CN105628913A - Kit for evaluating endometrial receptivity and using method thereof - Google Patents
Kit for evaluating endometrial receptivity and using method thereof Download PDFInfo
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Abstract
The invention discloses a kit for evaluating endometrial receptivity and a using method thereof, belonging to the medical and biological detection field. The kit comprises the following components: a positive control, a negative control, a first antibody, a second antibody, washing liquid, a fixing agent, a dehydrating agent, a hydrated agent, an embedding agent, a dewaxing transparent reagent, a buffer solution A, a buffer solution B, an antigen retrieval agent, a peroxidase inactivated agent, a non-specific protein blocking agent, a color-substrate solution, a composite dye solution, a differentiation solution, a blue return solution and a mounting medium. The invention also discloses the using method of the kit. The kit has the characteristics of good stability and high accuracy, provides a new evaluation index for the endometrial receptivity evaluation, can comprehensively evaluate the endometrial receptivity of dysgenesis patients, provides a reliable basis for individualized treatment, solves the problems of single detection index and poor detection method stability in the prior art, increases the successful rate of pregnancy of the dysgenesis patients, and has relatively high clinical application values and social benefits.
Description
Technical field
The present invention relates to a kind of test kit for assessing endometrium receptivity and using method thereof, belong to medical science and Biological Detection field.
Background technology
Assessing according to World Health Organization (WHO), there are about 1 couple of Mr. and Mrs at present and there is dysgenesia problem in every 7 couples of Mr. and Mrs, main manifestations is miscarriage or infertile. Pay close attention to the pith that healthy reproduction is national development. Research shows, the change of endometrium receptivity is probably one of principal element causing dysgenesia. Endometrium receptivity refers to that endometrium accepts the ability of embryo, refers to the state that endometrium is in a kind of embryo of permission location, sticks and invade. Be otherwise known as this period endometrial implantation window stage. If the endometrial receptivity of embryo is occurred abnormal by endometrium of implantation window stage, it is to embryo's generation rejection, and then does not allow the normal implantation of embryo to grow, and this state will cause miscarriage or the Averse pregnancy outcomes such as infertile. Therefore, the assessment of endometrium receptivity is significant for the diagnosis and treatment of dysgenesia patient.
At present, the evaluation measures of clinical neutron Endometrium endometrial receptivity is mainly morphological assessment. Its evaluation index includes three major types: the ultrasound modalities such as (1) endometrium thickness, echo and blood flow; (2) the biopsy form such as body of gland size, interstitial density and blood vessel abundance; (3) endometrium pinocytosis is dashed forward. But, there is certain dispute in morphological assessment endometrium receptivity. Because endometrium receptivity is interacted regulation and control by a series of cells and cytokine, the cytokine of cell and secretion thereof with embryo's contact process in play a significant role. But, endometrial Morphological characterization can not accurately reflect the expression of its cell and cytokine.
In this context, the new evaluation measures such as endometrial biopsy binding molecule detection starts development. Therefore, the research of endometrium receptivity biomarker increasingly comes into one's own. Research shows, endometrium immunocyte plays a significant role in its endometrial receptivity regulates and controls. Endometrium immunocyte mainly includes the four big classes such as natural killer cell, macrophage, T cell and dendritic cell. Further, all kinds of immunocytes are divided into again different subgroups, and in uterus film endometrial receptivity plays different effects in regulating. Natural killer cell, as immunocyte main in endometrium, by promoting trophoblast invasion, supporting spiral artery reconstruct and regulate and control the approach adjustment endometrium receptivities such as self killing ability, participates in Embryo implantation. Macrophage film in uterus local and system hormones regulate the lower conversion that can carry out subgroup, planting process both can have been removed pathogen, again can the inducing endometrial immunologic tolerance to embryo, and then maintain successful pregnancy. T cell mainly includes helper T lymphocyte, killer T cell and regulatory T cells. Wherein, helper T lymphocyte, mainly through secrete cytokines, regulates and controls endometrial differentiation, ready for Embryo implantation; Killer T cell participates in planting process mainly through the balance of regulation and control trophoblast invasion with secretion toxic granulations; And regulatory T cells plays immunosuppressive action, it is by suppressing the endometrium excessive inflammatory response to embryo, and then maintains continuation gestation. Dendritic cell, as important antigen presenting cell, can be divided into two classes according to its maturity: immature dendritic cell and mature dendritic cell. Immature dendritic cell plays immunologic tolerance effect in gestation, and mature dendritic cell plays immunologic rejection effect in gestation, and therefore, both balances film in uterus accepts to play equally in embryonic processes requisite effect. Above result of study shows, the Control factors of endometrium receptivity is not single, but various immunocyte interacts, mutually the result of balance.
