Method for detecting non-target cells in amniotic epithelial cells

Abstract

The invention discloses a method for detecting non-target cells in amniotic epithelial cells, which comprises reagents for detecting one or more of the following gene markers: FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7, LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44, CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7, GZMH, KLRC2 and the like. Wherein the non-target cells are selected from macrophage-like cells, monocyte-like cells, T cell-like cells, NK cell-like cells, myeloid-like cells and neutrophil-like cells, a detection method is provided for quality control of the amniotic epithelial cells, and guidance is also provided for a cell product preparation process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell detection, and particularly relates to a method for detecting non-target cells in amniotic epithelial cells. BACKGROUND

[0002] The amnion is located at the innermost side of the fetal membrane and is adjacent to the amniotic fluid, with a thickness of 0.08-0.12 mm. After the embryo is implanted, the amniotic sac gradually wraps the growing embryo into the amniotic cavity, and the epithelial layer of the amnion is covered on the surface of the amniotic sac. The amnion-derived epithelial cells are a group of heterogeneous cells, including epithelial cells mainly with barrier defense function and stromalized epithelial cells mainly with repair function. The amnion-derived epithelial cells have been widely used in clinical research and application for various diseases due to their functions such as anti-inflammatory, antibacterial, scar repair, immune regulation, analgesia, promotion of angiogenesis, cell differentiation and adhesion, and promotion of muscle and skeletal tissue regeneration, and the amnion-derived epithelial cells are easy to obtain and do not involve ethical issues, thus becoming an ideal tissue source.

[0003] At present, the amniotic epithelial cells have overall epithelial characteristics, but are obviously heterogeneous under subdivision, that is, in addition to the amniotic epithelial cells and stromalized epithelial cells, there are some epithelial cells in transition state and immune cells, and the most obvious heterogeneity is the immune cell-related subpopulation. Thus, a part of cells which are obviously different from the majority of cells in characteristics and functions are defined as non-target cells. Patent document 202411246025X discloses a mild separation method of amniotic epithelial cells, which comprises selecting moderate-strength trypsin to preliminarily digest the amniotic tissue for 2-5 times, each time for 5-20 minutes; the preliminarily digested amniotic tissue is soaked in an amniotic culture medium at 37°C for a period of time, the amniotic culture medium changes from thin and clear to turbid, showing a certain viscosity and a colloid solution state of thread-drawing, the epithelial cells of the amniotic tissue are loosened and detached through the collagen swelling phenomenon of the amniotic tissue itself, and the amniotic epithelial primary cells are mildly obtained through mild digestion combined with the collagen swelling characteristics, which is an effective method for obtaining good-state amniotic epithelial cells.

[0004] The amniotic epithelial cells are a group of heterogeneous cells, including epithelial cells mainly with barrier defense function and stromalized epithelial cells mainly with repair function, but it is known that the amnion as a part of the placenta contains macrophages or Hofbauer cells and granulocytes, but the NK-like cells and T cell-like cells in the amniotic epithelial cells and other characteristics are rarely reported. SUMMARY

[0005] In view of the above-mentioned deficiencies of the prior art, the purpose of the present application is to provide a method for detecting non-target cells in amniotic epithelial cells, which finds out a significantly heterogeneous subpopulation and defines it as a non-target cell group through single cell data integration analysis of amnion and placenta, further confirms the cell type and sorts out the related Gene Marker, and then detects the content of non-target cells in amniotic epithelial cells through these MRKER, so as to realize the quality control or sorting culture of amniotic cell products.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The present application provides a product for detecting non-target cells in amniotic epithelial cells, which comprises a reagent for detecting one or more of the following gene markers: FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7, LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44, CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7, GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G, ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1, OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG.

[0008] The non-target cells in the amniotic epithelial cells include one or more of macrophage-like cells (MACROPHAGE-LIKE CELL), monocyte-like cells (MONOCYTE-LIKE CELL), T cell-like cells (T-LIKE CELL), NK cell-like cells (NK-LIKE CELL), neutrophil-like cells (NEUTROPHIL_LIKE_CELL) and myelocyte-like cells (MYELOCYTE_LIKE_CELL).

[0009] As a preference, the gene marker combination of the macrophage-like cells is one or more of FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7.

[0010] As a preference, the gene marker combination of the monocyte-like cells is one or more of LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44.

