanism of uterine remodeling. In adenomyosis, the detection of Collagen IV deposition increased by ≥50% and the loss of Ecadherin polarity (Pearson coefficient <0.4) and diffuse distribution provides a precise criterion for disease phenotype.

[0052] 5. The present application uses LaminA nuclear membrane repair rate (Δ continuity index ≥0.4) and MUC1 regeneration speed (48h positive rate increase ≥30%) as potency indicators for endometrial repair drugs, promoting standardization of drug efficacy evaluation, and reasoning that LaminA continuity recovery rate ≥80% after drug intervention determines effectiveness, providing a clear threshold for preclinical research.

[0053] 6, The multi-time point protein expression profile provided by the method can guide the mechanical parameter design of the bionic uterus, such as simulating the uterine contraction frequency and optimizing the nutrient liquid composition, and can further support the development of artificial uterus and embryo culture technology. BRIEF DESCRIPTION OF DRAWINGS

[0054] Fig. 1 The fluorescence signals of tight junction related proteins (Ecadherin, ZO1 and MUC1) of the new-born mice at different time points and the estrus mice.

[0055] Fig. 2 The fluorescence signals of extracellular matrix related proteins (College IV, Laminin and Laminin A5) of the new-born mice at different time points and the estrus mice.

[0056] Fig. 3 The fluorescence signals of nuclear lamina related protein Lamin A of the new-born mice at different time points and the estrus mice. DETAILED DESCRIPTION

[0057] The present application aims to provide an immunofluorescence detection method and system based on dynamic detection of mouse uterus structure development at multiple time points, which synchronously captures the spatiotemporal expression profile of three proteins, not only reveals the three-level regulation mechanism of extracellular matrix-cell junction-nucleus in uterine remodeling, but also establishes a multi-index combined disease precise typing standard, and provides a new tool for regenerative medicine and targeted therapy. The present application will be further described below in combination with examples and / or drawings.

[0058] The method comprises the following steps:

[0059] 1 Experimental animal preparation: The sexually mature CD1 mice required for the experiment, the weight of the mice is between 26-30 grams. The indoor environment is controlled at 22-24℃, the humidity is maintained at 60%-70%, the light management is implemented for 12 hours of light (7:00-19:00) and 12 hours of darkness (19:00-7:00), and sufficient water and food supply ensures the normal intake of the mice. Generally, at 16:00 every day, the mice are put into the male mouse's cage at a ratio of 1:1, and the mice generally mate at 12 o'clock at night. At 9:00-10:00 the next morning, the female mice in the cage are checked for vaginal plugs to determine pregnancy. If the female mice have vaginal plugs, they are considered to be pregnant on the first day, and they are transferred to a new cage. The gestation period of mice is generally 19-21 days, and the birth of the mice is observed at the expected delivery time. The mice are marked as D1 (1 day old), D5 (5 days old), D10 (10 days old), D15 (15 days old), and D20 (20 days old) groups 24 hours after birth. Another group of sexually mature female mice (6-8 weeks old) is selected, and the estrus period is determined by vaginal smear method, and marked as estrus group.

[0060] 2 Tissue processing: After the mice are sacrificed by cervical dislocation at each time point, the uterine tissue is aseptically isolated and washed with PBS, then immediately placed in 4% PFA or 4℃ acetone for 8-16 hours. The fixation time is optimized based on the fluorescence signal-to-noise ratio of Collagen IV being ≥5. After dehydration with gradient ethanol (50%, 70%, 80%, 90%, and 100% ethanol for 5 minutes each), the tissue is embedded in paraffin containing 5% glycerol.

[0061] 3 Sectioning: The tissue embedded in paraffin is attached to a small wooden block, and the paraffin block is trimmed into a trapezoidal shape. The tissue sections are cut to a thickness of 5μm, and the cutting process is observed at all times. When the section is observed to be at the desired position, the section is placed in water on a 37℃ slide rack, and the section is attached to a polywater-treated glass slide. After the section is completely expanded, the excess water is removed and the excess water is absorbed with filter paper. The cut section is baked at 37℃ overnight and is ready for use.

[0062] 4 Immunofluorescence staining:

[0063] (1) Unfolding: Unfold the section on a 55℃ slide rack for 1 hour.

