A marker combination for identifying dp cells in vitro culture and application thereof

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to a combination of biomarkers for identifying cultured DP cells and their applications. The biomarker combination for identifying cultured DP cells includes the HES1 gene, the TINAGL1 gene, and the IL1B gene. This biomarker combination can be used to identify cultured DP cells.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a combination of biomarkers for identifying DP cells cultured in vitro and their applications. Background Technology

[0002] A normal hair follicle (HF) continuously goes through the anagen (growth) phase, catagen (regression) phase, and telogen (resting) phase. Dermal papilla cells (DPCs) are considered a unique type of mesenchymal stem cell with the potential for autologous stem cell therapy. They are located at the base of the hair follicle, in the hair bulb, and are surrounded by the dermal sheath and hair matrix cells.

[0003] Dermal papilla cells (DPs), as signaling centers for hair follicle stimulation (HF), play a crucial role in regulating hair growth, formation, and circulation. During in vitro isolation of DPs, the unique anatomical location and distinct biological characteristics of the dermal papilla make the extraction of pristine cells extremely difficult, posing a high risk of contamination. This contamination can negatively impact the purity of subsequently grown cells, resulting in the presence of numerous contaminating cells. Therefore, the identification of in vitro cultured DPs is essential for their subsequent application research.

[0004] Currently, methods for identifying diploid cells (DPs) in vivo are relatively mature. After being ex vivo, these cells lose neurohumoral regulation and intercellular interactions, living in a relatively stable environment lacking dynamic equilibrium. This makes them prone to weakened differentiation, a tendency towards morphological and functional homogenization, and transformation leading to immortality. Although in vitro cells differ from somatic cells, they do not lose their research significance. Ex vivo cells still carry a complete set of diploid genes. The behavior of cells in culture is merely a phenomenon caused by the switching on and off of corresponding genes, not an absolute defect. On the contrary, the loss of certain specific functions in cultured cells can provide clues about the expression and regulation of these genes. However, currently, there is no mature identification method for effectively identifying in vitro cultured DPs. Therefore, there is an urgent need to develop a method for accurately and rapidly identifying DPCs to support the identification of in vitro DPCs before their application. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of biomarkers for identifying DP cells in vitro and their applications, so as to solve or improve the problem of DP cell identification in vitro.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a combination of biomarkers for identifying DP cells cultured in vitro, wherein the biomarkers include the HES1 gene, the TINAGL1 gene, and the IL1B gene.

[0007] The present invention also provides an application of the marker combination for identifying DP cells in vitro as described above, which adopts the following technical solution: the application of the marker combination as described above in the in vitro isolation and culture of DP cells from hair follicle tissue.

[0008] The present invention also provides an in vitro cultured DP cell identification reagent, which adopts the following technical solution: an in vitro cultured DP cell identification reagent, the reagent comprising primers for detecting at least one of the HES1 gene, TINAGL1 gene and IL1B gene.

[0009] In a preferred embodiment of the DP cell identification reagent of the present invention, the primers used for detecting the HES1 gene are:

[0010] HES1-F: 5'-CAAGCTGGAGAAGGCGGACA-3';

[0011] HES1-R: 5'-TCGGTACTTCCCCAGCACACT-3';

[0012] The primers used to detect the TINAGL1 gene are:

[0013] TINAGL1-F: 5'-GGCCAGAGAGATACCGCCGG-3';

[0014] TINAGL1-R: 5'-ATGCGGAAGTGGCCCCTCTC-3';

[0015] The primers used to detect the IL1B gene are:

[0016] IL1B-F: 5'-CCGCGTCAGTTGTTGTGGCC-3';

[0017] IL1B-R: 5'-AGTCCCGGAGCGTGCAGTTC-3'.

[0018] The present invention also provides a DP cell identification kit, which adopts the following technical solution: a DP cell identification kit, the kit comprising the reagents described above.

[0019] The present invention also provides a method for identifying DP cells, which adopts the following technical solution: A method for identifying DP cells, which determines whether cells cultured in vitro are DP cells by detecting the expression levels of HES1, TINAGL1 and IL1B genes relative to housekeeping genes.

[0020] In a preferred embodiment of the DP cell identification method of the present invention, the expression level of at least one of the HES1 gene, TINAGL1 gene and IL1B gene is obtained by real-time RT-PCR detection.

