CN113804661A - Kit and method for evaluating endometrial receptivity - Google Patents
Kit and method for evaluating endometrial receptivity Download PDFInfo
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- G—PHYSICS
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention relates to a kit and a method for evaluating endometrial receptivity, wherein the kit comprises antigen retrieval liquid, a non-specific protein blocking agent, a primary antibody, a secondary antibody, antibody diluent, a TSA dye, a buffer solution, DAPI working solution, a sealing tablet, a fixing liquid, a dewaxing agent, a hydrating agent and sterilized deionized water. The invention aims at the marker staining of the uNK cell subset, marks out the specificity of the uNK subset, can more accurately and deeply analyze the state change of the NK subset which plays different functions on endometrial tissues, and can enrich the information obtained from a single tissue slice. In addition, the invention adopts a tyramide signal amplification technology, which allows the same sample to be subjected to multi-label complex staining by using different primary antibodies from the same species, and simultaneously, the extremely high sensitivity of the technology can detect trace target antigens, so that the dosage of the antibody can be obviously reduced, and the cost is saved.
Description
Technical Field
The invention relates to the field of medical and biological detection, in particular to a kit and a method for evaluating endometrial receptivity.
Background
The uterus is the site of pregnancy and it is necessary to ensure that there is no immunological rejection of the embryo, the half of the allograft, which relies on an immunologically tolerated environment maintained primarily by endometrial immune cells. Endometrial immune cells include natural killer (uNK) cells, T cells, macrophages, dendritic cells, and the like. Among them, the uNK cells are a group of immune cells with the highest number in the endometrium, especially in the middle secretion phase of the endometrium, the number of the uNK cells is increased sharply, and the maternal-fetal interface in the early pregnancy period accounts for more 70% of the total number of the lymphocytes. Meanwhile, the uNK cell can express receptors such as KIR and the like, can be combined with non-classical MHC-I molecules on the surface of a villous trophoblast cell, recognizes embryos and provides favorable conditions for embryo implantation, such as reduction of killing toxicity and reduction of apoptosis of trophoblast cells. The temporal and spatial distribution of the uNK cells means that they have a major influence on endometrial receptivity, i.e. the ability of the uterus to receive embryos.
In addition, the uNK cells play an important role in the growth and development of embryos and the birth weight of infants. The uNK cells can secrete molecules such as vascular endothelial growth factors, pleiotropic growth factors, bone glycine and the like, wherein the vascular endothelial growth factors are beneficial to angiogenesis of embryo planting sites, promote recasting of spiral artery blood vessels and are beneficial to invasion of embryo trophoblast cells; pleiotropic growth factors are involved in normal or pathological processes of angiogenesis, neurite outgrowth during brain development and regeneration of bone and cartilage; bone glycine plays an important role in the development of the heart and in regulating the thickness of collagen fibers in the skin and eyes.
At present, the relation between the uNK cells and the pathogenesis of certain pregnancy-related diseases, such as abortion, pregnancy hypertension, intrauterine fetal development retardation and the like, is not completely clarified, the action mechanism of the uNK cells is deeply explored, people can know the occurrence process of the pregnancy-related diseases, and a foundation is laid for effectively avoiding the risk of poor pregnancy and improving the success rate of pregnancy in the assisted reproduction and natural pregnancy process.
The diversity of NK cell functions arises from the fact that NK cells themselves are a heterogeneous population of cells composed of different subpopulations that differ in their biological function, surface receptor type and abundance. According to the difference of biological functions, NK cells can be divided into three subgroups of tolerance NK (surface receptor is mainly inhibitory receptor), toxicity NK (surface receptor is mainly activating receptor) and regulation NK (surface receptor is mainly activating receptor and target cell is highly expressed inflammatory factor). Phenotypically, the resistant NK is predominantly CD56brightCD16-、CD27-CD11b-NK cells, toxic NK is mainly CD56dimCD16+、CD27-CD11b+NK cells, regulated NK is mainly CD56brightCD16-、CD27+CD11b+/-NK cells. The various sub-populations of the uNK can interact with corresponding ligands on the surface of trophoblast cells or other intimal immune cells, etc., through their specific receptors, enabling the different sub-populations to exhibit different functions. For example CD56brightCD16-uNK cells for promoting embryo movement to uterusIntimal migration and endometrial angiogenesis, suppression of helper T cell 17(Th 17) activity, promotion of embryonic development, and the like; and CD56dimCD16+、CD27-CD11b+uNK cells, which secrete mainly toxic particles, act as cell killing, which can lead to pregnancy failure when the killing of trophoblast cells is too strong. Therefore, when analyzing the distribution and state of uNK on endometrium, we need to consider the heterogeneity of uNK, so as to obtain more comprehensive information of uNK, and provide more detailed and accurate reference information for understanding the action mechanism of uNK cells.
Currently, the common means of clinical assessment of uNK is mainly to label CD56 using the routine immunohistochemical staining (IHC) method+And (4) positive cells, and collecting images by using a microscope and carrying out quantitative analysis on the images. The method is difficult to carry out multi-target marking due to technical limitation, only one cell is marked by using a single marker generally, and target cells can be evaluated only in form and total population quantity, so that the obtained information is relatively general. In order to comprehensively monitor the state of the uNK cell subset of the patient, a plurality of target molecules need to be considered, so that it is important to develop a technical scheme capable of simultaneously labeling a plurality of target molecules on endometrial tissue.
Disclosure of Invention
The invention aims to provide a kit and a method for evaluating endometrial receptivity.
One technical solution for solving the above technical problems of the present invention is as follows: a kit for evaluating endometrial receptivity comprises antigen retrieval liquid, a non-specific protein blocking agent, a primary antibody, a secondary antibody, antibody diluent, a TSA dye, a buffer solution, DAPI working solution, a sealing tablet, a stabilizing liquid, a dewaxing agent, a hydrating agent and sterilized deionized water.
