CN110564834B - Marker for distinguishing gingival stem cells and deciduous tooth pulp stem cells and application thereof - Google Patents
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Abstract
The invention provides a marker for distinguishing gingival stem cells from deciduous tooth pulp stem cells and a distinguishing method thereof, wherein ANXA2 is used as the marker. The identification method is convenient and quick, has low cost, has higher reference value for the differentiation of gingival stem cells and deciduous tooth pulp stem cells and dental tissue engineering, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the field of stem cells, and particularly relates to a marker for distinguishing gingival stem cells and deciduous tooth pulp stem cells and a distinguishing method thereof.
Background
In recent years, with intensive research, various subpopulations of stem cells of oral origin have been identified one after another, such as gingival stem cells, deciduous tooth pulp stem cells, root tip papilla stem cells, periodontal ligament stem cells, and the like. However, the quality of the cells has great heterogeneity due to the different sources of oral stem cells; therefore, one of the major and difficult points of recent research at home and abroad is how to understand the surface markers of various stem cell sub-populations derived from oral cavity and how to distinguish and screen functional sub-populations quickly and accurately.
The gingival stem cells and the deciduous tooth pulp stem cells have similar growth and differentiation characteristics, and are relatively difficult to distinguish. At present, no mature method or product for rapidly distinguishing the two stem cells exists at home and abroad, and the separation and identification usually requires the joint detection of various CD molecules, production curves, cell morphology, osteogenic adipogenic differentiation tests and the like; the experiment and the test have high cost and high instrument and equipment dependence, and cannot meet the actual requirements of most of the existing medical institutions and scientific research institutions. In order to improve the efficiency of separating and identifying gingival stem cells and deciduous tooth pulp stem cells commonly used in dental tissue engineering, reduce the cost of cell identification and differentiation, and determine the efficacy of stem cells, it is necessary to develop a new marker and carry out industrialization, and research and prepare a new method for identifying gingival stem cells and deciduous tooth pulp stem cells, which has the advantages of good specificity, high sensitivity, convenient use, low price and high efficiency.
Annexin 2 (Annexin A2, ANXA 2) is a member of the Annexin family and is involved in cell cycle regulation, cell growth and proliferation, and development of inflammation. No one currently uses it for differentiating between gingival stem cells and deciduous dental pulp stem cells.
Disclosure of Invention
The invention aims to provide a group of specific primers for rapidly identifying gingival stem cells and deciduous tooth dental pulp stem cells and a method for rapidly identifying the gingival stem cells and the deciduous tooth dental pulp stem cells.
Specifically, the invention provides an application of ANXA2 in distinguishing gingival stem cells from deciduous tooth pulp stem cells.
Provides application of a reagent for detecting the expression level of ANXA2 in preparing a kit for distinguishing gingival stem cells from deciduous tooth pulp stem cells.
Preferably, the agent is a primer to ANXA2 mRNA or an antibody to ANXA2 protein, wherein the primer sequences are as set forth in SEQ ID NOs: 1-2.
Preferably, the kit also comprises a primer for detecting internal reference GAPDH, and the sequence of the primer is shown as SEQ ID NOs: 3-4.
Also provided is a method for rapidly differentiating gingival stem cells from deciduous tooth pulp stem cells, the method comprising detecting the expression level of ANXA2 mRNA or the expression level of ANXA2 protein in a cell sample to be differentiated, wherein the gingival stem cells are significantly high in expression level, and the deciduous tooth pulp stem cells are significantly low in expression level.
Preferably, the method specifically comprises the following steps:
(1) Extracting total cell RNA and then carrying out reverse transcription to obtain cDNA;
(2) Fluorescent quantitative PCR amplification: the expression level of ANXA2 mRNA was detected using primers for ANXA2 with the internal reference GAPDH gene and primers for ANXA2 with the sequences shown in SEQ ID NO:1-2, primers for GAPDH are SEQ ID NO:3-4;
(3) And (4) judging a result: the expression level is obviously higher than that of gingival stem cells, and obviously lower than that of deciduous tooth pulp stem cells.
