CN111122520A - Embryo implantation detection kit and application and use method thereof - Google Patents
Embryo implantation detection kit and application and use method thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses an embryo implantation detection kit and an application and a use method thereof. Discloses application of the decidua uteri NK cells Siglec-7 as a detection index in an embryo detection kit, wherein the decidua uteri NK cells Siglec-7 in a decidua uteri sample are detected as the detection index. The kit comprises a buffer solution, a fixing agent, a dehydrating agent, an embedding agent, a dewaxing agent, an antigen repairing agent, a fluorescent reagent, a primary antibody, a secondary staining solution and a blocking tablet. The embryo implantation capability is detected by using the detection tool disclosed by the invention, so that the implantation capability of the endometrial embryo can be further evaluated. Provides a possible detection and clinical diagnosis mode for the female patient with the unknown clinical spontaneous abortion and provides a powerful reference for discussing influence factors which are not disclosed by the patient with the unknown spontaneous abortion.
Description
Technical Field
The invention relates to a kit and application thereof, in particular to an embryo implantation detection kit taking uterine decidua NK cells Siglec-7 as a detection index, and application and a use method thereof.
Background
The incidence of recurrent spontaneous abortion in pregnancy is 1% -5%, and the etiology is complex. Except for the reasons of chromosome abnormality, endocrine diseases, genital tract abnormality and the like, the reason of 70 percent of repeated spontaneous abortion is still unknown. Conventional imaging and morphological tests do not reveal the functional state of the endometrium.
The invention patent with the application number of CN201710649484.6 and named as a method and a kit for detecting chromosome abnormality before embryo implantation is disclosed by the national intellectual property office in 2017, 09 and 22, and particularly, the traditional library construction steps are optimized by improving a single cell amplification method, so that the detection accuracy is improved, and the false positive rate of chromosome karyotypes and CNV is reduced.
The invention discloses an invention patent with application number CN201710569713.3 named as embryo implantation pre-chromosome abnormality detection kit by the national intellectual property office in 2017, 10, 20 and discloses an embryo implantation pre-chromosome abnormality detection kit, which consists of a single cell amplification reagent, a fragmentation reagent, a library construction reagent, a DNA purification reagent and a negative and positive quality control product. The invention also discloses a detection method of the kit. Compared with the prior art, the method for detecting the chromosome before implantation by using the embryo blastula stage cells is established, an innovative single cell amplification technology is adopted, the single cell amplification uniformity (more than or equal to 97%) and the coverage (more than or equal to 92%) are improved, and the method has the advantages of quick detection, wide coverage, high resolution, high precision and moderate flux.
The current research finds that abortion caused by unknown repeated reasons is mainly related to abnormal immunological tolerance of the mother tread. Aiming at the detection of the immune tolerance abnormality of the female tire tread, no efficient and accurate detection mode exists at present.
Disclosure of Invention
The invention aims to solve the problem that how to detect the immune tolerance abnormality of the tread of a mother tyre does not exist in the prior art, provides an embryo implantation detection kit taking the uterine decidua NK cells Siglec-7 as a detection index and an application and a use method thereof, and the kit can rapidly detect the immune tolerance abnormality of the tread of the mother tyre through the expression detection of the Siglec-7.
In order to achieve the above object, the technical solution of the present invention is as follows:
according to the invention, the uterine decidua NK cell Siglec-7 is used as a detection index for the first time, and is specifically applied to the embryo implantation detection kit, so that the embryo implantation detection kit is obtained, and the activity of Siglec-7 in a uterine decidua tissue sample is detected to serve as a basis for judging embryo implantation capacity.
The kit is used for detecting the uterine decidua NK cells Siglec-7 in the uterine decidua sample as a detection index.
The embryo implantation detection kit comprises a reagent for detecting the expression of the decidua uteri NK cell Siglec-7.
The kit comprises a positive control, a negative control, a buffer solution, a fixing agent, a dehydrating agent, an embedding agent, a dewaxing agent, an antigen repairing agent, a fluorescent reagent, a primary antibody, a secondary antibody, a complex dye solution and a blocking tablet.
