A one-step duplex RT-Q-PCR method, primers and TaqMan probes for analyzing the bioactivity and stability of mesenchymal stem cell products

Abstract

The present invention discloses a one-step dual RT-Q-PCR method, primers and TaqMan probes for analyzing the biological activity and stability of mesenchymal stem cell products, and belongs to the field of quality analysis of pharmaceutical products. The present invention provides a one-step analysis method for the mRNA of VEGF165 and / or HGF, the target of biological activity and stability analysis of mesenchymal stem cell products, and their expression levels relative to the internal reference gene GAPDH mRNA. The one-step method of dual detection of VEGF165 and HGF mRNA is performed by reverse transcription reaction and TaqMan probe Q-PCR amplification reaction. The coefficient of variation of biological repeated detection of samples is significantly reduced from more than 40% of the two-step method to less than 20%.

Description

Technical Field

[0001] The invention relates to a one-step double RT-Q-PCR method, primers and TaqMan probe for analyzing the biological activity and stability of a mesenchymal stem cell product, and belongs to the field of quality analysis of pharmaceutical products. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent mesenchymal stem cells that originate from the mesoderm. They are found in the perinatal tissues of infants (such as the placenta and umbilical cord), the dental pulp of adults, fat, and other parts of the body. Mesenchymal stem cells have excellent drug properties such as in vitro self-replication, low immunogenicity, and non-tumor formation. As a cutting-edge new drug for the treatment of organ degenerative diseases, metabolic diseases, autoimmune diseases, and infections caused by aging and injury, they have great application prospects and drug development value [1]. The biological activity and stability of mesenchymal stem cell products in the process of mesenchymal stem cell drug development are the key links and parameters for mesenchymal stem cell drug development quality research and product quality control, which directly affect the safety and efficacy of drugs. Establishing a convenient, fast, stable and reliable method for analyzing biological activity and stability is the basis for ensuring product safety and efficacy. Therefore, in the research and development of MSC drugs and products, it is crucial to develop quality analysis methods for bioactivity, stability, etc. that meet the requirements of convenience, speed, stability and reliability. Cellular drugs are new drugs with complex cell structures. It is generally difficult to directly apply the analytical methods of chemical drugs, traditional Chinese medicines, and biological macromolecule drugs to operate them. It is urgent to develop convenient, fast, stable and reliable analytical methods to characterize the bioactivity and stability of mesenchymal stem cell products [2].

[0003] One of the most important biological mechanisms of mesenchymal stem cells is their active factor secretion effect. Growth factors including VEGF165 and HGF play important roles in immune regulation, angiogenesis, tissue repair, etc. Convenient, sensitive, accurate and specific detection of the expression level of VEGF165 and HGF mRNA (meseenger RNA) in cells is one of the important means of quality control and activity detection of mesenchymal stem cells [3,4]. At the same time, whether cytokine mRNA including VEGF165 and HGF can be used as a target for stability analysis of mesenchymal stem cell products has not been reported yet.

[0004] Mesenchymal stem cells first obtain mRNA encoding active proteins by transcription from genomic DNA, so the detection of mRNA expression levels theoretically has the potential to become a target for analyzing the biological activity of mesenchymal stem cells and product stability. When the VEGF gene sequence is transcribed, multiple spliceosomes are formed, which are translated into different proteins to exert different biological functions. Detecting the expression levels of different transcripts is one of the important means of studying common gene functions. Fluorescence quantitative RT-Q-PCR (reverse transcription-quantitative-polymerase chain reaction) based on TaqMan probes is currently a relatively sensitive, convenient, accurate and specific method for detecting gene transcript expression.

[0005] Fluorescence quantitative Q-PCR technology adds chemical fluorescent substances to the PCR reaction system, monitors the changes in the fluorescence signal, and then performs quantitative analysis on the PCR amplification products. Q-PCR is divided into fluorescent dye method and fluorescent probe method (TaqMan probe). The dye can bind to any double-stranded DNA and lacks specificity, which will affect the accuracy of the quantitative results. The probe method (TaqMan probe) solves the problem of non-specific binding and avoids the interference of fluorescent signal of primer dimers and non-specific amplification products in the fluorescent dye method. It is more accurate. At the same time, the probe method (TaqMan probe) can detect samples with expression as low as single copy, with high sensitivity.

