Patent Application
20210231679
The present application relates to the technical field of biological cell identification, and in particular to a marker, an identification method, a detection kit of fetal trophoblast cells and use thereof.
At present, no passable therapy is available for genetic diseases with chromosomal abnormalities. Therefore, first-trimester detection of abnormalities of fetal genetic information through prenatal diagnosis and active intervention can effectively reduce the rate of birth defects. Currently, prenatal diagnosis methods used in clinical practice are mainly invasive examinations, such as chorionic villus biopsy, amniocentesis, and fetal cord blood testing. However, these invasive examinations cause a risk of infection and miscarriage. Although pre-implantation prenatal diagnosis can be performed on test tube babies, it is only suitable for test tube babies and technically demanding. The non-invasive examinations currently used in clinical practice include B-ultrasound and maternal serum biochemical examinations, but these methods are only auxiliary screening methods of prenatal genetic diseases with limited scope of application rather than prenatal diagnosis methods. Therefore, non-invasive prenatal diagnosis in the first trimester has attracted more and more attention.
The methods reported so far mainly included separating fetal cells and fetal episomal gene fragments from the peripheral blood of pregnant women for detection. Fetal cells in the peripheral blood of pregnant women include fetal nucleated red blood cells and fetal trophoblast cells. However, because the number of fetal cells in the peripheral blood of pregnant women is extremely small and volatile, it is often difficult to isolate a large number of fetal cells for reliable detection. The episomal gene fragments in the peripheral blood of pregnant women have been approved for the detection of triploids of some chromosomes (such as chromosomes 13, 18, 21, etc.). However, the complete genome information of the fetus is unavailable, so the types of genetic diseases that can be detected are very limited, and it is a screening method rather than a diagnostic method.
In 1971, Shettles et al. found fetal trophoblast cells in the cervical mucus of women in the first trimester, which initiated the study of fetal trophoblast cells in the cervix for prenatal diagnosis. The exfoliated trophoblast cells originate from the current pregnancy and are not affected by the previous pregnancy. The trophoblast cells so obtained contain the complete genomic information of the fetus and can be used to detect the chromosomal state of the fetus in the current pregnancy. More importantly, trophoblast cells are relatively rich in cervix uteri, and they can be present as early as in the first trimester, which paves the way for prenatal first-trimester diagnosis. Further studies have shown that trophoblast cells express human leukocyte antigen G (HLA-G), which can be used for the enrichment and separation of fetal trophoblast cells in cervical mucus. Other studies have also reported other markers for identifying fetal trophoblast cells, such as CK7, β-HCG and so on. However, because the cervical exfoliated cells collected in the cervix are complex, consisting of squamous epithelial cells, cervical columnar epithelial cells, cervical glandular epithelial cells, fibroblasts, red blood cells, white blood cells, lymphocytes, and a small amount of trophoblast cells. The markers and detection methods currently available are difficult to accurately detect fetal trophoblast cells in such a complicated cell sample. Therefore, there is a need to identify more sensitive and accurate markers and to develop corresponding detection methods and kits.
The present application provides a marker, an identification method, a detection kit of fetal trophoblast cells and use thereof, which solves the problems of poor sensitivity and low accuracy in identification of fetal trophoblast cells.
In order to solve the above technical problems, the present present application provides the use of hexokinase 2 (HK2) in the identification or isolation of fetal trophoblast cells, wherein HK2 is used as a biomarker of fetal trophoblast cells.
Optionally, the fetal trophoblast cells are fetal trophoblast cells in cervical mucus.
The hypoxic environment in the uterus in early pregnancy was known to facilitate cell proliferation and angiogenesis in the placenta, and cause the expression of glycolysis-related genes in trophoblast cells. During embryonic development, the extra-villous trophoblast cells cast off to the cervical canal and are wrapped in cervical mucus. These cast-off trophoblast cells obtain energy mainly through glycolysis. On the other hand, it is also known that glucose is firstly phosphorylated under the catalysis of hexokinase (HK) after being transported into cells by glucose transporter (GLUT) on the cell membrane surface during glycolysis. There are 4 subtypes of human HK that have been discovered, which are coded by HK1, HK2, HK3 and HK4. HK1 is widely expressed in almost all mammalian tissues. HK2 is usually expressed in insulin-sensitive tissues (such as fat, bone, and myocardium), but it is highly expressed in a variety of tumor tissues and regulates glucose metabolism by binding with ion channels on the outer mitochondrial membrane. HK3 is often expressed at low levels, while the expression of HK4 is restricted to the pancreas and liver.
The present application also provides a method for identifying fetal trophoblast cells, comprising staining a sample containing fetal trophoblast cells with a fluorescently labeled HK2 antibody substance, and then performing fluorescence detection, and HK2 positive cells are identified as target cells.
