CN111304153A - Method for separating trophoblast cells - Google Patents
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- CN111304153A CN111304153A CN201911298031.9A CN201911298031A CN111304153A CN 111304153 A CN111304153 A CN 111304153A CN 201911298031 A CN201911298031 A CN 201911298031A CN 111304153 A CN111304153 A CN 111304153A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The present invention provides a method for isolating trophoblast cells, the method comprising the steps of: screening the obtained specific antigen according to the specific antigens expressed on the surfaces of various types of trophoblast cells obtained by searching documents; confirming the expression of the antigen on the trophoblast cells through immunohistochemistry, and determining an antibody combination system through immunofluorescence; adding a coupling agent and an antibody combination into the immunomagnetic beads for coupling; collecting a placenta trophoblast sample, preparing the sample into a sample cell suspension, and adding immunomagnetic beads with specific antibodies for incubation; and washing the immunomagnetic beads incubated in the step to obtain the separated and purified placenta trophoblast cells. Compared with the traditional amniocentesis and villus puncture, the method has the advantages of no wound, short material taking time, low risk of infection and abortion and similar reliability of detection results.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to a kit for separating trophoblast cells and application thereof.
Background
Prenatal diagnosis is one of important means for reducing birth defects and improving population quality, and at present, the main means for clinical prenatal diagnosis is to perform karyotype analysis after obtaining a fetal sample through amniocentesis or umbilical vein puncture. The method is invasive in operation, long in analysis time, limited in diagnosis time in the middle and late pregnancy, low in acceptance of pregnant women and not favorable for clinical treatment.
With the development and the increasing maturity of high-throughput sequencing technology, the fetal free nucleic acid is subjected to large-scale parallel sequencing by means of the high-throughput sequencing technology, and the chromosome aneuploidy can be accurately detected, the current noninvasive prenatal diagnosis technology mainly refers to the technology, but the technology still has certain limitation and is mainly reflected in three aspects: firstly, only chromosome ploidy can be detected in the current clinical application, and the detection range is extremely limited; secondly, the placenta/maternal ploidy chimeric interference is caused, so that false positive is easily generated, and in addition, the false negative condition is difficult to completely avoid due to Z value calculation; thirdly, the detection capability of chromosome deletion is insufficient, and single base (point, insertion, deletion and the like) mutation conditions cannot be detected; fourthly, there is also individual variability in the content of fetal free nucleic acid.
Disclosure of Invention
The present invention is directed to overcoming the above-mentioned deficiencies of the prior art and to providing a method for isolating trophoblast cells.
In order to achieve the purpose, the invention adopts the technical scheme that: a method of isolating trophoblast cells, the method comprising the steps of;
(1) screening the obtained specific antigen according to the specific antigens expressed on the surfaces of various types of trophoblast cells obtained by searching documents;
(2) confirming the expression of the antigen of the step (1) on the trophoblast cells through immunohistochemistry, and determining an antibody combination system through immunofluorescence;
(3) adding a coupling agent and the antibody combination in the step (2) into immunomagnetic beads for coupling;
(4) collecting a placenta trophoblast sample, preparing the sample into a sample cell suspension, and adding the immunomagnetic beads coupled with the specific antibody prepared in the step (3) for incubation;
(5) and (4) washing the immunomagnetic beads incubated in the step (4) to obtain separated and purified placenta trophoblast cells.
Preferably, the antibody combination of step (2) is HLA-G + CK7 antibody, HLA-G + CK18 antibody and HLA-G + β -HCG antibody.
Preferably, the immunomagnetic beads in the step (3) are carboxyl magnetic beads, epoxy magnetic beads or tosyl magnetic beads.
Preferably, the incubation conditions of step (4) are: reacting at 2-8 deg.c for 20 min.
The invention also provides the application of the method for separating the trophoblast cells in paternity test.
The invention has the beneficial effects that: compared with the traditional amniocentesis and villus puncture, the method has the advantages of no wound, short material taking time, low risk of infection and abortion and similar reliability of detection results. The complete genome information of the fetus can be obtained, so that the detection of all genetic diseases is possible. The requirements on technicians and laboratory equipment are relatively low, the method can be developed in most medical institutions, and the method can be popularized in a large range.