[0006] The prior art lacks an analytical method for the biological activity and stability of mesenchymal stem cells, and there is no report on the use of RT-Q-PCR method technology for stably, accurately and specifically detecting the intracellular expression levels of VEGF165 and HGF mRNA to analyze the biological activity and stability of mesenchymal stem cell products. The present invention is developed based on this.

[0007] References:

[0008] 1.Liu P, Qian Y, Liu X, Zhu

[0009] 2. National Drug Administration Drug Evaluation Center: Technical Guidelines for Pharmaceutical Research and Evaluation of Human Mesenchymal Stem Cell Products (Trial) No. 33, 2023

[0010] 3. van Santen VJB, Bastidas Coral AP, Hogervorst JMA, Klein-Nulend J, Bakker AD. Biologically Relevant In Vitro 3D-Model to Study Bone Regeneration Potential ofHuman Adipose Stem Cells. Biomolecules. 2022Jan21; 12(2):169.

[0011] 4.Kojima H,Kushige H,Yagi H,Nishijima T,Moritoki N,Nagoshi N,NakanoY,Tanaka M,Hori S,Hasegawa Y,Abe Y,Kitago M,Nakamura M,KitagawaY.Combinational Treatment Involving Decellularized Extracellular MatrixHydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapyin Pancreatitis.Cell Transplant.2023Jan-Dec;32:9636897231170437. Summary of the invention

[0012] Mesenchymal stem cells have a complex mechanism of action and can produce a variety of biologically active components. The biological analysis targets and analytical detection methods that fully reflect the biological activity and stability of mesenchymal stem cells are still insufficient. Therefore, developing multiple analytical methods to analyze a variety of different product attributes can more accurately characterize the biological activity and stability of the product. This combination of analytical methods includes different biological analysis targets and methods. The present invention is based on the development of RT-Q-PCR method to detect the mRNA expression level of VEGF165 and HGF to fill the gap in the biological activity and stability analysis method of mesenchymal stem cell products.

[0013] After VEGF DNA is transcribed, numerous transcripts of different lengths are formed. The prior art lacks primers and probes for specific transcripts, and is unable to specifically identify a variety of spliceosome transcripts produced by VEGF gene transcription. Among them, VEGF165 mRNA is one of the VEGF transcripts with important biological activity. The purpose of the present invention is to design, synthesize and establish primers, probes and a method for simultaneous detection specifically for VEGF165 mRNA and another important active cytokine HGF, accurately, sensitively, conveniently and reliably simultaneously detect the expression levels of VEGF165 and HGF mRNA in mesenchymal stem cells, thereby establishing a convenient, fast, stable and reliable analysis method for the biological activity and stability of mesenchymal stem cell products.

[0014] In particular, this method designed probe sequences and primer sequences simultaneously for the unique sequence of VEGF165 among the 7 known mRNA transcripts of VEGF, avoiding the other 6 transcript sequences, improving the specificity of the method while meeting the requirements of stability, sensitivity, accuracy and precision. In addition, the present invention can realize one-step dual detection of mRNA expression levels of VEGF165 and HGF genes through the strategy of labeling probes with different fluorescent dyes and signal detection, and the detection efficiency is significantly improved. The method also verifies the stability of the expression levels of VEGF165 and HGF in continuous culture of mesenchymal stem cells, confirms that these two cytokines can be used as targets for biological activity and stability analysis of mesenchymal stem cell products, and is more convenient while ensuring the stability, accuracy and precision of the method. It is also confirmed that the detection of these two targets can provide new biological activity and stability detection targets and methods in addition to cell viability by detecting the mRNA expression levels of VEGF165 and HGF at different storage periods and different cell viabilities of the products after harvesting and storing mesenchymal stem cells. In summary, the present invention provides a convenient, fast, stable and reliable new method for analyzing the biological activity and stability of mesenchymal stem cell products.

[0015] The first object of the present invention is to provide an analytical target for detecting the biological activity and stability of a mesenchymal stem cell product, wherein the analytical target is VEGF165 and / or HGF.