[0011] As preferred, the specific genetic marker combination of the T cell-like cell is one or more of CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7.

[0012] As preferred, the specific genetic marker combination of the NK cell-like cell is one or more of GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G.

[0013] As preferred, the specific genetic marker combination of the myelocyte-like cell is one or more of ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1.

[0014] As preferred, the specific genetic marker combination of the neutrophil-like cell is one or more of OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG.

[0015] The present application also provides a method for detecting non-target cells in amniotic epithelial cells, comprising the following steps: detecting the expression level of one or more of the following genetic markers in the amniotic epithelial cells by any of the detection products: FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7, LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44, CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7, GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G, ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1, OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG; and then detecting the expression amount of the non-target cells in the amniotic epithelial cells by Q-PCR, and then detecting the content of the non-target cells by relative quantification method or absolute quantification method.

[0016] As preferred, the non-target cells in the amniotic epithelial cells comprise one or more of macrophage-like cells, monocyte-like cells, T cell-like cells, NK cell-like cells, neutrophil-like cells, and myelocyte-like cells.

[0017] As preferred, the macrophage-like cells are detected by detecting the expression level of one or more of the specific genetic markers FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7.

[0018] and / or detecting monocyte-like cells by detecting the expression level of one or more of the specific gene markers combination LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44;

[0019] and / or detecting T cell-like cells by detecting the expression level of one or more of the specific gene markers combination CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7;

[0020] and / or detecting NK cell-like cells by detecting the expression level of one or more of the specific gene markers combination GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G;

[0021] and / or detecting myelocyte-like cells by detecting the expression level of one or more of the specific gene markers combination ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1;

[0022] and / or detecting neutrophil-like cells by detecting the expression level of one or more of the specific gene markers combination OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] Through single-cell sequencing (scRNA-seq) of fresh isolated amniotic epithelial cells produced at full term, and integrated analysis of single-cell data of placental tissue, on the one hand, the heterogeneity of amniotic membrane is understood, and on the other hand, by analyzing the single-cell sequencing data, several heterogeneous populations are determined, the characteristic differential genes of each population are verified in the expression amount of the transcriptome sample of the amniotic membrane, and the non-target cell populations in the amniotic epithelial cells, including macrophage-like cells, monocyte-like cells, T cell-like cells, NK cell-like cells, neutrophil-like cells and myelocyte-like cells, are determined, and the corresponding cell population characteristic GENE MARKERS are obtained, thereby providing a detection method for quality control of amniotic epithelial cells, and providing guidance for cell product preparation process. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For the clustering of non-target cell subpopulations in the examples, the cell population circled by the red solid line is obviously separated from the whole of the remaining cell populations.

[0026] Figure 2Cell type definition was performed for SINGLE R in the example, where Cluster 10 was defined as MACROPHAGE-LIKE CELL, Cluster 18 was defined as MONOCYTE-LIKE CELL, Cluster 17+8 was defined as MYELOCYTE-LIKE CELL, Cluster 20 was defined as T-LIKE CELL, Cluster 21 was defined as NK-LIKE CELL, Cluster 13 was defined as NEUTROPHIL-LIKE CELL.

[0027] Figure 3 Highly expressed genes MARKER (AM23030EP00, AM23030EP0, AM23030EP1, AM23031EP00, AM23031EP0, AM23031EP1, AM23032EP00, AM23032EP0, AM23032EP1) were screened from the transcriptome data of 9 samples from different batches of cells of three donors in the example, Cluster 10 specific differential genes FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7 had higher expression in the transcriptome samples, defined as MACROPHAGE-LIKE CELL.

[0028] Figure 4 Cluster 18 specific differential genes LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44 had higher expression in the transcriptome samples in the example, defined as MONOCYTE-LIKE CELL.

[0029] Figure 5 Cluster 20 specific differential genes CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7 had higher expression in the transcriptome samples in the example, defined as T-LIKE_CELL.

[0030] Figure 6 Cluster 21 specific genes GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G had higher expression in the transcriptome samples in the example, defined as NK-LIKE CELL.

[0031] Figure 7For the example, Cluster 17+8 specific differential genes have higher expression in ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1 in the transcriptome sample, defined as MYELOCYTE_LIKE_CELL.

