Method for evaluating immunomodulatory potential of human mesenchymal stem cells and detection kit thereof

Abstract

The invention provides a method for evaluating immunomodulatory potential of human mesenchymal stem cells and a detection kit thereof, and belongs to the technical field of cell drugs. According to the method for evaluating the immunoregulation potential of the human mesenchymal stem cells, the immunoregulation capacity of the mesenchymal stem cells is evaluated by detecting the relative expression quantity of IDO mRNA of the mesenchymal stem cells in a stimulation group, and the higher the relative expression quantity of IDO mRNA is, the higher the immunoregulation capacity of the mesenchymal stem cells is, and the better the quality of the mesenchymal stem cells is. The screening method provided by the invention has the characteristics of rapidness, high efficiency, simplicity in operation and low cost, and is suitable for being applied to large-scale screening of the mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of cell medicines, and specifically relates to a method for evaluating the immunoregulatory potential of human mesenchymal stem cells and a detection kit thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent stem cells that originate from the early mesoderm and ectoderm of development. They can be isolated from a variety of tissues such as bone marrow, fat, synovium, bone, muscle and umbilical cord, and have the potential to differentiate into osteoblasts, chondroblasts, adipocytes, bone marrow stroma, nerve cells, liver cells, pancreatic islet cells and cardiomyocytes. They are easy to source, easy to separate, culture, expand and purify, and still have stem cell characteristics after multiple passages and expansions, and do not have the characteristics of immune rejection.

[0003] Mesenchymal stem cells can not only promote the regeneration and repair of damaged tissues, but also have good immunoregulatory potential. In addition, they can also be used as carriers for gene therapy and have a wide range of uses. However, there are many methods for preparing mesenchymal stem cells, such as tissue block adherent culture method, umbilical cord homogenate collagenase digestion method and modified collagenase digestion method. Mesenchymal stem cells prepared by different methods can directly affect the product quality of mesenchymal stem cells. Studies have found that mesenchymal stem cells not only have the potential for multidirectional differentiation, but more importantly, they have unique immunosuppression and immunoregulatory potential, and are widely used in clinical treatment. However, current research does not have a good method to evaluate the immunoregulatory potential of mesenchymal stem cells prepared in different ways. Therefore, it is of great significance to develop a method that can effectively evaluate the immunoregulatory potential of mesenchymal stem cells. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for evaluating the immunoregulatory potential of human mesenchymal stem cells, and to screen and obtain human mesenchymal stem cells with high immunoregulatory potential by measuring the relative expression level of IDO mRNA.

[0005] The present invention provides a method for evaluating the immunoregulatory potential of human mesenchymal stem cells, comprising the following steps:

[0006] The human mesenchymal stem cells to be tested are divided into a stimulation group and a control group, wherein the human mesenchymal stem cells in the stimulation group are cultured after being treated with IFN-γ, and the human mesenchymal stem cells in the control group are cultured without being treated with IFN-γ;

[0007] The total genomic RNA of human mesenchymal stem cells in the stimulation group and the control group was extracted, and the expression level of IDO mRNA was determined by RT-qPCR amplification technology to obtain the expression level of IDO in human mesenchymal stem cells in the stimulation group and the control group respectively;

[0008] The relative expression of IDO mRNA in the stimulated human mesenchymal stem cells was calculated. The relative expression of IDO in the human mesenchymal stem cells was positively correlated with the immunoregulatory potential of the human mesenchymal stem cells.

[0009] Preferably, during the IFN-γ treatment, the working concentration of IFN-γ is 18 to 50 ng / ml.

[0010] Preferably, the IFN-γ treatment time is 45 to 52 hours.

[0011] Preferably, the qPCR amplification primers include the nucleotide sequences shown in SEQ ID NO:9 to SEQ ID NO:10;

[0012] The reaction system of the qPCR amplification was 5 μL of 2×TsingKe Master qPCR Mix-SYBR (+UDG), 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer and 4.2 μL of cDNA template.

[0013] Preferably, the culture medium is MSC-T4 complete culture medium.

[0014] Preferably, the reaction procedure of the qPCR amplification is 50°C 2 min; 95°C 2 min; 95°C 15 sec, 55°C 30 sec, 72°C 30 sec, 40 cycles or 50°C 2 min; 95°C 2 min; 95°C 15 sec, 60°C 30 sec, 40 cycles.