[0064] (2) De-waxing: Place the section in cyclohexane or xylene for 10 minutes each, followed by 5 minutes of anhydrous ethanol twice, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, 50% ethanol for 5 minutes, and distilled water for 5 minutes.

[0065] (3) Repair: Microwave repair in citric acid repair solution or EDTA repair solution (pH 6.0 citric acid buffer is used for repair when detecting nuclear membrane-base membrane structure protein (Lamin A / Laminin A5); pH 8.0 EDTA buffer is used for repair when detecting cell junction protein (Ecadherin / ZO-1)) for 10 minutes, then naturally cool at room temperature, and then rinse in 1xPBS for 3 times, 5 minutes each time.

[0066] (4) Cell membrane punching (depending on whether the signal molecule is expressed on the cell membrane or in the cell): Place the section in 0.1% triton x-100 solution (100 μl triton x-100 + 9.9 ml 1xPBS) for 10 minutes.

[0067] (5) Blocking: Prepare 10% goat serum or 5% horse serum + 1% BSA, draw a wax circle around the section two millimeters away, add the blocking solution to the section, and then block at 37°C for 1 hour.

[0068] (6) Discard the blocking solution, and combine the primary antibody for targeting cell junction and matrix proteins and incubate overnight at 4°C. The antibody combination includes Ecadherin antibody (1:200), ZO-1 antibody (1:300), MUC1 antibody (1:500), and corresponding Alexa Fluor 488 / 594 secondary antibody.

[0069] (7) The next day, recover the primary antibody, and rinse in 1xPBS for 3 times, 5 minutes each time.

[0070] (8) Prepare 1% BSA, dilute the fluorescent secondary antibody (the secondary antibody is selected according to the primary antibody, and the secondary antibody is a fluorescent antibody) with 1% BSA at 1:200, add the secondary antibody to the section, and then place it at 37°C for 30 minutes. After the time is up, discard the secondary antibody, and rinse in 1xPBS for 3 times, 5 minutes each time.

[0071] (9) Counterstain with 6-diamidino-2-phenylindole dihydrochloride or propidium iodide at 37°C for 10 minutes. After the time is up, discard, and rinse in 1xPBS for 3 times, 5 minutes each time.

[0072] (10) Use an anti-fluorescence quencher or ProLong Gold anti-quenching mounting agent to mount the section, and then take pictures by laser scanning confocal microscope.

[0073] 5Image analysis: Laser confocal microscope collected images, ImageJ calculated fluorescence intensity and the proportion of positive area, Western blot verified the total protein amount. Image acquisition parameters include: Ecadherin (488 nm excitation / 525 nm emission), Lamin A (594 nm excitation / 617 nm emission), DAPI (405 nm excitation / 461 nm emission); the analysis area is limited to the middle of the uterine horn.

[0074] The system comprises:

[0075] 1Sample pretreatment control module: integrated dewaxing, repair, punching and sealing, antibody incubation time and temperature control, supporting 96 samples / batch processing; newborn mouse uterine tissue is fragile, dewaxing time is shortened to 80% of the conventional time, antibody incubation chamber is partitioned and temperature controlled, and the optimal reaction temperature of different proteins is adapted, E-cadherin 4°C vs Lamin A 25°C.

[0076] 2Imaging module: multi-channel confocal microscope (FITC / TRITC / Cy5 / DAPI synchronous acquisition); multi-channel confocal microscope can realize visualization of protein subcellular localization, such as Lamin A in the annular continuous structure of nuclear membrane (estrus stage marker), and quantitative determination of Collagen IV basement membrane localization rate ≥ 90%.

[0077] 3Data analysis module: the data analysis module is built-in ResNet-50 model, and the training set of ResNet-50 model contains more than 1000 labeled images of each development stage. After inputting the image, the uterine development stage score is output, and the output score is associated with Western blot data such as Lamin A continuity index vs protein expression. At the same time, the fluorescence intensity calculation, Pearson colocalization analysis and Western blot data integration algorithm are integrated, and the Western blot data integration algorithm and Pearson colocalization analysis such as Ecadherin and Lamin A colocalization rate ≥ 0.7 are combined to improve the result repeatability and efficiency.