[0021] In a preferred embodiment of the DP cell identification method of the present invention, the criterion for determining DP cells in vitro is the ΔCt value; the ΔCt value of DP cells meets the following criteria: the ΔCt value of the HES1 gene ≤ 11.8, the ΔCt value of the TINAGL1 gene ≤ 11.2, and the ΔCt value of the IL1B gene ≤ 4.4; the ΔCt value is calculated as follows: Ct value of the gene to be tested - Ct value of the housekeeping gene; the housekeeping gene is the GAPDH gene.

[0022] In a preferred embodiment of the DP cell identification method of the present invention, the primers for detecting the GAPDH gene are:

[0023] GAPDH-F:5'-AGCCACATCGCTCAGACACC-3';

[0024] GAPDH-R:5'-GTACTCAGCGCCAGCATCG-3'.

[0025] Beneficial effects:

[0026] The inventors discovered for the first time through experiments that the HES1, TINAGL1, and IL1B genes can be used to identify DP cells. The experiments verified that the identification of DP cells in vitro can be achieved by detecting the relative expression levels of the HES1, TINAGL1, and IL1B genes.

[0027] The DP cell identification method of the present invention is simple, easy to implement, and low in cost. By developing and manufacturing DP cell identification products, it is possible to conveniently, quickly, accurately, and in large quantities identify DP cells cultured in vitro, which is of great significance for clinical treatment and other applications, and fills the gap in related content in the prior art. Attached Figure Description

[0028] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Wherein:

[0029] Figure 1 Microscopic images of the cell morphology of primary isolated DP cells and low-purity DP cells provided in embodiments of the present invention; wherein, Figure 1 A (0.1 mm) and 1B (100 μm) are morphological images of primary isolated DP cells; Figure 1C (0.1 mm) and 1D (100 μm) are morphological images of low-purity DP cells isolated from primary culture.

[0030] Figure 2 Comparison of alkaline phosphatase (ALP) immunofluorescence results between primary isolated DP cells and low-purity DP cells; from top to bottom: GFP fluorescence image; nuclear-stained DAPI fluorescence image; fluorescence image of the two superimposed;

[0031] Figure 3 RNA seq results for identifying characteristic genes in screened DP cells;

[0032] Figure 4 The images show agarose gel electrophoresis of the amplification products; where ② is the electrophoresis image of the IL1B gene PCR product, ③ is the electrophoresis image of the TINAGL1 gene PCR product, and ⑤ is the electrophoresis image of the HES1 gene PCR product. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0034] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed.

[0035] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be used in this invention.

[0036] The present invention will now be described in detail with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0037] This invention addresses the problem that the effectiveness of in vitro identification of DP cells needs further improvement by providing a combination of biomarkers for identifying in vitro cultured DP cells. The biomarker combination for identifying in vitro cultured DP cells provided in this embodiment includes the HES1 gene, the TINAGL1 gene, and the IL1B gene.

[0038] Among them, the HES1 gene (hes family bHLH transcription factor 1, Gene ID: 3280) belongs to the proneural basic helix-loop-helix (BHLH) gene family. BHLH is a group of transcription factors involved in cell differentiation. As a downstream target gene of notch protein, HES1 transmits notch signals, which can maintain various immature cells in an undifferentiated state, regulate the cell response to differentiation-inducing factors, maintain a stable number of undifferentiated cells, and keep stem cells in a proliferative state.

[0039] The TINAGL1 gene (tubulointerstitial nephritis antigen-like 1, Gene ID: 64129), recombinant human tubulointerstitial nephritis antigen-like protein 1 (TINAGL1), can inhibit two major signaling pathways: epidermal growth factor receptor (EGFR) and integrin pathway. Mutations in the EGFR gene can lead to a surge in EGFR signaling activity, which in turn sends growth-promoting signals to cells. The integrin signaling pathway is involved in regulating cell migration to new locations and adhesion to other cells.

[0040] The IL-1B gene (interleukin 1 beta, Gene ID: 3553) contains IL-1B, a key pro-inflammatory cytokine involved in various autoimmune inflammatory responses and cellular activities, including cell proliferation, differentiation, and apoptosis. IL-1B is essential for cellular defense and tissue repair in almost all tissues and is associated with pain, inflammation, and autoimmunity. IL-1B also participates in neuroprotection, tissue remodeling, and repair.