But, time at present both at home and abroad using endometrium immunocyte as its endometrial receptivity evaluation index, it being mostly detection one of which immunocyte, and detection method is different, analysis result disunity, undulatory property are big. According to these results, patient uterine's uterine cavity lesion can not be carried out accurate evaluation by clinician, so perfect therapeutic scheme cannot be formulated. Therefore, developing reliable and stable endometrium panimmunity cell detection kit can carry out accurate evaluation to endometrium receptivity, and the diagnosis and treatment for patient provide reliable basis.
Detection method currently for endometrium immunocyte quantity mainly has flow cytometry and two kinds of methods of Immunohistochemical detection. But, the positioning scenarios of endometrial cell can not be analyzed by flow cytometry, and there is the problem such as cell and antigen loss in the process prepare cell suspension. Therefore, Cell immunohistochemical staining method becomes better selection. Clinically, immunohistochemical staining is widely applied in pathologic diagnosis of tumor, relates to field and includes the discriminating of tumor tissues origin, the judgement of undifferentiated malignant tumor character, the discriminating of various morphological system tumor, determines tumor original site etc. Its principle is the antibody on cell with labelling or in-house corresponding antigens positions, qualitative or detection by quantitative, eye-catching positive color is presented through histochemical color reaction, under optical microscope, fluorescence microscope or ultramicroscope, carry out artificial counting, or adopt microscope collection image binding of pathological picture analyzing software that it is carried out quantitative analysis.
Summary of the invention
An object of the present invention, is to provide a kind of test kit for endometrium receptivity assessment. The test kit of the present invention can the endometrium receptivity of comprehensive assessment dysgenesia patient, provide new Testing index for the etiological diagnosis of reproduction impaired patients, the formulation for its personalized medicines scheme provides reliable basis.
This invention address that the technical scheme of above-mentioned first technical problem is as follows: a kind of test kit for assessing endometrium receptivity, including following component: positive control, negative control, primary antibodie, two anti-, washing liquid, fixative, dehydrant, hydrated agent, embedding medium, dewaxing and clarifier, buffer A, buffer B, antigen retrieval agent, peroxidase deactivator, nonspecific proteins blocker, nitrite ion, redye liquid, differentiation liquid, return blue liquid and mountant.
Further, described positive control is normal women of child-bearing age endometrial tissue paraffin mass, and described negative control is that the 5%BSA without primary antibodie applies the endometrial tissue paraffin mass educated.
Further, described primary antibodie is the antibody of anti-human natural killer cell surface molecular CD56, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, one in the antibody of anti-human mature dendritic cell surface molecular CD83, described two to resist the IgG antibody for horseradish peroxidase-labeled, described washing liquid be normal saline, and described fixative is formalin, and described dehydrant is concentration is 70%v/v, 80%v/v, 95%v/v, the graded ethanol of 100%v/v, described hydrated agent is concentration is 100%v/v, 95%v/v, 95%v/v, the graded ethanol of 80%v/v, described embedding medium is paraffin, and described dewaxing and clarifier are dimethylbenzene, described buffer A is PBS, described buffer B is PBST, and described antigen retrieval agent is the 1mMTris/EDTA solution of pH9.0, and described peroxidase deactivator is 3%H2O2Solution, described nonspecific proteins blocker is 5%BSA, and described nitrite ion is DAB, and described after stain is hematoxylin, and described differentiation liquid is 1%v/v hydrochloride alcohol, described in return blue liquid be 1%v/v ammonia, described mountant is neutral gum.