[0015] Preferably, the relative expression level of IDO mRNA in human mesenchymal stem cells in the stimulation group is calculated according to Formula I:

[0016]

[0017] Among them, actin is the internal reference gene.

[0018] The present invention provides a kit for implementing the method for evaluating the immunoregulatory potential of human mesenchymal stem cells, comprising primers for detecting the expression amount of IDO mRNA of human mesenchymal stem cells;

[0019] The primers include at least one pair of primers: a first primer pair, a second primer pair and a third primer pair;

[0020] The nucleotide sequences of the first primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 2;

[0021] The nucleotide sequences of the second primer pair are shown in SEQ ID NO:3 to SEQ ID NO:4;

[0022] The nucleotide sequences of the third primer pair are shown in SEQ ID NO:5 to SEQ ID NO:6.

[0023] Preferably, a qPCR detection premix is ​​included.

[0024] Preferably, it also includes at least one of the following: an internal reference primer, an RNA extraction reagent and / or a reverse transcription reagent.

[0025] The present invention provides a method for evaluating the immunoregulatory potential of human mesenchymal stem cells, and the immunoregulatory ability of mesenchymal stem cells is evaluated by detecting the relative expression of IDO mRNA in the stimulation group. The higher the relative expression of IDO mRNA, the stronger the immunoregulatory ability of the mesenchymal stem cells and the better the quality. When the relative expression of IDO mRNA of the human mesenchymal stem cells in the stimulation group is ≥10, the human mesenchymal stem cells to be detected have high immunoregulatory potential. The screening method provided by the present invention is fast and efficient, simple to operate, low in cost, and suitable for large-scale evaluation or screening of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the optimization result of IFN-γ stimulation concentration of 0 to 20 ng / ml;

[0027] Figure 2 The results are optimized for IFN-γ stimulation concentrations ranging from 0 to 100 ng / ml;

[0028] Figure 3 This is the result of optimizing the IFN-γ stimulation time;

[0029] Figure 4 Results were compared between the IFN-γ stimulated group and the control group. DETAILED DESCRIPTION

[0030] The present invention provides a method for evaluating the immunoregulatory potential of human mesenchymal stem cells, comprising the following steps: dividing the human mesenchymal stem cells to be detected into a stimulation group and a control group, wherein the human mesenchymal stem cells in the stimulation group are cultured after being treated with IFN-γ, and the human mesenchymal stem cells in the control group are cultured without being treated with IFN-γ;

[0031] Extracting total genomic RNA from human mesenchymal stem cells in the stimulation group and the control group, and determining the expression of IDO mRNA using RT-qPCR amplification technology to obtain the expression of IDO in the human mesenchymal stem cells in the stimulation group and the control group, respectively; the qPCR amplification primers include the nucleotide sequences shown in SEQ ID NO:9 to SEQ ID NO:10;

[0032] The relative expression of IDO mRNA in the stimulated human mesenchymal stem cells was calculated. The relative expression of IDO in the human mesenchymal stem cells was positively correlated with the immunoregulatory potential of the human mesenchymal stem cells.

[0033] The present invention randomly divides human mesenchymal stem cells to be detected into a stimulation group and a control group. The human mesenchymal stem cells in the stimulation group are treated with IFN-γ and cultured, while the human mesenchymal stem cells in the control group are not treated with IFN-γ and cultured.

[0034] In the present invention, the human mesenchymal stem cells to be detected are preferably human mesenchymal stem cells derived from amniotic membrane and / or human mesenchymal stem cells derived from umbilical cord. The density of the human mesenchymal stem cells is preferably (1-1.5)×10 6 / ml, more preferably 1.2×10 6 / ml. The timing of the IFN-γ treatment is preferably to culture the human mesenchymal stem cells for 24 hours. The culture method is preferably at 37°C, 5% CO 2 Environment. After culturing for 24 hours, replace with fresh culture medium and perform IFN-γ treatment. During the IFN-γ treatment, the working concentration of IFN-γ is 18 to 50 ng / ml, can be 20 to 40 ng / ml, and can also be 25 to 30 ng / ml. Different concentrations of IFN-γ have an effect on the expression of IDO in human mesenchymal stem cells. Experiments have shown that the higher the treatment concentration of IFN-γ, the higher the expression of IDO. However, considering that high concentrations of IFN-γ treatment will affect the survival rate of human mesenchymal stem cells, taking into account the survival rate of human mesenchymal stem cells and the expression of IDO, a working concentration of 18 to 22 ng / ml is preferably a suitable treatment concentration of IFN-γ. The time of the IFN-γ treatment is preferably 45 to 52 hours, more preferably 48 hours. Different treatment times have an effect on the expression of IDO in human mesenchymal stem cells. Experiments have shown that the higher the treatment concentration of IFN-γ, the higher the expression of IDO. However, considering that long-term IFN-γ treatment will affect the survival rate of human mesenchymal stem cells, a treatment time of 45 to 52 hours is selected as the preferred treatment time for IFN-γ.

