CN116804226A - Specific primer group, kit and rapid distinguishing method for distinguishing umbilical cord mesenchymal stem cells and deciduous tooth dental pulp stem cells - Google Patents
Specific primer group, kit and rapid distinguishing method for distinguishing umbilical cord mesenchymal stem cells and deciduous tooth dental pulp stem cells Download PDFInfo
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- CN116804226A CN116804226A CN202311076094.6A CN202311076094A CN116804226A CN 116804226 A CN116804226 A CN 116804226A CN 202311076094 A CN202311076094 A CN 202311076094A CN 116804226 A CN116804226 A CN 116804226A
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Abstract
The application discloses a specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, a kit and a rapid distinguishing method, and belongs to the technical field of kit detection. The primer group is based on the obvious difference of the expression of RNF7 genes in umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells, and provides that RNF7 can be used as a special marker of stem cells for distinguishing and identifying umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells. The rapid distinguishing method is rapid and simple, can identify umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells in a short time, is low in cost, has high reference value and is suitable for popularization and application. The kit has high sensitivity, and can detect only 15pg of template cDNA. The primer groups in the kit are high-specificity primers which are verified to be successful by a large number of experiments, are combined with 2 specific fragments of the target gene, and have high specificity and sensitivity.
Description
Technical Field
The application belongs to the technical field of kit detection, and particularly relates to a specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, a kit and a rapid distinguishing method.
Background
Mesenchymal stem cells are a good biological material and are currently used for diabetes, alzheimer's disease, parkinson's disease, cerebral apoplexy, amyotrophic lateral sclerosis, multiple sclerosis, liver cirrhosis, pulmonary fibrosis, graft versus host disease, and the like. The mesenchymal stem cells have good biological performance, have the advantages of high proliferation, multi-lineage differentiation, immunoregulation, no material availability, no ethical problem and the like, are excellent materials of regenerative medicine, and are widely applied to tissue regeneration engineering. The human deciduous tooth pulp stem cells (hSHED) are mesenchymal stem cells separated from dental pulp tissues of the natural deciduous teeth of children, have various excellent performances, and have a faster proliferation rate compared with the bone marrow mesenchymal stem cells, are characterized in that the human deciduous tooth pulp stem cells can be differentiated into dentin cell fines under the induction condition, are mainly applied to dental tissue regeneration engineering, and have relevant clinical tests in recent years and have been developed to obtain positive effects. Human umbilical cord mesenchymal stem cells (hUCMSCs) are the most widely used mesenchymal stem cells, are easily available in materials and have excellent biological properties, and have been developed in preclinical and clinical studies of a plurality of diseases such as idiopathic pulmonary fibrosis, knee osteoarthritis, type 2 diabetes, ischemic stroke, spinal cord injury, liver cirrhosis, systemic lupus erythematosus, ulcerative colitis and the like at home and abroad so far, and are hot spots of stem cell therapy.
The umbilical cord mesenchymal stem cells and the deciduous tooth pulp stem cells have the characteristic of high-speed proliferation, can be differentiated into osteoblasts, adipocytes, nerve cells, muscle cells, chondrocytes, hepatocyte-like cells and the like under the induction condition, and positively express markers CD105, CD73 and CD90. As a mesenchymal stem cell, umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells all exhibit obvious mesenchymal characteristics, appear into a fibrous shape under a microscope, have no difference in cell morphology, and are positively expressed by flow cytometry detection and do not express hematopoietic stem cell markers, so that it is difficult to distinguish the two cells in morphology and surface characteristics. However, there are great differences in the function and research applications of umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells, which are more prone to dental tissue engineering, whereas umbilical cord mesenchymal stem cells are more prone to anti-inflammatory diseases, so it is very necessary to distinguish between the two stem cells, which is the basis of stem cell therapy. However, there is no method and product available on the market to effectively differentiate umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells.
Disclosure of Invention
The first object of the application is to provide a specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, wherein the primer group is based on the obvious difference of the expression of RNF7 genes in umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells, and the RNF7 can be used as a special marker of stem cells for distinguishing and identifying umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells.
The second object of the present application is to provide a method for rapidly distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, which uses a kit or a primer set, is rapid and simple in distinguishing, can identify umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells in a short time, is low in cost, has a high reference value for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells and dental tissue engineering, and is suitable for popularization and application.