[0016] In one embodiment, the method evaluates the biological activity and stability of the mesenchymal stem cell product by detecting the mRNA expression levels of VEGF165 and HGF relative to the internal reference gene.

[0017] In one embodiment, the GenBank number of the VEGF165 is AF486837.1, and the NCBI accession number of the HGF is NM_000601.6.

[0018] In one embodiment, the reference gene is selected from β-actin, GADPH, 18S rRNA or other stably expressed genes.

[0019] In one embodiment, the mesenchymal stem cell product includes umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells.

[0020] The second object of the present invention is to provide application of the analytical target in quality detection of mesenchymal stem cell products.

[0021] In one embodiment, the application is to detect the mRNA expression levels of VEGF165 and HGF relative to the internal reference gene using RT-Q-PCR.

[0022] In one embodiment, the reference gene is selected from β-actin, GADPH, 18S rRNA or other stably expressed genes.

[0023] The third object of the present invention is to provide a Q-PCR primer and probe set for detecting the analytical target, wherein the primer and probe set include the following (a) and / or (b):

[0024] (a) a VEGF165 upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, a VEGF165 downstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a VEGF165 probe having a nucleotide sequence as shown in SEQ ID NO.3;

[0025] (b) an HGF upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, an HGF downstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and an HGF probe having a nucleotide sequence as shown in SEQ ID NO.6.

[0026] In one embodiment, the primer set includes a GAPDH upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, a GAPDH downstream primer having a nucleotide sequence as shown in SEQ ID NO.8, and a GAPDH probe having a nucleotide sequence as shown in SEQ ID NO.9.

[0027] In one embodiment, the 5' end of the probe is modified with a reporter group.

[0028] In one embodiment, the reporter group comprises FAM, HEX, VIC, ROX or Cy5.

[0029] In one embodiment, the 3' end of the probe is modified with a quencher group.

[0030] In one embodiment, the quencher group is BHQ1, BHQ2 or TAMRA.

[0031] In one embodiment, the mesenchymal stem cell product includes umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells.

[0032] The fourth object of the present invention is to provide a kit for detecting the biological activity and stability of mesenchymal stem cell products, wherein the kit contains the Q-PCR primer and probe set.

[0033] In one embodiment, the detection kit contains a one-step Q-PCR reagent, a negative control, and a positive control.

[0034] In one embodiment, the one-step Q-PCR reagents include a reaction buffer and a mixed enzyme.

[0035] In one embodiment, the enzyme mixture includes MMLV RNA reverse transcriptase and Taq DNA polymerase.

[0036] The fifth object of the present invention is to provide an analytical method for detecting the biological activity and stability of mesenchymal stem cell products. The analytical method is not intended for the diagnosis of diseases. The analytical method uses the Q-PCR primers, probes and commercial reagents to perform a one-step RT-Q-PCR reaction for detection and analysis.

[0037] In the one-step RT-Q-PCR, cDNA synthesis (reverse transcription reaction) and Q-PCR reaction are carried out in a single reaction tube. In the two-step method, cDNA synthesis is first carried out in one reaction tube, and then the obtained cDNA is used as a template for the subsequent Q-PCR reaction in another reaction tube.

[0038] In one embodiment, the method comprises the following steps:

[0039] S1. Extract total RNA from samples;

[0040] S2, using the total RNA extracted from S1 as a template, and using the primer and probe set to perform a one-step RT-Q-PCR amplification reaction;

[0041] S3. Analysis of ΔCq and 2 –ΔCq The expression of VEGF165 and HGF mRNA in the samples was calculated.

[0042] In one embodiment, the final concentration of the primer is 0.1-1.0 μM, and the final concentration of the probe is 0.05-0.5 μM.

[0043] In one embodiment, the reaction conditions of the method are as follows: a reverse transcription temperature of 50 to 54° C., a reverse transcription time of 10 to 20 s, an annealing temperature of 52 to 60° C., and an annealing time of 20 to 40 s.