[0041] Through experiments, the inventors discovered that, compared to low-purity DP cells ("low-purity DP cells" refers to DP cells isolated and cultured from hair follicle cells that contain a large number of cells other than DP cells, such as keratinocytes, immune cells, and vascular cells), primary DP cells have higher expression levels of HES1, TINAGL1, and IL1B genes. The expression of HES1, TINAGL1, and IL1B genes is related to stem cell proliferation, cell proliferation, tissue repair, and remodeling. The inventors further identified HES1, TINAGL1, and IL1B genes as biomarkers that can be used to identify DP cells. Experiments verified that the relative expression levels of HES1, TINAGL1, and IL1B genes can be used to identify DP cells cultured in vitro.

[0042] The experimental results showed that the HES1, TINAGL1, and IL1B genes were all highly expressed in DP cells compared to low-purity DP cells. These genes are suitable as biomarkers for identifying DP cells.

[0043] The present invention also proposes an application of the marker combination as described above, in an embodiment of the present invention, the application of the marker combination as described above in the in vitro isolation and culture of DP cells from hair follicle tissue.

[0044] The present invention also proposes an in vitro cultured DP cell identification reagent, wherein the reagent of the present invention includes primers for detecting at least one of the HES1 gene, TINAGL1 gene and IL1B gene.

[0045] In a preferred embodiment of the present invention, the primers used for detecting the HES1 gene are:

[0046] HES1-F: 5'-CAAGCTGGAGAAGGCGGACA-3';

[0047] HES1-R: 5'-TCGGTACTTCCCCAGCACACT-3'. This primer for detecting the HES1 gene has high specificity and sensitivity, and can accurately detect the expression level of the HES1 gene.

[0048] In a preferred embodiment of the present invention, the primers used for detecting the TINAGL1 gene are:

[0049] TINAGL1-F: 5'-GGCCAGAGAGATACCGCCGG-3';

[0050] TINAGL1-R: 5'-ATGCGGAAGTGGCCCCTCTC-3'. This primer for detecting the TINAGL1 gene has high specificity and sensitivity, and can accurately detect the expression level of the TINAGL1 gene.

[0051] In a preferred embodiment of the present invention, the primers used for detecting the IL1B gene are:

[0052] IL1B-F: 5'-CCGCGTCAGTTGTTGTGGCC-3';

[0053] IL1B-R: 5'-AGTCCCGGAGCGTGCAGTTC-3'. This primer for detecting the IL1B gene has high specificity and sensitivity, and can accurately detect the expression level of the IL1B gene.

[0054] The present invention also proposes a DP cell identification kit, wherein the DP cell identification kit of the present invention includes the reagents described above.

[0055] This invention also proposes a method for identifying DP cells. The method, as described in this embodiment, determines whether cultured cells are DP cells by detecting the expression levels of HES1, TINAGL1, and IL1B genes relative to housekeeping genes. This method for identifying cultured DP cells determines whether the cells to be identified are DP cells by specifically detecting the relative expression levels of HES1, TINAGL1, and IL1B genes within the cells to be identified. This method can accurately identify DP cells from a group of cells to be identified, including DP cells and low-purity DP cells, which is of great significance for the qualitative analysis and clinical application of DP cells.

[0056] The identification method of this invention is simple, easy to implement, and low in cost. By developing and manufacturing identification products for in vitro cultured DP cells, DP cells can be identified conveniently, quickly, accurately, and in large quantities, which is of great significance for clinical treatment and other applications, and fills the gap in related content in the prior art.

[0057] In a preferred embodiment of the present invention, the expression level of at least one of the HES1 gene, TINAGL1 gene, and IL1B gene is obtained by real-time RT-PCR. By using real-time RT-PCR, the content of the target gene can be quantitatively detected in real time.

[0058] In a preferred embodiment of the present invention, the criterion for judging the in vitro cultured DP cells is the ΔCt value; the ΔCt value of the DP cells meets the following criteria: the ΔCt value of the HES1 gene is ≤11.8, the ΔCt value of the TINAGL1 gene is ≤11.2, and the ΔCt value of the IL1B gene is ≤4.4; the ΔCt value is calculated as follows: the Ct value of the gene to be tested - the Ct value of the housekeeping gene.