[0078] Example 1: Immunofluorescence detection of dynamic changes of mouse uterine structure

[0079] 1.1 Experimental animal grouping and treatment

[0080] Select CD1 strain female mice (n=30) and male mice 1:1 to cage, confirm pregnancy by vaginal plug, and take D1, D5, D10, D15, D20 young mouse uterine tissue (n=5 for each group) after delivery;

[0081] Another 6-8 weeks old female mice (n=10) were taken as estrus group, through vaginal smear combined with ELISA detection of serum E2 / P4 to confirm estrus stage (estrus: E2 concentration 42.5±3.8 pg / ml).

[0082] 1.2 Tissue processing and section preparation.

[0083] The uterus was fixed with 4% PFA for 12 hours (verified Collagen IV signal-to-noise ratio = 5.3±0.4), and then dehydrated with gradient ethanol and embedded with 5% glycerol paraffin; the section thickness was 4 μm, and polylysine slides were used, which were dried at 37°C and stored for later use. The gradient dehydration program is as follows in Table 1:

[0084] 1.3 Immunofluorescence staining

[0085] Microwave repair after deparaffinization: the Lamin A detection group was antigen repaired with pH 6.0 citric acid buffer for 10 minutes; the Ecadherin detection group was antigen repaired with pH 8.0 EDTA buffer for 12 minutes; the antibody incubation system is as follows in Table 2 (taking E-cadherin as an example)

[0086] Antibody incubation: Ecadherin (1:200, Abeam ab76055), ZO-1 (1:300, Invitrogen 40-2200), MUC1 (1:500, SantaCruz sc-7313) 4°C incubation for 16 hours;

[0087] Blocking: 10% goat serum or 5% horse serum + 1% BSA was prepared, and a wax circle was drawn around the section two millimeters away, the blocking solution was added to the section, and then blocked at 37°C for 1 hour. Discard the blocking solution, add the primary antibody and incubate overnight at 4°C. The blocking solution screening experiment used D10 uterus samples (n=3) to test different blocking systems (Table 3):

[0088] Staining: multiple staining steps were taken in the staining stage, the first round of staining (Lamin A):

[0089] According to the pretreatment sequence of sodium citrate repair, 0.1% Triton X-100 permeabilization, and goat serum blocking, the primary antibody: Anti-Lamin A / C (Abeam ab108595, 1:500) was treated at 4°C for 16h, and the secondary antibody: Alexa Fluor 594 (1:200) was incubated in the dark for 30min.

[0090] Second round of staining (E-cadherin): avoid cross-reaction by EDTA repair at pH8.0, primary antibody: Anti-E-cadherin (Cell Signaling 3195, 1:200), secondary antibody: Alexa Fluor 488 (1:200). Alexa Fluor 594 secondary antibody (1:500, Thermo Fisher A21207) was used to incubate for 30 minutes in the dark, and DAPI was used to stain the nucleus and mount the slide.

[0091] According to the above steps, the contrast test of D10 mice and estrus mice was selected, and the test results are shown in Figs. 1-3 As shown in the test results, it is found that:

[0092] D10 group: uterine epithelial cells are arranged loosely, Ecadherin, ZO-1 and MUC1 fluorescence intensity is weak; Collagen IV is mainly distributed in the interstitial area, and the fluorescence intensity is weak; Laminin is mainly distributed in the interstitial area and the basement membrane area, and has been preliminarily formed; Laminin A5 has no fluorescence signal; Lamin A also has no fluorescence signal;

[0093] Estrus group: epithelial cells are tightly connected, Ecadherin, ZO-1 and MUC1 fluorescence intensity is significantly enhanced; Collagen IV fluorescence intensity is significantly enhanced and enriched in the interstitial area, Laminin fluorescence intensity is significantly enhanced and mainly distributed in the interstitial area and the basement membrane area; Laminin A5 fluorescence intensity is significantly enhanced and mainly enriched in the basement membrane; Lamin A nuclear membrane is continuously distributed in a ring shape.