[0044] Beneficial effects:

[0045] In order to better ensure the biological activity and stability of mesenchymal stem cell products in more dimensions, the present invention first clarifies the analytical targets VEGF165 mRNA and HGF mRNA that can be used to identify the biological activity and stability of mesenchymal stem cell products, and detects their expression levels relative to the internal reference gene GAPDH mRNA to identify the quality of mesenchymal stem cell products. Further, the present invention provides a convenient, fast, stable and reliable one-step double RT-Q-PCR analysis method for the biological activity and stability of mesenchymal stem cell products, using the TaqMan probe method to increase the specificity of the fluorescent signal during Q-PCR amplification, and designing a TaqMan probe (nucleotide sequence as shown in SEQ ID NO.3) on the characteristic sequence of the VEGF165 mRNA transcript sequence that is different from other transcripts, and selecting the matching region of the probe to increase the accuracy of the detection, and designing different fluorescently labeled HGF detection probes (nucleotide sequence as shown in SEQ ID NO.6) in a reaction system, and using the reverse transcription reaction and Q-PCR amplification reaction in one step to complete the dual detection of VEGF165 and HGF mRNA, saving time and improving work efficiency. Comparing the one-step dual RT-Q-PCR analysis method provided by the present invention with the two-step analysis method, the one-step method significantly improves the stability of the method, and the coefficient of variation (CV, coefficient of variation) of biological repeated detection of samples is significantly reduced from more than 40% of the two-step method to less than 20%, and the time required for the reverse transcription step is also saved.

[0046] The present invention proposes a new and convenient one-step double RT-Q-PCR analysis method in the field of biological activity and stability analysis of mesenchymal stem cell products, which meets the requirements of method stability, precision and accuracy. It is a technical method with broad application prospects in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 VEGF165, GAPDH and HGF amplification curves of different generations of umbilical cord mesenchymal stem cells (P6, P7, P8);

[0048] Figure 2 Amplification curves of VEGF165, GAPDH and HGF in different generations of adipose-derived mesenchymal stem cells (P7, P8, P9). DETAILED DESCRIPTION

[0049] The detection core reagents of the present invention include VEGF165, HGF and internal reference gene GAPDH cDNA upstream primers, downstream primers and probes. The three probes use three different fluorescent dyes and are synthesized by a chemical method. The primers and probes are purified by simple reverse phase column desalting chromatography (de-salting, DSL) and high performance liquid chromatography (HPLC) respectively. The synthesized powder is diluted with DEPC (diethypyrocarbonate, diethyl pyrocarbonate) water to a final concentration of 10 μM, and 100 μL of the powder is packaged and stored at -20°C.

[0050] Table 1 Primers involved in the following examples

[0051]

[0052]

[0053] The methods involved in the following embodiments

[0054] 1. Preparation of test samples:

[0055] Total RNA was extracted from Trizol cell lysis buffer (commercial product). The specific process is as follows:

[0056] (1) Wash the cells once with 1 mL PBS, add 1 mL Trizol, and place on ice for 5-10 min to lyse the cells. After the cells are dissolved in Trizol, transfer them to an EP tube;

[0057] (2) Then, 200 μL of chloroform was added to each tube, vortexed, and thoroughly mixed, and then placed on ice for 5 min and centrifuged at 12,500 g for 15 min at 4°C;

[0058] (3) After centrifugation, carefully pipette 400 μL of the chloroform layer into a new clean EP tube, add the same volume of isopropanol, invert the EP tube, mix gently, and let stand at room temperature for 10 min;

[0059] (4) Centrifuge at 12500 g for 10 min at 4°C, carefully discard the supernatant, add 1 mL of pre-cooled anhydrous ethanol, shake well to wash, and centrifuge at 12500 g for 5 min at 4°C;

[0060] (5) Carefully pour off the ethanol, add 1 mL of pre-cooled anhydrous ethanol repeatedly, shake well to wash, and centrifuge at 12500 g for 5 min at 4°C;

[0061] (6) Carefully pour out the ethanol in the EP tube, turn the tube upside down in a fume hood, and dry it at room temperature for 15 minutes to allow the anhydrous ethanol to evaporate completely;

[0062] (7) Add 20 μL of DEPC water to dissolve the RNA precipitate at the bottom of the tube;

[0063] (8) NANO DROP 2000c (Thermo SCIENTIFIC) was used to detect RNA concentration and purity. When the absorbance ratio at 260 nm and 280 nm (A260 / A280) > 1.8, the RNA purity was qualified and the RNA could be used for the next step of RT-Q-PCR or frozen at -80 °C to avoid degradation.