Further, the antigen retrieval solution comprises Tris/EDTA; the non-specific protein blocking agent comprises bovine serum albumin; the primary antibody comprises an antibody for resisting human natural killer cell surface molecules CD56, an antibody for resisting human leukocyte surface molecules CD45, an antibody for resisting human natural killer cell subtype surface molecules CD27, an antibody for resisting human natural killer cell subtype surface molecules CD11B, an antibody for resisting human natural killer cell subtype surface molecules CD16 and an antibody for resisting human natural killer cell subtype surface molecules CD49 alpha; the secondary antibody comprises an IgG antibody marked by horseradish peroxidase; the antibody diluent comprises an IHC antibody diluent; the TSA dye comprises a multi-target immunofluorescence staining kit, wherein the multi-target immunofluorescence staining kit comprises a fluorescent dye PPD480, a fluorescent dye PPD520, a fluorescent dye PPD570, a fluorescent dye PPD650 and a signal amplification solution; the buffer comprises a 1 XTSST buffer; the DAPI working solution comprises DAPI powder dissolved in deionized water; the mounting agent comprises a water-soluble anti-fluorescence quenching mounting agent; the strengthening liquid comprises 10% (volume fraction) of neutral formalin; the dewaxing agent comprises xylene; the hydrating agent comprises ethanol with gradient concentration of 100%, 95% and 75% (volume fraction).
Another technical solution of the present invention for solving the above technical problems is as follows: a method of assessing endometrial receptivity comprising the steps of:
(1) preparation of reagents: configuring each reagent in the kit for evaluating the endometrial receptivity;
(2) dewaxing and hydrating: putting the glass slide with the specimen into three parts of dimethylbenzene in sequence, and soaking for 5 minutes respectively to finish dewaxing; then sequentially placing the mixture into two portions of 100% ethanol, soaking for 5 minutes respectively, then placing the mixture into 95% ethanol, soaking for 3 minutes, finally placing the mixture into 75% ethanol, soaking for 2 minutes, washing for 3 times by using sterilized deionized water, and completing hydration after 1 minute of each time;
(3) reinforcing the sample: putting the slide obtained in the step (2) into 10% neutral formalin for soaking for 10 minutes, washing for 3 times by using sterilized deionized water, and finishing reinforcement after 1 minute of each time;
(4) first antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling with high fire in a microwave oven (about 1min, measured as standard), putting the glass slide to be repaired with low fire for 10 min, taking out and naturally cooling to room temperature;
(5) serum blocking: washing the repaired slide for 1 time by using sterilized deionized water, washing the repaired slide for 1 time by using a 1X TBST buffer solution, throwing off excessive water, drawing a tissue by using a grouping pen ring, dropwise adding a non-specific protein blocking agent, and carrying out oscillation incubation for 10 minutes at room temperature for moisture retention (the room temperature is 15-25 ℃, the moisture retention is that the proper humidity is kept, and the conditions are conventionally understood and the same below);
(6) primary antibody incubation: discarding confining liquid, dropwise adding primary antibody working solution prepared by diluting one of the primary antibodies with antibody diluent, incubating at room temperature under humid conditions and shaking for 30min (specifically adjusted according to antibody specificity), and washing with 1 XTSST buffer solution for 3 times, each time for 2 min;
(7) and (3) secondary antibody incubation: removing the washing solution, adding the secondary antibody working solution dropwise, incubating for 10 min with shaking at room temperature under moisture retention, and washing for 2 min each time for 3 times with 1X TBST buffer solution;
(8) TSA incubation staining: removing washing liquor, dropwise adding a fluorescent dye working solution corresponding to an antibody of the primary antibody in the TSA dye, incubating for 10 minutes under a moisturizing condition at room temperature and shaking, and washing for 2 minutes by using a 1X TBST buffer solution;
(9) and (3) second antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling with high fire in a microwave oven (about 1min, measured as standard), placing the repairing glass slide for incubation, waiting for 10 min with low fire, taking out and naturally cooling to room temperature;
(10) repeating the steps (5) to (9) to perform the next round of antibody staining until all staining of one antibody group in the first antibody is completed;
(11) sealing: removing washing liquid, dripping DAPI working solution, incubating for 10 min at room temperature under moisture, washing for 2 min with 1XTBST buffer solution, dripping water-soluble fluorescence quenching blocking tablet, sealing, and fixing cover glass with colorless nail polish;
(12) processing and analyzing the multi-label dyeing result: and acquiring a spectral image by using an imaging microscope, identifying and analyzing the image by using case picture analysis software, and realizing quantitative analysis of the endometrial natural killer cell subpopulation on the same tissue slice.
Further, the step (1) of reagent preparation comprises:
a) and (3) sterilizing with deionized water: preparing 1800mL of deionized water, subpackaging into 6 bottles, wherein the liquid filling amount is less than 2/3, 300 mL/bottle, marking the sterilization date, and sterilizing for 30 minutes under the conditions of 0.15Mpa and 121 ℃;
b) xylene: analytically pure, and using the stock solution;
c) gradient concentration 100%, 95%, 75% ethanol: the 100% ethanol is directly used by 100mL of absolute ethanol of stock solution; the 95% ethanol is prepared from 5mL of sterilized deionized water and 95mL of absolute ethanol; the 75% ethanol is prepared from 25mL of sterilized deionized water and 75mL of absolute ethanol;
d) 10% neutral formalin: the stock solution is used, 100 mL;
e) antigen retrieval solution: dissolving 1 bag of 1.58g Tris/EDTA in 1000mL ultrapure water, adding 0.5mL tween-20, monitoring the pH value of the solution by using pH test paper, and adjusting the pH value to 9.0 (usually by using hydrochloric acid) to obtain an antigen retrieval solution;
f)1 × TBST buffer: mixing 50mL of 20-time mother liquor with 950mL of sterilized deionized water to prepare 1000mL of 1X TBST buffer solution, and bottling for later use;
g) a first antibody: respectively taking an antibody resisting human natural killer cell surface molecule CD56, an antibody resisting human leukocyte surface molecule CD45, an antibody resisting human natural killer cell subtype surface molecule CD27, an antibody resisting human natural killer cell subtype surface molecule CD11b, an antibody resisting human natural killer cell subtype surface molecule CD16 and an antibody resisting human natural killer cell subtype surface molecule CD49 alpha, and diluting the antibodies to corresponding multiples by using an antibody diluent according to an antibody specification to obtain an anti-working solution;
h) secondary antibody: selecting a secondary antigen solution matched with the TSA dye to be directly used as a secondary antibody working solution;
i) TSA dye: the working solution of the fluorescent dye is prepared by diluting mother solution of various fluorescent dyes by 100 times in a signal amplification solution;
j) DAPI working solution: and adding DAPI powder into deionized water to dissolve according to the concentration of 20mg/mL to prepare a storage solution, storing at 4 ℃ in a dark place, and diluting a working solution in deionized water by 100 times by using the storage solution to prepare the working solution.