The third object of the application is to provide a kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, which has high sensitivity and can be detected only by 15pg template cDNA. The primer group in the kit is 3 pairs of primers designed according to the specific expression molecule RNF7 in cells, is a high-specificity primer which is verified to be successful by a large number of experiments, is combined with 2 specific fragments of the target gene, and has high specificity and sensitivity.
The application is realized by the following technical scheme:
a specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells comprises three pairs of primer groups of RNF7 genes, specifically:
SEQ ID No.1：- CCAGGTGATGGATGCCTGTCTTA-/>；
SEQ ID No.2：- TCTCCCCAGACCACAACACA-/>；
SEQ ID No.3：- TGGGACGTGGAGTGCGATA-/>；
SEQ ID No.4：- CTTGACATCTAAGACAGGCATCCA-/>；
SEQ ID No.5：- AAGTCGGGAGGCGACAAGA-/>；
SEQ ID No.6：- TGACATCTAAGACAGGCATCCATC-/>。
a kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, comprising the specific primer group.
Preferably, the kit further comprises an RNA extraction reagent, an internal reference gene primer, a reverse transcription reagent and a qPCR reagent.
Preferably, the reference gene is GAPDH;
the internal reference gene primer specifically comprises:
SEQ ID No.7：- CCATGGGGAAGGTGAAGGTC-/>；
SEQ ID No.8：- AGTGATGGCATGGACTGTGG-/>。
a method for rapidly distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, which uses the primer set or the kit for rapid distinguishing.
Preferably, the method comprises the following steps:
s1, culturing a cell sample to be distinguished, extracting total RNA by adopting a Trizol method, quantitatively and reversely transcribing into cDNA,
s2, performing fluorescent quantitative PCR amplification reaction by using the primer group or the primer in the kit;
s3, pass through 2 （-ΔΔCt） The relative expression level of the RNF7 gene was calculated.
Preferably, in the step S1, the amount of the template cDNA is 15pg.
Preferably, in the step S2, the primer concentration is 2 pmol/[ mu ] L.
Compared with the prior art, the application has at least the following technical effects:
the application provides a specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, which is based on the obvious difference of the expression of RNF7 genes in umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells, and provides that RNF7 can be used as a special marker of stem cells for distinguishing and identifying umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells. The method is performed by calculating the p-value using the difference sum.
The method for rapidly distinguishing the umbilical cord mesenchymal stem cells from the deciduous tooth pulp stem cells is rapid and simple in distinguishing by using the kit or the primer set, can identify the umbilical cord mesenchymal stem cells and the deciduous tooth pulp stem cells in a short time, is low in cost, has high reference value for distinguishing the umbilical cord mesenchymal stem cells from the deciduous tooth pulp stem cells and dental tissue engineering, and is suitable for popularization and application.
And thirdly, the kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells has high sensitivity and can be detected by only 15pg template cDNA. The primer group in the kit is 3 pairs of primers designed according to the specific expression molecule RNF7 in cells, is a high-specificity primer which is verified to be successful by a large number of experiments, is combined with 2 specific fragments of the target gene, and has high specificity and sensitivity.
The fourth application is the kit developed by analyzing and verifying bioinformatics after analyzing the protein expression spectrogram from the high-resolution mass spectrum. Because the cells used in the early stage of the application are strictly quality controlled and the curative effect of the indication is evaluated, one of the important applications is to solve the problem that the screening expression quantity of the hSHED does not reach the standard when umbilical cord mesenchymal is used as a reference. In addition, the molecule also has important biological function, is a guarantee of definite curative effect, and is not only suitable for laboratory research phases of basic research of various stem cell products, but also suitable for cell quality control phases of clinical treatment before clinic and even in the future. The technical content of the application belongs to basic research and preclinical technical disclosure.
Drawings
FIG. 1 shows the relative expression levels of mRNA of RNF7 from umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells in test example 1;
FIG. 2 shows the relative expression levels of mRNA of RNF7 in 20 different stem cells of test example 2;
FIG. 3 shows the relative expression levels of mRNA of RNF7 in 10 different stem cells in test example 3.
Detailed Description
Embodiments of the present application will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the application, but are not intended to limit the scope of the application to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
Test example 1: identification of identification marker of umbilical cord mesenchymal stem cells and deciduous tooth dental pulp stem cells
1. Total RNA extraction of umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells
The test used umbilical cord mesenchymal stem cells and exfoliated deciduous tooth pulp stem cell samples from 5 different individuals, respectively.
