WO2022242285A1 - 一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法 - Google Patents

一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法 Download PDF

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WO2022242285A1
WO2022242285A1 PCT/CN2022/081234 CN2022081234W WO2022242285A1 WO 2022242285 A1 WO2022242285 A1 WO 2022242285A1 CN 2022081234 W CN2022081234 W CN 2022081234W WO 2022242285 A1 WO2022242285 A1 WO 2022242285A1
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hla
cells
sorting
cell
channel
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PCT/CN2022/081234
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French (fr)
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郑焱
马佳艳
丰晓威
刘梦羽
陈佩璇
管秩生
谢龙旭
葛毅媛
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广州凯普医药科技有限公司
广东凯普生物科技股份有限公司
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Priority to BR112022024743-5A priority Critical patent/BR112022024743B1/pt
Priority to AU2022277079A priority patent/AU2022277079A1/en
Priority to US18/009,740 priority patent/US11796443B2/en
Priority to JP2022573784A priority patent/JP7368642B2/ja
Priority to EP22803614.1A priority patent/EP4144834A4/en
Priority to KR1020227043777A priority patent/KR102626620B1/ko
Publication of WO2022242285A1 publication Critical patent/WO2022242285A1/zh

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    • G01N33/56966—Animal cells

Definitions

  • the invention belongs to the technical field of cell separation. More specifically, it relates to a method for separating and screening trophoblast cells from exfoliated cells of the cervix of pregnant women.
  • the pre-pregnancy, prenatal and neonatal three-level prevention and control system is one of the important means to reduce birth defects and improve the quality of the population in my country. Among them, prenatal screening and diagnosis are the most complicated and difficult links.
  • amniocentesis or umbilical vein puncture used clinically for prenatal diagnosis, and non-invasive prenatal screening technologies such as fetal cell-free nucleic acid sequencing have various deficiencies;
  • the risk of abortion and miscarriage is high, the analysis time is long, the detection items and scope are limited, and the diagnosis time is limited to the second and third trimesters, the acceptance of pregnant women is low, and it is not conducive to clinical treatment.
  • the detection range of fetal cell-free nucleic acid sequencing technology is extremely limited, and only 3-5 specified chromosomal ploidies can be detected. False positives and false negatives are unavoidable.
  • the ability to detect common mutations is insufficient, and the content of fetal cell-free nucleic acid in maternal blood also exists. issues of individual differences.
  • the applicant's patent application CN111304153A discloses a method for isolating trophoblast cells.
  • the method determines the specific antigens expressed on the surface of trophoblast cells and uses the immunomagnetic bead technology with corresponding specific antibodies to extract cells from placental trophoblast samples. Placental trophoblast cells were isolated and purified from the suspension.
  • this technique Compared with traditional amniocentesis and chorionic villus puncture, this technique has the advantages of non-invasiveness, and the sampling time is earlier, the risk of infection and miscarriage is low, and the test results have similar reliability.
  • the disclosed applicable antibody combinations are limited, and the accuracy and specificity need to be improved.
  • the applicant team has been conducting continuous research and development on this project.
  • the purpose of the present invention is to provide a method for separating and screening trophoblast cells from exfoliated cells of the cervix of pregnant women based on flow cytometry or microfluidic technology. Simultaneous labeling of antigens and identification and sorting of characteristic fluorescent signals have greatly improved the accuracy, and the specificity is also better than the previous immunomagnetic bead separation scheme. It also has the advantage of taking into account both the number and quality of cells. The obtained cells The quantity is large, the specificity is good, and the sensitivity is good.
  • a method for separating trophoblast cells comprising the steps of:
  • the specific antibody is a combination of antibodies corresponding to specific antigens expressed on the surface or intracellularly of trophoblast cells; preferably, the combination of specific antibodies is HLA-G+CK7, HLA-G+CK18, HLA-G+ ⁇ -HCG, CD31+HPL, MMP9+CD31, HLA-G+HPL, HLA-G+MMP9, HLA-G+CD31, HLA-G+P, CD31+P, HLA-G+CDH5, CD31+CDH5, CD31+CK7+ HLA-G, HLA-G+CK18+CD31, HLA-G+ ⁇ -HCG+CD31, CD31+HPL+HLA-G, MMP9+CD31+HLA-G, CD31+P+HLA-G or HLA-G+ Combination of CDH5+CD31;
  • step (3) Sorting the cell suspension that has been incubated in step (2) by using a flow cytometer to obtain isolated and purified placental trophoblast cells;
  • microfluidic sorting chip to subject the cell suspension incubated in step (2) to fluorescent-labeled microfluidic cell sorting to obtain isolated and purified placental trophoblast cells.
  • the structure of the microfluidic sorting chip used in step (3) is: including a substrate and a cover sheet attached thereto; selecting but not limited to acrylic as the basic material to be made by injection molding technology;
  • One side of the substrate is provided with a main channel, a side channel A and a side channel B, and the two side channels correspond to the left and right ends of the main channel respectively;
  • the other side of the substrate is provided with a C inlet, an S inlet, an N outlet and a T outlet; the four ports all penetrate the other side of the substrate and communicate with the flow channel; and the position of the C inlet corresponds to the left end of the main channel, The position of the S inlet corresponds to the end of the side channel A, the position of the N outlet corresponds to the right end of the main channel, and the position of the T outlet corresponds to the end of the side channel B;
  • a deflection electrode device is also provided in the main channel at the confluence of the N outlet and the T outlet.
  • the channel widths of all main channels, side channel A and side channel B are no more than 1000 ⁇ m, and the depths are no more than 500 ⁇ m.
  • the channel widths of all the main channel, side channel A and side channel B are 500-1000 ⁇ m.
  • the channel widths of all the main channel, side channel A and side channel B are 1000 ⁇ m.
  • the C inlet can pass into the mixed cell sample to be sorted, the S inlet can pass into the buffer, the T outlet is for collecting target cells, and the N outlet is for collecting non-target cells.
  • the mixed sample containing the target cells flows into the main channel of the chip from the C inlet, and the buffer solution enters the side channel of the chip at the S inlet.
  • the incoming channel is controlled by the opening and closing of the electrode, the target cells are sorted and reach the T outlet, while the non-target cells continue to reach the N outlet along the main channel, and the sorting is completed.
  • the primary antibody incubation condition in step (2) is: 4°C for 30-90min (preferably 4°C, 60min), and the secondary antibody-fluorescence labeling complex incubation condition is 2°C-8°C for 20min.
  • the specific method of step (2) is: through incubation, the primary antibody, secondary antibody-fluorescent labeling complex is specifically linked to the target antigen step by step, and washing and centrifugation techniques are used in the middle to avoid cross-contamination.