Further, the addition amount of the non-specific protein blocker in the step (5) is 150uL, the addition amount of the primary antibody working solution in the step (6) is 150uL, the addition amount of the secondary antibody working solution in the step (7) is 150uL, and the addition amount of the fluorescent dye working solution in the step (8) is 150 uL.
Further, two slides with specimens are taken and subjected to steps (2) to (11), respectively, primary antibody incubations of the two slides are carried out using different antibody sets, and the obtained staining results are subjected to the treatment and analysis of step (12) together.
Furthermore, the antibody group corresponding to the primary antibody on one slide is an antibody against human natural killer cell subtype surface molecule CD49 alpha, an antibody against human natural killer cell subtype surface molecule CD16, an antibody against human natural killer cell surface molecule CD56 and an antibody against human leukocyte surface molecule CD45, and the antibody group corresponding to the primary antibody on the other slide is an antibody against human natural killer cell subtype surface molecule CD27, an antibody against human natural killer cell subtype surface molecule CD11B, an antibody against human natural killer cell surface molecule CD56 and an antibody against human leukocyte surface molecule CD 45.
Furthermore, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD49 α was 2000 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD16 was 100 times, the dilution factor of the working solution of the antibody against human natural killer cell surface molecule CD56 was 100 times, the dilution factor of the working solution of the antibody against human leukocyte surface molecule CD45 was 300 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD27 was 3000 times, and the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD11B was 500 times.
Further, an antibody against human natural killer cell subtype surface molecule CD49 α corresponds to fluorochrome 570, an antibody against human natural killer cell subtype surface molecule CD16 corresponds to fluorochrome 650, an antibody against human natural killer cell surface molecule CD56 corresponds to fluorochrome 520, an antibody against human leukocyte surface molecule CD45 corresponds to fluorochrome 480, an antibody against human natural killer cell subtype surface molecule CD27 corresponds to fluorochrome 570, and an antibody against human natural killer cell subtype surface molecule CD11B corresponds to fluorochrome 650.
The invention has the beneficial effects that: the technical scheme provided by the invention breaks through the limitation of insufficient dye types in multi-color dyeing of the traditional immunohistochemistry, can realize multiple fluorescent immunohistochemical dyeing on one tissue section, and can simultaneously mark multiple target molecules, thereby evaluating the endometrial receptivity by utilizing multiple indexes. The invention aims at the marker staining of the uNK cell subset, marks out the specificity of the uNK subset, can more accurately and deeply analyze the state change of the NK subset which plays different functions on endometrial tissues, and can enrich the information obtained from a single tissue slice. In addition, the invention adopts a tyramide signal amplification technology, which allows the same sample to be subjected to multi-label complex staining by using different primary antibodies from the same species, and simultaneously, the extremely high sensitivity of the technology can detect trace target antigens, so that the dosage of the antibody can be obviously reduced, and the cost is saved.
Multiple fluorescence immunohistochemistry (mhhc) detection (fluorescent dye labeling) can solve the problem of multi-target labeling. However, how to reasonably select the target point will significantly affect the difficulty and stability of subsequent staining and data analysis. In general, the representative phenotype of NK cells is CD3-CD56+NK can be classified as CD3 according to the expression intensity of CD56 in combination with CD16-CD56brightCD16-，CD3-CD56brightCD16+，CD3-CD56dimCD16-，CD3-CD56dimCD16+Four subgroups; CD3-CD56+The combination of CD11b and CD27 can mark NK cells with different developmental stages and different functions, namely CD3-CD56+CD11b-CD27-、CD3-CD56+CD11b+CD27-、CD3-CD56+CD11b+CD27+、CD3-CD56+CD11b-CD27+. In order to eliminate the problem of nonspecific staining of endometrial glands, the invention adds a labeled CD45 molecule, and in addition, the invention verifies that the phenotype is CD45 through flow cytometry+CD3-CD56+Innovatively reduced to CD45+CD56+。CD45+CD56+CD49a+The phenotype can be used as a supplementIndicating CD3-CD56brightCD16-、CD3-CD56+CD11b-CD27-And CD3-CD56+CD11b-CD27+Phenotype. In order to reduce the difficulty of constructing a staining system, the marker molecules to be incorporated are divided into two groups of staining systems, wherein the two groups of staining systems are combined into one group of CD45, CD49a, CD56 and CD16 and the other group of staining systems is combined into two groups of staining systems, namely CD45, CD56, CD27 and CD11b, so that the optimization of experimental conditions is realized, the stability of the staining systems is ensured, and the characteristics of most NK cells are also ensured to be covered.
In addition to the identification of marker molecules, another difficulty is process flow optimization. mIHC is a technique of Tyramine Signal Amplification (TSA) and realizes multicolor marking by multiple rounds of single staining. Compared with an IHC method, the mIHC process is more complicated, and the following key quality control points are screened out through repeated searching:
1. screening for antibodies suitable for mhhc: a single dye is selected to carry out marker staining on CD45, CD49a, CD56, CD16, CD27 and CD11b antibodies with different clone numbers respectively, and compared with a DAB staining image of conventional IHC, the effectiveness of the antibodies on endometrial tissue is confirmed.