[0032] Figure 8 For the example, Cluster 13 specific differential genes have higher expression in OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG in the transcriptome sample, defined as NEUTROPHIL_LIKE_CELL.

[0033] Figure 9 For the example, amplification curve of gene marker in non-target cells was detected by qPCR.

[0034] Figure 10 For the example, melting curve of gene marker in non-target cells was detected by qPCR.

[0035] Figure 11 For the example, relative expression of gene marker in non-target cells was detected by qPCR.

[0036] Figure 12 For the example, correlation verification results of lymphocyte proliferation inhibition rate and non-target cell content. DETAILED DESCRIPTION

[0037] In order to more fully understand the technical solutions, objects and advantages of the present application, the technical effects produced by the present application will be further described in detail below in combination with the drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, not all. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present application all belong to the protection scope of the present application.

[0038] Example 1

[0039] In this embodiment, the non-target cell subpopulation in amniotic epithelial cells was confirmed, and the quality control was performed by GENE MARKER detection of cells.

[0040] Materials: three fresh amniotic membrane tissues of full-term cesarean section.

[0041] Sample acquisition method: The same as the patent document 202411246025X "A gentle separation method of amniotic epithelial cells" to separate and prepare cell samples. In short, the sample was washed to remove blood stains, digested three times, and then incubated in amniotic medium overnight. The cells were gently stirred, centrifuged, washed, and counted. 5x10^6 cells were collected for single-cell sequencing, and the rest was 5.5x10^4 / cm 2 After inoculation into the treated culture flask and subcultured to EP1, 5x10^6 cells were collected for single-cell sequencing.

[0042] Data acquisition: Six samples were taken for detection and processed by Cell ranger to obtain matrix data. Placental single-cell data GSE173193 (C1, C2) was downloaded from the GOE website. All matrix data was analyzed using RSeurat V5, and after data integration, dimensionality reduction, and clustering processes, the non-target cell subpopulation clustering situation was obtained as shown in Figure 1 The red solid line circled cell population was obviously separated from the rest of the cell population. The characteristic genes of each subpopulation were found by the FindMarker() function to confirm the subpopulation attributes and GENE MARKER. Except for Cluster 16, which was a proliferation characteristic cell population, the rest were immune-related cell subpopulations, as shown in Table 1, which only showed the TOP 40 GENE MARKER.

[0043] Table 1

[0044]

[0045]

[0046] Cell type classification and definition were performed using SINGLE R, as shown in Figure 2 Cluster 10 was defined as MACROPHAGE-LIKE CELL, Cluster 18 was defined as MONOCYTE-LIKE CELL, Cluster 17+8 was defined as NEUTROPHIL-LIKE CELL, Cluster 20 was defined as T-LIKE CELL, Cluster 21 was defined as NK-LIKE CELL, and Cluster 13 was defined as MYELOCYTE-LIKE CELL.

[0047] To further screen the gene MARKER from the expression level, the transcriptome data of 9 samples of different batches of cells of three donors (AM23030EP00, AM23030EP0, AM23030EP1, AM23031EP00, AM23031EP0, AM23031EP1, AM23032EP00, AM23032EP0, AM23032EP1) were analyzed to further screen the highly expressed GENE MARKER, and the results are shown in Figures 3 to 8

[0048] As shown in Figure 3 , the Cluster 10 specific differential genes have higher expression of FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7 in the transcriptome sample, and are defined as MACROPHAGE-LIKE CELL.

[0049] As shown in Figure 4 , the Cluster 18 specific differential genes have higher expression of LGALS2, LILRB4, HLA-DMB, HLA-DMA, CD74, RNASE6, ANPEP, CD44 in the transcriptome sample, and are defined as MONOCYTE-LIKE CELL.

[0050] As shown in Figure 5 , the Cluster 20 specific differential genes have higher expression of CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7 in the transcriptome sample, and are defined as T-LIKE_CELL.

[0051] As shown in Figure 6 , the Cluster 21 specific genes have higher expression of GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G in the transcriptome sample, and are defined as NK cell.

[0052] As shown in Figure 7 , the Cluster 17+8 specific differential genes have higher expression of ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1 in the transcriptome sample, and are defined as neutrophil, named MYELOCYTE_LIKE_CELL.