The two of the purpose of the present invention, are to provide the using method of mentioned reagent box. The using method of the test kit of the present invention, can draw quantitative result stable, 8 kinds of immunocytes of endometrium accurately, can be used for the assessment of endometrium receptivity.
This invention address that the technical scheme of above-mentioned second technical problem is as follows: the using method of a kind of test kit for assessing endometrium receptivity, comprise the steps:
(1) take testing sample, positive control and negative control, put in normal saline, remove the clot on testing sample, positive control and negative control respectively, be then placed in 10% formalin of 8mL and fix 24h, arrive fixed preparation respectively;
(2) by step (1) gained fixed preparation, after carrying out processed, dimethylbenzene transparence and paraffin embedding, it is placed in cold bench, after paraffin, forms sample to be tested piece of tissue, positive control tissue block and negative control tissue block respectively;
(3) by step (2) gained sample to be tested piece of tissue, positive control tissue block and negative control tissue block, it is placed on microtome and carries out serial section, each piece of tissue cuts to obtain 8 sections, totally 24 sections, it is placed in 37 DEG C of water and spreads out, then it is laid in respectively on microscope slide, is placed in 60 DEG C dry 1 hour, obtains dried section;
(4) by the dried section of step (3) gained, dewax with dewaxing agent, then carry out aquation with hydrated agent, and be rinsed by buffer A, obtain the section after aquation;
(5) by the section after step (4) gained aquation, get rid of unnecessary liquid, apply with endogenous peroxydase inactivator and educate, be then rinsed by buffer A, obtain the section after endogenous peroxydase inactivation;
(6) section after being inactivated by step (5) gained endogenous peroxydase, puts into and carries out Microwave method 15min in antigen retrieval buffers, places room temperature cooling, is then rinsed by buffer A, cuts into slices after obtaining antigen retrieval;
(7) by the section after step (6) gained antigen retrieval, getting rid of unnecessary liquid, addition nonspecific proteins blocker room temperature is applied and is educated 20min, the section after being closed;
(8) section after step (7) gained being closed, except 8 sections of negative control, non-specific protein blocker is all got rid of in all the other sections, it is separately added into the antibody of anti-human natural killer cell surface molecular CD56, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, the antibody of anti-human mature dendritic cell surface molecular CD83, all apply in 37 DEG C of water-baths and educate 60min, then rinse by buffer A, each 3min, wash 3 times, buffer B is rinsed, each 3min, wash 1 time, obtain the section after primary antibodie,
(9) by the section after step (8) gained primary antibodie, getting rid of unnecessary liquid, add two and resist, 37 DEG C of water-baths are applied and are educated 30min, get rid of two and resist, and then use buffer A to rinse, and each 3min washes 3 times, obtain two anti-after section;
(10) section after being resisted by step (9) gained two, gets rid of unnecessary liquid, develops the color with nitrite ion, the section after being developed the color;
(11) section after being developed the color by step (10) gained, gets rid of nitrite ion, adds and redyes liquid and redye 4min, differentiation liquid differentiation 20s, return blue agent and return blue 30s, obtains the section after returning basket;
(12) step (11) gained is returned the section after basket, gets rid of unnecessary liquid, process with dehydrant and clarifier, obtain the section after transparence;
(13) by the section after step (12) gained transparence, get rid of unnecessary liquid, carry out mounting by mountant, obtain immunohistochemical staining section;
(14) step (13) gained immunohistochemical staining is cut into slices, it is placed on the object stage of imaging microscope and gathers picture, pathological picture analysis software tissue staining degree, statistics positive cell number, total cell number and positive cell is utilized to account for the percentage ratio of total cell number, the endometrium receptivity of assessment sample to be tested.