  • the specific method of step (3) is: the cell resuspension that has been incubated is passed into the C inlet of the microfluidic sorting chip, and the buffer is passed into the S inlet; then the microfluidic sorting chip is placed on the cell The sorter runs the sorting program. After the program ends, collect the samples at the T outlet to obtain the sorted trophoblast cells.
  • the cell sorting liquid phase system in step (3) is 0.2%-0.4% Triton-X-100 (preferably 0.3% Triton-X-100). More preferably, a PBS configuration is utilized.
  • the conditions for sorting by flow cytometer in step (3) adjust the sample loading speed to 1000-2000 events/second, and the collection rate to 5.0.
  • the best system for the cell suspension in step (1) is 1xPBS containing 0.2%-0.4% FBS (preferably 1xPBS containing 0.3% FBS).
  • the method is useful in constructing human STR identification, human chromosome ploidy detection, thalassemia gene detection, deafness gene detection, whole exome gene sequencing, chromosome microdeletion/duplication detection (high-throughput sequencing method), or chromosome structure
  • variation detection high-density chip method
  • the method for isolating placental trophoblast cells based on flow cytometry or microfluidic technology has the advantage of noninvasively obtaining specimens, and the sampling time is earlier, and infection and abortion The risk is low, and the test results have higher reliability and wider coverage.
  • This type of specimen can obtain the complete genome nucleic acid sample of the fetus, making it possible to detect and analyze all genetic diseases, and basically realize the coverage of genetic disease detection (from chromosome ploidy, chromosome structural variation CNV, mitochondria, microdeletion/duplication, single gene mutation, SNP/STR genetic signature test, etc.).
  • the method of the present invention can obtain considerable cells (thousands, and the lowest quality control standard designed is >2000 positive cells), and can realize the use of conventional molecular testing techniques to detect specimens without special operations, which is beneficial to technicians and The requirements for laboratory equipment are not high, which reduces the technical threshold and cost of use. It can be carried out in more medical institutions and can be widely promoted.
  • the method provided by the present invention is a parallel screening scheme based on multiple markers, which can realize simultaneous labeling and identification and sorting of more multiple antigens at the same time, and the accuracy is greatly improved.
  • technologies such as the magnetic bead method, which have the advantage of taking into account both the quantity and quality of cells.
  • the obtained cells are not only large in number, but also cause little damage to the cells.
  • the obtained cells are of high quality, with good detection specificity and sensitivity.
  • Figure 1 is a schematic diagram of the substrate structure of the microfluidic sorting chip; (a) and (b) are the two sides of the substrate respectively.
  • Figure 2 shows the single fluorescent marker positive cell population selected in flow cytometry sorting.
  • Figure 3 is the double fluorescent marker positive cell population selected in flow cytometry sorting.
  • Figure 4 shows channel 1 marked by FAM, "822-” is the remaining specimen after sorting, "822+” is the sorted specimen, and "822" is the specimen before sorting.
  • Figure 5 shows HEX labeled lane 2.
  • Figure 6 shows TAMRA labeled lane 3.
  • Figure 7 is ROX labeled lane 4.
  • Fig. 8 is a schematic diagram of the results of Y-STR detection of samples containing Y chromosomes in syncytiotrophoblast cells after sorting.
  • Figure 9 shows the results of deafness gene detection on the sorted specimens.
  • Figure 10 shows the results of Mediterranean genetic testing on sorted specimens.
  • Fig. 11 is a diagram of a pedigree carrying a pathogenic mutation in a detected family.
  • Figure 12 is a schematic diagram of the whole chromosome.
  • Figure 13 is a schematic diagram of abnormal chromosomes.
  • Fig. 14 is a photo of positive cells sorted by the method of the present invention.
  • Fig. 15 is a comparison photo of positive cells and negative cells before and after sorting by the method of the present invention.
  • Fig. 16 is a photo of the positive cells sorted by the control magnetic bead method.
  • the present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • FIG. 1 The schematic diagram of the microfluidic sorting chip used to separate and screen trophoblast cells from the exfoliated cells of the cervix of pregnant women is shown in Figure 1.
  • the structural design of the microfluidic sorting chip is described as follows: the chip is prepared using but not limited to acrylic as the basic material, and the pipe shape in Figure 1 is formed on one side of the substrate by injection molding technology. The width of the pipe does not exceed 1000 ⁇ m and the depth does not exceed 500 ⁇ m, and use the other side of the substrate for lamination to form a complete chip.
  • the microfluidic sorting chip includes a substrate and a cover attached thereto;
  • a main channel, a side channel A and a side channel B are provided on one side of the substrate, and the two side channels are respectively close to the left and right ends of the main channel; the widths of all the channels are the same 1000 ⁇ m;
  • the other side of the substrate is provided with C inlet (cell sample liquid inlet), S inlet (buffer liquid inlet), N outlet (non-target cell liquid storage hole) and T outlet (target cell liquid storage hole) ;
  • C inlet cell sample liquid inlet
  • S inlet buffer liquid inlet
  • N outlet non-target cell liquid storage hole
  • T outlet target cell liquid storage hole
  • the C inlet can be used for the mixed cell samples to be sorted
  • the S inlet can be used for the buffer solution
  • the T outlet is for the collection of target cells
  • the N outlet is for the collection of non-target cells.
  • a deflection electrode device is also provided in the main channel for cell sorting, and the specific position of the deflection electrode device is located at the confluence of the N outlet and the T outlet; according to the presence or absence of the flow cell signal, the electrode can be turned on/off. Negatively charged cells are deflected in the electromagnetic field created by the electrodes and enter designated conduits leading to N or T outlets.
  • the mixed sample containing the target cells flows into the main channel of the chip from the C inlet, and the buffer solution enters the side channel of the chip at the S inlet.
  • the incoming channel is controlled by the opening and closing of the electrode, the target cells are sorted and reach the T outlet, while the non-target cells continue to reach the N outlet along the main channel, and the sorting is completed.
  • the chip After the sorting is completed, the chip is discarded immediately, and the chip needs to be replaced before each sorting to ensure that the sorting environment is clean, controllable and free of cross-contamination.
  • Example 2 Separation and screening of trophoblast cells from cervical exfoliated cells of pregnant women based on microfluidic sorting chip
  • the sorting method of trophoblast cell comprises the following steps:
  • step 2 Using the microfluidic sorting chip of Example 1, the cell suspension incubated in step 2 was subjected to fluorescence-labeled microfluidic cell sorting; specific methods include the following (15)-(18).