Prior to the assay, the operating environment is treated with an RNase inhibitor. (in the following operations, with a mask, a bottle mouth is horizontally taken out of a conical bottle containing a gun head, a centrifuge tube and the like, and then immediately covered, so as to avoid environmental pollution.)
1) Pouring out the culture medium in the culture dish/bottle, washing 2-3 times with PBS, adding 0.7 mL of Trizol solution, blowing off the cells attached to the culture dish/bottle with a gun, directly transferring the cells into a 1.5 mL centrifuge tube, adding 0.5 mL of Trizol into the culture dish to wash the culture dish, harvesting the remaining cells which are not completely blown off, transferring the cells into the same centrifuge tube, shaking the cells, and standing for 5min at room temperature.
2) 200 mu L of chloroform is directly added, vortex shaking is carried out for 15 and S, and the mixture is placed for 2 to 3 minutes at room temperature after being evenly mixed.
3) Centrifuge at 12000 rpm for 20 min at 4 ℃.
4) After centrifugation, the mixture was separated into three phases, the upper aqueous phase was aspirated about 400. Mu.L, and the yellow tip was aspirated, with mRNA dissolved therein, into a new 1.5 mL centrifuge tube.
5) Add 500 μl isopropanol, mix well, blow with gun, and leave at room temperature for 10 min.
6) Centrifugation was performed at 12000 rpm for 20 min at 4℃and RNA formed white pellets which settled to the bottom of the tube, and the supernatant was discarded.
7) Adding 0.5-1.0 mL absolute ethyl alcohol, rinsing, and pouring out for 2 times.
8) Blow-drying in a sterile operating table at room temperature for 5-10 min.
9) The water was treated with 20. Mu.L DEPC and homogenized.
10 OD value was measured to quantify RNA concentration and purity.
2. Fluorescent quantitative PCR:
preparing a reaction system at a 200 mu L PCR tube: primer mixture 1 [ mu ] L, master Mix mixture 18 [ mu ] L, template cDNA1 [ mu ] L, and capping and sealing.
Putting the reaction into a fluorescence quantitative PCR instrument, and performing a first-step pre-denaturation reaction for 10min at 95 ℃; secondly, denaturing at 95 ℃ for 10s; thirdly, annealing at 60 ℃ for 30s; fourthly, extending at 72 ℃ for 32 seconds; the second step to the fourth step are circulated for 40 times; entering a dissolving procedure, and sequentially carrying out reaction at 95 ℃ for 15s; reacting at 60 ℃ for 1min; reacting at 95 ℃ for 15s; reacting at 60 ℃ for 15s;
the primer mixture contains the 3 pairs of specific primers (SEQ ID Nos. 1 to 6) described above, wherein the concentration of all the primers is 2 pmol/. Mu.L.
The primer is obtained through a large number of experimental optimization, has high sensitivity and high specificity, and the sensitivity test shows that the amplification sensitivity of the RNF7 primer can reach 15pg.
RNF7 primer set:
SEQ ID No.1：- CCAGGTGATGGATGCCTGTCTTA-/>
SEQ ID No.2：- TCTCCCCAGACCACAACACA-/>
SEQ ID No.3：- TGGGACGTGGAGTGCGATA-/>
SEQ ID No.4：- CTTGACATCTAAGACAGGCATCCA-/>
SEQ ID No.5：- AAGTCGGGAGGCGACAAGA-/>
SEQ ID No.6：- TGACATCTAAGACAGGCATCCATC-/>
internal reference GAPDH primer set:
SEQ ID No.7：- CCATGGGGAAGGTGAAGGTC-/>
SEQ ID No.8：- AGTGATGGCATGGACTGTGG-/>
the reaction solution was mainly a1× UltraSYBR Mixture mixture containing GAPDH as a reference genome (Δct), the Ct values were normalized, and each sample was compared with each other using the established efficiency-based Δct method. To evaluate expression intensity, a normalized Ct value (Δct) is converted to a normalized expression value according to the following formula: en=2 (- Δct), 2 (- Δct) was calculated, the test was repeated 3 times, and then statistical analysis was performed.
3. Results: the average value of the sample group of deciduous tooth dental pulp stem cells was compared with the average value of the sample group of umbilical cord mesenchymal stem cells.