[0035] After IFN-γ treatment, the genomic RNA of the mesenchymal stem cells is preferably extracted after culturing for 45 to 50 hours, and more preferably after culturing for 48 hours. The culture medium is MSC-T4 complete medium. The culture conditions are preferably 37°C, 5% CO 2. After the culture, single cells are preferably prepared. The single cells are preferably prepared by digestion using a trypsin solution with a mass concentration of 0.25%. The digestion time is 2.5 to 3.5 minutes, more preferably 3 minutes. After digestion, MSC-T4 complete culture medium is preferably added to terminate the digestion. After digestion, single cells are separated and resuspended. When resuspending, the solution used is preferably MSC-T4 complete culture medium.

[0036] The present invention has no particular limitation on the method for extracting RNA from human mesenchymal stem cells, and any RNA extraction method known in the art can be used. In the embodiment of the present invention, a kit method is used to extract RNA from cells.

[0037] In the present invention, after obtaining RNA, the RNA is preferably reverse transcribed. The present invention has no particular limitation on the reverse transcription method, and any reverse transcription method known in the art can be used.

[0038] In the present invention, the reaction system of the qPCR amplification is 2×TsingKe Master qPCR Mix-SYBR (+UDG) 5 μL, 10 μM forward primer 0.4 μL, 10 μM reverse primer 0.4 μL and cDNA template 4.2 μL. The reaction procedure of the qPCR amplification is 50°C 2min; 95°C 2min; 95°C 15sec, 55°C 30sec, 72°C 30sec, 40 cycles or 50°C 2min; 95°C 2min; 95°C 15sec, 60°C 30sec, 40 cycles. The primers for qPCR amplification include the nucleotide sequences shown in SEQ ID NO: 9 to SEQ ID NO: 10.

[0039] In the present invention, the relative expression level of IDO mRNA in the human mesenchymal stem cells in the stimulation group is calculated according to Formula I:

[0040]

[0041] Among them, actin is the internal reference gene.

[0042] In the present invention, when the relative expression amount of IDO of the human mesenchymal stem cells in the stimulation group is ≥10, the human mesenchymal stem cells to be detected have high immunoregulatory potential.

[0043] The present invention provides a kit for implementing the method for evaluating the immunoregulatory potential of human mesenchymal stem cells, comprising primers for detecting the expression amount of IDO mRNA of human mesenchymal stem cells;

[0044] The primers include at least one pair of primers: a first primer pair, a second primer pair and a third primer pair;

[0045] The nucleotide sequences of the first primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 2;

[0046] The nucleotide sequences of the second primer pair are shown in SEQ ID NO:3 to SEQ ID NO:4;

[0047] The nucleotide sequences of the third primer pair are shown in SEQ ID NO:5 to SEQ ID NO:6.

[0048] In the present invention, the kit preferably includes a qPCR detection premix, an internal reference primer, an RNA extraction reagent and / or a reverse transcription reagent. The qPCR detection premix includes Master qPCR Mix-SYBR (+UDG). The internal reference may include an actin gene. The internal reference primer may include a nucleotide sequence such as SEQ ID NO: 7 and SEQ ID NO: 8. The present invention has no particular limitation on RNA extraction reagents or reverse transcription reagents, and RNA extraction reagents and reagents or reverse transcription reagents known in the art may be used.