On the basis of technique scheme, the present invention can also do following improvement.
Further, the endometrium of the ex vivo human body of step (1) described testing sample.
Further, the concrete operations of step (14) described imaging microscope and pathological picture analysis software evaluation endometrium receptivity are as follows: imaging microscope gathers 200 times of pictures in nonoverlapping 20 visuals field; The picture of 8 kinds of immunocytes of gained is imported in pathological picture quantified system analysis and is analyzed. In pathological picture quantified system analysis, what set cell is sized to 55��400 pixels, and nuclear haematoxylin dyeing intensity need to more than 0.15, and cell total in gathered picture is identified and counts by system synthesis two indices; Tissue positive cells presents eye-catching brown color, and system is by the optical density value of display positive cell brown color, and system will be greater than all cells of this value and is identified as positive cell and counts; Positive cell rate is calculated according to positive cell number and total cell number. The 8 of sample to be tested kinds of immunocyte rates and term of reference are compared, and then judges the endometrium receptivity of sample to be tested.
Wherein, natural killer cell rate term of reference is 0.97��21.15%; Macrophage rate term of reference is 0.5��2.65%, T cell rate term of reference is 1.12��9.10%, helper T lymphocyte rate term of reference is 0.10��0.50%, killer T cell rate term of reference is 0.81��5.45%, regulatory T cells rate term of reference is 0.05��0.26%, immature dendritic cell rate term of reference is 0.006��0.140%, and mature dendritic cell rate term of reference is 0.07��4.01%. When arbitrary immunocyte rate of sample to be tested exceedes its term of reference, the endometrium receptivity pointing out this sample to be tested is abnormal.
The invention has the beneficial effects as follows:
1. the test kit of the present invention, for the index that the assessment offer of endometrium receptivity is more excellent, the classification diagnosis for reproduction impaired patients provides foundation.
2. the using method of the test kit of the present invention, the detection for endometrium panimmunity cell provides stable, quantitative analysis method accurately.
3. the test kit of the present invention, reliable basis is provided for the accurate comprehensive assessment patient uterine's uterine cavity lesion of clinician, therapeutic scheme for formulating individuation provides powerful guarantee, successful pregnancy rate for improving dysgenesia patient makes tremendous contribution, has higher clinical value and social benefit.
Accompanying drawing explanation
Fig. 1 is the detection application of the assessment endometrium receptivity test kit of the present invention.
In figure, (A) for endometrium natural killer cell dyeing picture, (B) for endometrium macrophage dyeing picture, (C) for endometrium T cell dyeing picture, (D) for endometrium helper T lymphocyte dyeing picture, (E) for endometrium killer T cell dyeing picture, (F) for endometrium regulatory T cells dyeing picture, (G) for endometrium immature dendritic cell dyeing picture, (H) is endometrial maturation dendritic cell dyeing picture.
Detailed description of the invention
Below in conjunction with accompanying drawing, principles of the invention and feature being described, example is served only for explaining the present invention, is not intended to limit the scope of the present invention.
A kind of test kit for assessing endometrium receptivity, including following component: positive control, negative control, primary antibodie, two anti-, washing liquid, fixative, dehydrant, hydrated agent, embedding medium, dewaxing and clarifier, buffer A, buffer B, antigen retrieval agent, peroxidase deactivator, nonspecific proteins blocker, nitrite ion, redye liquid, differentiation liquid, return blue liquid and mountant.
Described positive control is normal women of child-bearing age endometrial tissue paraffin mass, and described negative control is that the 5%BSA without primary antibodie applies the endometrial tissue paraffin mass educated.