Drawings
FIG. 1 is a schematic of FAM labeled channel 1, where "697 +" is the sorted sample, "697-" is the sample remaining after sorting, and "697" is the sample before sorting.
FIG. 2 is a schematic of HEX labeled channel 2, where "697 +" is the sorted specimen, "697-" is the specimen remaining after sorting, and "697" is the specimen before sorting.
FIG. 3 is a schematic of TAMRA labeled channel 3, where "697 +" is the specimen sorted, "697-" is the specimen remaining after sorting, and "697" is the specimen before sorting.
Fig. 4 is a schematic diagram of the ROX-labeled channel 4, where "697 +" is the sorted specimen, "697-" is the specimen remaining after sorting, and "697" is the specimen before sorting.
FIG. 5 is a schematic diagram showing the result of Y-STR detection of a specimen containing Y chromosome in sorted trophoblast cells.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1
First, antibody selection
According to the specific antigens expressed on the surfaces of various types of trophoblast cells obtained by searching the literature, the antigens are shown in a table 1:
table 1: screening the resulting antigen
The identification of trophoblast cells is not internationally unified, but the more approved molecular markers include Cytokeratin (CK), vimentin, human chorionic gonadotropin (β -hCG), human placental prolactin (hPL), matrix metalloproteinase 9(MMP9), and the like, all of which are expressed on extravillous trophoblast cells, amnion endothelial cells and fetal vascular wall endothelial cells in the embryo and maternal interface during the whole pregnancy, the trophoblast cells are cells with secretory characteristics and secrete a plurality of hormones, mainly β -hCG and hPL, wherein β -hCG is mainly a specific protein secreted by the trophoblast cells, and the isolated cell purity identification is carried out by research, CK-7 is expressed on all of the above 3 types of trophoblast cells, is not expressed on interstitial cells, but CK-7 is also expressed on the glandular epithelium, is not suitable for being used in uterus epithelial cells, is obviously expressed in uterine epithelial cells, and is taken as a sample with high-specificity of trophoblast antibody, and is taken as a high-specificity anti-CK antibody, and a high-7 antibody is expressed in a cytoplasm sample, and can be used for counteracting the identification of the trophoblast cells.
Therefore, based on the characteristics of each trophoblast cell, HLA-G, CK-7, CK-18, β -HCG were selected as the subjects and their locations in the cells were determined.
Determination of antibody combination System
The expression of the retrieved antigen on trophoblast cells was confirmed by immunohistochemistry, and the antibody combination system was determined by immunofluorescence. The specific steps and results are as follows:
1. preparing a cell wax block:
shaking the cervical exfoliated cells obtained from the cotton swab, placing the shaken cells in a 15ml centrifuge tube, and centrifuging at 2000rpm for 10 min. The supernatant was discarded, and 95% absolute ethanol was added thereto, followed by centrifugation at 2000rpm for 5 min. After the supernatant was discarded, 5% agar was added, and after it was solidified, dehydration was carried out in a dehydrator, and paraffin embedding was carried out to prepare a wax block.
2. Immunohistochemical staining:
dewaxing and hydrating the baked slide, performing antigen retrieval, boiling a sodium citrate buffer solution in an autoclave, putting the slide into the autoclave, timing for 3min after air injection, naturally cooling, and washing with PBS. PBS was removed, reagent A (endogenous peroxidase) was added, incubated at room temperature for 10min, and washed 3 times with PBS, each for 3 min. PBS was removed, reagent B (normal goat serum working solution) was added, incubated at room temperature for 10min, washed 3 times with PBS, each for 3 min. PBS was removed, reagent monoclonal antibody (PBS was used instead of monoclonal antibody in blank control), water bath at 37 ℃ for 1h, PBS was washed 3 times for 3min each. PBS was removed, reagent C (biotin-labeled goat anti-mouse IgG polymer working solution) was added, incubation was carried out at room temperature for 10min, and PBS was washed 3 times, each for 3 min. PBS was removed, reagent D (horseradish-enzyme-labeled streptavidin working solution) was added, incubation was carried out at room temperature for 10min, and PBS was washed 3 times, each for 3 min. PBS was removed, reagent DAB was added, incubation at room temperature for 10min, and color development was observed. Hematoxylin was stained for 2s, differentiated with hydrochloric acid and alcohol, and rewound with tap water. Dehydrated transparent, neutral gum sealing.