[0064] 2. Fluorescence quantitative RT-Q-PCR detection:

[0065] (1) Quantitative PCR was performed in each reaction well using 1 μg RNA as template.

[0066] (2) Set up three biological replicates for each cell generation, and set up three technical replicates for each biological replicate sample Q-PCR test. All components except RNA in the Q-PCR reaction system were added to a tube, and then the corresponding RNA samples were added separately to reduce pipetting errors.

[0067] (3) Set up 3 negative control wells.

[0068] (4) Prepare the mixed solution shown in Table 2 below in the RT-Q-PCR tube, so that the final concentration of the primer in the system is 0.5 μM, and the final concentration of the probe is 0.1 μM. All reagents other than the primers and probes are purchased commercially and meet the quality standards;

[0069] Table 2 RT-Q-PCR reaction system (20 μL)

[0070]

[0071] (5) The reaction procedure is shown in Table 3 below:

[0072] Table 3 RT-Q-PCR reaction procedure

[0073]

[0074] (6) Data processing and analysis: The amplification curve should be a standard S-shape, without wave-like jitter or other abnormal phenomena. The negative control well should have no amplification or the Cq value should be greater than 35. Use the software supporting the instrument to export the Q-PCR amplification curve and the Cq (quantification cycle) value of the corresponding sample gene amplification. The qPCR test obtains the Cq value of each target gene and internal reference of the sample. The Cq value of each target gene minus the corresponding internal reference Cq value is ΔCq. The Cq value of each target gene minus the corresponding internal reference Cq value is ΔCq. For each sample, the Cq difference between technical replicate wells is ≤1, and the data between replicate wells ΔCq ≤0.5 are selected as acceptable data. Calculate the ΔCq mean value and ΔCq standard deviation value. The target gene expression value is calculated according to 2 -ΔCq Algorithmic calculation.

[0075] Example 1: One-step RT-Q-PCR reaction to detect the expression levels of VEGF165 and HGF relative to the internal reference gene GAPDH mRNA in umbilical mesenchymal stem cells (UC-MSCs)

[0076] Extract total RNA and test its quality and concentration;

[0077] Umbilical cord mesenchymal stem cells were subcultured from the 6th to the 8th generation, with three parallel samples collected for each generation. 6 The cells were placed in 2 mL centrifuge tubes, 1 mL of Trizol was added to each tube for lysis, and the cells were stored at -80°C.

[0078] RNA was extracted and its quality and concentration were determined. The specific results are shown in Table 4 below:

[0079] Table 4 Quantification of RNA in samples of umbilical cord mesenchymal stem cells at different generations

[0080] Sample No. Concentration (ng / μl) A260 / A280 UC-P6-1 1224.6 2.04 UC-P6-2 593.9 2.05 UC-P6-3 568.9 2.00 UC-P7-1 1518.6 2.02 UC-P7-2 560.4 2.02 UC-P7-3 851.9 2.02 UC-P8-1 1283.7 2.05 UC-P8-2 883.4 2.03 UC-P8-3 945.3 2.03

[0081] One-step RT-Q-PCR detection: 3 biological replicates per generation, 3 technical replicates per biological replicate, equivalent to 9 replicates per generation, Q-PCR amplification curve is as follows Figure 1 shown.

[0082] Data processing and analysis: The data acceptance criteria are the same as above. The expression levels of VEGF165 in cells at passages P6, P7, and P8 relative to the internal reference gene GAPDH mRNA are shown in Tables 5 and 6 below. The ΔCq data fluctuated slightly, and the ΔCq coefficient of variation was within 10%. The analysis results 2 –ΔCqThe coefficient of variation was also within 20%, which is much lower than the 30% requirement for the stability coefficient of variation of the Q-PCR method in the pharmaceutical industry's consensus on cell product analysis (Recommendations on qPCR / ddPCR assay validation by GCC, Bioanalysis (2022) 14 (12), 853–863), indicating that the stability and precision of this method fully meet the detection requirements. At the same time, the coefficient of variation CV of VEGF165 mRNA from the 6th generation to the 8th generation of umbilical cord mesenchymal stem cells was only 4.73%. The data showed that the stability of VEGF165 mRNA expression can be used as a target for the biological activity and stability analysis of mesenchymal stem cell products:

[0083] Table 5 Calculation table of ΔCq (Cq(VEGF165)-Cq(GAPDH)) of three technical replicates of three biological replicates of umbilical cord mesenchymal stem cells at different generations

[0084]

[0085] Table 6 Calculation table of VEGF165 relative to GAPDH mRNA expression levels in different generations of umbilical cord mesenchymal stem cells

[0086]

[0087]

[0088] The results of HGF expression levels relative to the internal reference gene GAPDH mRNA in P6, P7, and P8 cells are shown in Tables 7 and 8. The coefficients of variation of ΔCq were all within 10%. –ΔCq The coefficient of variation was also within 20%, indicating that the stability and precision fully met the detection requirements. At the same time, the coefficient of variation CV of HGF generations from the 6th generation to the 8th generation of umbilical cord mesenchymal stem cells was only 5.22%. The data showed that the stability of HGF mRNA expression can be used as a target for the biological activity and stability analysis of mesenchymal stem cell products:

[0089] Table 7 Calculation table of ΔCq (Cq (HGF) - Cq (GAPDH)) of three technical replicates of three biological replicates of different generations of umbilical cord mesenchymal stem cells

[0090]

[0091] Table 8 Calculation table of HGF relative to GAPDH mRNA expression levels in different generations of umbilical cord mesenchymal stem cells

[0092]

[0093]

[0094] Example 2: One-step RT-Q-PCR reaction to detect the expression levels of VEGF165 and HGF relative to the internal reference gene GAPDH mRNA in adipose tissue mesenchymal stem cells (AD-MSCs)

[0095] AD-MSC cells were subcultured from the 7th to the 9th generation, with three parallel samples collected for each generation. 6 The cells were placed in 2 mL centrifuge tubes, 1 mL of Trizol was added to each tube for lysis, and the cells were stored at -80°C.

[0096] RNA was extracted and its quality and concentration were measured. The specific results are shown in Table 9 below:

[0097] Table 9 Quantification of RNA in samples of adipose-derived mesenchymal stem cells at different generations

[0098] Sample No. Concentration (ng / μl) A260 / A280 AD-P7-1 1239.8 2.00 AD-P7-2 1319.0 1.99 AD-P7-3 1216.5 2.00 AD-P8-1 1331.4 2.01 AD-P8-2 1354.0 2.00 AD-P8-3 1262.4 2.02 AD-P9-1 1616.0 1.99 AD-P9-2 1454.5 1.98 AD-P9-3 1327.3 2.01

[0099] One-step RT-Q-PCR detection: 3 biological replicates per generation, 3 technical replicates per biological replicate, equivalent to 9 replicates per generation, Q-PCR amplification curve is as follows Figure 2 shown.

[0100] Data processing and analysis: The data acceptance criteria are the same as above. The expression levels of VEGF165 in cells at passages P7, P8, and P9 relative to the internal reference gene GAPDH mRNA are shown in Tables 10 and 11 below. The ΔCq data fluctuated slightly, and the ΔCq coefficient of variation was within 10%. The analysis results 2 –ΔCq The coefficient of variation was also within 20%, indicating that the repeatability and precision fully met the detection requirements. At the same time, the coefficient of variation CV of VEGF165 mRNA from the 7th to the 9th generation of mesenchymal stem cells was 22.67%. The data showed that the expression of VEGF165 mRNA was stable, further proving that it can be used as a target for the biological activity and stability analysis of mesenchymal stem cell products:

[0101] Table 10 Calculation table of ΔCq (Cq (VEGF165) - Cq (GAPDH)) of three technical replicates of three biological replicates of different generations of adipose-derived mesenchymal stem cells

[0102]

[0103]

[0104] Table 11 Calculation of VEGF165 relative to GAPDH mRNA expression levels in adipose-derived mesenchymal stem cells at different generations

[0105]