[0053] As shown in Figure 8 ​As shown, Cluster 13-specific differentially expressed genes OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, and GMFG were highly expressed in the transcriptome sample. This cell population was defined as NEUTROPHIL_LIKE_CELL.

[0054] Based on the expression abundance of the gene MARKER, one or more combinations of the above genes were selected as GENE MARKER in each non-target cell population. The expression level of non-target cells in amniotic epithelial cells was detected by qPCR. The primers used are shown in Table 2. The content of non-target cells was detected by relative quantification or absolute quantification.

[0055] Table 2

[0056]

[0057]

[0058] Example 2

[0059] I. Materials

[0060] Two amniotic membrane samples were taken from full-term cesarean sections and separated by different personnel according to the above method. The freshly separated samples were named AM25001EP00 and AM25002EP00, respectively, and the samples after passage culture were named AM25001EP1 and AM25002EP1. PBMC was selected as a positive control and 293T as a negative control.

[0061] II. Reagents

[0062] 1. Cell culture media: amniotic cells (basal medium + 5% ELITE + 10ng / mL EGF growth factor), PBMCs (Zhuhai Beso ALYS505N-0 medium), 293T cells (DMEM + 10% serum).

[0063] 2. Nucleic acid extraction reagent: Adazol Total RNA Extraction Reagent (Adamas life).

[0064] 3. qPCR kits: Best Biotech All-in-one RT SuperMix for qPCR (With gDNAEraser); 2×Universal SYBR qPCR Master Mix (Best Biotech); Adazol Total RNAExtraction Reagent (Adamas Life).

[0065] 4. Primer synthesis: synthesized by Shanghai Biotech, select CD68, CD3D, KLRD1 / CD94, CEACAM8 to detect the corresponding non-target cells respectively.

[0066] III. Equipment

[0067] CO2 cell incubator Thermo 371, Roche 96 qPCR instrument, HF safe A2 biosafety cabinet, microspectrophotometer (NanoDrop One).

[0068] IV. Method

[0069] 1. Collect EP00 and EP1 amniotic cell samples and positive control cells PBMC and negative control cells 293T.

[0070] 2. RNA extraction and reverse transcription to obtain cDNA template.

[0071] 3. SYBR Green dye method for qPCR detection.

[0072] 4. Relative expression calculation

[0073] The relative expression level of the target gene in different samples (EP00, EP1, PBMC, 293T) is calculated by 2^(-ΔΔCt).

[0074] V. Results

[0075] The qPCR amplification curve can be seen, the curve has obvious exponential growth period, the overall trend is consistent, the repeatability is good Figure 9 ); qPCR melting curve can be seen, peak group is concentrated, dispersion is small, the consistency of amplification products between samples is good Figure 10 ); qPCR is used to detect the relative expression of non-target cells. By detecting one or several genes, the type of non-target cells in the sample and the expression of related genes are determined. The reference gene GAPDH is defined as 100% expression, and the relative expression of other genes is shown in Figure 11 . Due to the low content of neutrophils in the separation process of PBMC, the expression of CEACAM8 in PBMC is low.

[0076] Genetic testing of two samples revealed that sample AM25002EP00 showed more expression of non-target cells than AM25001EP00, including macrophage-like cells, T-like cells, NK-like cells, and neutrophil-like cells. Sample AM25002EP00, due to logistical reasons, underwent prolonged tissue exposure, resulting in increased oxidative stress and inflammatory cytokine stimulation, leading to greater upregulation of immune-related genes. The test results are consistent with this, indicating the feasibility of the detection method. In both samples, after a period of culture and passage to the EP1 generation, EP00 cells underwent post-stress repair during culture, resulting in a significant reduction in the expression levels of T-like cells, NK-like cells, and neutrophil-like cells, which is also in line with expectations.

[0077] Placental macrophages, known as Hofbauer cells (HBCs), are often classified as anti-inflammatory M2 macrophages. However, influenced by the surrounding environment, they can transform into a mixed phenotype of pro-inflammatory M1 and anti-inflammatory M2 macrophages. Sample AM25001 initially showed a relatively low number of macrophages, but this increased after culture, suggesting that it may be predominantly M2 macrophages. AM25002, on the other hand, showed high expression levels before culture but decreased after culture, possibly indicating that its main macrophages are M1 macrophages, which decreased after culture and repair. Based on the above, it can be determined that the detection method for non-target cells in amniotic epithelial cells in this invention is reliable. The separation process or culture method can be optimized based on the expression of non-target cells to meet practical application needs.