3. Immunofluorescence:
dewaxing and hydrating the baked slide, repairing antigens, putting the baked slide into a sodium citrate buffer boiling in an autoclave, timing for 3min after air injection, naturally cooling, washing by PBS, removing the PBS, adding diluted primary antibodies (a mouse anti-human CK7 monoclonal antibody, a mouse anti-human CK18 monoclonal antibody, a mouse anti-human β -HCG monoclonal antibody and a rabbit anti-human HLA-G monoclonal antibody which are purchased from abcam company), incubating at 4 ℃ overnight, washing by PBS for 3 times, washing for 3min each time, adding secondary antibodies (fluorescently-labeled goat anti-mouse and goat anti-rabbit antibodies) after water absorption paper is dried, washing for 3 times at 37 ℃ in a wet box for one hour, dripping DAPI 5min for 3min each time, washing for 4 times by PBS, drying by water absorption paper after 5min each time, sealing by a sealing liquid containing a fluorescence quencher, observing under a fluorescence microscope, determining HLA-G positioning in cell membranes, CK 395, CK 64, β -HCG, HCG 63 and HLA-6778 after immunohistochemical experiments, and determining HLA-G + HCG.
Third, DNA extraction and paternity test
Extracting DNA in the separated trophoblast cells and cervical exfoliated cells of the pregnant women, performing PCR amplification by using a read D-21 paternity test kit, performing capillary electrophoresis analysis on PCR products by using a 3500xL sequencer, performing fluorescence calibration on the instrument by using G5-Matrix Standard, writing corresponding Panels and bins files, and performing result analysis by using software GeneMapper ID version 3.0. The method comprises the following specific steps:
1. sorting trophoblast cells:
(1) and (3) uniformly mixing the cervical exfoliated cell preservation solution on a shaking and uniformly mixing device for 5 min.
(2) And taking out the preservation solution, placing the preservation solution into a 15ml centrifuge tube, adding 3ml of 1xPBS into a bottle of the preservation solution, shaking and uniformly mixing, taking out and placing the mixture into the 15ml centrifuge tube.
(3) Centrifuge at 3000rpm for 10min, and discard the supernatant.
(4) 1ml of 1 XPBST was added, mixed well and transferred to a 1.5ml EP tube, centrifuged at 3000rpm for 5min and the supernatant discarded.
(5) Repeat step 4 twice.
(6) Adding 200ul 0.5% Triton X-100, mixing, and permeating at room temperature for 20 min.
(7) Repeat step 4 three times.
(8) Adding primary antibodies, namely adding 200ul of the diluted mouse anti-human CK7 monoclonal antibody, the diluted mouse anti-human CK18 monoclonal antibody, the diluted mouse anti-human β -HCG monoclonal antibody and the diluted rabbit anti-human HLA-G monoclonal antibody (all the antibodies are purchased from abcam company), uniformly mixing, and standing at 4 ℃ overnight.
(9) Repeat step 4 three times.
(10) Adding a secondary antibody: the diluted biotin-labeled goat anti-rabbit and goat anti-mouse antibodies are 200ul, mixed evenly and reacted for 1h at 37 ℃.
(11) Step 4 was repeated three times and resuspended in 200ul buffer (DPBS + 0.1% BSA +2mM EDTA).
(12) After 25ul beads and 50ul buffer are fully mixed, the mixture is centrifuged at 600Xg for 10min, the supernatant is discarded, and after being resuspended by 25ul buffer, the mixture is added into the mixture obtained in the step (11).
(13) Reacting at 2-8 deg.c for 20 min.
(14) Add 1ml buffer and mix, after standing for 2min on the magnetic frame, discard the supernatant (leave 200ul contrast).
(15) Repeating step (14) two to three times.
(16) After adding 200ul of Buffer preheated at 37 ℃ for resuspension, 4ul of Release Buffer was added and mixed well.