Described primary antibodie is the antibody of anti-human natural killer cell surface molecular CD56, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, one in the antibody of anti-human mature dendritic cell surface molecular CD83, described two to resist the IgG antibody for horseradish peroxidase-labeled, described washing liquid be normal saline, and described fixative is formalin, and described dehydrant is concentration is 70%v/v, 80%v/v, 95%v/v, the graded ethanol of 100%v/v, described hydrated agent is concentration is 100%v/v, 95%v/v, 95%v/v, the graded ethanol of 80%v/v, described embedding medium is paraffin, and described dewaxing and clarifier are dimethylbenzene, described buffer A is PBS, described buffer B is PBST, and described antigen retrieval agent is the 1mMTris/EDTA solution of pH9.0, and described peroxidase deactivator is 3%H2O2Solution, described nonspecific proteins blocker is 5%BSA, and described nitrite ion is DAB, and described after stain is hematoxylin, and described differentiation liquid is 1%v/v hydrochloride alcohol, described in return blue liquid be 1%v/v ammonia, described mountant is neutral gum.
The using method of a kind of test kit for assessing endometrium receptivity, comprises the steps:
(1) using the endometrium of ex vivo human body as testing sample, positive control and negative control, put in 20mL normal saline, remove the clot on testing sample, positive control and negative control respectively, it is then placed in 10% formalin of 8mL and fixes 24h, arrive fixed preparation respectively;
(2) by step (1) gained fixed preparation, clean with deionized water, it is sequentially placed into the 75% alcohol-pickled 2h of 250mL, 80% alcohol-pickled 2h, 95% alcohol-pickled 1.5h, 100% alcohol-pickled 1h carries out dehydration, then the xylene solution being sequentially placed into 250mL soaks 20min, another part of xylene solution soaks 20min, proceed to another part of xylene solution immersion 20min again and carry out transparence, then proceed in paraffin and embed, embedding is placed on cold bench, after paraffin, form sample to be tested piece of tissue respectively, positive control tissue block and negative control tissue block,
(3) by step (2) gained sample to be tested piece of tissue, positive control tissue block and negative control tissue block, it is placed on microtome and carries out serial section, slice thickness is 4 ��m, each piece of tissue cuts to obtain 8 sections, totally 24 sections, are placed in 37 DEG C of water and spread out, are then laid on microscope slide respectively, it is placed in 60 DEG C dry 1 hour, obtains dried section;
(4) by the dried section of step (3) gained, put in color jar and dewax in the following order: 250mL dimethylbenzene soaks 10min, another part of 250mL dimethylbenzene soaks 5min, anhydrous alcohol and dimethylbenzene equal-volume mixed solution soak 3min, aquation is carried out according to the following steps: 100% alcohol-pickled 2min after dewaxing, 95% alcohol-pickled 2min, the alcohol-pickled 2min of another part 95%, 80% alcohol-pickled 2min, then PBS rinses 3min, obtains the section after aquation;
(5) by the section after step (4) gained aquation, unnecessary liquid is got rid of, with the 3%H of 100 �� L2O2Apply and educate 15min, be then rinsed with PBS, each 3min, wash 3 times, obtain the section after endogenous peroxydase inactivation;
(6) section after being inactivated by step (5) gained endogenous peroxydase, puts into and carries out Microwave method 15min in 550mL antigen retrieval buffers, and room temperature places cooling, then it is rinsed with PBS, each 3min, washes 3 times, cuts into slices after obtaining antigen retrieval;
(7) by the section after step (6) gained antigen retrieval, get rid of unnecessary liquid, add 100 �� L 5%BSA room temperature apply educate 20min, the section after being closed;