[0094] The experimental results show that the maturation of the uterus structure of estrus mice is closely related to the strengthening of tight connection, the directional deposition of collagen and the stabilization of nuclear lamina. The quantitative evaluation of the development stage found in the test is shown in Table 4:

[0095] Table 4 Comparison of markers of D10 group and estrus group

[0096] Example 2: Evaluation of the pathological characteristics of adenomyosis

[0097] In 6-week-old BALB / c female mice, 17β-estradiol silicone rods were implanted subcutaneously, with a sustained release of 0.5 mg for 60 days. Other experimental procedures were the same as in Example 1. Pathological characteristics on the 30th day: Collagen IV deposition: the fluorescence intensity of ectopic lesion area reached 3200±450 AU, and the amount of Collagen IV deposition increased by ≥50%. The general normal endometrium was 1050±210 AU. Loss of polarity: the Pearson coefficient of E-cadherin polarity distribution decreased to 0.28±0.07, and the general normal was 0.81±0.04.

[0098] In this embodiment, Collagen IV is a protein that mainly acts on blood vessel walls and nerve endings. If the Collagen IV deposition increases by ≥50%, it means that the deposition of Collagen IV in the model has increased by more than 1.5 times. Loss of Ecadherin polarity (Pearson coefficient <0.4): cadherin is an activated cell membrane protein located between the basement membrane and the keratinized epithelium. Loss of polarity can mean that these cadherin molecules are no longer arranged in the expected order. If it is also diffusely distributed, it means that the protein does not gather in a specific location, but is dispersed throughout the structure. Therefore, Collagen IV deposition, cadherin dispersion, and loss of polarity all indicate that there are significant changes in cell structure under certain abnormal conditions, such as adenomyosis of the uterus. The activity of matrix metalloproteinase during the estrus period has a significant correlation with the nuclear rearrangement of Lamin A (r>0.8, p<0.01). In addition, it was found in the experiment that Collagen IV deposition, cadherin dispersion, and loss of polarity (Pearson coefficient <0.4) indicated that these factors together caused abnormal changes in the structure of the uterus, providing a precise criterion for disease characteristics. In this process, if the drug intervention can simultaneously reverse the damage in this embodiment and meet the following conditions: Lamin A continuity recovery ≥80%, E-cadherin and Lamin A co-localization Pearson coefficient ≥0.7, it can be considered effective and has potential clinical value.

[0099] Example 3: Quantitative study of Laminin A5 basement membrane dynamics and MMP-Lamin A cascade regulation during the estrus cycle

[0100] The experimental design is shown in the steps in Example 1. The quantitative observation and analysis of LamA5 basement membrane dynamics are as follows:

[0101] Estrus period characteristics: LamA5 is in a continuous linear distribution in the basement membrane, forming a dense network structure to support epithelial polarity; developmental stage comparison: D10 mouse LamA5 localization is fragmented, with a low co-localization rate with Collagen IV (Pearson coefficient <0.3), confirming that the basement membrane is immature; dynamic tracking: from the proestrus to the estrus period, the fluorescence intensity of LamA5 increased by 2.8 times (p<0.001), indicating the peak of basement membrane maturation (Table 5):

[0102] Table 5 Changes in the localization of LamA5 and Collagen IV during the estrus cycle

[0103] Data from ImageJ JACoP plugin, calculating LaminA connectivity index and co-localization rate.

[0104] 1. Validation of the correlation between MMP activity and LaminA nuclear rearrangement

[0105] Enzyme activity detection: MMP-9 activity in estrous endometrium was significantly increased (320% higher than diestrous, p<0.001). Nuclear rearrangement quantification: LaminA in estrous endometrium changed from diffuse distribution to continuous ring structure, with a connectivity index ≥0.85. Correlation analysis: MMP-9 activity vs LaminA continuity: r=0.87, p=0.002, MMP-9 activity vs LaminA5 co-localization rate: r=0.91, p=0.001. This indicates that basement membrane degradation is directly related to nuclear skeleton reconstruction.

[0106] This example explains that MMPs degrade basement membrane components such as Collagen IV in estrus, remove the physical barrier, and promote the reassembly of LamA5 into a continuous network; the new basement membrane activates nuclear membrane proteins such as LaminA through integrin signaling, ultimately establishing epithelial cell polarity. This process is inhibited in early development (D10) due to insufficient MMP activity. This example demonstrates:

[0107] 1. LamA5 basement membrane assembly is a marker of functional maturation of the uterus in estrus, with a continuity rate >90% defining the critical threshold for estrus.