The positive control is an abnormal decidua uteri tissue with high expression of Siglec-7, and the negative control is a normal decidua uteri tissue with low level of Siglec-7.
A low level as referred to herein means that there is little or negligible positive expression.
The kit comprises PBS buffer solution, PBST buffer solution, PBT buffer solution, 4% paraformaldehyde, tetrahydrofuran, xylene I, xylene II, sodium citrate, citric acid and 30% H2O2Bovine serum albumin and DAPI.
The preparation method of the reagent comprises the following steps: adding 3g of sodium citrate and 0.4g of citric acid into 1000ml of distilled water; the conventional preservation concentration of H2O2 is 30%, the H2O2 is preserved in a sealed and dark way, and the H2O2 is prepared by self when in use: 2ml of 30 percent H2O2 is added into 18ml of distilled water to be prepared for use; bovine serum albumin is powder, is stored at 4 ℃, and needs to be prepared by self when in use: PBT dissolve 0.3g BSA, end up to 10ml with PBT constant volume, PBT =0.1% Triton X-100 in PBS; DAPI was stored at-20 ℃ in 1:50 in PBS.
The invention also provides a using method of the embryo implantation detection kit, which comprises the following steps:
x1. preprocessing the sample;
x2. dehydrating;
x3. embedding;
x4. paraffin sections;
x5, baking slices and baking slices;
x6, dewaxing and rehydration;
x7, antigen retrieval;
x8. removal of endogenous enzymes;
x9, sealing;
x10. primary antibody incubation and secondary antibody incubation;
x11, counterdyeing;
x12. mounting and comparing with positive control and negative control.
The sample pretreatment of the invention comprises the following steps: taking 5-15 g of a molt tissue sample, and removing villi on the surface of the molt; and washing the mixture for 2-3 times by using PBS buffer solution, and then fixing the mixture in 4% paraformaldehyde for 8-12 hours.
The dehydration of the invention is as follows: the sample treated in X1 was dehydrated by passing through pure water for 2h, 50% ethanol for 2h, and 75% ethanol in this order.
The embedding of the invention is as follows: samples from X2 were dehydrated with 75% ethanol and tetrahydrofuran at a 1:1 volume ratio and then embedded routinely.
The paraffin section of the invention comprises: the sample obtained by X3 is cut into thin slices with the thickness of 3-6 μm.
The baking sheet and the baking sheet of the invention are as follows: and (3) placing the slices with the thickness of 3-6 microns obtained in the X4 in a system at 37 ℃ and 65 ℃ for 8-12 h respectively.
The dewaxing and rehydration method comprises the following steps: sequentially dewaxing the slices in the X5 by using xylene I and xylene II for 10-15 min; then carrying out 100% ethanol I rehydration for 2-5 min, 100% ethanol II rehydration for 2-5 min, 90% ethanol I rehydration for 2-5 min, 90% ethanol II rehydration for 2-5 min and 70% ethanol rehydration for 2-5 min in sequence; and finally, rehydrating in distilled water twice for 2-5 min each time.
The antigen retrieval of the invention is as follows: taking an antigen retrieval box, and adding 0.01mol/L sodium citrate into the antigen retrieval box, wherein the pH value is 6.0; it can be used for antigen retrieval.
The method for removing endogenous enzymes comprises the following steps: the flakes from the X6 treatment were placed in a wet box and 3% H was added2O2Incubating for 10 minutes at room temperature, washing for 3-5 times with PBS buffer, 5-10 min each time, washing for 10min with 0.1% Triton X-100, and washing for 3-5 times with PBST buffer, 5-10 min each time.
The closure of the invention is as follows: adding 3% BSA into the wet box, and incubating and sealing for 30-45 min at room temperature.
The primary antibody incubation and the secondary antibody incubation are as follows: keeping the Purified anti-human CD3284 ℃ for 8-12 h, taking out, rewarming for 45min, washing for 3-5 times by PBST buffer solution, and each time for 5-10 min; incubating for 1-2 h at room temperature or 4 ℃ by Mouse 594; PBST buffer washing 3 times, each time 5 ~ 10 min.