Optimization of antibody and dye matching: firstly, because different dyes have different excitation wavelengths, emission wavelengths and signal intensities and different titers and specificities of different antibodies, the optimal matching of the antibodies and the dyes is completed according to the principle that the high titer corresponds to the low-intensity signal dyes and the compensation among the dyes is the least. The invention finally selects and uses PPD480 matched with CD45, PPD520 matched with CD56, PPD570 matched with CD49 alpha/CD 27 and PPD650 matched with CD16/CD 11B. Meanwhile, since the TSA method is more sensitive than the conventional immunohistochemical method, optimization of the dilution concentration of the antibody is required. At present, commercial antibody specifications generally recommend the optimal concentration for conventional immunohistochemical IHC, and in the prior antibody condition verification experiment, because the TSA method has a signal amplification effect, the method generally takes the recommended concentration of IHC as a starting point to perform gradient dilution on the antibody, so as to find out the concentration which can meet the requirements of images with high signal-to-noise ratio and is also optimal in specificity. Therefore, not only is the system optimized, but also the cost of the antibody in the follow-up formal experiment is reduced.
2. Antibody labeling order and antigen retrieval conditions were explored: the two combination schemes of the staining system need to label 4 antibodies, so that the antigen is necessarily repaired for many times and the design before and after the staining sequence is involved. Before the final dyeing condition is confirmed, the method searches for the antigen repairing condition; factors influencing the repair conditions mainly comprise the pH value of the repair solution and the selection of repair time, and different antibodies also show sensitivity to the times of antigen repair. In practical operation, the invention firstly uses the repair liquid with different pH values to repair the antigen, compares the dyeing results of the same antibody, and tries multiple times of repair on the basis of the comparison results to observe the result phenotypes of different antibodies. After obtaining the above two key results, the present invention continues to fine-tune the repair time and design the position of different antibodies in the TSA multi-round staining. For example: the method comprises the steps of placing the antibody which is easily affected by multi-round repair on the first round of dyeing, placing the antibody which is not affected by multi-round repair on the last round of dyeing, and finally obtaining the optimal experimental condition, so that the antibody performance is consistent with that of the conventional IHC and is not interfered by other antibody signals. Meanwhile, a single-channel image obtained after splitting the multispectral image is compared with an antibody single-staining test result, the expression position and the expression pattern of the antibody are mainly observed, and whether the experimental conditions are optimal or not is verified.
Drawings
FIG. 1 and FIG. 2 are combined to form a digital pathological image, in which FIG. 1 is the image collected by the panoramic pathological imaging analysis system, FIG. 2 is the result obtained by the digital analysis of the panoramic pathological imaging analysis system, in which (i) the number of the pattern cells is CD16+CD45+CD56dim uNK, model # cell CD16-CD45+CD56brightuNK and No. C cell of CD49a+CD45+CD56+The cell type II is a single weak positive cell of CD56, the cell type III is a single strong positive cell of CD56, the cell type III is a single positive cell of CD16, the cell type III is a single positive cell of CD49a, and the cell type III is a single positive cell of CD 45;
FIGS. 3 and 4 are two digital pathological diagrams of the combination of the present invention, wherein FIG. 3 is a panoramic pathological diagramThe image analysis system collects the picture, fig. 4 is the result of the digital analysis of the panoramic pathology image analysis system, wherein, the number pattern cell is CD11b+CD27-CD45+CD56+uNK, model # cells are CD11b-CD27+CD45+CD56+uNK and No. C cell of CD11b-CD27-CD45+CD56+uNK, model cell No. 4 is CD56 single positive cell, model cell No. 4 is CD27 single positive cell, model cell No. sixty percent is CD11b single positive cell, model cell No. seventy percent is CD45 single positive cell.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1
The invention relates to a kit for evaluating endometrial receptivity, which comprises antigen repairing liquid, a non-specific protein blocking agent, a primary antibody, a secondary antibody, antibody diluent, a TSA dye, a buffer solution, a DAPI working solution, a sealing tablet, a reinforcing liquid, a dewaxing agent, a hydrating agent and sterilized deionized water.
Specifically, the antigen retrieval solution comprises Tris/EDTA; the non-specific protein blocking agent comprises bovine serum albumin; the primary antibody comprises an antibody for resisting human natural killer cell surface molecules CD56, an antibody for resisting human leukocyte surface molecules CD45, an antibody for resisting human natural killer cell subtype surface molecules CD27, an antibody for resisting human natural killer cell subtype surface molecules CD11B, an antibody for resisting human natural killer cell subtype surface molecules CD16 and an antibody for resisting human natural killer cell subtype surface molecules CD49 alpha; the secondary antibody comprises an IgG antibody marked by horseradish peroxidase; the antibody diluent comprises an IHC antibody diluent; the TSA dye comprises a multi-target immunofluorescence staining kit, wherein the multi-target immunofluorescence staining kit comprises a fluorescent dye PPD480, a fluorescent dye PPD520, a fluorescent dye PPD570, a fluorescent dye PPD650 and a signal amplification solution; the buffer comprises a 1 XTSST buffer; the DAPI working solution comprises DAPI powder dissolved in deionized water; the mounting agent comprises a water-soluble anti-fluorescence quenching mounting agent; the strengthening liquid comprises 10% (volume fraction) of neutral formalin; the dewaxing agent comprises xylene; the hydrating agent comprises ethanol with gradient concentration of 100%, 95% and 75% (volume fraction).