  • Two, specifically, the sorting method of trophoblast cells comprises the following steps:
  • Add primary antibody add mouse anti-human CK7 monoclonal antibody, mouse anti-human CK18 monoclonal antibody, mouse anti-human ⁇ -HCG monoclonal antibody, mouse anti-human MMP9 monoclonal antibody, mouse anti- Human CDH5 monoclonal antibody, mouse anti-human P monoclonal antibody, mouse anti-human hPL monoclonal antibody, rabbit anti-human HLA-G monoclonal antibody, rabbit anti-human CD31 monoclonal antibody (abcam company) 200 ⁇ l, after mixing, 4 °C, 60min;
  • step 4 Repeat step 4 three times and resuspend with 200 ⁇ l buffer (DPBS+0.1%BSA+2mM EDTA);
  • step (14) Repeat step (13) two to three times, add 200 ⁇ l 1 ⁇ PBST to resuspend, and obtain cell suspension;
  • the sorting method of trophoblast cells comprises the following steps:
  • cervical exfoliated cell liquid sample is prepared into sample cell suspension; Concrete method is shown in (1)-(5) among the embodiment 2;
  • step 3 Use a flow cytometry sorter (BDFACSAria II, USA) to sort the cell suspension incubated in step 2 with fluorescent markers, including the following steps:
  • Figure 2 shows the results of single fluorescent marker sorting
  • Figure 3 shows the results of double fluorescent marker sorting.
  • the trophoblast cells were sorted and separated for the sample (cervical exfoliated cells).
  • the sorted trophoblast cells (the trophoblast cells obtained in the 17th step of cell sorting according to the method of Example 2), and the cells obtained in the 18th step of cell sorting according to the method of Example 2 (cervical Exfoliated cells (remaining cells of trophoblast cells removed) were centrifuged at 12000 rpm for 3 min.
  • PCR reaction program 50°C, 10min; 96°C, 4min; (94°C, 5sec; 60°C, 1min10sec) ⁇ 27cycles; 60°C, 30min; 15°C, save.
  • the cervical exfoliated cells for deafness susceptibility gene detection, specifically 9 mutation sites (mtDNA1494, mtDNA1555, SLC26A4-IVS7(-2), SLC26A4) of deafness-related genes (GJB2, GJB3, SLC26A4 and mtDNA) -2168, GJB2-35, GJB2-176, GJB2-235, GJB2-299, GJB3-538) for detection.
  • Example 2 Using the method described in Example 2 and a commercial kit ( ⁇ - and ⁇ -thalassemia gene detection kit (PCR+membrane hybridization method), Chaozhou Kaipu Biochemical Co., Ltd., National Food and Drug Administration (quasi) word 2012 No.
  • ⁇ - and ⁇ -thalassemia gene detection kit PCR+membrane hybridization method
  • Chaozhou Kaipu Biochemical Co., Ltd., State Food and Drug Administration (Quasi) Word 2012 No. 3400399) for follow-up testing, see the instructions for specific operations.
  • the original results are shown in Figure 10, and the analysis of the results is shown in Table 4.
  • Example 2 In the known deafness family (father, mother (16 weeks of pregnancy), eldest son (deaf child), young son (deaf child)), according to the method described in Example 2, obtain the cervical exfoliated cell specimen of pregnant woman and amniotic fluid samples, pregnant women and other family members collected whole blood samples, and extracted DNA (pregnant women after cervical exfoliated cells were sorted according to the extraction method described in Example 4, amniotic fluid and whole blood samples using human whole blood genomic DNA extraction reagents Kit (Kaipu Biotechnology, China) for extraction (source of samples: human samples tested by Kaipu Medical Laboratory, and human genome DNA obtained using human whole exome detection kit (Aiji Taikang Biotechnology (Beijing) Co., Ltd. , Cat. No. T086V4) for whole exome sequencing (WES):
  • Genomic DNA is treated with transposase Tn5 to generate a 300bp fragment to build a DNA library; adapters P5, P7, index1, and 2 are added to both ends; DNA fragments of appropriate length are selected, amplified and purified; and exon probes with biotin Hybridization of the library; using the strong binding ability of biotin and streptavidin, combine the magnetic beads with streptavidin with the probes bound to the target library; adsorb the magnetic beads and remove the supernatant; The DNA on the magnetic beads was eluted, and the library was amplified by PCR; the quality of the library was identified; sequencing was carried out on the machine.
  • High-throughput sequencing technology was used to detect the whole exome, and Sanger sequencing was used to verify the detected pathogenic or suspected pathogenic loci.
  • the samples sent for inspection were amniotic fluid samples at 16 weeks of pregnancy. Two older brothers were deaf and dumb, and both parents were normal.
  • the CDH23 gene c.8363T>C (p.Leu2788Pro) heterozygous mutation and the GJB2 gene c.109G>A (p.Val27Ile) heterozygous mutation were detected in the sample. See Figure 11 for the family tree.
  • the test results can find out the cause of the disease and the carrying status of the disease-causing mutation in the family.
  • the trophoblast cells sorted by the method in Example 2 were extracted using the method described in Example 4 for extracting DNA from cervical exfoliated cells of pregnant women, and the obtained human genomic DNA was fragmented, pre-amplified, amplified, and PCR product After purification, it is labeled with two different fluorescent dyes (normal samples are labeled green with Cy3, and patient samples are labeled red with Cy5);
  • the fluorescent product After the fluorescent product is purified, it is injected into the chip. After cleaning, the chip is scanned and the results are obtained.
  • FIG. 13 the horizontal axis is a schematic diagram of chromosome zones, and the vertical axis is the signal ratio (expressed in log2ratio) between the sample and the standard sample.
  • the horizontal axis is a schematic diagram of chromosome zones, and the vertical axis is the signal ratio (expressed in log2ratio) between the sample and the standard sample.
  • the vertical axis is the signal ratio (expressed in log2ratio) between the sample and the standard sample.
  • the sample which was 22q11.21 deletion.
  • the start-end position of the abnormal fragment [UCSC hg19] was arr22q11.21(19006943_21461068)x1, and the abnormal fragment size was 2.454Mb.
  • the relevant disease area is Pathogenic (pathogenicity, ACMG classification), and this abnormality covers 106 ISCA genes such as TBX1, CRKL, GP1BB, SLC25A1, DGCR10, TSSK1A, GSC2, and CLTCL1. This region is abnormally located in the 22q11.2recurrent(DGS/VCFS)region(includes TBX1).