Preferably, wherein step (1) is:
pouring out a cell culture medium, washing with PBS for 2-3 times, adding Trizol solution, blowing off cells attached to a culture dish/bottle by using a gun, directly transferring to a 1.5mL centrifuge tube, adding Trizol to wash the culture dish, harvesting the remaining cells which are not completely blown off, transferring to the same centrifuge tube, shaking, standing at room temperature, adding chloroform, standing at room temperature after shaking, centrifuging, absorbing an upper aqueous phase, adding isopropanol, mixing, standing at room temperature, centrifuging, discarding a supernatant, adding absolute ethyl alcohol, rinsing for 2 times, blow-drying, adding DEPC (diethyl phthalate) treatment water, mixing, and measuring an OD (optical density) value to quantify the concentration and purity of RNA. And (3) sucking the reaction product, using an RT-PCR kit to obtain cDNA, and terminating the reaction at a high temperature after the reaction at 37 ℃.
Preferably, the step (2) comprises the following steps:
prepare the reaction system in a 200. Mu.l PCR tube: 1 mu L of primer mixed solution, 18 mu L of Master Mix mixture and 1 mu L of template cDNA are covered and sealed, and placed in a fluorescent quantitative PCR instrument for a first step of pre-denaturation reaction at 95 ℃ for 10min; secondly, performing denaturation at 95 ℃ for 10s; thirdly, annealing at 60 ℃ and reacting for 30s; fourthly, extending the temperature of 72 ℃ for 32s; circulating the second step to the fourth step for 40 times; entering a dissolving program, and sequentially carrying out reactions at 95 ℃ for 15s; reacting at 60 ℃ for 1min; reacting for 15s at 95 ℃; reacting at 60 ℃ for 15s.
Preferably, wherein the concentration of the primer is 2 pmol/. Mu.l; the Master Mix was a1 × Ultra SYBR Mix.
Preferably, wherein step (2) further comprises:
ct values were normalized using GAPDH as reference gene and using the established efficiency-based Δ Δ Ct squareThe expression intensity is evaluated by the method, the normalized Ct value delta Ct is converted into a normalized expression value, and 2- ΔΔCt And (4) performing mapping analysis.
The invention has the following positive effects:
(1) The method is quick, simple and convenient, can identify the gingival stem cells and the deciduous tooth pulp stem cells in a short time, does not need expensive and complicated instruments and operations, and only needs a fluorescence quantitative PCR instrument which is prepared in a laboratory and a medical structure.
(2) The invention has high sensitivity, and can detect only 15pg template cDNA.
(3) The 2 pairs of primers designed according to the specific expression protein in the cell are high-specificity primers which are successfully verified by a large amount of experiments, are combined with 2 specific segments of the target gene, and have high specificity and sensitivity.
(4) The identification method is convenient and quick, has low cost, has higher reference value for distinguishing the gingival stem cells from deciduous tooth pulp stem cells and dental tissue engineering, and is suitable for popularization and application.
Drawings
FIG. 1 is a comparison of ANXA2 mRNA expression levels in two cells.
Fig. 2 is a comparison of ANXA2 protein expression levels in two cells.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Example 1 screening of identification markers for gingival Stem cells and deciduous tooth pulp Stem cells
1. Total RNA extraction of gingival stem cells and deciduous tooth pulp stem cells
Gingival stem cell samples and corresponding primary tooth pulp stem cell samples from 5 different groups of individuals were used in this experiment.
Before the experiment, the operating environment was treated with RNA inhibitors.
In the following operation, a conical bottle with a mask, a gun head, a centrifuge tube and the like is horizontally taken from the bottle mouth, and then the bottle mouth is immediately covered to avoid environmental pollution.
1) Pouring out the culture medium in the culture dish/bottle, washing with PBS for 2-3 times, adding 0.7mL of Trizol solution (red), blowing off the cells attached to the culture dish/bottle by using a gun, directly transferring to a 1.5mL centrifuge tube, adding 0.5mL of Trizol in the culture dish, washing the culture dish to obtain the remaining cells which are not completely blown off, transferring to the same centrifuge tube, shaking, and standing at room temperature for 5min.
2) Directly adding 200 μ L chloroform, vortex shaking for 15S, mixing, standing at room temperature for 2-3min (or adding chloroform to separate protein, DNA, and lipid completely). )
3) Centrifuge at 12000rpm for 20min at 4 ℃.