[0049] In one embodiment of the present invention, the first primer pair, the second primer pair and the third primer pair are used to evaluate the immunoregulatory potential of human mesenchymal stem cells. The results show that the first primer pair, the second primer pair and the third primer pair have higher amplification efficiency, so that the relative expression level of mesenchymal stem cells in the detection stimulation group is significantly improved compared with the detection results of the primer pairs shown in SEQ ID NO: 9 and SEQ ID NO: 10, which indicates that the kit can improve the accuracy of the evaluation of the immunoregulatory potential of mesenchymal stem cells.

[0050] The following is a detailed description of a method for evaluating the immunoregulatory potential of human mesenchymal stem cells and a detection kit thereof provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The sources of the instruments and reagents involved in the embodiments of the present invention are specifically described in Tables 1 and 2.

[0052] Table 1 Description of main instrument sources

[0053] instrument brand model High-speed refrigerated centrifuge Thermo Fisher Scientific (China) Co., Ltd. HeraeusFresco17 Micro-volume spectrophotometer Thermo Fisher Scientific (China) Co., Ltd. Nanodropone Real-time fluorescence quantitative PCR instrument Hoffmann-La Roche LightCycler480II Vortex mixer Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd. MX-F

[0054] Table 2 Description of the sources of main reagents

[0055]

[0056]

[0057] Example 1

[0058] Screening method for IFN-γ stimulation concentration

[0059] According to 1×10 6 / well inoculation volume Human amnion-derived MSCs were inoculated into six-well plates, and MSC-T4 complete medium was added to each well to 3 ml, and cultured for 24 hours. Stimulation group and control group were set up for the six-well plate, in which IFN-γ with a final concentration of 20ng / ml, 10ng / ml, and 5ng / ml was added to the stimulation group, and IFN-γ was not added to the control group. Three wells were made for each concentration and placed at 37°C and 5% CO 2 The cells were cultured in an incubator, and the cells were collected after 48 hours. The relative expression of IDO mRNA was detected after RNA extraction. The specific method is as follows:

[0060] After culturing for 48 h, the supernatant was discarded, and the cells were washed twice with D-PBS, 0.25% trypsin was added at 1 ml / well, and the cells were placed in CO 2 After 3 minutes of digestion in the incubator, add 1 ml of complete medium to terminate the digestion. Transfer the terminated cells to a 15 ml centrifuge tube, centrifuge at 400 g for 3 minutes, remove the supernatant, resuspend with 1 ml of MSC-T4 complete medium, and count the cells according to YR-QC-SOP-23 "Standard Operating Procedures for Viable Cell Counting, Viability and Diameter Detection" for later use. 6 Place the cell sample in a 1.5ml RNase free EP tube, centrifuge at 1500rpm for 5min, and remove the supernatant. Animal tissue / cell total RNA extraction kit preparation: Add 48ml of anhydrous ethanol to the rinse solution RW bottle. Extract RNA according to the following steps:

[0061] ① Add 350 μl of lysis buffer RLA and 10 μl of proteinase K, vortex, centrifuge at 12,000 rpm for 4 min, and collect the supernatant.

[0062] ② Add the obtained supernatant to the genomic DNA removal column, centrifuge at 12,000 rpm for 30 seconds, and retain the filtrate.

[0063] ③ Slowly add 70% ethanol (1 times the volume of the supernatant) to the above filtrate, mix well and transfer to the RNase-Free adsorption column CR4 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0064] ④ Add 700 μl of deproteinization solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0065] ⑤ Add 500 μl of rinsing solution RW with ethanol added to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 50 seconds, and discard the waste liquid.

[0066] ⑥ Add 500 μl of rinsing solution RW with ethanol to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 50 seconds, and discard the waste liquid.

[0067] ⑦ Centrifuge at 12,000 rpm (~13,400 × g) for 2 min, discard the waste liquid, and place the RNase-Free adsorption column CR4 at room temperature for 2 min.

[0068] ⑧ Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube and drop 30 μl RNase-Free ddH 2 0, placed at room temperature for 2 min, centrifuged at 12,000 rpm for 2 min to obtain RNA solution. The RNA concentration of the sample to be tested was determined using a micro-spectrophotometer, and 1 μg of RNA was taken for subsequent detection.

[0069] 4) RNA reverse transcription: ① Take out gDNA remover, 10×gDNA remover Buffer, and RNase-free water from the reverse transcription kit, place them on ice to melt, and centrifuge for a few seconds before use. Take the qPCR eight-tube strip and prepare the reverse transcription reaction system, as shown in Table 3.