  • Deletions in the proximal (A-D) region of 22q11.2 are associated with DiGeorge/Velocardioffacial (DGS/VCFS) syndrome, usually with congenital heart disease, cardiac abnormalities, characteristic facial features, DD/ID, behavioral problems, immunodeficiency and hypocalcemia (PMID 25217958).
  • DGS/VCFS DiGeorge/Velocardioffacial
  • This region is abnormally located in the 22q11.2recurrent region(central,B/C-D)(includes CRKL).
  • the clinical phenotypes that may result from the deletion of this region include: deformed facial features, growth restriction/short stature, central nervous system abnormalities/seizures, Developmental delay, intellectual disability, skeletal abnormalities, cardiovascular deficits, urogenital abnormalities, and immunodeficiency/recurrent infections (PMID 25123976).
  • the results show that the sorted cell sample can effectively detect the variation of chromosome structure and detect the corresponding mutation.
  • Example 8 Comparison of method for separating and screening trophoblast cells from cervical exfoliated cells of pregnant women based on microfluidic sorting chip and magnetic bead method
  • the inventive method is with embodiment 2.
  • the process of sorting trophoblast cells by magnetic bead method is as follows:
  • step 4 Repeat step 4 three times and resuspend with 200 ⁇ l buffer (DPBS+0.1%BSA+2mM EDTA).
  • step (14) Repeat step (14) two to three times.
  • step (18) Repeat step (18) three times to finally obtain trophoblast cells.
  • Figure 14 is a photograph of the positive cells sorted by the method of the present invention
  • Figure 15 is the positive cells before and after the method of the present invention.
  • Figure 16 is a photo of the positive cells sorted by the magnetic bead method. It can be clearly seen from the comparison that the order of magnitude of the number of cells screened by the two methods has obvious differences, and the method of the present invention is significantly better than that of the magnetic beads. Statistics show that the number of positive cells sorted by the method of the present invention can reach about 3000-13000 cells.

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Abstract

提供了一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法，所述方法基于特定的滋养层细胞表面或胞内表达的特异性抗原及组合，并利用设计的微流控分选芯片或流式细胞仪，对胎盘滋养层样本的细胞悬浮液进行细胞分选，获得分离纯化的胎盘滋养层细胞。与传统方法相比，所述方法具有无创获取标本，特异性好，取材时间较早，导致感染及流产的风险低的优势，能实现同时对多个抗原进行同步标记和特征荧光信号的识别和分选，准确性得到较大提升，检测结果又具有更高的可信度和更广的覆盖范围。与现有磁珠法技术相比，所述方法获得细胞数量多，特异性好，灵敏性好，能够应用于多种检测用途和应用场景。

Description

一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法 技术领域
本发明属于细胞分离技术领域。更具体地，涉及一种从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法。
背景技术
孕前、产前和新生儿三级防控体系是我国降低出生缺陷、提高人口素质的重要手段之一，其中产前筛查和诊断是最为复杂且难度最高的环节。
目前临床上用于产前诊断的羊膜腔穿刺术或者脐静脉穿刺术，以及胎儿游离核酸测序等无创产前筛查技术都存在各种不足；羊膜腔穿刺术或者脐静脉穿刺术取材方法操作感染和流产的风险高、分析时间长，检测项目和范围有限，而且诊断时间限制在孕中晚期，孕妇的接受度低且不利于临床处理。胎儿游离核酸测序技术检验范围极其有限，且存在仅可检测3-5条指定染色体倍性，假阳性、假阴性情况难以避免，对常见变异检出能力不足，母血胎儿游离核酸的含量也存在个体差异性等问题。