(8) section after step (7) gained being closed, except negative control 8 is cut into slices, 5%BSA is all got rid of in all the other sections, it is separately added into the antibody of each 60 anti-human natural killer cell surface molecular CD56 of �� L, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, one in the antibody of anti-human mature dendritic cell surface molecular CD83, all apply in 37 DEG C of water-baths and educate 60min, then rinse with PBS, each 3min, wash 3 times, PBST rinses, each 3min, wash 1 time, obtain the section after primary antibodie,
(9) by the section after step (8) gained primary antibodie, getting rid of unnecessary liquid, every upper 60 �� L bis-that add of section resist, and 37 DEG C of water-baths are applied and educated 30min, get rid of two and resist, and then use PBS to rinse, and each 3min washes 3 times, obtain two anti-after section;
(10) section after being resisted by step (9) gained two, gets rid of unnecessary liquid, and every upper 60 �� LDAB nitrite ions that add of section develop the color, the section after being developed the color;
(11) section after being developed the color by step (10) gained, gets rid of nitrite ion, and dropping 60uL haematoxylin is redyed in 1% ammonia of 1% hydrochloride alcohol differentiation 20s, 60uL of 4min, 60uL and returned blue 30s, obtains the section after returning basket;
(12) step (11) gained is returned the section after basket, get rid of unnecessary liquid, proceed to the 70% alcohol-pickled 2min of 250mL successively, 80% alcohol-pickled 2min, 95% alcohol-pickled 2min, 100% alcohol-pickled 2min carry out dehydration, then the xylene solution proceeding to 250mL soaks 3min, another part of xylene solution 10min, another part of xylene solution 5min carries out Vitrification management, obtains the section after transparence;
(13) by the section after step (12) gained transparence, get rid of unnecessary liquid, add 10 �� L neutral gums and carry out mounting, obtain immunohistochemical staining section;
(14) step (13) gained immunohistochemical staining is cut into slices, it is placed on the object stage of imaging microscope and gathers picture, and the percentage ratio of total cell number, the endometrium receptivity of assessment sample to be tested is accounted for pathological picture analysis software tissue staining degree, statistics positive cell number, total cell number and positive cell.
The concrete operations adopting imaging microscope and pathological picture analysis software evaluation endometrium receptivity are as follows: adopt 200 times of pictures that imaging microscope gathers nonoverlapping 20 visuals field; The picture of 8 kinds of immunocytes of gained is imported in pathological picture quantified system analysis and is analyzed. In pathological picture quantified system analysis, what set cell is sized to 55��400 pixels, and nuclear haematoxylin dyeing intensity need to more than 0.15, and cell total in gathered picture is identified and counts by system synthesis two indices; Tissue positive cells presents eye-catching brown color, and system is by the optical density value of display positive cell brown color, and system will be greater than all cells of this value and is identified as positive cell and counts; Positive cell rate is calculated according to positive cell number and total cell number. The 8 of sample to be tested kinds of immunocyte rates and term of reference are compared, and then judges the endometrium receptivity of sample to be tested.
Wherein, natural killer cell rate term of reference is 0.97��21.15%; Macrophage rate term of reference is 0.5��2.65%, T cell rate term of reference is 1.12��9.10%, helper T lymphocyte rate term of reference is 0.10��0.50%, killer T cell rate term of reference is 0.81��5.45%, regulatory T cells rate term of reference is 0.05��0.26%, immature dendritic cell rate term of reference is 0.006��0.140%, and mature dendritic cell rate term of reference is 0.07��4.01%. When arbitrary immunocyte rate of sample to be tested exceedes its term of reference, the endometrium receptivity pointing out this sample to be tested is abnormal.