Example 2
The invention relates to a method for evaluating endometrial receptivity, which comprises the following steps:
(1) preparation of reagents: configuring each reagent in the kit for assessing endometrial receptivity as described in example 2;
(2) dewaxing and hydrating: putting the glass slide with the specimen into three parts of dimethylbenzene in sequence, and soaking for 5 minutes respectively to finish dewaxing; then sequentially placing the mixture into two portions of 100% ethanol, soaking for 5 minutes respectively, then placing the mixture into 95% ethanol, soaking for 3 minutes, finally placing the mixture into 75% ethanol, soaking for 2 minutes, washing for 3 times by using sterilized deionized water, and completing hydration after 1 minute of each time;
(3) reinforcing the sample: putting the slide obtained in the step (2) into 10% neutral formalin for soaking for 10 minutes, washing for 3 times by using sterilized deionized water, and finishing reinforcement after 1 minute of each time;
(4) first antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling with high fire in a microwave oven (about 1min, measured as standard), putting the glass slide to be repaired with low fire for 10 min, taking out and naturally cooling to room temperature;
(5) serum blocking: washing the repaired slide for 1 time by using sterilized deionized water, washing the repaired slide for 1 time by using a 1X TBST buffer solution, throwing off excessive water, drawing a tissue by using a grouping pen ring, dropwise adding a non-specific protein blocking agent, and carrying out oscillation incubation for 10 minutes at room temperature for moisture retention (the room temperature is 15-25 ℃, the moisture retention is that the proper humidity is kept, and the conditions are conventionally understood and the same below);
(6) primary antibody incubation: discarding confining liquid, dropwise adding primary antibody working solution prepared by diluting one of the primary antibodies with antibody diluent, incubating at room temperature under humid conditions and shaking for 30min (specifically adjusted according to antibody specificity), and washing with 1 XTSST buffer solution for 3 times, each time for 2 min;
(7) and (3) secondary antibody incubation: removing the washing solution, adding the secondary antibody working solution dropwise, incubating for 10 min with shaking at room temperature under moisture retention, and washing for 2 min each time for 3 times with 1X TBST buffer solution;
(8) TSA incubation staining: removing washing liquor, dropwise adding a fluorescent dye working solution corresponding to an antibody of the primary antibody in the TSA dye, incubating for 10 minutes under a moisturizing condition at room temperature and shaking, and washing for 2 minutes by using a 1X TBST buffer solution;
(9) and (3) second antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling with high fire in a microwave oven (about 1min, measured as standard), placing the repairing glass slide for incubation, waiting for 10 min with low fire, taking out and naturally cooling to room temperature;
(10) repeating the steps (5) to (9) to perform the next round of antibody staining until all staining of one antibody group in the first antibody is completed;
(11) sealing: removing washing liquid, dripping DAPI working solution, incubating for 10 min at room temperature under moisture, washing for 2 min with 1XTBST buffer solution, dripping water-soluble fluorescence quenching blocking tablet, sealing, and fixing cover glass with colorless nail polish;
(12) processing and analyzing the multi-label dyeing result: and acquiring a spectral image by using an imaging microscope, identifying and analyzing the image by using case picture analysis software, and realizing quantitative analysis of the endometrial natural killer cell subpopulation on the same tissue slice.
Specifically, the preparation method of the slide with the specimen in the step (2) comprises the following steps:
(01) taking a sample: scraping endometrium tissue with endometrium curet under aseptic condition for LH 7-9 days (luteal metaphase), and placing into aseptic bottle containing PBS;
(02) tissue fixation: removing blood clots around the tissue as clean as possible, and then placing the specimen in a neutral fixing liquid bottle of 10% formalin for overnight fixation;
(03) tissue dehydration: placing the fully fixed specimen on a tissue embedding box padded with lens wiping paper, and then placing the tissue embedding box into a dehydrator for dehydration;
(04) tissue embedding: after the tissue is dehydrated, putting the dehydrated tissue into a mold with a fixed shape, and injecting molten wax to fix and mold the tissue;
(05) tissue section: placing the fixed and formed tissue wax block in a slicer, setting the slice thickness to be 4 mu m, slicing, picking up a sample slice, placing the sample slice in a 37 ℃ slice spreading pool, fishing out the slice by using a clean glass slide or a glass slide treated by a slice adhesive, placing the sample slice on a 60 ℃ slice drying rack for drying for 10-15min, and finally placing the sample slice in a 60 ℃ slice drying box for drying for 1h to obtain the glass slice with the sample.
Specifically, the step (1) of preparing the reagent comprises the following steps:
a) and (3) sterilizing with deionized water: preparing 1800mL of deionized water, subpackaging into 6 bottles, wherein the liquid filling amount is less than 2/3, 300 mL/bottle, marking the sterilization date, and sterilizing for 30 minutes under the conditions of 0.15Mpa and 121 ℃;
b) xylene: analytically pure, use of stock solution (operation in fume hood);
c) gradient concentration 100%, 95%, 75% ethanol: the 100% ethanol is directly used by 100mL of absolute ethanol of stock solution; the 95% ethanol is prepared from 5mL of sterilized deionized water and 95mL of absolute ethanol; the 75% ethanol is prepared from 25mL of sterilized deionized water and 75mL of absolute ethanol;
d) 10% neutral formalin: the stock solution is used, 100 mL;
e) antigen retrieval solution: dissolving 1 bag of 1.58g Tris/EDTA in 1000mL ultrapure water, adding 0.5mL tween-20, monitoring the pH value of the solution by using pH test paper, and adjusting the pH value to 9.0 (usually by using hydrochloric acid) to obtain an antigen retrieval solution;
f)1 × TBST buffer: mixing 50mL of 20-time mother liquor with 950mL of sterilized deionized water to prepare 1000mL of 1X TBST buffer solution, and bottling for later use;
g) a first antibody: respectively taking an antibody resisting human natural killer cell surface molecule CD56, an antibody resisting human leukocyte surface molecule CD45, an antibody resisting human natural killer cell subtype surface molecule CD27, an antibody resisting human natural killer cell subtype surface molecule CD11b, an antibody resisting human natural killer cell subtype surface molecule CD16 and an antibody resisting human natural killer cell subtype surface molecule CD49 alpha, and diluting the antibodies to corresponding multiples (such as 100-fold dilution: 1ul antibody +99ul antibody diluent) by using an antibody diluent according to an antibody specification to obtain an anti-working solution;
h) secondary antibody: selecting a secondary antigen solution matched with the TSA dye to be directly used as a secondary antibody working solution;
i) TSA dye: the working solution of the fluorescent dye is prepared by diluting mother solution of various fluorescent dyes by 100 times in a signal amplification solution;
j) DAPI working solution: and adding DAPI powder into deionized water to dissolve according to the concentration of 20mg/mL to prepare a storage solution, storing at 4 ℃ in a dark place, and diluting a working solution in deionized water by 100 times by using the storage solution to prepare the working solution.