申请人的专利申请CN111304153A披露了一种分离滋养层细胞的方法，该方法通过确定滋养层细胞表面表达的特异性抗原，利用带有对应特异性抗体的免疫磁珠技术，从胎盘滋养层样本细胞悬浮液中分离纯化胎盘滋养层细胞。该技术与传统的羊膜腔穿刺和绒毛穿刺相比，具有无创的优势，且取材时间较早，感染及流产的风险低，检测结果又具有相似的可信度。另一方面看，该技术仍有改进的空间，所披露适用的抗体组合有限，准确性和特异性扔有待更好的提升。申请人团队对该项目一直在进行持续的研究开发。
发明内容
本发明的目的是提供一种基于流式细胞分离或微流控技术从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法，不仅克服传统方法的问题和缺陷，还可实现同时对更多多个抗原进行同步标记和特征荧光信号的识别和分选，准确性得到较大提升，同时特异性也优于之前的免疫磁珠法分离方案，还具有能够兼顾细胞数量和质量的优势，所获得细胞数量多，特异性好，灵敏性好。
本发明上述目的通过以下技术方案实现：
一种分离滋养层细胞的方法，包括如下步骤：
(1)将宫颈脱落细胞液样本制备成样本细胞悬浮液；
(2)向样本细胞悬浮液中加入特异性抗体进行孵育；
所述特异性抗体是对应滋养层细胞表面或胞内表达的特异性抗原的抗体组合；优选地特异性抗体组合为HLA-G+CK7、HLA-G+CK18、HLA-G+β-HCG、CD31+HPL、MMP9+CD31、HLA-G+HPL、HLA-G+MMP9、HLA-G+CD31、HLA-G+P、CD31+P、HLA-G+CDH5、CD31+CDH5、CD31+CK7+HLA-G、HLA-G+CK18+CD31、HLA-G+β-HCG+CD31、CD31+HPL+HLA-G、MMP9+CD31+HLA-G、CD31+P+HLA-G或HLA-G+CDH5+CD31的组合；
(3)利用流式细胞仪将步骤(2)孵育完成的细胞重悬液进行分选，即可获得分离纯化的胎盘滋养层细胞；
或利用微流控分选芯片，将步骤(2)孵育完成的细胞重悬液进行荧光标记微流控细胞分选，即可获得分离纯化的胎盘滋养层细胞。
优选地，步骤(3)所用微流控分选芯片的结构为：包括基片和与之贴合的盖片；选用包括但不限于亚克力作为基本材质通过注塑成型技术制成；
所述基片的一面上设有主流道、侧流道A和侧流道B，两个侧流道分别对应靠近主流道的左、右两端部；
所述基片的另一面上设有C入口、S入口、N出口和T出口；四个口均贯穿到基片另一面与流道连通；且C入口的位置对应于主流道的左端部，S入口的位置对应于侧流道A端部，N出口的位置对应于主流道的右端部，T出口的位置对应于侧流道B端部；
所述主流道内、在N出口与T出口汇合处，还设有偏转电极装置。
优选地，所有主流道、侧流道A和侧流道B的流道宽度均不超过1000μm，深度均不超过500μm。
更优选地，所有主流道、侧流道A和侧流道B的流道宽度均为500-1000μm。
更优选地，所有主流道、侧流道A和侧流道B的流道宽度均为1000μm。
微流控分选芯片中，C入口可通入待分选混合细胞样品、S入口可通入缓冲液、T出口为目标细胞收集，N出口为非目标细胞收集。
分选过程中，含有目标细胞的混合样品由C入口处流入芯片主流道，缓冲液于S入口进入芯片侧流道，二者在流道交汇处混合后继续沿主流道向同一方向 流动。混合细胞流经偏转电极装置时，以电极开闭控制进入的管道，目标细胞被分选而到达T出口，而非目标细胞则沿主流道继续到达N出口，分选完成。
另外，优选地，步骤(2)中一抗孵育条件为：4℃反应30-90min(优选4℃，60min)，二抗-荧光标记复合物孵育条件为2℃-8℃反应20min。
优选地，步骤(2)的具体方法是：通过孵育将一抗、二抗-荧光标记复合物先后分步与目标抗原进行特异性连接，中间使用洗涤和离心分离技术避免交叉污染。
优选地，步骤(3)的具体方法是：孵育完成的细胞重悬液通入微流控分选芯片的C入口，并在S入口通入缓冲液；然后将微流控分选芯片放置于细胞分选仪并运行分选程序，程序结束后，搜集T出口的标本，获得分选的滋养层细胞。
优选地，步骤(3)中细胞分选液相体系为0.2％-0.4％Triton-X-100(优选0.3％Triton-X-100)。更优选地，利用PBS配置。
优选地，步骤(3)所述利用流式细胞仪进行分选的条件：调整上样速度为1000-2000events/秒，收集速率为5.0。
优选地，步骤(1)中细胞悬浮液的最佳体系为含0.2％-0.4％FBS的1xPBS(优选含0.3％FBS的1xPBS)。
另外所述方法在构建人类STR鉴定、人类染色体倍性检测、地中海贫血基因检测、耳聋基因检测、全外显子组基因测序、染色体微缺失/重复检测(高通量测序法)、或染色体结构变异检测(高密度芯片法)的产品方面的应用，也应当在本发明的保护范围之内。
本发明具有以下有益效果：
本发明提供的基于流式细胞分离或微流控技术分离胎盘滋养层细胞的方法，与传统的羊膜腔穿刺和绒毛穿刺相比，具有无创获取标本的优势，且取材时间较早，感染及流产的风险低，检测结果又具有更高的可信度和更广的覆盖范围。通过该种类的标本可获取胎儿完整的基因组核酸样本，使检测分析所有遗传性疾病成为可能，基本实现遗传病检测覆盖(从染色体倍性、染色体结构变异CNV、线粒体、微缺失/重复、单基因突变、SNP/STR遗传特征检验等)。
本发明方法可获得相当可观的细胞(数千个，设计的质控最低标准是>2000个阳性细胞)，可实现使用常规分子检验技术来检测标本，而不需要特殊的操作，对技术人员和实验室设备的要求不高，降低了技术门槛和使用成本，可在更多的 医疗机构开展，能够得到大范围的推广。
同时本发明提供的方法是一种可基于多重标记并行的筛选方案，能实现同时对更多多个抗原进行同步标记和特征荧光信号的识别和分选，准确性得到较大提升，相比现有磁珠法等技术，具有能够兼顾细胞数量和质量的优势，所获得细胞不仅数量多，而且对细胞损伤小，所得细胞质量优，检测特异性好、灵敏性好。
附图说明
图1为微流控分选芯片的基片结构示意图；(a)图和(b)图分别为基片两个面。
图2为流式细胞分选中选择的单荧光标记阳性细胞群。
图3为流式细胞分选中选择的双荧光标记阳性细胞群。
图4为FAM标记的通道1，“822-”为分选后剩余标本，“822+”为分选出的标本，“822”为分选前标本。
图5为HEX标记的通道2。
图6为TAMRA标记的通道3。
图7为ROX标记的通道4。
图8为对分选后合体滋养层细胞中含有Y染色体的标本进行Y-STR检测的结果示意图。
图9为分选标本进行耳聋基因检测的结果。
图10为分选标本进行地中海基因检测的结果。
图11为检测家系致病突变携带家系图。
图12为全染色体示意图。
图13为异常染色体示意图。
图14为本发明方法分选所得阳性细胞的照片。
图15为本发明方法分选前后、阳性细胞与阴性细胞对比照片。
图16为对照磁珠法分选所得阳性细胞的照片。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1微流控分选芯片设计
用于从孕妇宫颈脱落细胞中分离筛选滋养层细胞的微流控分选芯片的示意图如图1所示。