[0059] In a preferred embodiment of the present invention, the housekeeping gene is the GAPDH gene.

[0060] In a preferred embodiment of the present invention, the primers for detecting the GAPDH gene are:

[0061] GAPDH-F:5'-AGCCACATCGCTCAGACACC-3';

[0062] GAPDH-R:5'-GTACTCAGCGCCAGCATCG-3'.

[0063] The following detailed description of the biomarkers for identifying in vitro cultured DP cells and their applications is provided through specific embodiments.

[0064] The reagents used in the following examples are shown in Table 1 below:

[0065] Table 1

[0066]

[0067]

[0068] Example 1: Isolation and culture of primary DP cells in vitro

[0069] Wash the skin tissue or hair follicle tissue three times with PBS (remove as much of the epidermis as possible from the skin tissue with a scalpel), and use micro forceps to completely peel off the hair follicles and place them into a culture dish containing AmnioMAX (human amniotic fluid cell complete culture medium).

[0070] Add 5 drops of AmnioMAX medium to a culture dish, placing 2 hair follicles in each drop. Use a 1mL syringe to cut off the dermal papilla from the hair follicle. With your left hand, use the syringe needle to hold the dermal sheath outside the hair follicle head in place. With your right hand, use the syringe needle to poke the center of the hair follicle head to flip it over. Cut off the dermal papilla along the indentation and fix it to the well plate with a needle. Slowly add 2mL of medium along the wall (do not blow up the dermal papilla). The remaining portion consists of low-purity DP cells (low-purity DP cells contain fewer DP cells). Similarly, slowly add 2mL of medium along the wall for primary cell isolation and culture. Change the medium on the fourth day, and then every four days thereafter. Cell observation: Remove the cells to be passaged from the CO2 incubator and observe cell growth under a microscope. Randomly select two fields of view and photograph them under low and high magnification respectively. Cell density of 80%-90% confirms that passage is possible.

[0071] Cell digestion: Wipe the outer surface of the culture flask with a lint-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture flask back and forth, and remove the old culture medium using a pipette. Wash the cell surface once with an appropriate amount of PBS. Add an appropriate amount of digestive enzyme to each culture flask and digest for 3-10 minutes at room temperature. After gentle shaking, the cells appear as quicksand to the naked eye. Under a microscope, the cells appear as spherical shapes, indicating complete digestion. Add an appropriate amount of stop solution to terminate the digestion. Transfer the liquid to a 50mL centrifuge tube, rinse the culture flask with an appropriate amount of PBS, transfer the liquid to a 50mL centrifuge tube, and centrifuge. Centrifugation parameters: 400g, 5min.

[0072] Cell seeding and culture: After centrifugation, discard the supernatant (to obtain primary isolated DP cells and low-purity DP cells).

[0073] Primary DP cells isolated from culture were resuspended in complete culture medium, and 20 μL was used for cell counting. Based on the count results, cells were counted at a rate of 8000-20000 cells / cm². 2The cells were seeded into culture flasks at a specific density. They were then passaged in a 37°C, 5.0% CO2 incubator. P3 and P5 generation cells were obtained sequentially.

[0074] Example 2: Cell morphology observation of primary isolated DP cells and low-purity DP cells

[0075] The cell morphology of primary isolated DP cells and low-purity DP cells was observed under an inverted microscope, and the results are as follows: Figure 1 As shown, the primary isolated DP cells exhibit uniform morphology, conforming to typical DP cell characteristics. The cells are spindle-shaped with high morphological uniformity, arranged in whorls, bundles, or radial patterns. Adherent cells are primarily spindle-shaped fibroblasts. In contrast, the low-purity DP cells isolated from the primary culture exhibit scattered and heterogeneous morphology, including both spindle-shaped and fibrous cells, as well as cobblestone-like cells, which do not conform to typical DP cell characteristics.

[0076] Example 3 ALP Immunofluorescence Identification

[0077] Cell seeding: Place cultured primary cells (1 × 10⁶ cells per well) into a plate. 4 Cells were seeded in 24-well plates and cultured to approximately 30% confluence (about 2 days), with duplicate wells set up.