As it is shown in figure 1, the application in the assessment of endometrium immunocyte immue quantitative detection reagent box film endometrial receptivity in uterus, wherein, (A) for natural killer cell, presenting brown color around its nucleus, be uniformly distributed in endometrial stromal cell, cell rate is 5.82%; (B) for macrophage, presenting brown color around its nucleus, be uniformly distributed in endometrial stromal cell, cell rate is 0.7%; (C) for T cell, presenting brown color around its nucleus, be uniformly distributed in endometrial stromal cell, cell rate is 2.19%; (D) for helper T lymphocyte, presenting brown color around its nucleus, be uniformly distributed in endometrial stromal cell, cell rate is 0.3%; (E) for killer T cell, presenting brown color around its nucleus, be uniformly distributed in endometrial stromal cell, cell rate is 1.64%; (F) for regulatory T cells, presenting brown color in its nucleus, be sporadicly distributed in endometrial stromal cell, cell rate is 0.02%; (G) for immature dendritic cell, presenting brown color around its nucleus, be distributed in the position near body of gland, cell rate is 0.044%; (H) for mature dendritic cell, presenting brown color around its nucleus, and cell presents two kinds of distribution modes, be i.e. individual cells distribution and cluster distribution, cell rate is 3.04%. Wherein, regulatory T cells rate is 0.02%, and lower than term of reference 0.05��0.26%, the endometrium receptivity pointing out this sample is abnormal.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (5)
1. the test kit being used for assessing endometrium receptivity, it is characterized in that, including following component: positive control, negative control, primary antibodie, two anti-, washing liquid, fixative, dehydrant, hydrated agent, embedding medium, dewaxing and clarifier, buffer A, buffer B, antigen retrieval agent, peroxidase deactivator, nonspecific proteins blocker, nitrite ion, redye liquid, differentiation liquid, return blue liquid and mountant.
2. a kind of test kit for assessing endometrium receptivity according to claim 1, it is characterized in that, described positive control is normal women of child-bearing age endometrial tissue paraffin mass, and described negative control is that the 5%BSA without primary antibodie applies the endometrial tissue paraffin mass educated.
3. a kind of test kit for assessing endometrium receptivity according to claim 1 and 2, it is characterised in that described primary antibodie is the antibody of anti-human natural killer cell surface molecular CD56, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, one in the antibody of anti-human mature dendritic cell surface molecular CD83, described two to resist the IgG antibody for horseradish peroxidase-labeled, described washing liquid be normal saline, and described fixative is formalin, and described dehydrant is concentration is 70%v/v, 80%v/v, 95%v/v, the graded ethanol of 100%v/v, described hydrated agent is concentration is 100%v/v, 95%v/v, 95%v/v, the graded ethanol of 80%v/v, described embedding medium is paraffin, and described dewaxing and clarifier are dimethylbenzene, described buffer A is PBS, described buffer B is PBST, and described antigen retrieval agent is the 1mMTris/EDTA solution of pH9.0, and described peroxidase deactivator is 3%H2O2Solution, described nonspecific proteins blocker is 5%BSA, and described nitrite ion is DAB, and described after stain is hematoxylin, and described differentiation liquid is 1%v/v hydrochloride alcohol, described in return blue liquid be 1%v/v ammonia, described mountant is neutral gum.
4. the using method being used for assessing the test kit of endometrium receptivity as described in any one of claims 1 to 3, it is characterised in that comprise the steps:
(1) take testing sample, positive control and negative control, put in normal saline, remove the clot on testing sample, positive control and negative control respectively, be then placed in 10% formalin of 8mL and fix 24h, arrive fixed preparation respectively;
(2) by step (1) gained fixed preparation, after carrying out processed, dimethylbenzene transparence and paraffin embedding, it is placed in cold bench, after paraffin, forms sample to be tested piece of tissue, positive control tissue block and negative control tissue block respectively;
(3) by step (2) gained sample to be tested piece of tissue, positive control tissue block and negative control tissue block, it is placed on microtome and carries out serial section, each piece of tissue cuts to obtain 8 sections, totally 24 sections, it is placed in 37 DEG C of water and spreads out, then it is laid in respectively on microscope slide, is placed in 60 DEG C dry 1 hour, obtains dried section;
(4) by the dried section of step (3) gained, dewax with dewaxing agent, then carry out aquation with hydrated agent, and be rinsed by buffer A, obtain the section after aquation;
