Kit for identifying gingival stem cells and periodontal ligament stem cells and use method thereof

Abstract

The invention discloses a kit for identifying gingival stem cells and periodontal ligament stem cells. The kit comprises a primer for amplifying a GAMT gene and a primer for amplifying a reference gene, the invention also discloses a method for identifying gingival stem cells and periodontal ligament stem cells by using the kit, which comprises the following steps: extracting total RNA (Ribonucleic Acid) of a biological sample to be detected, carrying out reverse transcription to obtain cDNA (Complementary Deoxyribonucleic Acid), carrying out real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection by using the kit, and finally identifying the gingival stem cells and periodontal ligament stem cells according to the relative expression and judging whether the biological sample to be detected is gingival stem cells or periodontal ligament stem cells. The GAMT gene is remarkably low in expression in the gingival stem cells and remarkably high in expression in the periodontal ligament stem cells, the relative expression quantity of the gene is detected through the kit, the gingival stem cells and the periodontal ligament stem cells can be accurately and effectively identified according to differential expression of the gene, and the GAMT gene is suitable for application and popularization.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a kit for identifying gingival stem cells and periodontal ligament stem cells and a method for using the same. Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells that have been very active in basic stem cell research and clinical translation in recent years, and dental stem cells are a type of mesenchymal stem cells that can differentiate into at least three different cell lines. So far, a variety of dental stem cells have been isolated and identified, mainly including: dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle stem cells, stem cells from the apical papilla of the tooth root, gingival stem cells, etc. The isolation and identification of numerous dental stem cells have enabled the rapid development of oral tissue engineering, which is of great significance for changing the function and appearance of patients.

[0003] In addition to possessing the basic stem cell properties of mesenchymal stem cells, the greatest advantage of dental stem cells is the convenience of obtaining samples. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) can be obtained from the tissue of impacted wisdom teeth that need to be removed with gingivectomy at one time, which is convenient and easy to obtain and is widely used. They are important seed cells in oral tissue engineering. Since the clinical application effects of GMSCs and PDLSCs are different, developing an effective method to identify the two types of stem cells is a prerequisite for clinical application transformation. Existing methods for identifying gingival stem cells and periodontal ligament stem cells, such as those based on cell morphology, osteogenic and adipogenic differentiation tests, and flow cytometry identification, have problems such as high cost, complex procedures, and high equipment requirements, and cannot truly and efficiently identify gingival stem cells and periodontal ligament stem cells simply. Summary of the Invention

[0004] The first object of the present invention is to provide a kit for identifying gingival stem cells and periodontal ligament stem cells to make up for the deficiencies that gingival stem cells and periodontal ligament stem cells are not easy to distinguish and the existing identification methods are complex and costly. The kit can effectively identify gingival stem cells and periodontal ligament stem cells. Based on the kit for identifying gingival stem cells and periodontal ligament stem cells, the present invention also aims to provide a method for using the kit to identify gingival stem cells and periodontal ligament stem cells.

[0005] To solve the above technical problems, the present invention specifically adopts the following technical solutions: In a first aspect, the present invention provides a kit for identifying gingival stem cells and periodontal ligament stem cells, the kit comprising primers for amplifying the GAMT gene and primers for amplifying a reference gene.

[0006] Further, the primers for amplifying the GAMT gene are nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2.

[0007] Further, the reference gene includes GAPDH.

[0008] Further, the primers for amplifying the reference gene are nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4.

[0009] In a second aspect, the present invention further provides a method for identifying gingival stem cells and periodontal ligament stem cells using the kit as described in the first aspect, the steps being as follows: Step 1: Extract total RNA from the biological sample to be tested and reverse transcribe it to obtain cDNA; Step 2: Using the cDNA described in Step 1 as a template, perform real-time fluorescence quantitative PCR detection using the kit to obtain the relative expression level of the GAMT gene; Step 3: Based on the relative expression level of the GAMT gene, determine whether the biological sample to be tested is a gingival stem cell or a periodontal ligament stem cell; When the expression level of the GAMT gene is significantly high, the biological sample to be tested is a periodontal ligament stem cell; when the expression level of the GAMT gene is significantly low, the biological sample to be tested is a gingival stem cell.