[0078] Example 3: Detection of non-target cells

[0079] I. Materials

[0080] Nine amniotic epithelial cell samples from full-term cesarean section patients were taken from the cell bank and were identified as AM24001EP0, AM24002EP0, AM24004EP0, AM23013EP0, AM23034EP0, AM23038EP0, AM23020EP0, AM23026EP0, and AM23039EP0.

[0081] II. Reagents

[0082] 1. Cell culture medium: amniotic cells (basal medium + 5% ELITE + 10ng / mL EGF growth factor); PBMC (Zhuhai Beso ALYS505N-0 medium + 10μg / mL ConA + 1000UI / mL IL-2).

[0083] 2. Nucleic acid extraction reagent: Adazol Total RNA Extraction Reagent (Adamas life).

[0084] 3. qPCR kit: Boster All-in-one RT SuperMix for qPCR (With gDNA Eraser) (Boster Biological Technology); 2x Universal SYBR qPCR Master Mix (Boster Biological Technology); Adazol Total RNA Extraction Reagent (Adamas life).

[0085] 4. Primer synthesis: synthesized by Shanghai Sungene, and CSF1R, CD74, CD3G, KLRC2, IFITM1, and CEACAM8 were selected to detect the expression of corresponding non-target cells.

[0086] III. Equipment

[0087] CO2 cell incubator: Thermo 371; Roche 96 qPCR instrument; HFsafe A2 biosafety cabinet; microspectrophotometer (NanoDrop One); 6-well plate; electric pipette; 10 mL pipette; 15 mL centrifuge tube.

[0088] IV. Methods

[0089] (I) Non-target cell detection

[0090] 1. Resuscitate 9 cell samples taken from the cell bank

[0091] Prepare a 37°C constant temperature water bath in advance, take the 9 amniotic epithelial cells stored in liquid nitrogen, transfer them from the liquid nitrogen transfer tank to the cell culture room, and place them in the water bath in batches for resuscitation. Gently shake the cell bank during resuscitation to ensure that the frozen tube completely melts within 2 minutes. After resuscitation, disinfect the outer wall of the frozen tube with 75% alcohol, and transfer the cell suspension to 9 centrifuge tubes containing 15 mL of preheated culture medium in the safety cabinet, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and retain the cell pellet, add 2 mL of fresh culture medium to resuspend the cells, count them, and then inoculate the cells into 6-well plates at 5x10^5 per well. Place in a 37°C, 5% CO2 incubator for culture, and replace the culture medium after the cells adhere. A total of 6 6-well plates, 36 wells, of which 9 wells were digested after 3 days of culture for detection of non-target gene expression, and the remaining 27 sample wells were used for co-culture with PBMC.

[0092] Three PBMC samples, sample numbers: IF00000440, IF00001394, IF00001441, were resuscitated in the same way and inoculated at 1 x 106 / well in 81 wells of 14 6-well plates. 27 wells were blank group and 54 wells were stimulation proliferation group, of which 27 wells were simple stimulation proliferation group and 27 wells of 2.7 x 107 cells were used to inoculate 27 amniotic epithelial cell wells for co-culture, ensuring that the culture medium volume of each well was 2 mL, and then 2 mL of liquid was supplemented every day. After 96 h, the suspended PBMCs were collected for AO / PI counting (Note: the blank culture medium should be flushed into each well for 3 times to achieve accurate counting).

[0093] 2. RNA extraction and reverse transcription to obtain cDNA template

[0094] RNA extraction: Take cells in logarithmic growth phase and operate according to the instructions of the extraction kit. First, add lysis solution to fully lyse the cells, use a pipette to repeatedly blow and beat until there are no obvious cell clumps, then add chloroform, shake vigorously for 15 seconds, and then stand at room temperature for 3 minutes. Centrifuge at 4°C, 12000 rpm for 15 minutes, carefully aspirate the upper colorless aqueous phase into a new centrifuge tube, add an equal volume of isopropanol, mix gently after inverting, then stand at room temperature for 10 minutes. Centrifuge again at 4°C, 12000 rpm for 10 minutes, discard the supernatant, add 75% ethanol to wash the RNA precipitate, centrifuge at 4°C, 7500 rpm for 5 minutes, discard the ethanol, then air dry the precipitate at room temperature, and finally dissolve the RNA with RNase-free water. Use a UV spectrophotometer to detect the concentration and purity of the RNA, and ensure that the OD260 / OD280 ratio is between 1.8 and 2.0.