[0106] The results of HGF expression levels relative to the internal reference gene GAPDH mRNA in P7, P8, and P9 cells are shown in Tables 12 and 13. The coefficients of variation of ΔCq were all within 10%, 2 –ΔCq The coefficient of variation was also within 20%, indicating that the repeatability and precision fully met the detection requirements. At the same time, the coefficient of variation CV of HGF mRNA from the 7th to the 9th generation of adipose-derived mesenchymal stem cells was 21.30%. The data showed that the expression of HGF mRNA was stable, further indicating that HGF mRNA can be used as a target for the biological activity and stability analysis of mesenchymal stem cell products:

[0107] Table 12 Calculation table of ΔCq (Cq (HGF) - Cq (GAPDH)) of three technical replicates of three biological replicates of different generations of adipose-derived mesenchymal stem cells

[0108]

[0109] Table 13 Calculation table of HGF relative to GAPDH mRNA expression levels in adipose-derived mesenchymal stem cells at different generations

[0110]

[0111] Example 3: One-step RT-Q-PCR reaction to detect the expression levels of VEGF165 and HGF relative to the internal reference gene GAPDH mRNA at different storage periods and different cell viabilities after harvesting and storage of umbilical cord mesenchymal stem cells (UC-MSCs)

[0112] P4 UC-MSC cells were cultured until the confluence reached 80% and then harvested. The cell viability was 99.7% as determined by trypan blue staining. The cells were divided into 3 groups, with 2×10 6 cells, take three 15 ml centrifuge tubes, and add 2 × 10 6The cells were resuspended to 3 ml with 0.9% NaCl solution, and the three groups of cells were treated as follows: Group 1 was added with 5% human albumin (HSA) and placed in a 4°C refrigerator for 12 h; Group 2 was placed in a 4°C refrigerator for 12 h without human albumin (HSA); Group 3 was added with 5% human albumin (HSA) and placed in a 4°C refrigerator for 24 h. After the corresponding time, the cells in each group were observed and recorded for cell viability, centrifuged at 1800 rpm for 5 min at room temperature, the supernatant was removed, 1 ml Trizol was added to each group, pipetted and mixed, and transferred to a 2 ml centrifuge tube, RNA was extracted and the quality and concentration were determined, and then one-step RT-Q-PCR reaction was used to detect the expression levels of VEGF165 and HGF relative to the internal reference gene GAPDH mRNA at different storage periods and different cell viabilities after UC-MSC harvest, with 3 technical replicates in each group; the specific results of RNA quality and concentration determination of each group of samples are shown in Table 14 below;

[0113] The cell viability, average value of ΔCq technical repeats, standard deviation of ΔCq technical repeats, and 2 –ΔCq The average of technical replicates, 2 –ΔCq The standard deviation (SD) of technical repeats is shown in Tables 15 and 16 below. The results show that with the extension of storage time after cell collection (12h to 24h in the experimental group) and whether HSA protective agent is added, the cell viability gradually decreases. The mRNA expression levels of VEGF165 and HGF relative to the GAPDH reference gene also show a downward trend as the storage time increases from 12h to 24h. VEGF165 mRNA decreases from 0.0208±0.000651 to 0.0190±0.00109 and 0.0207±0.000843, and HGF mRNA decreases from 0.0406±0.00570 to 0.0355±0.00300 and 0.0340±0.00415, indicating that the detection of VEGF165 and HGF relative to the GAPDH reference gene mRNA expression levels can provide new targets and analysis methods for the biological activity and stability analysis of mesenchymal stem cells in addition to cell viability detection:

[0114] Table 14 RNA quantification of UC-MSC cell samples in three groups with different storage periods and different cell viabilities

[0115] Sample No. Concentration (ng / μl) A260 / A280 1 882.4 1.95 2 875.5 1.98 3 654.9 1.93

[0116] Table 15 Calculation table of VEGF165 relative to GAPDH mRNA expression levels in UC-MSC at different storage periods and different cell viabilities

[0117]

[0118] Table 16 Calculation table of HGF relative to GAPDH mRNA expression levels at different storage periods and different cell viabilities of UC-MSC

[0119]

[0120] Comparative Example 1: Two-step reaction to detect the mRNA expression levels of UC-MSC VEGF165 and HGF relative to the internal reference gene GAPDH