4) After centrifugation, the mixture was separated into three phases, and the upper aqueous phase (the volume of the aqueous layer was about 60% of the amount of Trizol added) was pipetted into another centrifuge tube (1.5 mL) at about 400. Mu.L (a yellow tip, in which mRNA was dissolved), taking care not to contact the intermediate phase.
5) Adding 500 μ L (equal volume of upper water phase theoretically absorbed in the above step) of isopropanol, mixing (blowing with gun), and standing at room temperature for 10min.
6) The mixture was centrifuged at 12000rpm for 20min at 4 ℃ to form white RNA pellets on the bottom of the tube, and the supernatant was discarded.
7) Adding 0.5-1.0mL of absolute ethyl alcohol, rinsing, pouring off, and repeating for 2 times.
8) Blow-drying for 5-10min in sterile operation table at room temperature
9) Treating water (sterile water) with 20. Mu.L DEPC, and blowing well
10 OD260/OD280 of 1.8-2.0.
2. Fluorescent quantitative PCR:
prepare the reaction system in a 200. Mu.l PCR tube: 1 mu L of primer mixed solution, 18 mu L of Master Mix mixture and 1 mu L of template cDNA are sealed by a cover, the reaction is put into a fluorescence quantitative PCR instrument, and the first step of pre-denaturation is carried out for 10min at 95 ℃; secondly, performing denaturation at 95 ℃ for 10s; thirdly, annealing at 60 ℃ and reacting for 30s; fourthly, extending the temperature of 72 ℃ for 32s; circulating the second step to the fourth step for 40 times; entering a dissolving program, and sequentially carrying out reactions at 95 ℃ for 15s; reacting at 60 ℃ for 1min; reacting at 95 ℃ for 15s; reacting at 60 ℃ for 15s;
the primer mixture contained the 2 specific primers (SEQ ID Nos. 1-2) described above, wherein the concentration of all primers was 2 pmol/. Mu.l.
The primer is obtained by optimizing a large number of tests, has high sensitivity and high specificity, and sensitivity tests show that the amplification sensitivity of the ANXA2 primer can reach 15pg.
ANXA2(SEQ ID Nos.1－2)：
SEQ ID No.1：5’-AGGAGACCTGGAAAATGC-3’
SEQ ID No.2：5’-ATCAGGACCTTATCTCGCGT-3’
Internal reference (SEQ ID Nos. 3-4):
SEQ ID No.3：5’-GAGCGAGATCCCTCCAAA-3’
SEQ ID No.4：5’-ATGACGAACATGGGGGCATC-3’
the reaction solution was mainly a Mixture containing 1 × UltraSYBR mix, ct values were normalized using GAPDH as a reference genome (Δ Ct), and each sample was compared to using the established efficiency-based Δ Δ Ct method. To evaluate expression intensity, normalized Ct values (Δ Ct) were converted to normalized expression values according to the following formula: EN =2^ (- Δ Ct), calculate 2^ (- Δ Δ Ct), plot analysis.
3. Example results
The average value of the gingival stem cell sample group and the average value of the primary tooth pulp stem cell sample group were compared. The mRNA expression test results showed that the expression of ANXA2 was high in the gingival stem cell group and that the expression of ANXA2 was low in the deciduous tooth pulp stem cell group (fig. 1).
We performed protein level re-verification on the above experimental results by selecting a group of samples, and further confirmed the accuracy of the results of our molecular experiments by Western Blot method (fig. 2).
The above experiment suggests that: ANXA2 is significantly higher in gingival stem cells than in deciduous tooth pulp stem cells, both at mRNA levels and protein expression levels. ANXA2 can be used as an effective surface marker molecule to rapidly distinguish and identify gingival stem cells and deciduous tooth dental pulp stem cells.
Example 2 further validation of the use of ANXA2 in the identification of gingival and deciduous dental pulp stem cells
To further confirm whether ANXA2 could reliably identify gingival stem cells and deciduous tooth pulp stem cells, we expanded the sample group, and in cell samples different from the 20 gingival stem cells and the 20 deciduous tooth pulp stem cells of example 1, detection of expression levels of ANXA2 and expression levels of GAPDH and calculation of expression values were performed again according to the method in example 1. The results showed that the expression level of ANXA2 in gingival stem cells was significantly higher than that in deciduous dental pulp stem cells (P < 0.01).