[0078] Fixation: After removing the culture medium, the cells were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then washed three times with PBS, soaking for 5 minutes each time.

[0079] Blocking: Add 1 mL of PBS (containing 3% BSA / 0.1% Triton) to each well and block at room temperature for 1 hour. Adding primary antibody: After discarding the blocking buffer, add diluted ALP primary antibody (diluted with PBS / 3% BSA), incubate overnight at 4°C, and wash 3 times with PBS for 5 minutes each time.

[0080] Add secondary antibody: Add diluted secondary antibody and incubate at room temperature in the dark for 1 hour. Wash with PBS 3 times, 5 minutes each time.

[0081] DAPI nucleostained nuclei: stain nuclei with DAPI staining solution at room temperature for 5 minutes.

[0082] Observation and photography: Images were captured using a fluorescence microscope equipped with a digital camera.

[0083] like Figure 2 As shown, primary isolated DP cells exhibit higher ALP immunofluorescence intensity compared to low-purity DP cells, consistent with the ALP staining characteristics of DP cells.

[0084] Example 4: RNA-seq detection of primary isolated DP cells and low-purity DP cells

[0085] RNAseq was performed on three batches of primary DP cells and low-purity DP cells isolated and cultured in vitro according to Example 1 and screened according to Examples 2 and 3.

[0086] Based on the detection results obtained from RNA seq, characteristic genes that are specifically highly expressed in DP cells compared to low-purity DP cells and are related to germinal function were screened.

[0087] The results are as follows Figure 3 As shown in Table 2 below, the functional classification of highly expressed genes in the RNA seq results is presented. Based on the detection results obtained from RNA seq, characteristic genes that are specifically highly expressed in DP cells compared to low-purity DP cells and are related to germinal function were screened. It was found that genes HES1, TINAGL1, and IL1B were highly expressed in all three batches of primary DP cells, and these genes are related to cell proliferation and tissue repair.

[0088] Table 2 shows the functional classification of highly expressed genes in the RNA seq results.

[0089]

[0090] Example 5: Primers were designed and screened for the identified highly expressed genes.

[0091] Total RNA was extracted from primary DP cell samples. The RNA extraction steps are as follows:

[0092] 1) Transfer the cell suspension to a 1.5 mL EP centrifuge tube, centrifuge at 500 g for 5 min, and discard the supernatant. Add 1 mL TRIZOL and pipette repeatedly 15 times to completely lyse the cells.

[0093] 2) Add 200 μL of chloroform and shake vigorously until it turns milky white. Let it stand at room temperature for 2 minutes.

[0094] 3) Place in a low-temperature high-speed centrifuge, centrifuge at 4℃, 13800g for 15 minutes.

[0095] 4) After centrifugation, remove the EP tube and find that the liquid in the tube is divided into three layers. Carefully transfer the upper layer of liquid into a new 1.5mL EP tube.

[0096] 5) Add 500 μL of cold isopropanol to a new EP tube and invert it to precipitate the RNA.

[0097] 6) Place in a low-temperature high-speed centrifuge at 4°C, 13800g, and centrifuge for 10 minutes.

[0098] 7) Discard the supernatant, mix with 500 μL of 75% ethanol, and centrifuge at 13800 g for 3 min at 4 °C.

[0099] 8) Discard the supernatant, allow the alcohol to evaporate at room temperature, and dissolve the RNA in 20 μL of DEPC water.

[0100] The RNA from primary DP cell samples was reverse transcribed into cDNA. The experimental steps are as follows:

[0101] 1) cDNA first-strand synthesis

[0102] Preheat the PCR instrument to 65°C. Prepare the reaction system in each PCR tube on ice according to the table below. The reaction system is shown in Table 3 below:

[0103] Table 3

[0104] Primers Random primer 1μL water <![CDATA[DEPC treated H2O]]> Up to 12μL Total volume / 12μL

[0105] Mix the reaction tubes, centrifuge briefly, place them in a preheated PCR instrument, incubate at 65°C for 5 minutes, and immediately return to ice.