[0095] Prepare the reaction system, with a total reaction volume of 20 μL, including 500 ng RNA template, 4 μL 5 x reverse transcription buffer, 1 μL reverse transcriptase, 1 μL primer mixture, and RNase-free water. Mix the reaction system gently, centrifuge briefly, then place it in a PCR instrument, set the reaction program: 37°C incubation for 15 minutes for reverse transcription, 85°C heating for 5 seconds (inactivate reverse transcriptase), and after the reaction is completed, obtain the cDNA template, which can be used immediately or stored at -20°C.

[0096] 3. qPCR detection by SYBR Green dye method

[0097] Prepare primers, including the reference gene GAPDH, SYBR Green qPCR mixture, cDNA template, and enzyme-free water in advance. Prepare the qPCR reaction system on ice. Set up three replicates for each sample, with a total reaction volume of 20 μL, containing 10 μL of SYBR Green mixture, 1 μL of cDNA template, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), and 8 μL of enzyme-free water. Gently mix the prepared reaction system, avoiding air bubbles, and briefly centrifuge. Transfer the mixture to a 96-well qPCR plate and seal the wells with a sealing film to prevent evaporation and contamination. Place the 96-well plate in a qPCR instrument and set the reaction program: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing extension for 30 seconds, for a total of 40 cycles. Finally, perform melting curve analysis by slowly increasing the temperature from 65℃ to 95℃. Copy the results for further analysis.

[0098] 4. Calculation of relative expression levels

[0099] After the experiment, the Ct values ​​of the target gene and internal reference gene for each sample were obtained using the Light Cycler software built into the qPCR instrument. The ΔCt value for each replicate well was calculated (ΔCt = target gene Ct value - internal reference gene Ct value), and the average of the three replicates was taken as the ΔCt value for that sample. Using one well as a control, the ΔΔCt values ​​for the other wells were calculated (ΔΔCt = sample well ΔCt value - control well ΔCt value). The relative expression level of the target gene in different samples was calculated using the 2^(-ΔΔCt) method.

[0100] (II) Effects of non-target cell expression levels in amniotic epithelial cells on immune cell proliferation inhibition experiments

[0101] 1. Grouping

[0102] 1.1 PBMC Blank Group

[0103] PBMCs (1×10^6 cells / well) served as a baseline control, reflecting the natural proliferation state of PBMCs under unstimulated conditions.

[0104] 1.2 PBMC-stimulated proliferation group

[0105] PBMCs (Zhuhai Beso ALYS505N-0 medium + 10 μg / mL ConA + 1000 UI / mL IL-2) were used as the baseline group to reflect the maximum proliferative capacity of PBMCs under strong stimulation conditions.

[0106] 1.3 Co-culture group

[0107] PBMCs (Zhuhai Beso ALYS505N-0 medium + 10 μg / mL ConA + 1000 UI / mL IL-2) + AMECs (5 × 10^5 cells / well) were used as the experimental group to detect the inhibitory effect of AMECs on PBMC proliferation, including direct contact and paracrine coordination.

[0108] 2. Result Processing

[0109] After culture, gently pipette the cells in the wells to completely collect PBMCs; take 20 μL of each sample, add 20 μL of AO / PI staining solution at room temperature in the dark, mix thoroughly, and incubate for 2 min; then take another 20 μL and add it to a counting chamber, and count the cells using a Countstar automated cell counter:

[0110] Inhibition rate = [(Control wells - Co-culture wells) / (Control wells - Blank wells)] × 100%

[0111] V. Results

[0112] 1. qPCR results

[0113] The qPCR amplification curves show a clear exponential growth phase, with a consistent overall trend and good reproducibility. Figure 9 The qPCR melting curves show concentrated peak clusters with low dispersion, indicating good consistency of amplification products among samples. Figure 10 The results of gene expression level detection in non-target cells are shown in Table 3 and... Figure 11 As shown.