微流控分选芯片的结构设计描述如下：芯片选用包括但不限于亚克力作为基本材质进行制备，通过注塑成型技术在一面基材上形成图1之管道形状，管道宽度不超过1000μm，深度不超过500μm，并使用另一面基材进行贴合，形成完整芯片。
具体地，微流控分选芯片包括基片和与之贴合的盖片；
如图1所示，所述基片的一面上设有主流道、侧流道A和侧流道B，两个侧流道分别靠近主流道的左、右两端部；所有流道宽度均为1000μm；
所述基片的另一面上设有C入口(细胞样品进液孔)、S入口(缓冲液进液孔)、N出口(非目标细胞储液孔)和T出口(目标细胞储液孔)；四个口均贯穿到基片另一面与流道连通；且C入口的位置对应于主流道的左端部，S入口的位置对应于侧流道A端部，N出口的位置对应于主流道的右端部，T出口的位置对应于侧流道B端部；
C入口可通入待分选混合细胞样品、S入口可通入缓冲液、T出口为目标细胞收集，N出口为非目标细胞收集。
另外，所述主流道内还设有偏转电极装置用于细胞分选，偏转电极装置的具体位置位于N出口与T出口汇合处；根据流动细胞信号的有无，可控制开启/关闭电极。带负电的细胞在电极形成的电磁场中发生偏转，进入指定的通向N出口或T出口的管道。
分选过程中，含有目标细胞的混合样品由C入口处流入芯片主流道，缓冲液于S入口进入芯片侧流道，二者在流道交汇处混合后继续沿主流道向同一方向流动。混合细胞流经偏转电极装置时，以电极开闭控制进入的管道，目标细胞被分选而到达T出口，而非目标细胞则沿主流道继续到达N出口，分选完成。
分选完成后，芯片随即弃用，每次分选前需要重新更换芯片，以保证分选环境洁净、可控和无交叉污染。
实施例2基于微流控分选芯片从孕妇宫颈脱落细胞中分离筛选滋养层细胞
一、滋养层细胞的分选方法包括如下步骤：
1、将宫颈脱落细胞液样本制备成样本细胞悬浮液；具体方法包括如下文 (1)-(5)所示；
2、向样本细胞悬浮液中加入特异性抗体进行孵育；具体方法包括如下文(6)-(14)所示；
3、利用实施例1的微流控分选芯片，将步骤2孵育完成的细胞重悬液进行荧光标记微流控细胞分选；具体方法包括如下文(15)-(18)所示。
其中，特异性抗体的组合如表1所示：
表1：滋养层细胞上表达的抗原及抗体组合
Figure PCTCN2022081234-appb-000001
二、具体地，滋养层细胞的分选方法包括如下步骤：
(1)将细胞保存液(宫颈脱落细胞)在震荡混匀器上混匀，5min；
(2)将保存液取出置于15ml离心管中，在保存液的瓶中再加入3ml的1×PBS， 震荡混匀后，取出置于15ml离心管中；
(3)3000rpm离心10min，弃去上清；
(4)加入1ml的1×PBST，混匀后移入1.5mlEP管中，3000rpm离心5min后弃去上清；
(5)重复步骤4两次，制备出细胞悬浮液；
(6)加入200μl的0.3％的Triton X-100，混匀后室温通透20min；
(7)重复步骤4三次；
(8)加入一抗：分别加入按照比例稀释后的鼠抗人CK7单克隆抗体、鼠抗人CK18单克隆抗体、鼠抗人β-HCG单克隆抗体、鼠抗人MMP9单克隆抗体、鼠抗人CDH5单克隆抗体、鼠抗人P单克隆抗体、鼠抗人hPL单克隆抗体、兔抗人HLA-G单克隆抗体、兔抗人CD31单克隆抗体(abcam公司)200μl，混匀后，4℃，60min；
(9)重复步骤4三次；
(10)加入二抗-荧光标记复合物：按比例稀释的羊抗兔及羊抗鼠抗体200μl，混匀后，37℃，60min；
(11)重复步骤4三次后用200μl buffer(DPBS+0.1％BSA+2mM EDTA)重悬；
(12)2℃-8℃反应20min；
(13)加入1ml的1×PBST，混匀后移入1.5mlEP管中，3000rpm离心5min后弃去上清；
(14)重复步骤(13)两到三次，加入200μl 1×PBST重悬，得细胞重悬液；
(15)将获得的细胞重悬液通入实施例1的微流控分选芯片的C入口，并在S入口通入1×PBST；
(16)将微流控芯片放置于细胞分选仪载物台上固定，并开启电源设定指定程序，运行；
(17)程序结束后，搜集T出口的标本，获得分选的滋养层细胞。
(18)搜集N出口的标本，为宫颈脱落细胞中去除了滋养层细胞的剩余细胞。
实施例3基于流式细胞仪从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法
滋养层细胞的分选方法包括如下步骤：
1、将宫颈脱落细胞液样本制备成样本细胞悬浮液；具体方法同实施例2中 的(1)-(5)所示；
2、向样本细胞悬浮液中加入特异性抗体进行孵育；具体方法同实施例2中的(6)-(14)所示；其中，特异性抗体的组合同上表1所示；
3、利用流式细胞分选仪(BDFACSAria II型，美国)将步骤2孵育完成的细胞重悬液进行荧光标记分选，包括如下步骤：
1)打开流式细胞仪，按照操作说明进行每日开机操作；
2)对仪器进行液流调整，使液流断点位置位于窗口中上部；
3)分选液路调整，确认液滴延迟，调整上样速度到1000-2000events/秒；
4)选择5ml流式管作为收集装置，分选细胞数目选择3000，方向为左，添加要分选的细胞群，依次设门，放入收集管；
5)将孵育完成的细胞悬浮液放入进样仓，设置5.0的收集速率，上样；
6)在300-500V范围内调整电压，使荧光染料之间补偿尽量小，调整设门位置，使阳性细胞位于中央，将门内细胞进行收集；收集管中的细胞即为选中的目的细胞。
按照给定标记组合获得目标细胞群，如图2、图3。其中图2为单荧光标记分选结果，图3为双荧光标记分选结果。
实施例4方法的应用案例---人类STR鉴定
1、按照上述实施例2的方法，针对样本(宫颈脱落细胞)分选分离滋养层细胞。
2、将分离后的滋养层细胞与孕妇的宫颈脱落细胞进行DNA提取：
1)将分选后的滋养层细胞(按照实施例2方法进行细胞分选的第17步获得的滋养层细胞)，与按照实施例2方法进行细胞分选的第18步获得的细胞(宫颈脱落细胞中去除了滋养层细胞的剩余细胞)，分别12000rpm离心3min。
2)弃去上清，加200μl溶液P重悬沉淀。
3)加入20μl的蛋白酶K、200μl溶液L，混匀。
4)56℃温浴20min，颠倒混匀。
5)加入200μl无水乙醇，充分混匀后，转入吸附柱，10000rpm离心1min，弃去收集管中废液。
6)加500μl的W1,10000rpm离心1min，弃去收集管中废液。
7)加500μl的W2,10000rpm离心1min，弃去收集管中废液。
8)加500μl的W2,10000rpm离心1min，弃去收集管中废液。
9)空管离心12000rpm，3min，弃去收集管。
10)将吸附柱放入新的1.5ml离心管，开盖静置2min，加50μl的TE，静置5min后，12000rpm离心2min，弃去柱子。
11)测定提取的DNA浓度及纯度。