If the value of fluorescence intensity is above a threshold, the result is set positive, and if the value below the threshold, the result negative. The threshold can be determined by normal methods and technical measures in the art. For example, when the background is basically the same, an absolute value can be directly set to determine the threshold, while when the background influence is strong, the average value plus quintuple standard deviation is used to determine the threshold.
Optionally, the sample is obtained from processed cervical mucus or peripheral blood of a pregnant woman. The process is preparing liquefied cervical mucus or the enriched peripheral blood into a single-cell suspension, and spreading the single-cell suspension, in the form of single cells, onto a microwell array chip or a glass slide, preferably a microwell array chip.
Optionally, the fluorescently labeled HK2 antibody substance is an HK2 antibody, or a combination of HK2 primary antibody and secondary antibody, wherein the HK2 antibody is labeled with fluorescein or other fluorescent substances, and the HK2 secondary antibody in the combination of the HK2 primary antibody and the secondary antibody is labeled with fluorescein or other fluorescent substances.
Optionally, the identification method further includes: staining the sample with a nuclear stain and performing fluorescence detection, wherein the cells positive for HK2 and positive for nuclear staining are identified as target cells. Nuclear staining is used to eliminate the interference of non-cellular substances on the fluorescence detection result.
After the nucleus is stained, cells with nucleus can be visually identified, and clearly distinguished from non-nuclear substance which, however, does not develop color.
Optionally, the identification method further includes: staining the sample with a nuclear stain and observing the morphology of the nucleus, and excluding cells with regular form of round or elliptical nucleus from target cells. Experiments showed that cells with round or elliptical nucleus are not fetal trophoblast cells.
The present application also provides a kit for detecting fetal trophoblast cells, which includes a fluorescently labeled HK2 antibody substance.
Optionally, the kit further includes a nuclear stain.
Optionally, the detection kit for fetal trophoblast cells further includes a microwell array chip to disperse the sample in single cells, facilitating subsequent detection and separation
Optionally, the fluorescently labeled HK2 antibody substance is HK2 antibody, or a combination of HK2 primary antibody and secondary antibody, wherein the HK2 antibody is labeled with fluorescein or other fluorescent substances, and the HK2 secondary antibody in the combination of the HK2 primary antibody and the secondary antibody is labeled with fluorescein or other fluorescent substances.
Optionally, the nuclear stain is selected from nuclear fluorescent dyes, preferably 4′,6-diamidino-2-phenylindole (DAPI) or Hoechst series dyes.
Optionally, the detection kit for fetal trophoblast cells is used to detect fetal trophoblast cells in a biological sample of a pregnant woman, wherein the biological sample is a cervical mucus sample or a peripheral blood sample.
The present application also provides the use of the above-mentioned fetal trophoblast cell markers in the manufacture of a product for diagnosing fetal genetic diseases during pregnancy.
The present application described herein can be used to sensitively and reliably detect fetal trophoblast cells in cervical mucus samples or blood samples of women in the first trimester, and is especially useful for prenatal diagnosis in the first trimester.
The marker, the identification method, the detection kit of fetal trophoblast cells and use thereof are described in the following in conjunction with examples. It should be understood that these examples are only used to illustrate present application and not to limit the scope of present application.
As shown in
(a) Collecting cervical mucus samples: using a cell brush to collect cervical mucus from women in the first trimester;
(b) Digesting mucus: cervical mucus is liquefied and prepared into a single-cell suspension.
(c) Cell spreading: spreading the cells in the form of single cells, for example, spreading on a microwell array chip or glass slide;
(d) Cell fixation, permeabilization, and staining: the cells are fixed, permeabilized and stained to identify trophoblast cells derived from the fetus, wherein the reagent used for staining is a certain concentration of fluorescently labeled hexokinase 2 (HK2) antibody substance;
(e) Imaging and image analysis: fluorescence analysis is performed to determine HK2 positive cells with high HK2 expression, which is preliminarily determined as fetal trophoblast cells;
(f) Collecting the target cells: the target cells were obtained through various equipment, such as a micromanipulator;
(g) Gene amplification and sequencing: single-cell sequencing or other detection methods known in the art for determining fetal cells is performed to determine whether the cells are fetal-derived trophoblast cells.
The fluorescently labeled HK2 antibody substance is a HK2 antibody directly labeled with fluorescein or other fluorescent substances (such as quantum dots), or a combination of a non-labeled HK2 primary antibody and a secondary antibody labeled with fluorescein or other fluorescent substances.
In an embodiment, the threshold for HK2 positivity is the average value plus quintuple standard deviation of the HK2 signal values of all or part of the cells in the sample.