Specifically, the addition amount of the non-specific protein blocker in the step (5) is 150uL, the addition amount of the primary antibody working solution in the step (6) is 150uL, the addition amount of the secondary antibody working solution in the step (7) is 150uL, and the addition amount of the fluorescent dye working solution in the step (8) is 150 uL.
Specifically, two slides with specimens are taken and respectively subjected to the steps (2) to (11), primary antibody incubations of the two slides adopt different antibody groups, and the obtained staining results are processed and analyzed together in the step (12).
Specifically, the antibody group corresponding to the primary antibody on one slide is an antibody against human natural killer cell subtype surface molecule CD49 alpha, an antibody against human natural killer cell subtype surface molecule CD16, an antibody against human natural killer cell surface molecule CD56 and an antibody against human leukocyte surface molecule CD45, and the antibody group corresponding to the primary antibody on the other slide is an antibody against human natural killer cell subtype surface molecule CD27, an antibody against human natural killer cell subtype surface molecule CD11B, an antibody against human natural killer cell surface molecule CD56 and an antibody against human leukocyte surface molecule CD 45.
Specifically, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD49 α is 2000 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD16 is 100 times, the dilution factor of the working solution of the antibody against human natural killer cell surface molecule CD56 is 100 times, the dilution factor of the working solution of the antibody against human leukocyte surface molecule CD45 is 300 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD27 is 3000 times, and the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD11B is 500 times.
Specifically, the antibody against human natural killer cell subtype surface molecule CD49 α corresponds to fluorochrome 570, the antibody against human natural killer cell subtype surface molecule CD16 corresponds to fluorochrome 650, the antibody against human natural killer cell surface molecule CD56 corresponds to fluorochrome 520, the antibody against human leukocyte surface molecule CD45 corresponds to fluorochrome 480, the antibody against human natural killer cell subtype surface molecule CD27 corresponds to fluorochrome 570, and the antibody against human natural killer cell subtype surface molecule CD11B corresponds to fluorochrome 650.
The experimental conditions for the treatment of two specimen-bearing slides according to the present invention are shown in tables 1 and 2:
TABLE 1
TABLE 2
The step (12) of processing and analyzing the multi-marker staining result specifically comprises the following steps: and imaging by adopting a fluorescence microscope, and acquiring a panoramic color picture of the 4-color multicolor fluorescence immunohistochemical slide by using a panoramic pathology scanning system. And the panoramic pathology analysis system identifies the combined positive signals on the picture, counts the percentage of the positive signals, obtains the distribution and the percentage of different functional uNK cell subsets in the endometrial environment of the patient and evaluates the endometrial receptivity. The uNK with tolerance function of the uNK cell subset adopted by the invention comprises a combination of CD16-CD45+CD56bright、CD49a+CD45+CD56+uNK, CD11b of COMBINATION II-CD27+CD45+CD56+、CD11b-CD27-CD45+CD56+uNK; the uNK for killing bacteria comprises a combination of CD16+CD45+CD56dim, CD11b combination two+CD27-CD45+CD56+uNK。
The following are examples of practical applications of the present invention:
patient 1 and patient 2 underwent endomembrane biopsy at mid-luteal stage, and the obtained endomembrane tissue was formalin-fixed, paraffin-embedded, sectioned, and subjected to the above examination and panoramic pathological analysis to obtain results.
Two patients were treated with assisted reproduction in follow-up, patient 1 successfully obtained a clinical pregnancy positive, and patient 2 failed. The single positive cell rate of CD56 of patient 1 is 3.33%, the single positive cell rate of CD56 of patient 2 is 1.13%, and the traditional single-index detection result cannot obviously reflect the difference among patients, is relatively general, and cannot accurately evaluate the immune state of the patient. The results of the multi-index test of the present invention show that patient 1 has tolerance uNK, CD16-CD45+CD56bright、CD49a+CD45+CD56+、CD11b-CD27+CD45+CD56+、CD11b-CD27-CD45+CD56+uNK for CD45+CD56+The percentages of the total uNK population were 19.16%, 38.83%, 4.56%, 51.24%, respectively, and patient 2 corresponded to 10.07%, 16.82%, 2.31%, 38.61%, respectively; killer uNK, CD16 of patient 1+CD45+CD56dim、CD11b+CD27-CD45+CD56+uNK for CD45+CD56+The percentage of the total uNK population was 19.82% and 45.61%, respectively, and the percentage of patient 2 was 35.58% and 55.06%, respectively, and it can be seen that the uNK subpopulation of patient 1 was predominantly tolerated and patient 2 u was predominantly toleratedThe NK subgroup is mainly lethal, and the endometrial environment is enriched with different functional uNK subgroups which can influence the endometrial receptivity, thereby causing different pregnancy fates.
Patient 3 underwent G-CSF uterine cavity infusion immunotherapy during assisted reproductive therapy. The intimal biopsy is performed in the mid-luteal phase before and after perfusion, and the obtained intimal tissue is fixed by formalin, embedded by paraffin, sliced, examined and subjected to panoramic pathological analysis to obtain a result.