The counterdyeing of the invention comprises the following steps: staining the cell nucleus with a fluorescent reagent at room temperature for 5 minutes; PBST buffer washing 3 ~ 5 times, each time 5 ~ 10 min.
The seal sheet of the invention is observed as follows: and (5) observing after mounting, observing the dyeing degree, and judging whether the immune tolerance of the female tread of the sample to be detected is abnormal or not by combining the results of positive control and negative control.
The invention has the following beneficial effects:
the invention provides a novel detection tool for detecting embryo implantation ability and a novel detection method for evaluating embryo implantation ability. The positive rate of fluorescence staining Siglec-7 of the tissue section of the endometrial sample of the patient with unexplained abortion is obviously increased compared with the normal level, and the intimal state of the patient is not favorable for embryo implantation.
The invention provides a new detection index for detecting embryo implantation capability and provides application of siglec-7 as a detection index in an embryo implantation detection kit. Through the detection of siglec-7 expression, the implantation capacity of the endometrial embryo can be further evaluated. Provides a possible detection and clinical diagnosis mode for the female patient with the unknown clinical spontaneous abortion and provides a powerful reference for discussing influence factors which are not disclosed by the patient with the unknown spontaneous abortion.
Thirdly, whether the decidua uteri sample is normal or not is judged by detecting whether the decidua uteri NK cell Siglec-7 in the decidua uteri sample is active or not, if the decidua uteri sample is active, fluorescence can be generated by separation of a fluorescence group, and the corresponding decidua uteri sample is abnormal; i.e. the intimal state is not conducive to embryo implantation. If the group is not active, the fluorescence-carrying group can not be separated to generate fluorescence, and the corresponding molting sample of uterus is normal, namely the endometrial state is favorable for embryo implantation.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
The current research finds that abortion caused by unknown repeated reasons is mainly related to abnormal immunological tolerance of the mother tread. Natural killer cells, NK cells, account for 80% of uterine lymphocytes and play an important role in uterine pregnancy immunity. Siglec-7, a sialic acid binding immunoglobulin-like lectin 7, is an important inhibitory receptor on the surface of NK cells, distributed mainly in peripheral blood NK cells.
We have found that: siglec-7 is expressed in human endometrium NK cells, and abnormal expression will result in disruption of the balance between embryonic trophoblast cells and the inner membrane. However, the corresponding diagnosis indexes of the NK cell function of the uterus are not available at present, and meanwhile, the evaluation of the implantation capability of the endometrial embryo has limitations. Therefore, Siglec-7 can be used as a molecular marker to evaluate the implantation ability of the inner membrane embryo.
The invention provides application of the uterine decidua NK cells Siglec-7 as a detection index in an embryo implantation detection kit.
The positive rate of fluorescence staining Siglec-7 of the tissue section of the endometrial sample of the patient with unexplained abortion is obviously increased compared with the normal level, and the intimal state of the patient is not favorable for embryo implantation.
Example 2
The invention provides an embryo implantation detection kit, which takes the expression degree of decidua uteri NK cells Siglec-7 in a decidua uteri sample as a detection index.
Further, the embryo implantation detection kit comprises a reagent for detecting the expression of the decidua uteri NK cell Siglec-7.
Specifically, the embryo implantation detection kit comprises: PBS buffer solution, PBST buffer solution, PBT buffer solution, 4% paraformaldehyde, tetrahydrofuran, xylene I, xylene II, sodium citrate, citric acid and 30% H2O2Bovine serum albumin and DAPI.
The preparation method of the reagent comprises the following steps: adding 3g of sodium citrate and 0.4g of citric acid into 1000ml of distilled water; the conventional preservation concentration of H2O2 is 30%, the H2O2 is preserved in a sealed and dark way, and the H2O2 is prepared by self when in use: 2ml of 30 percent H2O2 is added into 18ml of distilled water to be prepared for use; bovine serum albumin is powder, is stored at 4 ℃, and needs to be prepared by self when in use: PBT dissolve 0.3g BSA, end up to 10ml with PBT constant volume, PBT =0.1% Triton X-100 in PBS; DAPI was stored at-20 ℃ in 1:50 in PBS.