3、将分离后的滋养层细胞与孕妇的宫颈脱落细胞中的DNA提取后，使用阅微D-21人类STR鉴定试剂盒进行PCR扩增，使用3500xL测序仪对PCR产物进行毛细管电泳分析，并用G5-Matrix Standard对仪器进行荧光校准，同时编写了相应的Panels，bins文件，并用软件GeneMapper ID version 3.0来进行结果分析。
4、进行STR检测
1)PCR扩增，扩增体系如表2所示：
表2：PCR扩增体系
Figure PCTCN2022081234-appb-000002
PCR反应程序：50℃，10min；96℃，4min；(94℃，5sec；60℃，1min10sec)×27cycles；60℃，30min；15℃，保存。
2)STR检测结果
按照说明书将Hidi 8.7μl内参0.3μl混匀，加入1ml扩增后产物，使用3500xL测序仪对PCR产物进行毛细管电泳分析，并用G5-Matrix Standard对仪器进行荧光校准，同时编写了相应的Panels，bins文件，并用软件GeneMapper ID version3.0来进行结果分析，结果如图4～7所示，结果表明，标本中除孕妇本人的DNA外，确实存在另一独立个体DNA信息，且与孕妇本人具有较强的亲缘关系。
3)Y-STR检测
将STR中检测到Y染色体的标本使用阅微40Y试剂盒进行Y-STR检测，结 果如图8所示，结果表明该标本中存在男性DNA。
实施例5方法的应用案例---耳聋易感基因检测
使用实施例2所述方法和商品化试剂盒(耳聋易感基因检测试剂盒(PCR+导流杂交法)，潮州凯普生物化学有限公司，国械注准20153401698)对孕妇的宫颈脱落细胞(样本来源：广州凯普医学检验所)进行耳聋易感基因检测，具体对耳聋相关基因(GJB2,GJB3,SLC26A4和mtDNA)的9个突变位点(mtDNA1494、mtDNA1555、SLC26A4-IVS7(-2)、SLC26A4-2168、GJB2-35、GJB2-176、GJB2-235、GJB2-299、GJB3-538)进行检测。
用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取，之后用商品化试剂盒(耳聋易感基因检测试剂盒(PCR+导流杂交法)，潮州凯普生物化学有限公司，国械注准20153401698)进行后续检测，具体操作详见说明书。原始结果如图9所示，结果分析见表3。
表3耳聋结果分析
155M纯合 176M纯合 235M纯合
299M纯合 1494M纯合 1555M纯合
7445M纯合 538M纯合 2168M纯合
IVS-M纯合 1229M纯合 正常样本
空白对照 空白对照 空白对照
结果表明，该试验检测结果与临床检测结果一致。
实施例6方法的应用案例---地中海贫血相关基因检测检测
使用实施例2所述方法和商品化试剂盒(α-和β-地中海贫血基因检测试剂盒(PCR+膜杂交法)，潮州凯普生物化学有限公司，国食药监械(准)字2012第3400399号)对孕妇的宫颈脱落细胞(样本来源：凯普医学检验所检测人源标本的剩余核酸样本)进行耳聋易感基因检测检测，具体对常见的3种α-地贫缺失型(-- SEA、-α 3.7、-α 4.2)，2种α-地贫突变型(CS、QS)及11种β-地贫突变型(CD14-15、CD17、CD27-28、CD41-42、CD43、CD71-72、-28、-29、IVS-I-1、IVS-II-654、βEN)进行检测。
用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取，之后用商品化试剂盒(α-和β-地中海贫血基因检测试剂盒(PCR+膜杂交法)，潮州凯普 生物化学有限公司，国食药监械(准)字2012第3400399号)进行后续检测，具体操作详见说明书。原始结果如图10所示，结果分析见表4。
表4地贫结果分析
东南亚缺失(--SEA/--SEA) 左侧缺失型(-α4.2/αα) QS突变杂合子
左侧缺失型(-α4.2/αα) 41-42M杂合子 17M杂合子
正常样本 正常样本 右侧缺失型(-α3.7/-α3.7)
右侧缺失型(-α3.7/-α3.7) 正常样本 左侧缺失型(-α4.2/αα)
正常样本 正常样本 654M杂合子
结果表明，该试验检测结果与临床检测结果一致。
实施例7方法的应用案例---全外显子组基因测序
1、在已知的耳聋家系中(父、母(孕16周)、大儿子(聋儿)、小儿子(聋儿))，按照实施例2中所述方法，获得孕妇的宫颈脱落细胞标本和羊水标本，孕妇本人以及其他家系成员采集全血标本，并提取DNA(孕妇宫颈脱落细胞分选后标本按照实施例4所述提取方法获得，羊水和全血标本使用人类全血基因组DNA提取试剂盒(凯普生物，中国)进行提取(样本来源：凯普医学检验所检测人源标本，获得的人基因组DNA使用人全外显子组检测试剂盒(艾吉泰康生物科技(北京)有限公司，货号T086V4)进行全外显子测序(WES)：
将基因组DNA使用转座酶Tn5处理生成300bp片段建成DNA文库；两端加接头P5,P7,index1，2；选取适合长度DNA片段，并扩增纯化；与带有生物素的外显子探针库进行杂交；利用生物素biotin与链霉亲和素的强结合能力，将带有链霉亲和素的磁珠与已结合目标文库的探针结合起来；吸附磁珠，去除上清液；洗脱掉磁珠上的DNA，PCR扩增文库；鉴定文库质量；上机测序。
2、全外显子测序结果
使用高通量测序技术进行全外显子组检测，对检测到的致病或疑似致病位点使用Sanger测序进行验证。送检样本为孕16周羊水标本，有两个哥哥为聋哑，父母均正常。样本检出CDH23基因c.8363T>C(p.Leu2788Pro)杂合突变，GJB2基因c.109G>A(p.Val27Ile)杂合突变。家系图谱见图11。
结果表明，分选后的脱落细胞标本可有效检出致病突变，与羊水标本相比完全一致。检测结果可发现，该家系的致病原因和致病突变的携带情况。
实施例8方法的应用案例---全基因组DNA拷贝数变异(CNV)检测
1、采用Phalanx Biotech公司CytoOneArray染色体芯片及配套检测试剂盒，根据比较基因组杂交技术的原理(aCGH)检测全基因组DNA拷贝数变异：
实施例2方法分选的滋养层细胞，用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取，获得的人基因组DNA经片段化、扩增前处理、扩增及PCR产物纯化后，用两种不同的荧光染料进行标记(正常样品用Cy3标记呈现绿色，患者样品用Cy5标记呈现红色)；
荧光产物经纯化后注入芯片，芯片经清洗后进行扫描并获得结果。
2、全基因组DNA拷贝数变异(CNV)检测结果
全染色体示意图见图12，异常染色体的示意图见图13。图13中横轴为染色体区带的示意图，纵轴为样本与标准样本间的信号比值(以log2ratio表示)。当染色体拷贝数有显著差异时以不同颜色来表示。染色体发生扩增的区域(Gain)以蓝色表示，染色体发生缺失的区域(Loss)以红色表示。图中黑线的长度与数值分别代表每个区段(Segment)的大小与信号的平均值。