[0070] Table 3 Reverse transcription reaction system

[0071] Components RNA template gDNAremover 10xgDNAremoverBuffer RNase-free water Total volume Dosage 1 μg 1μL 1μL Add to 10 μL 10μL

[0072] ② Mix gently with a pipette tip, centrifuge briefly, incubate at 42℃ for 2 min, and then incubate at 60℃ for 5 min.

[0073] ③The mixture was quickly cooled on ice, and after a brief centrifugation, the following components were added according to Table 4:

[0074] Table 4 Reaction system

[0075]

[0076]

[0077] ④ Mix gently with a pipette tip, centrifuge briefly, and set the reaction conditions according to Table 5 for reverse transcription reaction.

[0078] Table 5 Reverse transcription conditions

[0079] condition time 25℃ 10min 55℃ 15min 85℃ 5min

[0080] 5) qPCR amplification:

[0081] ① Take the required number of fluorescent eight-tube strips, prepare the qPCR reaction system according to Table 6 using the cDNA obtained by reverse transcription of RNA, and treat the IDO and ACTIN groups respectively.

[0082] Table 6 qPCR reaction system

[0083] Components Usage 2 x TsingKe*Master qPCR Mix-SYBR(+UDG) 5μL 10 μM forward primer 0.4μL 10 μM reverse primer 0.4μL DNA template (<100ng} 4.2μL

[0084] Among them, IDO:

[0085] Forward primer: 5-AGACTGCTGGTGGAGGACATG-3' (SEQ ID NO: 9);

[0086] Reverse primer: 5'-AAAGGACAAACTCACGGACTG-3' (SEQ ID NO: 10);

[0087] β-actin: forward primer: 5′-GTCACCTTCACCGTTCCAGTTTT-3′ (SEQ ID NO: 7);

[0088] Reverse primer: 5'-CTTAGTTGCGTTACACCCTTTCTT-3' (SEQ ID NO: 8).

[0089] ②After removing the bubbles, cover the tube and centrifuge briefly to perform PCR amplification reaction.

[0090] ③PCR amplification (LightCycler480) (SYBR Green I channel), the specific reaction procedure is shown in Table 6:

[0091] Table 6 PCR amplification reaction program

[0092]

[0093] According to the CT values ​​of ACTIN and IDO groups, the relative expression of IDO mRNA in the treatment group was obtained by substituting them into formula I.

[0094]

[0095] The results of the relative expression of IDO mRNA under different IFN-γ stimulation concentrations are shown in Figure 1 Compared with the control group, the relative expression of IDO mRNA in mesenchymal stem cells increased with the increase of IFN-γ stimulation concentration.

[0096] Example 2

[0097] The relative expression of IDO mRNA in human amnion-derived mesenchymal stem cells was determined according to the method of Example 1, except that 20 ng / ml, 50 ng / ml and 100 ng / ml of IFN-γ were used to stimulate the mesenchymal stem cells for 48 h. At the same time, the growth of the stimulated mesenchymal stem cells was observed under a microscope, and when a decrease in the number of adherent cells was observed, cell debris or floating dead cells in the culture supernatant were considered to be apoptosis of the mesenchymal stem cells.

[0098] The relative expression of IDO mRNA in mesenchymal stem cells under different IFN-γ stimulation concentrations is shown in Figure 2 . After 48 hours of IFN-γ stimulation with 20ng / ml, 50ng / ml and 100ng / ml, the relative expression of IDO mRNA was 861.08, 1595.73 and 3224.81, respectively. Compared with the control group, IFN-γ stimulation can increase the relative expression of IDO mRNA. However, with the increase of stimulation concentration, the number of stem cells that cause apoptosis due to IFN-γ stimulation will also increase significantly. In some extreme cases (such as a batch of stem cells that are particularly sensitive to stimulation and intolerant), most stem cells apoptosis, which is not conducive to the subsequent collection and detection analysis of mRNA. Therefore, taking into account the effectiveness of the stimulation and the tolerance of the stem cells themselves, the stimulation concentration of 20ng / ml was selected.

[0099] Example 3

[0100] The relative expression of IDO mRNA in mesenchymal stem cells was determined according to the method of Example 1, except that 20 ng / ml IFN-γ was used to stimulate the mesenchymal stem cells, and the stimulation time was 24 h, 48 h and 72 h, respectively.