Example 3
The invention also provides a detection method of the embryo detection kit, which comprises the following steps:
x1. sample pretreatment
Taking 5-15 g of a molt tissue sample, and removing villi on the surface of the molt; and washing the mixture for 2-3 times by using PBS buffer solution, and then fixing the mixture in 4% paraformaldehyde for 8-12 hours.
X2. dehydration
The sample treated in X1 was dehydrated by passing through pure water for 2h, 50% ethanol for 2h, and 75% ethanol in this order.
X3. embedding
Samples from X2 were dehydrated with 75% ethanol and tetrahydrofuran at a 1:1 volume ratio and then embedded routinely.
X4. Paraffin section
The sample obtained by X3 is cut into thin slices with the thickness of 3-6 μm.
X5. baking sheet and baking sheet
And (3) placing the slices with the thickness of 3-6 microns obtained in the X4 in a system at 37 ℃ and 65 ℃ for 8-12 h respectively.
X6. dewaxing and rehydration
Sequentially dewaxing the slices in the X5 by using xylene I and xylene II for 10-15 min; then carrying out 100% ethanol I rehydration for 2-5 min, 100% ethanol II rehydration for 2-5 min, 90% ethanol I rehydration for 2-5 min, 90% ethanol II rehydration for 2-5 min and 70% ethanol rehydration for 2-5 min in sequence; and finally, rehydrating in distilled water twice for 2-5 min each time.
X7. antigen retrieval
Taking an antigen retrieval box, and adding 0.01mol/L sodium citrate into the antigen retrieval box, wherein the pH value is 6.0; it can be used for antigen retrieval.
X8. removal of endogenous enzymes
The flakes from the X6 treatment were placed in a wet box and 3% H was added2O2Incubating for 10 minutes at room temperature, washing for 3-5 times with PBS buffer, 5-10 min each time, washing for 10min with 0.1% Triton X-100, and washing for 3-5 times with PBST buffer, 5-10 min each time.
X8. sealing
Adding 3% BSA into the wet box, and incubating and sealing for 30-45 min at room temperature.
X9 Primary antibody incubation and Secondary antibody incubation
Keeping the Purified anti-human CD3284 ℃ for 8-12 h, taking out, rewarming for 45min, washing for 3-5 times by PBST buffer solution, and each time for 5-10 min; incubating for 1-2 h at room temperature or 4 ℃ by Mouse 594; PBST buffer washing 3 times, each time 5 ~ 10 min.
X10. counterdyeing
Staining the cell nucleus with a fluorescent reagent at room temperature for 5 minutes; PBST buffer washing 3 ~ 5 times, each time 5 ~ 10 min.
X11. sealing sheet
And (5) observing after mounting, observing the dyeing degree, and judging whether the immune tolerance of the female tread of the sample to be detected is abnormal or not by combining the results of positive control and negative control.
If the active gene exists, the separation with the fluorescent group can generate fluorescence, and the corresponding uterine decidua sample is abnormal; i.e. the intimal state is not conducive to embryo implantation. If the group is not active, the fluorescence-carrying group can not be separated to generate fluorescence, and the corresponding molting sample of uterus is normal, namely the endometrial state is favorable for embryo implantation.
Examples 4 to 8
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (8)
1. Application of uterus decidua NK cells Siglec-7 as a detection index in an embryo implantation detection kit.
2. An embryo implantation detection kit according to claim 1, characterized in that: the kit is used for detecting the uterine decidua NK cells Siglec-7 in the uterine decidua sample as a detection index.
3. The embryo implantation detection kit of claim 2, wherein: the kit comprises a reagent for detecting the expression of the decidua uteri NK cell Siglec-7.
4. The embryo implantation detection kit according to claim 2 or 3, wherein: the kit comprises a positive control, a negative control, a buffer solution, a fixing agent, a dehydrating agent, an embedding agent, a dewaxing agent, an antigen repairing agent, a fluorescent reagent, a primary antibody, a secondary antibody, a complex dye solution and a blocking tablet.