Further, in order to reduce or eliminate the interference of some non-cellular impurities (e.g. cell debris, bubbles, non-cellular particles, etc.) contained in, or introduced into the sample that adsorb the fluorescent-labeled antibody substance of HK2 with the fluorescence analysis, the embodiment of the method of present application further includes using the nuclear dye (e.g. nuclear fluorescent dyes, preferably 4′,6-diamidino-2-phenylindole (DAPI) or Hoechst series dyes) to stain the sample cells simultaneously with, before or after the HK2 staining. In this case, cells that are positive for both nuclear staining and HK2 staining may be preliminarily identified as fetal trophoblast cells.
In addition to eliminating the interference of non-cellular impurities, the nuclear dye staining described above can also show the morphological characteristics of the cell nucleus to help further improve the accuracy of determining fetal trophoblast cells. The present inventor found that HK2-positive and DAPI-positive cells include cells with different nucleus morphologies, in which most of the cells with irregular (for example, not basically round or elliptical) nucleuses were identified as fetal trophoblast cells by genetic analysis, and the cells with substantially round or irregular nucleuses were usually identified as not fetal trophoblast cells. Single-cell sequencing has verified that the accuracy of the method for detecting fetal trophoblast cells provided by present application can be as high as 70-85%.
Based on the above detection method, present application also provides a kit for isolating and identifying fetal trophoblast cells from a cervical mucus sample of a pregnant woman. In an embodiment, the kit includes the above-mentioned hexokinase 2 (HK2) fluorescently labeled antibody substance, a nuclear stain and a microwell array chip. The fluorescently labeled HK2 antibody may be, but is not limited to, FITC labeled HK2 antibody, and the nuclear stain may be 4′,6-diamidino-2-phenylindole (DAPI). The microwell array chip includes a plurality of microwells that can accommodate cells and can be addressed to accurately locate and obtain target cells.
Since trophoblast cells are relatively rich in cervical mucus and there are usually no red blood cells, they can generally be detected without the need of enrichment process.
Although present application is achieved using cervical mucus samples, a skilled person in the art can expect that the method and kit of present application can also be applied to the detection of fetal trophoblast cells from peripheral blood samples of pregnant women after reading the specification. In the case of peripheral blood, since the number of leukocytes is large while the number of trophoblast cells is very small, the trophoblast cells may optionally be appropriately enriched and then subjected to detection steps such as staining and fluorescence imaging. Enrichment operations such as the removal of red blood cells by red blood cell lysis, density gradient centrifugation, or the removal or partial removal of white blood cells based on CD45 antibody-coated magnetic spheres are known in the art.
The following examples describe in detail the whole process and analysis results of the detection and identification of fetal trophoblast cells using a specific detection kit and the above-mentioned identification method of fetal trophoblast cells to illustrate the effectiveness and superiority of present application.
Identification and Analysis of Fetal Cells in Cervical Mucus for Prenatal Diagnosis
This embodiment includes the following steps:
(1) Using a sterile cell brush to obtain the cervical mucus from 5 women in early pregnancy (6-8 weeks of gestation) (all patients gave informed consent). The brush rotates about 10 times, and the cell brush with the mucus collected by the brush was stored in 2 ml Hank's Balanced Salt Solution (HBSS), respectively numbered as Samples 1-5, and placed in a 4-Degree ice box and transported to the laboratory.
(2) The cell brush was taken out and placed in 0.25% pancreatin (containing chelating agent) and incubated at 37° C. for 3-5 minutes to digest the mucus, and the cell dispersion was observed under a microscope. Fetal bovine serum (FBS) was used to stop the digestion of pancreatin when cells were sufficiently dispersed.
(3) The 400 g suspension obtained in the previous step was centrifuged for 5 minutes and washed with HBSS 3 times. If the cell suspension contains large-sized squamous epithelial cells or impurities, as observed under a microscope, the cell suspension can be filtered with a filter with a pore size of 40-70 μm.
(4) The prepared cell suspension was counted and loaded on the microwell array chip at a ratio of 1-1.5×105 cells per microwell array chip. In the embodiment, the microwell array chip containing a total of 400 numbered areas, and a total of about 140,000 addressable microwells was used to accommodate and locate cells, wherein the diameter of each micropore was 30 μm, so that the cells were evenly distributed on the chip and sink into the micropores of the chip. As shown in
(5) 200 μl 2% paraformaldehyde (PFA) was added to the chip and the cells were fixed at room temperature for 15 minutes, and then the chip was rinsed 3-5 times with 400 μl of phosphate buffered saline (PBS).
(6) PFA-fixed cells were permeabilized with 200 μL 0.5% Triton-100 at room temperature for 15 minutes, and then the chip was rinsed 3-5 times with 400 μl of PBS.