[0106] 2) Add the following reagents to the PCR tube after the first-strand synthesis system has been reacted on ice, as shown in Table 4 below:

[0107] Table 4

[0108] Ribolock RNase Inhibitor(20U / μL) 1μL 10mMdNTP Mix 2μL RevertAid M-MμlV RT(200U / μL) 1μL Total volume 20μL

[0109] Mix, briefly centrifuge, and incubate in a PCR instrument at 25°C for 5 min, 42°C for 90 min, 70°C for 5 min, and 4°C forever.

[0110] PCR amplification of cDNA (Polymerase chain reaction is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as a special DNA replication outside the organism. The biggest feature of PCR is that it can greatly increase a small amount of DNA): PCR amplification of extracted cDNA was performed using RT-qPCR primers. The primer sequences used are shown in Table 5.

[0111] Table 5 Primer sequences used for screening genes with high expression characteristics.

[0112]

[0113] The RT-qPCR experimental steps are as follows:

[0114] 1) The cDNA after reverse transcription is diluted 10 times to obtain the template for real-time quantitative PCR reaction.

[0115] 2) Remove the SuperRealPreMix Plus (SYBR Green) and primers stored at -30 to -10℃ and thaw at room temperature.

[0116] The RT-PCR reaction procedure is shown in Table 6 below:

[0117] Table 6

[0118]

[0119]

[0120] 3) Prepare the PCR reaction system in the PCR wells. The content of each component in each well after preparation is shown in Table 7 below:

[0121] Table 7

[0122] 2×SuperReal PreMixPlus 10μL upstream primer 0.6μL Downstream primer 0.6μL 50×ROX Reference Dye 2μL Samples 0.6μL

[0123] 4) After the PCR reaction system is prepared, place the PCR tubes into the RT-PCR instrument and run the pre-set program.

[0124] 5) After the PCR reaction is complete, export the experimental data, tidy up the lab bench, turn off the RT-PCR instrument, and shut down the computer.

[0125] 6) Analyze the experimental data, using MSC as a reference sample, and calculate the logarithm of RQ (lgRQ) of each gene to be tested in RPE using the ΔΔCT method to determine whether it meets the screening criteria.

[0126] The results are shown in Table 8. The qPCR amplification results corresponding to primers NO.1-6 were better than those corresponding to primers NO.7-12. The qPCR products corresponding to primers NO.1-6 were selected for subsequent agarose gel electrophoresis experiments.

[0127] Table 8 shows the qPCR amplification results corresponding to the screening primers.

[0128]

[0129] Example 6: Agarose gel electrophoresis experiment of amplified products

[0130] The qPCR amplification products corresponding to the selected primers were subjected to agarose gel electrophoresis. At the same time, a DNA ladder of 50-500 bp was used as an electrophoresis marker to obtain the DNA molecule size of the PCR product corresponding to each selected primer. The size of the primer amplification fragment was compared with that of the DNA molecule. If they were consistent, it meant that the amplification product was the target amplification fragment and the corresponding primer was suitable.

[0131] The results are as follows Figure 4 As shown, the results revealed that the molecular size of the PCR amplification products corresponding to the selected primers was consistent with the target amplification fragment, indicating that the selected primers were suitable. Figure 4Lane ④ in the image is an electrophoresis diagram of the PCR product of the GPRC5A gene. Since the expression results of the GPRC5A gene do not meet the characteristics of a gene marker, it is not considered a characteristic gene of DP cells.

[0132] Example 7: Expression of HES1, TINAGL1, and IL1B genes in DP cells and low-purity DP cells by qPCR.

[0133] Three batches of primary DP cells and three batches of low-purity DP cells (isolated according to the method in Example 1) were used for RNA seq detection.

[0134] Cell sample RNA is reverse transcribed into cDNA;

[0135] PCR amplification of cDNA was performed using RT-qPCR. The selected primer sequences are as follows:

[0136] HES1-F: 5'-CAAGCTGGAGAAGGCGGACA-3' (SEQ ID NO. 1);

[0137] HES1-R: 5'-TCGGTACTTCCCCAGCACACT-3' (SEQ ID NO. 2).

[0138] TINAGL1-F: 5'-GGCCAGAGAGATACCGCCGG-3' (SEQ ID NO.3);

[0139] TINAGL1-R: 5'-ATGCGGAAGTGGCCCCTCTC-3' (SEQ ID NO. 4).