5. The embryo implantation detection kit of claim 4, wherein: the positive control is abnormal decidua uteri tissue with high expression of Siglec-7, and the negative control is normal decidua uteri tissue with low level of Siglec-7.
6. The embryo implantation detection kit according to claim 2 or 3, wherein: the kit comprises PBS buffer solution, PBST buffer solution, PBT buffer solution, 4% paraformaldehyde, tetrahydrofuran, xylene I, xylene II, sodium citrate, citric acid and 30% H2O2Bovine serum albumin and DAPI.
7. The method of using the embryo implantation test kit of claim 4, wherein: the method comprises the following steps:
x1. preprocessing the sample;
x2. dehydrating;
x3. embedding;
x4. paraffin sections;
x5, baking slices and baking slices;
x6, dewaxing and rehydration;
x7, antigen retrieval;
x8. removal of endogenous enzymes;
x9, sealing;
x10. primary antibody incubation and secondary antibody incubation;
x11, counterdyeing;
x12. mounting and comparing with positive control and negative control.
8. The method of using the embryo implantation test kit of claim 7, wherein:
the sample pretreatment comprises the following steps: taking 5-15 g of a molt tissue sample, and removing villi on the surface of the molt; washing with PBS buffer solution for 2-3 times, and fixing in 4% paraformaldehyde for 8-12 h;
the dehydration comprises the following steps: dehydrating the sample obtained by processing in the X1 by using pure water for 2h, 50% ethanol for 2h and 75% ethanol in sequence;
the embedding is as follows: dehydrating the sample obtained by processing in X2 by using 75% ethanol and tetrahydrofuran in a volume ratio of 1:1, and then conventionally embedding;
the paraffin section is as follows: cutting the sample obtained by X3 into slices with the thickness of 3-6 mu m;
the baking sheet and the baking sheet are as follows: placing the slices with the thickness of 3-6 microns obtained in the X4 in a system at 37 ℃ and 65 ℃ for 8-12 hours respectively;
dewaxing and rehydration: sequentially dewaxing the slices in the X5 by using xylene I and xylene II for 10-15 min; then carrying out 100% ethanol I rehydration for 2-5 min, 100% ethanol II rehydration for 2-5 min, 90% ethanol I rehydration for 2-5 min, 90% ethanol II rehydration for 2-5 min and 70% ethanol rehydration for 2-5 min in sequence; finally, rehydrating the distilled water twice for 2-5 min each time;
the antigen retrieval is: taking an antigen retrieval box, and adding 0.01mol/L sodium citrate into the antigen retrieval box, wherein the pH value is 6.0; performing antigen retrieval;
the removal of endogenous enzymes is: the flakes from the X6 treatment were placed in a wet box and 3% H was added2O2Incubating for 10 minutes at room temperature, washing for 3-5 times with PBS buffer, 5-10 min each time, washing for 10min with 0.1% Triton X-100, and washing for 3-5 times with PBST buffer, 5-10 min each time;
the sealing is as follows: adding 3% BSA into the wet box, and incubating and sealing for 30-45 min at room temperature;
the primary antibody incubation and the secondary antibody incubation are as follows: keeping the Purified anti-human CD3284 ℃ for 8-12 h, taking out, rewarming for 45min, washing for 3-5 times by PBST buffer solution, and each time for 5-10 min; incubating for 1-2 h at room temperature or 4 ℃ by Mouse 594; PBST buffer solution washing 3 times, each time 5 ~ 10 min;
the counterdyeing comprises the following steps: staining the cell nucleus with a fluorescent reagent at room temperature for 5 minutes; PBST buffer solution washing 3-5 times, each time for 5-10 min;
the mounting observed was: and (5) observing after mounting, observing the fluorescence degree, and judging whether the immune tolerance of the female tread of the sample to be detected is abnormal or not by combining the results of positive control and negative control.
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| CN113804661A (en) * | 2021-08-12 | 2021-12-17 | 深圳市锦欣医疗科技创新中心有限公司 | Kit and method for evaluating endometrial receptivity |
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