[0108] 2. There is a strong positive correlation between increased MMP activity and LaminA nuclear rearrangement (r>0.8, p<0.01), indicating that extracellular matrix degradation directly drives nuclear skeleton reconstruction, and both maintain endometrial regenerative capacity.

[0109] This method can be extended to the study of infertility mechanisms and the development of endometrial repair drugs, such as the screening of compounds targeting the MMP-LaminA pathway. The efficacy of endometrial repair drugs can be evaluated based on LaminA nuclear membrane repair rate and MUC1 regeneration speed; LaminA nuclear membrane repair rate with a continuous index Δ value ≥0.4 as the effective standard to measure the degree of nuclear membrane integrity recovery; the proportion of MUC1 positive cells increased by ≥30% within 48 hours after intervention reflects the reconstruction of epithelial barrier function.

[0110] Finally, it should be noted that the above examples and embodiments are merely intended to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An immunofluorescence detection method based on multi-timepoint dynamic detection of mouse uterine structural development, characterized in that, Includes the following steps: (1) Preparation of experimental animals: Sexually mature female mice and male mice were selected and caged at a ratio of 1:1 to confirm the pregnancy status. Mice within 24 hours after birth were designated as group D1. Uterine tissue was collected consecutively at days 1, 5, 10, 15, and 20 after birth; female mice aged 6-8 weeks were selected and their estrous cycle stage was determined by vaginal smears as a control group. (2) Organizational processing: After euthanizing the mice, the uterus was separated, washed with PBS, and fixed with 4% PFA or acetone at 4°C for 8-24 hours. Continue dehydration with different gradients of ethanol, 1 hour for each gradient, then embed in paraffin; (3) Slice preparation: Cut the paraffin-embedded tissue into 3-5 μm continuous sections. Place the sections in preheated water on a slide stage at 34-37 ℃ and spread them. Attach the slides to polylysine glass slides and dry them at 37 ℃ overnight. (4) Immunofluorescence staining: The immunofluorescence staining procedure includes steps 4.1 spreading, 4.2 dewaxing, 4.3 repair, 4.4 cell membrane perforation, 4.5 blocking, and 4.6 incubation with primary and secondary antibodies. 4.2 Dewaxing: Dewaxing was performed twice with xylene. After dewaxing, the sections were treated in stages with anhydrous ethanol, alcohol of different concentration gradients and distilled water, with each treatment lasting 5-8 minutes. The 4.3 repair process described above: Microwave the sections with buffer for 8-12 minutes; use pH 6.0 citrate buffer to repair the nuclear membrane-basement membrane structural proteins Lamin A / Laminin A5; and use pH 8.0 EDTA buffer to repair the cell junction proteins Ecadherin / ZO-1. 4.4 Cell membrane perforation: Cell membrane perforation depends on the expression of signaling molecules on the cell membrane and their expression within the cell. The slides are permeated in 0.1% Triton X-100 cell permeabilizer solution for 8-12 minutes. The 4.5 blocking step: Prepare a blocking solution of 10% goat serum or 5% horse serum + 1% BSA and block at 37°C for 45 minutes to 1 hour; 4.6 Primary and Secondary Antibody Incubation: Discard the blocking solution. Block the primary antibody combination targeting cell junctions and matrix proteins at 2-4°C overnight for 16 hours. The next day, recover the primary antibody, wash with 1× PBS, prepare 1% BSA, and dilute the secondary antibody (fluorescent antibody) with 1% BSA at a ratio of 1:

200. After adding the secondary antibody to the slide, incubate at 37°C in the dark for 25-30 minutes, then discard the secondary antibody and wash with 1× PBS. Then, reverse stain with 20 μg / m 6-diamidino-2-phenylindole dihydrochloride, incubate at 37°C for 8-10 minutes, wash with 1× PBS, and mount with an anti-fluorescence quencher. (5) Image analysis: Images were acquired using a laser confocal microscope, and fluorescence intensity and the percentage of positive areas were calculated using ImageJ. The analysis area was limited to the middle segment of the uterine horn, and the total protein content was verified using Western blot.