After the perfusion of the patient 3, the rate of the single positive cells of the CD56 is increased from 2.81% to 6.44%, and the expected treatment effect of increasing the number of the uNK is achieved. However, the multi-index test results showed tolerance uNK, CD16 in patient 3 after perfusion-CD45+CD56bright、CD49a+CD45+CD56+、CD11b-CD27+CD45+CD56+、CD11b-CD27-CD45+CD56+uNK for CD45+CD56+The percentages of the total population of uNK were 1.81%, 33.18%, 10.28%, 50.27%, respectively, with the corresponding percentages before perfusion being 10.19%, 51.72%, 12.03%, 58.63%; lethal uNK after perfusion, CD16+CD45+CD56dim、CD11b+CD27-CD45+CD56+uNK for CD45+CD56+The percentages of the total population of uNK were 11.92%, 35.68%, respectively, and the corresponding percentages before perfusion were 4.26%, 25.91%. The results show that the total uNK number of the patient 3 is improved after treatment, but the ratio of the killer uNK subgroup in the uNK total population is increased, and the ratio of the tolerance uNK subgroup is reduced, so that the immune microenvironment is unfavorable for embryonic development. Therefore, the invention can provide more detailed pathological information for a clinician and provide beneficial guidance for clinical medication of the clinician.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A kit for evaluating endometrial receptivity is characterized by comprising antigen retrieval liquid, a non-specific protein blocking agent, a primary antibody, a secondary antibody, antibody diluent, a TSA dye, a buffer solution, DAPI working solution, a sealing tablet, a fixing liquid, a dewaxing agent, a hydrating agent and sterilized deionized water.
2. The kit for evaluating endometrial receptivity according to claim 1, wherein,
the antigen retrieval solution comprises Tris/EDTA;
the non-specific protein blocking agent comprises bovine serum albumin;
the primary antibody comprises an antibody for resisting human natural killer cell surface molecules CD56, an antibody for resisting human leukocyte surface molecules CD45, an antibody for resisting human natural killer cell subtype surface molecules CD27, an antibody for resisting human natural killer cell subtype surface molecules CD11B, an antibody for resisting human natural killer cell subtype surface molecules CD16 and an antibody for resisting human natural killer cell subtype surface molecules CD49 alpha;
the secondary antibody comprises an IgG antibody marked by horseradish peroxidase;
the antibody diluent comprises an IHC antibody diluent;
the TSA dye comprises a multi-target immunofluorescence staining kit, wherein the multi-target immunofluorescence staining kit comprises a fluorescent dye PPD480, a fluorescent dye PPD520, a fluorescent dye PPD570, a fluorescent dye PPD650 and a signal amplification solution;
the buffer comprises a 1 XTSST buffer;
the DAPI working solution comprises DAPI powder dissolved in deionized water;
the mounting agent comprises a water-soluble anti-fluorescence quenching mounting agent;
the reinforcing liquid comprises 10% of neutral formalin;
the dewaxing agent comprises xylene;
the hydrating agent comprises ethanol with gradient concentration of 100%, 95% and 75%.
3. A method of assessing endometrial receptivity comprising the steps of:
(1) preparation of reagents: configuring each reagent in the kit for assessing endometrial receptivity according to claim 2;
(2) dewaxing and hydrating: putting the glass slide with the specimen into three parts of dimethylbenzene in sequence, and soaking for 5 minutes respectively to finish dewaxing; then sequentially placing the mixture into two portions of 100% ethanol, soaking for 5 minutes respectively, then placing the mixture into 95% ethanol, soaking for 3 minutes, finally placing the mixture into 75% ethanol, soaking for 2 minutes, washing for 3 times by using sterilized deionized water, and completing hydration after 1 minute of each time;
(3) reinforcing the sample: putting the slide obtained in the step (2) into 10% neutral formalin for soaking for 10 minutes, washing for 3 times by using sterilized deionized water, and finishing reinforcement after 1 minute of each time;
(4) first antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling the liquid with high fire in a microwave oven, putting the glass slide to be repaired into the cup with low fire for 10 minutes, taking out the glass slide to be repaired, and naturally cooling the glass slide to room temperature;
(5) serum blocking: washing the repaired slide for 1 time by using sterilized deionized water, washing the repaired slide for 1 time by using a 1X TBST buffer solution, throwing off excessive water, drawing a tissue by using a composition pen ring, dripping a non-specific protein blocking agent, and incubating for 10 minutes by moisturizing and oscillating at room temperature;
(6) primary antibody incubation: discarding confining liquid, dropwise adding primary antibody working solution prepared by diluting one of the primary antibodies with antibody diluent, incubating at room temperature under humid conditions and shaking for 30min, and washing with 1X TBST buffer solution for 3 times, each time for 2 min;
(7) and (3) secondary antibody incubation: removing the washing solution, adding the secondary antibody working solution dropwise, incubating for 10 min with shaking at room temperature under moisture retention, and washing for 2 min each time for 3 times with 1X TBST buffer solution;
(8) TSA incubation staining: removing washing liquor, dropwise adding a fluorescent dye working solution corresponding to an antibody of the primary antibody in the TSA dye, incubating for 10 minutes under a moisturizing condition at room temperature and shaking, and washing for 2 minutes by using a 1X TBST buffer solution;
(9) and (3) second antigen retrieval: pouring the antigen repairing liquid into the repairing cup, boiling the glass with high fire in a microwave oven, placing the glass into the glass for incubation, taking out the glass and naturally cooling the glass to room temperature after the glass is incubated and the glass is cooled with low fire for 10 minutes;
(10) repeating the steps (5) to (9) to perform the next round of antibody staining until all staining of one antibody group in the first antibody is completed;
(11) sealing: removing washing liquid, dripping DAPI working solution, incubating for 10 min at room temperature under moisture, washing for 2 min with 1XTBST buffer solution, dripping water-soluble fluorescence quenching blocking tablet, sealing, and fixing cover glass with colorless nail polish;
(12) processing and analyzing the multi-label dyeing result: and acquiring a spectral image by using an imaging microscope, identifying and analyzing the image by using case picture analysis software, and realizing quantitative analysis of the endometrial natural killer cell subpopulation on the same tissue slice.