(17) Blowing and beating for 5-10 times with a sample adding gun after 15min at room temperature, standing for 2min on a magnetic frame, and collecting supernatant.
(18) Adding 200ul buffer, beating by blowing for 5-10 times, standing on magnetic frame for 2min, and collecting supernatant.
(19) Repeating the step (18) three times to finally obtain the trophoblast cells.
Example 2
1. Extracting DNA from cervical exfoliated cells of pregnant women:
(1) 200ul of the cells from step 14 (remaining cells of cervical exfoliated cells with trophoblast cells removed) were centrifuged at 12000rpm for 3min after sorting.
(2) The supernatant was discarded and 200ul of solution P was added to resuspend the pellet.
(3) Adding 20ul of proteinase K and 200ul of solution L, and mixing.
(4) Bathing at 56 deg.C for 20min, and mixing by reversing.
(5) Adding 200ul of anhydrous ethanol, mixing, transferring to adsorption column, centrifuging at 10000rpm for 1min, and discarding waste liquid in the collection tube.
(6) 500ul of W1 was added and centrifuged at 10000rpm for 1min, and the waste liquid in the collection tube was discarded.
(7) 500ul of W2 was added and centrifuged at 10000rpm for 1min, and the waste liquid in the collection tube was discarded.
(8) 500ul of W2 was added and centrifuged at 10000rpm for 1min, and the waste liquid in the collection tube was discarded.
(9) The tube was centrifuged at 12000rpm for 3min and the collection tube was discarded.
(10) Placing the adsorption column into a new 1.5ml centrifuge tube, opening the cover, standing for 2min, adding 50ul of TE, standing for 5min, centrifuging at 12000rpm for 2min, and discarding the column.
(11) The concentration and purity of the extracted DNA were determined.
2. Performing STR detection
(1) PCR amplification was performed in the following manner as shown in Table 2:
table 2: PCR amplification system
PCR reaction procedure: 50 ℃ for 10 min; 96 ℃ for 4 min; (94 ℃, 5 sec; 60 ℃, 1min, 10 sec). times.27 cycles; 30min at 60 ℃; and preserving at 15 ℃.
(2) STR detection result
0.3ul of Hidi 8.7ul internal reference is uniformly mixed according to the instruction, 1ml of amplified product is added, a 3500xL sequencer is used for carrying out capillary electrophoresis analysis on the PCR product, G5-Matrix Standard is used for carrying out fluorescence calibration on the instrument, corresponding Panels and bins files are compiled at the same time, and software GeneMapper ID version 3.0 is used for carrying out result analysis, and the result is shown in figures 1-4.
(3) Y-STR detection
The sample with the Y chromosome detected in the STR is subjected to Y-STR detection by using a read micro 40Y kit, and the result is shown in figure 5, and the result shows that male DNA exists in the sample.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A method of isolating trophoblast cells, comprising the steps of:
(1) screening the obtained specific antigen according to the specific antigens expressed on the surfaces of various types of trophoblast cells obtained by searching documents;
(2) confirming the expression of the antigen of the step (1) on the trophoblast cells through immunohistochemistry, and determining an antibody combination system through immunofluorescence;
(3) adding a coupling agent and the antibody combination in the step (2) into immunomagnetic beads for coupling;
(4) collecting a placenta trophoblast sample, preparing the sample into a sample cell suspension, and adding the immunomagnetic beads coupled with the specific antibody prepared in the step (3) for incubation;
(5) and (4) washing the immunomagnetic beads incubated in the step (4) to obtain separated and purified placenta trophoblast cells.
2. The method for isolating trophoblast cells according to claim 1, wherein the antibody combination of step (2) is an HLA-G + CK7 antibody, an HLA-G + CK18 antibody and an HLA-G + β -HCG antibody.
3. The method for isolating trophoblast cells according to claim 1, wherein the immunomagnetic beads of step (3) are carboxyl magnetic beads, epoxy magnetic beads or tosyl magnetic beads.
4. The method for isolating trophoblast cells according to claim 1, wherein the incubation conditions of step (4) are: reacting at 2-8 deg.c for 20 min.
5. Use of the method of isolating trophoblast cells of claim 1 in paternity testing.
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