[0140] IL1B-F: 5'-CCGCGTCAGTTGTTGTGGCC-3' (SEQ ID NO.5);

[0141] IL1B-R: 5'-AGTCCCGGAGCGTGCAGTTC-3' (SEQ ID NO. 6).

[0142] GAPDH-F: 5'-AGCCACATCGCTCAGACACC-3' (SEQ ID NO. 13);

[0143] GAPDH-R:5'-GTACTCAGCGCCAGCATCG-3'(SEQ ID NO.14)

[0144] The results are shown in Table 9:

[0145] Table 9. Results of HES1, TINAGL1, and IL1B gene expression detection in primary DP cells and low-purity DP cells.

[0146]

[0147]

[0148] As shown in Table 9 above, compared with low-purity DP cells, the HES1, TINAGL1, and IL1B genes were all highly expressed in all three batches of DP cells. Furthermore, the ΔCt values ​​of HES1, TINAGL1, and IL1B genes relative to GAPDH genes in DP cells were less than 11.8, 11.2, and 4.4, respectively, while the ΔCt values ​​of HES1, TINAGL1, and IL1B genes relative to GAPDH genes in low-purity DP cells were greater than 11.8, 11.2, and 4.4, respectively.

[0149] ΔCt=Ct(HES1 / TINAGL1 / IL1B)-Ct(GAPDH).

[0150] Example 8 Identification of DP cells

[0151] P3 generation DP cells (obtained by passage culture of primary DP cells isolated in Example 1), P5 generation DP cells (obtained by passage culture of primary DP cells isolated in Example 1), and low-purity DP cell samples (obtained by primary isolation in Example 1) were selected. RT-qPCR was used to detect the cDNA extracted and reverse transcribed from each sample, and the ΔCt value was calculated.

[0152] ΔCt=Ct(HES1 / TINAGL1 / IL1B)-Ct(GAPDH).

[0153] The primers for HES1, TINAGL1, and IL1B gene detection are as follows:

[0154] HES1-F: 5'-CAAGCTGGAGAAGGCGGACA-3' (SEQ ID NO. 1);

[0155] HES1-R: 5'-TCGGTACTTCCCCAGCACACT-3' (SEQ ID NO. 2).

[0156] TINAGL1-F: 5'-GGCCAGAGAGATACCGCCGG-3' (SEQ ID NO.3);

[0157] TINAGL1-R: 5'-ATGCGGAAGTGGCCCCTCTC-3' (SEQ ID NO. 4).

[0158] IL1B-F: 5'-CCGCGTCAGTTGTTGTGGCC-3' (SEQ ID NO.5);

[0159] IL1B-R: 5'-AGTCCCGGAGCGTGCAGTTC-3' (SEQ ID NO. 6).

[0160] The primers for GAPDH gene detection are as follows:

[0161] GAPDH-F: 5'-AGCCACATCGCTCAGACACC-3' (SEQ ID NO. 13);

[0162] GAPDH-R: 5'-GTACTCAGCGCCAGCATCG-3' (SEQ ID NO. 14).

[0163] By comparison, as shown in Table 10, the ΔCt values ​​of HES1, TINAGL1, and IL1B relative to GAPDH in P3 generation DP cells were 11.5, 9.9, and 4.0, respectively, which are less than the criteria of 11.8, 11.2, and 4.4. However, the two low-purity DP cell samples obtained from primary isolation (isolated and cultured according to the method in Example 1) and one P5 generation DP cell sample did not meet this criterion. Considering that the expression levels of HES1, TINAGL1, and IL1B genes may change with the number of passages, we proposed that ΔCt values ​​of HES1, TINAGL1, and IL1B relative to GAPDH of less than 11.8, 11.2, and 4.4 can be used to screen DP cells with good germinal function. DP cells screened by this criterion were used in subsequent mouse experiments.

[0164] Table 10. Gene expression identification results of P3 and P5 generation DP cells and primary isolated low-purity DP cells.