4. The method of claim 3, wherein the step (1) of preparing the reagent comprises:
a) and (3) sterilizing with deionized water: preparing 1800mL of deionized water, subpackaging into 6 bottles, wherein the liquid filling amount is less than 2/3, 300 mL/bottle, marking the sterilization date, and sterilizing for 30 minutes under the conditions of 0.15Mpa and 121 ℃;
b) xylene: analytically pure, and using the stock solution;
c) gradient concentration 100%, 95%, 75% ethanol: the 100% ethanol is directly used by 100mL of absolute ethanol of stock solution; the 95% ethanol is prepared from 5mL of sterilized deionized water and 95mL of absolute ethanol; the 75% ethanol is prepared from 25mL of sterilized deionized water and 75mL of absolute ethanol;
d) 10% neutral formalin: the stock solution is used, 100 mL;
e) antigen retrieval solution: dissolving 1 bag of 1.58g Tris/EDTA in 1000mL ultrapure water, adding 0.5mL tween-20, monitoring the pH value of the solution by using pH test paper, and adjusting the pH value to 9.0 to obtain an antigen retrieval solution;
f)1 × TBST buffer: mixing 50mL of 20-time mother liquor with 950mL of sterilized deionized water to prepare 1000mL of 1X TBST buffer solution, and bottling for later use;
g) a first antibody: respectively taking an antibody resisting human natural killer cell surface molecule CD56, an antibody resisting human leukocyte surface molecule CD45, an antibody resisting human natural killer cell subtype surface molecule CD27, an antibody resisting human natural killer cell subtype surface molecule CD11b, an antibody resisting human natural killer cell subtype surface molecule CD16 and an antibody resisting human natural killer cell subtype surface molecule CD49 alpha, and diluting the antibodies to corresponding multiples by using an antibody diluent according to an antibody specification to obtain an anti-working solution;
h) secondary antibody: selecting a secondary antigen solution matched with the TSA dye to be directly used as a secondary antibody working solution;
i) TSA dye: the working solution of the fluorescent dye is prepared by diluting mother solution of various fluorescent dyes by 100 times in a signal amplification solution;
j) DAPI working solution: and adding DAPI powder into deionized water to dissolve according to the concentration of 20mg/mL to prepare a storage solution, storing at 4 ℃ in a dark place, and diluting a working solution in deionized water by 100 times by using the storage solution to prepare the working solution.
5. The method of claim 3, wherein the nonspecific protein blocker is added in an amount of 150uL in step (5), the primary antibody working solution is added in an amount of 150uL in step (6), the secondary antibody working solution is added in an amount of 150uL in step (7), and the fluorochrome working solution is added in an amount of 150uL in step (8).
6. The method for evaluating endometrial receptivity according to any one of claims 3 to 5, wherein two slides with specimens are taken and subjected to steps (2) - (11), respectively, and the primary antibody incubations of the two slides are performed with different antibody sets, and the staining results obtained are processed and analyzed together in step (12).
7. The method of claim 6, wherein the set of antibodies against the primary antibody is antibodies against human natural killer cell subtype surface molecule CD49 α, antibodies against human natural killer cell subtype surface molecule CD16, antibodies against human natural killer cell surface molecule CD56, antibodies against human leukocyte surface molecule CD45, antibodies against human natural killer cell subtype surface molecule CD27, antibodies against human natural killer cell subtype surface molecule CD11B, antibodies against human natural killer cell surface molecule CD56, and antibodies against human leukocyte surface molecule CD 45.
8. The method according to claim 7, wherein the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD49 α is 2000 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD16 is 100 times, the dilution factor of the working solution of the antibody against human natural killer cell surface molecule CD56 is 100 times, the dilution factor of the working solution of the antibody against human leukocyte surface molecule CD45 is 300 times, the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD27 is 3000 times, and the dilution factor of the working solution of the antibody against human natural killer cell subtype surface molecule CD11B is 500 times.
9. The method of claim 7, wherein the antibody against human natural killer cell subtype surface molecule CD49 α corresponds to fluorochrome 570, the antibody against human natural killer cell subtype surface molecule CD16 corresponds to fluorochrome 650, the antibody against human natural killer cell surface molecule CD56 corresponds to fluorochrome 520, the antibody against human leukocyte surface molecule CD45 corresponds to fluorochrome 480, the antibody against human natural killer cell subtype surface molecule CD27 corresponds to fluorochrome 570, and the antibody against human natural killer cell subtype surface molecule CD11B corresponds to fluorochrome 650.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116046503A (en) * | 2023-04-03 | 2023-05-02 | 迈杰转化医学研究(苏州)有限公司 | Antigen retrieval liquid capable of enhancing specific staining effect and reducing staining background and application thereof |
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| CN105142651A (en) * | 2013-02-05 | 2015-12-09 | 人类起源公司 | Natural killer cells from placenta |
| US20170160171A1 (en) * | 2015-11-20 | 2017-06-08 | Oregon Health And Science University | Multiplex immunohistochemistry image cytometry |
| CN111122520A (en) * | 2018-10-31 | 2020-05-08 | 四川大学华西第二医院 | Embryo implantation detection kit and application and use method thereof |
| CN111579798A (en) * | 2020-05-29 | 2020-08-25 | 深圳市锦欣医疗科技创新中心有限公司 | Kit for evaluating endometrial receptivity and using method thereof |
| CN112143785A (en) * | 2020-10-21 | 2020-12-29 | 艾基诺米公司 | Method for evaluating endometrial receptivity of a patient and kit for carrying out the method |
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| CN105142651A (en) * | 2013-02-05 | 2015-12-09 | 人类起源公司 | Natural killer cells from placenta |
| US20170160171A1 (en) * | 2015-11-20 | 2017-06-08 | Oregon Health And Science University | Multiplex immunohistochemistry image cytometry |
| CN111122520A (en) * | 2018-10-31 | 2020-05-08 | 四川大学华西第二医院 | Embryo implantation detection kit and application and use method thereof |
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| CN116046503A (en) * | 2023-04-03 | 2023-05-02 | 迈杰转化医学研究(苏州)有限公司 | Antigen retrieval liquid capable of enhancing specific staining effect and reducing staining background and application thereof |
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