[0010] Compared with the prior art, the present invention has the following beneficial effects: By using the kit provided by the present invention for identifying gingival stem cells and periodontal ligament stem cells, it is possible to specifically quantitatively detect the relative expression levels of the markers of gingival stem cells and periodontal ligament stem cells. Based on the high or low relative expression levels of the markers, it is possible to efficiently and low-costly identify gingival stem cells and periodontal ligament stem cells, solving the problem that gingival stem cells and periodontal ligament stem cells are not easily distinguishable and the existing identification methods are complex and costly. The method provided by the present invention for identifying gingival stem cells and periodontal ligament stem cells using the above kit is simple and fast, and can provide a rapid and effective means for the identification of gingival stem cells and periodontal ligament stem cells, which is of great significance for applications such as clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0012] Figure 1 It is a result graph of the abundance of GAMT protein expression in gingival stem cells and periodontal ligament stem cells in Example 1.

[0013] Figure 2 It is a result graph of the relative mRNA expression levels of the GAMT gene in gingival stem cells and periodontal ligament stem cells in Example 2. Specific Embodiments

[0014] The following further describes the present invention in detail in conjunction with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0015] Unless otherwise specified in the following examples, other components are not included except for inevitable impurities. The reagents and instruments used in the examples are all conventional selections in the art unless otherwise specified. The experimental methods without specific conditions in the examples are carried out according to conventional conditions, such as those described in the literature, books or the methods recommended by the manufacturers. Unless otherwise specified, the experiments in the following examples are all set up with three repeated experiments, and the results are averaged.

[0016] Example 1 Screening of Markers for Gingival Stem Cells and Periodontal Ligament Stem Cells In this example, samples of gingival stem cells and periodontal ligament stem cells from 3 different individuals were used.

[0017] 1. Extraction of Total Cellular Proteins from Gingival Stem Cells and Periodontal Ligament Stem Cells 1) Collect the cells in an EP tube, wash the cells 2 times with 1 mL of pre-cooled PBS buffer, and discard the supernatant.

[0018] 2) Add 300 μL of RIPA lysis buffer and 30 μL of protease inhibitor, and lyse at 4°C for 15 minutes.

[0019] 3) Ultrasonically disrupt the cells at low temperature for 2 minutes.

[0020] 4) Centrifuge at 12000 rpm for 15 minutes at 4°C.

[0021] 5) Transfer the supernatant to another pre-chilled EP tube, which is the total cellular protein.

[0022] 2. Protein LC-MS / MS analysis Add dithiothreitol (DTT) to the protein to a final concentration of 20 mM, and incubate at 56 °C for 30 minutes to reduce the disulfide bonds of the protein; then add indole-3-acetic acid (IAA) to a final concentration of 25 mM, and let it stand in the dark for 1 hour to alkylate the thiol groups of the protein; add trypsin, and the mass ratio of trypsin to the protein in the solution is 1:50, and gently shake at 37 °C for 3 hours to digest the protein into peptide fragments. Add the digested sample solution to a desalting column for desalting treatment.

[0023] After desalting the peptide samples of each group, use the TMT labeling kit for labeling. Dissolve a single sample in 100 μL of TEAB buffer, mix well, and let it stand for 10 minutes; add the TMT reagent, vortex, and incubate for 1 hour; add 8 μL of 5% hydroxylamine to each vial to terminate the reaction, and incubate for 15 minutes.

[0024] Mix the labeled peptide samples and perform peptide separation and detection on LC-MS / MS.

[0025] The statistical results are as Figure 1 shown. The expression level of GAMT protein in the periodontal ligament stem cell group is higher, and the expression level of GAMT protein in the gingival stem cell group is lower.

[0026] Example 2 Identification of gingival stem cells and periodontal ligament stem cells In this example, samples of gingival stem cells and periodontal ligament stem cells from 3 different individuals were used.

[0027] 1. cDNA synthesis of gingival stem cells and periodontal ligament stem cells 1) Centrifuge the cells at 1000 rpm for 5 minutes at room temperature, discard the supernatant, wash twice with pre-chilled PBS buffer; add 1 mL of Trizol reagent; transfer the tissue or cell lysate to an RNase-free EP tube.

[0028] 2) Let it stand on ice for 5 minutes, add 200 μL of chloroform to each tube, mix well, and let it stand on ice for 10 minutes.

[0029] 3) Centrifuge at 13000 rpm for 15 minutes at 4 °C.

[0030] 4) Transfer the upper aqueous phase to a pre-chilled EP tube containing 500 μL of isopropanol, let it stand on ice for 10 minutes, centrifuge at 13000 rpm for 10 minutes, and discard the supernatant.

[0031] 5) Wash the RNA pellet twice with 1 mL of 75% ethanol, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.