[0165]

[0166] Example 9 Mouse Experiment

[0167] Two female BALB / c pregnant mice were raised to delivery. Epidermal / dermal mixed dressings were prepared from the newborn mice. P3 generation cells (DP cells-P3), primary low-purity DP cells (primary low-purity DP cells isolated in Example 1), and P5 generation cells (DP cells-P5) obtained from the primary DP cells isolated in Example 1 were cultured and passaged. Cells were harvested when the cell coverage in the culture dish reached 70-80%. Dressings were prepared from DP cells-P3, DP cells-P5, and primary low-purity DP cells respectively. Six Balb / c nude mice (without hair growth function) were anesthetized with isoflurane inhalation, fixed with their backs facing upwards, and two 5mm diameter incisions were made on each side of the back skin via metal puncture. Three mice received a matrix gel containing DP cells and P3 cells on the left side of their backs and a matrix gel containing mouse cells on the right side; two mice received a matrix gel containing DP cells and P5 cells on the left side of their backs and a matrix gel containing mouse cells on the right side; and one mouse received a matrix gel containing primary low-purity DP cells on the left side of its back and a matrix gel containing mouse cells on the right side. The corresponding dressings were then transplanted to the wound site, 20 μL per well (6 × 10⁶ cells per well). 4 Cells were then placed on a PET film, and a medical 3M film was applied to the skin surface after transplantation to fix the wound.

[0168] On day 7, the dressing and 3M membrane were removed. On days 7 and 14, microneedles were rolled 10 times again on the transplanted areas on both sides of the back. On day 35, hair growth was observed, and skin was taken from the back. The experiment showed that only the experimental group using matrix gel containing DP cells-P3 had new hair growth, while mice using matrix gel containing primary low-purity DP cells and DP cells-P5 showed no hair growth on their backs.

[0169] In summary, this invention, through RT-PCR and immunofluorescence experiments, demonstrates for the first time that the HES1, TINAGL1, and IL1B genes can be used to identify primary isolated DP cells and low-purity DP cells. Furthermore, when the identified, relatively pure DP cells were applied to wound sites in nude mice, the mice grew hair, indicating that this identification method can effectively screen DP cells with hair growth function.

[0170] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of a combination of HES1, TINAGL1, and IL1B gene markers in identifying dermal papilla cells in vitro.

2. Application of a biomarker combination consisting of the HES1 gene, TINAGL1 gene, and IL1B gene in the in vitro isolation and culture of dermal papilla cells from hair follicle tissue.

3. Application of amplification primers for HES1, TINAGL1 and IL1B genes in the preparation of reagents for identifying dermal papilla cells.

4. The application according to claim 3, characterized in that, The primers for amplifying the HES1 gene are: HES1-F: 5'-CAAGCTGGAGAAGGCGGACA-3'; HES1-R: 5'-TCGGTACTTCCCCAGCACACT-3'; The primers for amplifying the TINAGL1 gene are: TINAGL1-F: 5'-GGCCAGAGAGATACCGCCGG-3'; TINAGL1-R: 5'-ATGCGGAAGTGGCCCCTCTC-3'; The primers for amplifying the IL1B gene are: IL1B-F: 5'-CCGCGTCAGTTGTTGTGGCC-3'; IL1B-R: 5'-AGTCCCGGAGCGTGCAGTTC-3'.

5. Application of amplification primers for HES1, TINAGL1 and IL1B genes in the preparation of DP cell identification kits.

6. A method for identifying dermal papilla cells, characterized in that, By detecting the expression levels of HES1, TINAGL1, and IL1B genes relative to housekeeping genes, it can be determined whether the cells cultured in vitro are dermal papilla cells.

7. The method for identifying dermal papilla cells according to claim 6, characterized in that, The expression levels of at least one of the HES1, TINAGL1, and IL1B genes were determined by real-time RT-PCR.

8. The method for identifying dermal papilla cells according to claim 6, characterized in that, The ΔCt value is used to determine the quality of dermal papilla cells cultured in vitro. The ΔCt value of dermal papilla cells meets the following criteria: The ΔCt value for the HES1 gene is ≤11.8, the ΔCt value for the TINAGL1 gene is ≤11.2, and the ΔCt value for the IL1B gene is ≤4.

4. The ΔCt value is calculated as follows: Ct value of the gene to be tested - Ct value of the housekeeping gene; The housekeeping gene is the GAPDH gene.

9. The method for identifying dermal papilla cells according to claim 8, characterized in that, The primers for detecting the GAPDH gene are: GAPDH-F: 5'-AGCCACATCGCTCAGACACC-3'; GAPDH-R: 5'-GTACTCAGCGCCAGCATCG-3'.

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