As shown in FIG. 1, the relative expression level of mRNA of RNF7 in umbilical cord mesenchymal stem cells and deciduous tooth dental pulp stem cells was shown.
The mRNA expression test result shows that the RNF7 expression of the deciduous tooth dental pulp stem cell group is high, and the RNF7 expression of the umbilical cord mesenchymal stem cell group is low.
Test example 2: further verifying application of RNF7 in identification of mesenchymal stem cells of deciduous tooth umbilical cord and stem cells of deciduous tooth dental pulp
To further confirm whether RNF7 was indeed able to reliably identify umbilical cord mesenchymal stem cells and deciduous tooth pulp stem cells, the sample population was expanded, and in cell samples of 20 umbilical cord mesenchymal stem cells and 20 deciduous tooth pulp stem cells different from test example 1, detection of the RNF7 expression level and GAPDH expression level and calculation of the expression value were performed again in the same manner as in test example 1.
As shown in FIG. 2, the total of 20 umbilical cord mesenchymal stem cells and the RNF7 mRNA expressed by the exfoliated deciduous tooth pulp stem cells were expressed.
The results showed that the RNF7 expression level in the stem cells of dental pulp of deciduous teeth was significantly higher than that in the stem cells of umbilical cord mesenchymal stem cells (p < 0.01).
Test example 3: the test was further performed using another 10 groups of samples, each containing 1 sample of exfoliated deciduous tooth dental pulp stem cells and 1 sample of umbilical cord mesenchymal stem cells. The detection of the expression level of RNF7 and the expression level of GAPDH was performed separately without informing whether it is specifically deciduous tooth pulp stem cells or umbilical cord mesenchymal stem cells, and the determination of the high expression level is deciduous tooth pulp stem cells and the determination of the low expression level is umbilical cord mesenchymal stem cells, respectively, by judging which cell type the two samples in each group are based on the relative expression level of RNF 7.
As shown in FIG. 3, the total of 10 groups of umbilical cord mesenchymal stem cells and RNF 7-expressing amounts of deciduous tooth pulp stem cells were expressed with respect to each other.
The results show that the method can correctly distinguish umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells in 10 groups.
The above 3 sets of results are all validated for RNF7 gene expression, indicating the reliability of the method.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the application and is not intended to limit the scope of the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (8)
1. A specific primer group for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, which is characterized by comprising three pairs of primer groups of RNF7 genes, and specifically comprises:
SEQ ID No.1：- CCAGGTGATGGATGCCTGTCTTA-/>；
SEQ ID No.2：- TCTCCCCAGACCACAACACA-/>；
SEQ ID No.3：- TGGGACGTGGAGTGCGATA-/>；
SEQ ID No.4：- CTTGACATCTAAGACAGGCATCCA-/>；
SEQ ID No.5：- AAGTCGGGAGGCGACAAGA-/>；
SEQ ID No.6：- TGACATCTAAGACAGGCATCCATC-/>。
2. a kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, comprising the specific primer set of claim 1.
3. The kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells according to claim 2, further comprising an RNA extraction reagent, an internal reference gene primer, a reverse transcription reagent and a qPCR reagent.
4. The kit for distinguishing umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells according to claim 3, wherein the reference gene is GAPDH;
the internal reference gene primer specifically comprises:
SEQ ID No.7：- CCATGGGGAAGGTGAAGGTC-/>；
SEQ ID No.8：- AGTGATGGCATGGACTGTGG-/>。
5. a method for rapid differentiation of umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells, characterized in that rapid differentiation is performed using the primer set of claim 1 or the kit of any one of claims 2 to 4.
6. The method for rapidly differentiating umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells according to claim 5, comprising the steps of:
s1, culturing a cell sample to be distinguished, extracting total RNA by adopting a Trizol method, quantitatively and reversely transcribing into cDNA,
s2, performing fluorescent quantitative PCR amplification reaction by using the primer set as claimed in claim 1 or the primer in the kit as claimed in any one of claims 2 to 4;
s3, calculating the relative expression quantity of the RNF7 gene by 2 (-delta Ct).
7. The method according to claim 6, wherein the amount of the template cDNA in S1 is 15pg.
8. The method for rapidly differentiating umbilical cord mesenchymal stem cells from deciduous tooth pulp stem cells according to claim 6, wherein the primer concentration in S2 is 2 pmol/μl.
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