[0032] 6) Air-dry the RNA pellet in a fume hood.

[0033] 7) Dissolve the RNA in 50 μL of DEPC-treated deionized water to obtain total cellular RNA.

[0034] 8) Perform spectrophotometric analysis to determine the concentration and purity of the RNA sample. When the OD260 / OD280 is between 1.8 and 2.0, the RNA purity meets the standard; otherwise, the RNA extraction procedure needs to be repeated.

[0035] 9) Add 2 μL of SuperScript IV VILO cDNA Synthesis Premix to the RNA sample, and add RNase-free water to make the total volume of the reaction system 10 μL. Incubate at 37 °C for 15 minutes and then at 85 °C for 5 seconds to stop the reverse transcription and obtain cDNA.

[0036] 2. Real-time fluorescence quantitative PCR 1) Prepare the real-time fluorescence quantitative PCR reaction system: 10 μL of 2×SYBR Green qPCR Master Mix (real-time fluorescence quantitative PCR premix), 1 μL of the upstream primer of the GAMT gene, 1 μL of the downstream primer of the GAMT gene, 1 μL of the upstream primer of the internal reference gene GAPDH, 1 μL of the downstream primer of the internal reference gene GAPDH, 2 μL of cDNA template, and add ddH2O to make the total volume of the reaction system 20 μL.

[0037] 2) Perform the reaction in a real-time fluorescence quantitative PCR instrument: The first step is pre-denaturation at 95 °C for 5 minutes; the second step is denaturation at 95 °C for 20 seconds; the third step is annealing at 60 °C for 30 seconds; the fourth step is extension at 72 °C for 1 minute; the second to fourth steps are cycled 35 times; enter the melting program, and perform reactions at 95 °C for 15 seconds, 60 °C for 1 minute, 95 °C for 15 seconds, and 60 °C for 15 seconds in sequence.

[0038] The upstream primer and downstream primer of the GAMT gene are the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the upstream primer and downstream primer of the internal reference gene GAPDH are the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0039] According to the results of real-time fluorescence quantitative PCR, calculate the 2^(-ΔΔCt) value as the relative expression level of the GAMT gene, and compare the relative expression levels of the GAMT gene in the gingival stem cell group and the periodontal ligament stem cell group.

[0040] The results were as Figure 2 shown. The relative expression level of the GAMT gene in the periodontal ligament stem cell group was significantly high, and the relative expression level of the GAMT gene in the dental pulp stem cell group was significantly low.

[0041] In summary, the technical solution of the present invention can use a kit containing primers for amplifying the GAMT gene and primers for amplifying the reference gene to specifically quantitatively detect the relative expression level of the GAMT gene, a marker of dental pulp stem cells and periodontal ligament stem cells, and identify dental pulp stem cells and periodontal ligament stem cells according to the high or low relative expression level of the GAMT gene. This method is efficient, accurate, and convenient for detection.

[0042] It should be noted that in the present invention, nucleotide sequences having at least 85% identity with SEQ ID NO.1 and SEQ ID NO.2 can be used as primers for amplifying the GAMT gene, and nucleotide sequences having at least 85% identity with SEQ ID NO.3 and SEQ ID NO.4 can be used as primers for amplifying the reference gene, and all of them are considered to be included in the protection scope of the present invention.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for identifying gingival stem cells and periodontal ligament stem cells, characterized in that, The kit includes primers for amplifying the GAMT gene and primers for amplifying a reference gene.

2. The kit according to claim 1, wherein The primers for amplifying the GAMT gene are the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The kit according to claim 1, wherein The reference gene includes GAPDH.

4. The kit according to claim 3, wherein The primers for amplifying the reference gene are the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. A method for identifying gingival stem cells and periodontal ligament stem cells using the kit according to claim 1, characterized in that, It includes the following steps: Step 1: Extract the total RNA of the biological sample to be tested and reverse transcribe it to obtain cDNA; Step 2: Using the cDNA described in Step 1 as a template, perform real-time fluorescence quantitative PCR detection with the kit to obtain the relative expression level of the GAMT gene; Step 3: According to the relative expression level of the GAMT gene, determine whether the biological sample to be tested is a gingival stem cell or a periodontal ligament stem cell; When the relative expression level of the GAMT gene is significantly high, the biological sample to be tested is a periodontal ligament stem cell; when the relative expression level of the GAMT gene is significantly low, the biological sample to be tested is a gingival stem cell.

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