(7) The cells after permeabilization are blocked with 3% BSA and Fc receptor blocking agent to block non-specific sites of the cells, and incubated for 1 hour at room temperature.
(8) HK2 fluorescent antibody (e.g. fluorescein FITC-labeled HK2 antibody (1:100 dilution) was added to the chip and the cells were incubated for 1 hour at room temperature, and then the chip was rinsed 3-5 times with 400 μl of PBS.
(9) 200 μl DAPI was added to the chip and the cells were incubated for 10 minutes at room temperature, and then the chip was rinsed 3-5 times with 400 μl of PBS.
(10) The high-content imaging analysis system scanned the chip and analyzed the scan results, wherein the threshold for HK2 positive was the average value plus the quintuple standard deviation of the HK2 signal values of all or part of the cells on the chip. DAPI positive was confirmed when color development is observed.
It is worth noting that there are very few impurities on the chip that are HK2 positive but DAPI negative. Therefore, the nuclear dye can help us distinguish between cells and non-cellular impurities, so as to eliminate the interference of the very few non-cellular impurities that can adsorb the fluorescently labeled HK2 antibody substance. It can be seen from the staining images of the two chip coding regions shown in
(11) The target cells are selected by the micromanipulation system, and the genome of a single cell is amplified by the MALBAC single cell whole genome amplification kit.
(12) The primers are designed for the sex-determining gene SRY and Y chromosome is tested to determine the fetal origin of the isolated cells.
Among the 5 samples, the Y chromosome was detected in 3 samples, as shown in the agarose gel electrophoresis image in
Lane 1: DNA marker;
Lane 2: DNA extracted from a cell line derived from women was used as a negative control;
Lane 3: DNA extracted from a cell line derived from men was used as a positive control;
Lane 4: The Y chromosome of the target cell that is HK2-positive/DAPI-positive and meets the morphological characteristics (i.e., irregular nuclear shape) in Sample 1 is detected as positive, indicating that the fetus corresponding to this sample is a male fetus, and the cell can be identified as derived from the fetus; in later clinical testing, the fetus corresponding to this sample was identified as a male fetus, which was consistent with the experimental results.
Lane 5: The Y chromosome of the target cell that is HK2-positive/DAPI-positive and meets the morphological characteristics in Sample 2 is detected as negative, which may be because the fetus corresponding to this sample is a female fetus; in later clinical testing, the fetus corresponding to this sample was identified as a female fetus, which was consistent with the experimental results.
Lane 6: The Y chromosome of the target cell that is HK2-positive/DAPI-positive and meets the morphological characteristics in Sample 3 is detected as positive, indicating that the fetus corresponding to this sample is a male fetus, and the cell can be identified as derived from the fetus; in later clinical testing, the fetus corresponding to this sample was identified as a male fetus, which was consistent with the experimental results.
Lane 7: The Y chromosome of the target cell that is HK2-positive/DAPI-positive and meets the morphological characteristics in Sample 4 is detected as negative, which may be because the fetus corresponding to this sample is a female fetus; in later clinical testing, the fetus corresponding to this sample was identified as a female fetus, which was consistent with the experimental results.
Lane 8: The Y chromosome of the target cell that is HK2-positive/DAPI-positive and meets the morphological characteristics in Sample 5 is detected as positive, indicating that the fetus corresponding to this sample is a male fetus, and the cell can be identified as derived from the fetus; in later clinical testing, the fetus corresponding to this sample was identified as a male fetus, which was consistent with the experimental results.
Table 1 shows the statistical data of the number of target cells (HK2 positive, DAPI positive and meeting morphological characteristics) identified in the five samples in the embodiment and the ratio of the number of target cells to the total number of sample cells. It can be found that the proportion of target cells extracted from the sample cells is relatively high, and there are fewer omissions, that is, the sensitivity is high.
For the target cells of Samples 1, 3, and 5, single-cell sequencing can be used to verify the accuracy of the method for determining fetal trophoblast cells according to present application. The statistical data is shown in Table 2.
The data in Table 2 shows that the accuracy of the method is also high.
In summary, a relatively large number of target cells in the 5 samples were detected, indicating that the method is of high sensitivity. The single-cell sequencing on the target cells proved that the method has high accuracy at the same time.
It should be understood that, the above-mentioned examples are merely used to illustrate the technical solution of present application but not to limit the scope of present application, a skilled person in the art should understand that the technical solutions described in the above-mentioned examples can still be modified, or some or all of the technical features can be equivalently replaced, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the examples of present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811290842.X | Oct 2018 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/125798 | Dec 2018 | US |
| Child | 17229686 | US |