Further, the test was performed using another 10 sets of samples, each set of samples comprising 1 gingival stem cell sample and 1 deciduous tooth pulp stem cell sample. Specifically, the expression levels of ANXA2 and GAPDH were measured without informing whether they were gingival stem cells or deciduous tooth pulp stem cells, and the cell types of the two samples in each group were determined based on the relative expression level of ANXA2, and a high expression level was determined as gingival stem cells and a low expression level was determined as deciduous tooth pulp stem cells. The results showed that this method can correctly distinguish between gingival stem cells and deciduous tooth pulp stem cells in group 10.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Sequence listing
<110> Kangyan Bao (Beijing) Stem cell technology Co., ltd
<120> marker for distinguishing gingival stem cells and deciduous tooth pulp stem cells and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> upstream primer
<400> 1
aggagacctg gaaaatgc 18
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<212> DNA
<213> downstream primer
<400> 2
atcaggacct tatctcgcgt 20
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<211> 21
<212> DNA
<213> upstream primer
<400> 3
gagcgagatc cctccaaa 18
<210> 4
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<212> DNA
<213> downstream primer
<400> 4
atgacgaaca tgggggcatc 20
Claims (7)
- Use of anxa2 for differentiating gingival stem cells from deciduous dental pulp stem cells.
- 2. Application of reagent for detecting ANXA2 expression level in preparation of kit for distinguishing gingival stem cells from deciduous tooth pulp stem cells.
- 3. The use as claimed in claim 2, wherein the agent is a primer to ANXA2 mRNA or an antibody to ANXA2 protein, wherein the primer sequences are as set forth in SEQ ID NOs: 1-2.
- 4. A method for rapidly differentiating gingival stem cells from deciduous tooth pulp stem cells, the method comprising detecting the expression level of ANXA2 mRNA or ANXA2 protein in a cell sample to be differentiated, wherein a significantly high expression level is a gingival stem cell and a significantly low expression level is a deciduous tooth pulp stem cell.
- 5. The method of claim 4, comprising in particular the steps of:(1) Extracting total RNA of cells, and performing reverse transcription to obtain cDNA;(2) Fluorescent quantitative PCR amplification: the expression level of ANXA2 mRNA was detected using primers for ANXA2 with the internal reference GAPDH gene and primers for ANXA2 with the sequences shown in SEQ ID NO:1-2, primers for GAPDH are SEQ ID NO:3-4;(3) And (5) judging a result: the expression level is obviously higher than that of gingival stem cells, and obviously lower than that of deciduous tooth pulp stem cells.
- 6. The method according to claim 5, wherein the step (1) is specifically:pouring out the cell culture medium, washing with PBS for 2-3 times, adding Trizol solution, blowing off the cells attached to the culture dish by using a gun, directly transferring the cells into a 1.5mL centrifuge tube, adding Trizol to wash the culture dish to obtain the remaining cells which are not completely blown off, transferring the cells into the same centrifuge tube, shaking, standing at room temperature, adding chloroform, standing at room temperature after shaking and mixing, centrifuging, absorbing the upper-layer water phase, adding isopropanol and mixing, standing at room temperature, centrifuging, discarding the supernatant, adding absolute ethyl alcohol, rinsing for 2 times, blow-drying, adding DEPC (diethylhexyl phthalate) processing water for mixing, measuring the OD (optical density) value to quantify the RNA concentration and purity, absorbing the RNA, obtaining cDNA (complementary deoxyribonucleic acid) by using an RT-PCR (reverse transcription-polymerase chain reaction) kit, and terminating the reaction at 37 ℃ and then at high temperature.
- 7. The method of claim 5 or 6, wherein step (2) comprises the steps of:prepare the reaction system in a 200. Mu.l PCR tube: 1 mu L of primer mixed solution, 18 mu L of Master Mix mixture and 1 mu L of template cDNA are covered and sealed, and placed in a fluorescent quantitative PCR instrument for a first step of pre-denaturation reaction at 95 ℃ for 10min; second, denaturation is carried out for 10s at 95 ℃; thirdly, annealing at 60 ℃ and reacting for 30s; the fourth step is to extend the temperature of 72 ℃ for 32s; circulating the second step to the fourth step for 40 times; entering a dissolving program, and sequentially carrying out reactions at 95 ℃ for 15s; reacting at 60 ℃ for 1min; reacting for 15s at 95 ℃; reacting at 60 ℃ for 15s.
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