[0121] The two-step method was used to detect the mRNA expression levels of VEGF165 and HGF in umbilical cord mesenchymal stem cells at passages P6, P7, and P8 relative to the internal reference gene GAPDH. The two-step method refers to first performing a reverse transcription reaction and then performing a Q-PCR reaction. The test results are shown in Tables 17 and 18. The data show that the final result of the one-step RT-Q-PCR reaction test is 2 –ΔCq The CV values ​​of biological replicates were 0.30%, 8.02%, and 12.72% (Table 6), which were significantly lower than those of the two-step method (49.79%, 40.53%, and 57.96%). The final results of the one-step RT-Q-PCR reaction for HGF mRNA detection in P6, P7, and P8 generation cells of umbilical cord mesenchymal stem cells were 2 –ΔCq The CV values ​​of biological replicates of 9.47%, 17.78%, and 7.47% (Table 8) were significantly lower than those of the two-step method (105.79%, 107.99%, and 80.88%), indicating that the one-step RT-Q-PCR reaction detection was more stable and reliable. The coefficient of variation of biological replicate detection of samples was significantly reduced from more than 40% of the two-step method to less than 20%, with better repeatability, more accurate and precise results, and more suitable as an analytical method for the biological activity and stability of mesenchymal stem cell products:

[0122] Table 17 Calculation table of three technical replicates of three biological replicates of umbilical cord mesenchymal stem cells at different generations using the two-step method ΔCq (Cq (VEGF165) - Cq (GAPDH))

[0123]

[0124] Table 18 Two-step calculation table of VEGF165 relative to GAPDH mRNA expression levels in different generations of umbilical cord mesenchymal stem cells

[0125]

[0126] The results of HGF mRNA detection of umbilical cord mesenchymal stem cells are shown in Tables 19 and 20 below:

[0127] Table 19 Calculation table of ΔCq (Cq(HGF)-Cq(GAPDH)) of three technical replicates of three biological replicates of different generations of umbilical cord mesenchymal stem cells by two-step method

[0128]

[0129] Table 20 Two-step calculation table of HGF relative to GAPDH mRNA expression levels in different generations of umbilical cord mesenchymal stem cells

[0130]

[0131] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of analytical targets in quality detection of mesenchymal stem cell products, characterized in that: The application is to use RT-Q-PCR to detect the mRNA expression levels of VEGF165 and HGF relative to the internal reference gene; the internal reference gene is GAPDH; the quality detection of the mesenchymal stem cell product is the detection of the biological activity and stability of the mesenchymal stem cell product; The analysis targets are VEGF165 and HGF; the GenBank number of VEGF165 is AF486837.1, and the NCBI accession number of HGF is NM_000601.

6.

2. An analytical method for detecting the biological activity and stability of a mesenchymal stem cell product, characterized in that: The analysis method is not intended for the diagnosis of a disease, but is a method for detecting and analyzing a disease by performing an RT-Q-PCR reaction using a primer and a probe set; The primer and probe set includes: The nucleotide sequence of VEGF165 upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of VEGF165 downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of VEGF165 probe is shown in SEQ ID NO.3; The nucleotide sequence of an HGF upstream primer is shown in SEQ ID NO.4, the nucleotide sequence of an HGF downstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of an HGF probe is shown in SEQ ID NO.6; The nucleotide sequence of the GAPDH upstream primer is shown in SEQ ID NO.7, the nucleotide sequence of the GAPDH downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the GAPDH probe is shown in SEQ ID NO.

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3. The analysis method according to claim 2, characterized in that The 5' end of the probe is modified with a reporter group.

4. The analysis method according to claim 3, characterized in that The reporter group includes FAM, HEX, VIC, ROX or Cy5; the 3' end of the probe is modified with a quencher group.

5. The analysis method according to claim 4, characterized in that The quenching group is BHQ1, BHQ2 or TAMRA.

6. The analysis method according to claim 5, characterized in that The method comprises the following steps: S1. Extract total RNA from samples; S2, using the total RNA extracted from S1 as a template, and using the primer and probe set to perform a one-step RT-Q-PCR amplification reaction; S3. Analysis of ΔCq and 2 –ΔCq The expression of VEGF165 and HGF mRNA in the samples was calculated.

7. The analysis method according to claim 6, characterized in that The final concentration of the primers is 0.1-1.0 µM, and the final concentration of the probe is 0.05-0.5 µM.

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