2. The immunofluorescence detection method based on multi-timepoint dynamic detection of mouse uterine structure development according to claim 1, characterized in that: The determination of the estrous cycle stage is as follows: Proestrus assessment: Smear shows nucleated epithelial cells, with a few keratinized cells occasionally; Estrus determination: All cells are anucleate keratinized cells, or occasionally a small number of epithelial cells, with very few white blood cells; Determining the post-estrus phase: coexistence of leukocytes, keratinocytes, and nucleated epithelial cells; Determination of the interestrus period: a large number of white blood cells, a small number of epithelial cells and mucus.

3. The immunofluorescence detection method for mouse uterine structure development based on multi-timepoint dynamic detection as described in claim 1, characterized in that: The antibody combination consists of Ecadherin antibody 1:200, ZO-1 antibody 1:100, and MUC1 antibody 1:100 as the primary antibody, and Alexa Fluor 488 / 594 as the secondary antibody.

4. The immunofluorescence detection method based on multi-timepoint dynamic detection of mouse uterine structure development according to claim 1, characterized in that: The 6-diamidino-2-phenylindole dihydrochloride can be used in combination with DAPI, D9542, and Sigma-Aldrich, or it can be used with 5 μg / m of propidium iodide for reverse staining. The propidium iodide can also be used with PI, P4170, and Sigma-Aldrich.

5. The immunofluorescence detection method for mouse uterine structure development based on multi-timepoint dynamic detection according to claim 1, characterized in that: The method described above can establish thresholds for the following combinations of biomarkers during uterine development: During the D10 developmental stage, the identification criteria are: E-cadherin protein expression intensity between 500-800 AU, LaminA protein exhibiting a discontinuous distribution pattern, and Collagen IV localization rate in the basement membrane not exceeding 15%; During the estrus period, its characteristics are as follows: E-cadherin protein expression intensity reaches 2000 AU or higher, Lamin A protein forms a complete circular continuous structure, and Collagen IV localization rate in the basement membrane is not less than 90%.

6. The immunofluorescence detection method for mouse uterine structure development based on multi-timepoint dynamic detection according to claim 1, characterized in that: The method described can be used in reproductive biology research to track the dynamics of basement membrane formation of the nuclear membrane-basement membrane structural protein Laminin A5, and to quantify the temporal correlation between increased matrix metalloproteinase (MMP) activity during estrus and Lamin A nuclear rearrangement: r > 0.8, p < 0.

01. A value of r above 0.8 indicates a strong positive correlation between the two variables, meaning that when MMP activity increases, Lamin A nuclear rearrangement also increases accordingly.

7. The immunofluorescence detection method based on multi-timepoint dynamic detection of mouse uterine structure development according to claim 1, characterized in that: The method can be used to construct the pathological features of adenomyosis, which are as follows: Collagen IV interstitial anomalous deposition: Collagen IV deposition exceeds the normal value by 200%, and is diffuse rather than localized; Basement membrane disintegration: Collagen IV deposition was ≥50% higher than in the normal group and was diffusely distributed; Loss of nuclear stability: Lamin A exhibits fractures or fragmentation. E-cadherin polarity loss: its misalignment with cell polarity markers was determined by a Pearson colocalization coefficient < 0.

4.

8. The immunofluorescence detection method for mouse uterine structure development based on multi-timepoint dynamic detection according to claim 1, characterized in that: The method described above can be used to evaluate the efficacy of endometrial repair drugs based on the Lamin A nuclear membrane repair rate and the MUC1 regeneration rate. The Lamin A nuclear membrane repair rate is measured by a continuity index Δ value ≥ 0.4 as the effective standard to measure the degree of restoration of nuclear membrane integrity. An increase of ≥ 30% in the proportion of MUC1 positive cells within 48 hours after intervention reflects the reconstruction of epithelial barrier function.

9. A mouse uterine development detection system, characterized in that: The system includes: a sample preprocessing control module, a multi-channel confocal microscope imaging module, and a data analysis module integrating fluorescence intensity calculation, Pearson colocalization analysis, and Western blot data integration algorithms.

10. A mouse uterine development detection system according to claim 9, characterized in that: The data analysis module incorporates a ResNet-50 model, which outputs a uterine development stage score after inputting an image. The ResNet-50 model, combined with Western blot data integration algorithm and Pearson colocalization analysis, improves the repeatability and efficiency of the results.

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