[0114] Table 3: Results of gene expression level detection in non-target cells

[0115]

[0116]

[0117] 2. Results of immune cell proliferation inhibition rate

[0118] The data on the inhibition rate of immune cell proliferation are shown in Table 4.

[0119] Table 4

[0120]

[0121]

[0122] 3. Correlation test between lymphocyte proliferation inhibition rate and non-target cell content

[0123] The above data accorded with normal distribution, and the relationship between the non-target cell content and the lymphocyte proliferation inhibition rate was detected using Pearson Correlation, and the results are shown in Table 1. Figure 12 The results showed that the non-target cell content was negatively correlated with the lymphocyte proliferation inhibition rate, and the average negative correlation coefficient Cor was >0.95.

[0124] In summary, amniotic epithelial cells are a group of heterogeneous cells, including epithelial cells mainly with barrier defense function and interstitial cells mainly with repair function, but the most obvious heterogeneity is the immune cell-related subpopulation. It is known that amnion, as a part of placenta, contains macrophages or Hofbauer cells and granulocytes, but the immune cells such as NK-like cells and T cell-like cells in amniotic epithelium are not completely the same as peripheral blood mature immune cells, but are a kind of specialized cells that can perform immune functions. The detection method of non-target cells in amniotic epithelial cells in the present application can be used to evaluate the quality of amniotic epithelial cells, or to sort and classify culture, and has important guiding significance for the classification application of amniotic epithelial cells to different diseases in clinic.

[0125] The above only for the preferred embodiments of the present application, and is not intended to limit the present application, for those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A product for detecting a non-target cell in an amniotic epithelial cell, characterized by, The reagent comprises one or more of the following gene markers: FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7, LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44, CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7, GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G, ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1, OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG; wherein the non-target cells in the amnion epithelial cells comprise one or more of macrophage-like cells, monocyte-like cells, T cell-like cells, NK cell-like cells, myelocyte-like cells, and neutrophil-like cells.

2. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the macrophage-like cells is one or more of FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, and EGFL7.

3. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the monocyte-like cells is one or more of LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, and CD44.

4. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the T cell-like cells is one or more of CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, and TCF7.

5. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the NK cell-like cells is one or more of GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, and APOBEC3G.

6. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the myelocyte-like cells is one or more of ALPL, HCAR2, IFITM2, IFITM1, MYO1F, and AIF1.

7. The product for detecting non-target cells in the amniotic epithelial cells according to claim 1, wherein The specific gene marker combination of the neutrophil-like cells is one or more of OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, and GMFG.

8. A method for detecting non-target cells in amniotic epithelial cells, characterized by, comprising the steps of: detecting the expression level of one or more of the following gene markers: FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7, LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44, CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7, GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G, ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1, OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG, by the detection product of any one of claims 1 to 7, then detecting the expression level of the non-target cells in the amnion epithelial cells by Q-PCR, and then detecting the content of the non-target cells by relative quantification or absolute quantification; wherein the non-target cells in the amnion epithelial cells comprise one or more of macrophage-like cells, monocyte-like cells, T cell-like cells, NK cell-like cells, myelocyte-like cells and neutrophil-like cells.

9. The method of claim 8, wherein the non-target cells are selected from the group consisting of bacteria, fungi, viruses, and prions. detecting macrophage-like cells by detecting the expression level of one or more of the specific gene marker combination FCGBP, RNASE6, DAB2, STAB1, RAB3IL1, CSF1R, CD68, CD209, EGFL7; and / or detecting monocyte-like cells by detecting the expression level of one or more of the specific gene marker combination LGALS2, LILRB4, HLA-DMB, HLADMA, CD74, ANPEP, CD44; and / or detecting T cell-like cells by detecting the expression level of one or more of the specific gene marker combination CCR7, CD3G, CD3D, CD27, CD5, SPOCK2, TCF7; and / or detecting NK cell-like cells by detecting the expression level of one or more of the specific gene marker combination GZMH, KLRC2, KLRD1, GZMB, KLRF1, CCL5, APOBEC3G; and / or detecting myelocyte-like cells by detecting the expression level of one or more of the specific gene marker combination ALPL, HCAR2, IFITM2, IFITM1, MYO1F, AIF1; and / or detecting neutrophil-like cells by detecting the expression level of one or more of the specific gene marker combination OLFM4, CAMP, CEACAM8, RETN, HP, RFLNB, GMFG.

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