(5) by the section after step (4) gained aquation, get rid of unnecessary liquid, apply with endogenous peroxydase inactivator and educate, be then rinsed by buffer A, obtain the section after endogenous peroxydase inactivation;
(6) section after being inactivated by step (5) gained endogenous peroxydase, puts into and carries out Microwave method 15min in antigen retrieval buffers, places room temperature cooling, is then rinsed by buffer A, cuts into slices after obtaining antigen retrieval;
(7) by the section after step (6) gained antigen retrieval, getting rid of unnecessary liquid, addition nonspecific proteins blocker room temperature is applied and is educated 20min, the section after being closed;
(8) section after step (7) gained being closed, except 8 sections of negative control, non-specific protein blocker is all got rid of in all the other sections, it is separately added into the antibody of anti-human natural killer cell surface molecular CD56, the antibody of anti-human Macrophage Surface molecule CD68, the antibody of anti-human T cell surface molecular CD3, the antibody of anti-human helper T lymphocyte surface molecular CD4, the antibody of anti-human killer T cell surface molecular CD8, the antibody of anti-human regulatory T cells nuclear factor Foxp3, the antibody of anti-human immature dendritic cell surface molecular CD1a, the antibody of anti-human mature dendritic cell surface molecular CD83, all apply in 37 DEG C of water-baths and educate 60min, then rinse by buffer A, each 3min, wash 3 times, buffer B is rinsed, each 3min, wash 1 time, obtain the section after primary antibodie,
(9) by the section after step (8) gained primary antibodie, getting rid of unnecessary liquid, add two and resist, 37 DEG C of water-baths are applied and are educated 30min, get rid of two and resist, and then use buffer A to rinse, and each 3min washes 3 times, obtain two anti-after section;
(10) section after being resisted by step (9) gained two, gets rid of unnecessary liquid, develops the color with nitrite ion, the section after being developed the color;
(11) section after being developed the color by step (10) gained, gets rid of nitrite ion, adds and redyes liquid and redye 4min, differentiation liquid differentiation 20s, return blue agent and return blue 30s, obtains the section after returning basket;
(12) step (11) gained is returned the section after basket, gets rid of unnecessary liquid, process with dehydrant and clarifier, obtain the section after transparence;
(13) by the section after step (12) gained transparence, get rid of unnecessary liquid, carry out mounting by mountant, obtain immunohistochemical staining section;
(14) step (13) gained immunohistochemical staining is cut into slices, it is placed on the object stage of imaging microscope and gathers picture, pathological picture analysis software tissue staining degree, statistics positive cell number, total cell number and positive cell is utilized to account for the percentage ratio of total cell number, the endometrium receptivity of assessment sample to be tested.
5. the using method of a kind of test kit for assessing endometrium receptivity according to claim 4, it is characterised in that the endometrium of the ex vivo human body of step (1) described testing sample.
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Cited By (4)
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| CN109060488A (en) * | 2018-08-08 | 2018-12-21 | 三峡大学 | A kind of tissue returns blue liquid |
| 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 |
| CN111505311A (en) * | 2020-04-30 | 2020-08-07 | 深圳市锦欣医疗科技创新中心有限公司 | Biomarker for repeated embryo planting failure, screening method and application 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 |
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| CN105044348A (en) * | 2015-02-10 | 2015-11-11 | 桂林医学院附属医院 | SALL4 immunohistochemical detection kit for diagnosis of lung cancer |
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| CN105044348A (en) * | 2015-02-10 | 2015-11-11 | 桂林医学院附属医院 | SALL4 immunohistochemical detection kit for diagnosis of lung cancer |
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| ANN M. STEWART-AK等: ""Endometrial Leukocytes Are Altered Numerically and Functionally in Women with Implantation Defects", 《AMERICAN JOURANAL OF REPRODUCTIVE IMMUNOLOGY》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060488A (en) * | 2018-08-08 | 2018-12-21 | 三峡大学 | A kind of tissue returns blue liquid |
| CN109060488B (en) * | 2018-08-08 | 2021-03-23 | 三峡大学 | Tissue bluing liquid |
| 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 |
| CN111505311A (en) * | 2020-04-30 | 2020-08-07 | 深圳市锦欣医疗科技创新中心有限公司 | Biomarker for repeated embryo planting failure, screening method and application 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 |
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