样本检测到1处异常，为22q11.21缺失，异常片段起始-结束位置[UCSC hg19]为arr22q11.21(19006943_21461068)x1，异常片段大小2.454Mb。相关疾病区域为Pathogenic(致病性，ACMG分类)，此段异常涵盖TBX1、CRKL、GP1BB、SLC25A1、DGCR10、TSSK1A、GSC2、CLTCL1等106个ISCA基因。此区域异常位于22q11.2recurrent(DGS/VCFS)region(includes TBX1)。22q11.2近端(A-D)区域的缺失与DiGeorge/Velocardioffacial(DGS/VCFS)综合征相关，临床表现通常为先天性心脏疾病、心脏异常、特征性面部特征、DD/ID、行为问题、免疫缺陷和低钙血症(PMID 25217958)。此区域异常位于22q11.2recurrent region(central,B/C-D)(includes CRKL)，此区域缺失可能导致的临床表型包括：畸形的面部特征，生长受限/身材矮小，中枢神经系统异常/发作，发育迟缓，智力障碍，骨骼异常，心血管缺陷，泌尿生殖系统异常和免疫缺陷/反复感染(PMID 25123976)。
结果表明，该分选细胞标本可有效的进行染色体结构变异检测并检出相应的突变。
实施例8基于微流控分选芯片从孕妇宫颈脱落细胞中分离筛选滋养层细胞方法与磁珠法对比
(一)实验样本：
宫颈脱落细胞液样本2份，来自凯普医学检验所检测人源标本。
(二)采用磁珠法分选作为对照实验，对比两种方法效果的差异。
本发明方法同实施例2。
磁珠法分选滋养层细胞的过程如下：
(1)将采集到的孕妇宫颈脱落细胞保存液标本震荡混匀，5min。
(2)将保存液取出置于15ml离心管中，在保存液的瓶中再加入3ml的细胞分离液，震荡混匀后，取出置于15ml离心管中。
(3)3000rpm离心10min，弃去上清。
(4)加入1ml的1×PBST，混匀后移入1.5mlEP管中，3000rpm离心5min后弃去上清。
(5)重复步骤4两次。
(6)加入200μl的0.5％的Triton X-100，混匀后室温通透20min。
(7)重复步骤4三次。
(8)加入一抗200μl，混匀后，4℃过夜。
(9)重复步骤4三次。
(10)加入二抗200μl，混匀后，37℃反应1h。
(11)重复步骤4三次后用200μl buffer(DPBS+0.1％BSA+2mM EDTA)重悬。
(12)将25μl beads与50μl buffer充分混匀后600g离心10min弃去上清，用25μl buffer重悬后加入第(11)步的混合液中。
(13)2℃-8℃反应20min。
(14)加入1ml buffer混匀，磁力架上静置2min后，弃去上清(留200μl对比)。
(15)重复步骤(14)两到三次。
(16)加入200μl 37℃预热的buffer重悬后，加4μl Release Buffer混匀。
(17)室温15min后用加样枪吹打5-10次，磁力架上静置2min，收集上清。
(18)加入200μl的buffer，吹打5-10次，磁力架上静置2min，收集上清。
(19)重复步骤(18)三次，最终获得滋养层细胞。
(三)结果
分选得到的阳性细胞用舜宇RX50荧光显微镜进行照片拍摄，如图14-16所 示：图14为本发明方法分选所得阳性细胞的照片，图15为本发明方法分选前后，阳性细胞与阴性细胞对比照片，图16为磁珠法分选所得阳性细胞的照片，对比可以明显的看出，两种方法筛选后的细胞数的数量级具有明显的差异，本发明方法显著优于磁珠法，统计显示本发明方法分选所得阳性细胞数可达约3000-13000个细胞。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (9)

  1. 一种分离滋养层细胞的方法，其特征在于，包括如下步骤：
    (1)将宫颈脱落细胞液样本制备成样本细胞悬浮液；
    (2)向样本细胞悬浮液中加入特异性抗体进行孵育；
    所述特异性抗体是对应滋养层细胞表面或胞内表达的特异性抗原的抗体组合，即HLA-G+CK7、HLA-G+CK18、HLA-G+β-HCG、CD31+HPL、MMP9+CD31、HLA-G+HPL、HLA-G+MMP9、HLA-G+CD31、HLA-G+P、CD31+P、HLA-G+CDH5、CD31+CDH5、CD31+CK7+HLA-G、HLA-G+CK18+CD31、HLA-G+β-HCG+CD31、CD31+HPL+HLA-G、MMP9+CD31+HLA-G、CD31+P+HLA-G或HLA-G+CDH5+CD31的特异性抗体组合；
    (3)利用流式细胞仪将步骤(2)孵育完成的细胞重悬液进行荧光标记分选，获得分离纯化的胎盘滋养层细胞；
    或利用微流控分选芯片，将步骤(2)孵育完成的细胞重悬液进行荧光标记微流控细胞分选，获得分离纯化的胎盘滋养层细胞。
  2. 根据权利要求1所述的方法，其特征在于，所述微流控分选芯片包括基片和与之贴合的盖片；
    所述基片的一面上设有主流道、侧流道A和侧流道B，两个侧流道分别靠近主流道的左、右两端部；
    所述基片的另一面上设有C入口、S入口、N出口和T出口；四个口均贯穿到基片另一面与流道连通；且C入口的位置对应于主流道的左端部，S入口的位置对应于侧流道A端部，N出口的位置对应于主流道的右端部，T出口的位置对应于侧流道B端部；
    所述主流道内、在N出口与T出口汇合处，还设有偏转电极装置。
  3. 根据权利要求2所述的方法，其特征在于，所有主流道、侧流道A和侧流道B的流道宽度均不超过1000μm，深度均不超过500μm。
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中一抗孵育条件为：4℃反应30-90min；二抗-荧光标记复合物孵育条件为2℃-8℃反应20min。
  5. 根据权利要求1所述的方法，其特征在于，步骤(2)的具体方法是：通过孵育将一抗、二抗-荧光标记复合物先后分步与目标抗原进行特异性连接，中间使用洗涤和离心分离技术避免交叉污染。
  6. 根据权利要求3所述的方法，其特征在于，步骤(3)的具体方法是：孵育完成的细胞重悬液通入微流控分选芯片的C入口，并在S入口通入缓冲液；然后将微流控分选芯片放置于细胞分选仪并运行分选程序，程序结束后，搜集T出口的标本，获得分选的滋养层细胞。
  7. 根据权利要求1所述的方法，其特征在于，步骤(3)中细胞分选液相体系为0.2％-0.4％Triton-X-100。
  8. 根据权利要求1所述的方法，其特征在于，步骤(1)中细胞悬浮液的最佳体系为含0.2％-0.4％FBS的1xPBS。
  9. 权利要求1-8任一所述方法在构建人类STR鉴定、人类染色体倍性检测、地中海贫血基因检测、耳聋基因检测、全外显子组基因测序、染色体微缺失/重复检测、或染色体结构变异检测的产品方面的应用。
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