[0101] The relative expression of IDO mRNA in mesenchymal stem cells under different IFN-γ stimulation time is shown in Figure 3 . After stimulation with 20ng / ml IFN-γ for 24h, 48h, and 72h, the relative expression of IDO mRNA was 1.11, 199.70, and 582.72, respectively. Compared with the control group, IFN-γ stimulation can increase the relative expression of IDO mRNA. And with the extension of the stimulation time, the relative expression of IDO mRNA in mesenchymal stem cells also increases. However, with the extension of the stimulation time, the number of stem cells that cause apoptosis due to IFN-γ stimulation will also increase significantly. In some extreme cases (such as a batch of stem cells that are particularly sensitive to stimulation and intolerant), most stem cells apoptosis, which is not conducive to the subsequent collection and detection analysis of mRNA. Therefore, taking into account the effectiveness of the stimulation and the tolerance of the stem cells themselves, 48h stimulation was selected.

[0102] Example 4

[0103] High expression of IDO mRNA in amniotic mesenchymal stem cells after IFN-γ stimulation

[0104] Human amnion-derived MSCs were cultured at a rate of 1.2×10 6 Each well was inoculated into a six-well plate, and fresh complete medium was replaced after 24 h of culture. IFN-γ with a final concentration of 20 ng / ml was added to the stimulation group, and the plate was placed in a 37°C 5% CO 2 The cells were cultured in an incubator. After 48 h of culture, the supernatant was discarded and the cells were washed twice with D-PBS. 0.25% trypsin was added at 1 ml / well and placed in a CO 2 After digestion in the incubator for 3 minutes, 1 ml of complete medium was added to terminate the digestion. The cells that terminated the digestion were transferred to a 15 ml centrifuge tube, centrifuged at 400 g for 3 minutes, and then the supernatant was removed and the cell pellet was collected for RNA extraction.

[0105] Method for extracting MSC cell genomic RNA:

[0106] 1. Take the cell sample to be tested, extract RNA according to the instructions of the RNA extraction kit, measure the concentration, and take 1 μg for standby use;

[0107] 2. Take 1 μL of gDNA remover, 1 μL of 10×gDNA remover Buffer, RNase-free water (replenish to 10 μL) and 1 μg RNA from the reverse transcription kit according to the number of reaction tubes, mix gently for a few seconds, centrifuge briefly and set aside;

[0108] 3. Incubate at 42℃ for 2 min, then at 60℃ for 5 min. Quickly cool the mixture on ice. Centrifuge briefly and take 1μL of dNTP Mix and Goldenstar according to the number of reaction tubes. TM Randomer 1μL, 5xGoldenstar TM Buffer 4μL, DTT 1μL, Goldenstar TM RT6 1μL, RNase-free water 2μL, gently mix for a few seconds, centrifuge briefly and perform reverse transcription reaction according to the preset program;

[0109] 4. Dilute the reverse transcription product 4 times, treat with IDO and ACTIN groups respectively, take 2×TsingKe*Master qPCR Mix-SYBR(+UDG) 5μL, 10μM forward primer 0.4μL, 10μM reverse primer 0.4μL, Template DNA 4.2μL according to the number of reaction tubes, mix gently for a few seconds, remove bubbles and cover the tube, centrifuge instantly, and perform PCR amplification reaction;

[0110] 5. Analyze the results after amplification according to the preset procedure described in Example 1.

[0111] The results are shown in Table 7 and Figure 4 Compared with the control group, the expression level of IDO mRNA in the IFN-γ stimulation group was significantly increased.

[0112] Table 7 qPCR test results of the stimulation group and the control group

[0113]

[0114]

[0115] Note: The bold 35 is a reference CT value given by the instrument at the end of the qPCR test because the IDO mRNA expression level in the control group is low; / indicates that the expression level of this sample is extremely low and the CT value cannot be detected. These data are based on the results of collecting cells and extracting mRNA for testing after 20ng / ml stimulation for 48 hours.

[0116] Example 5

[0117] The known primers for detecting IDO gene expression in Example 1 (SEQ ID NO: 9 and SEQ ID NO: 10) and three pairs of independently designed new primers were used for experiments. The experimental method is shown in Example 4. Only the PCR reaction conditions are shown in Table 8 below.

[0118] The specific sequences of the three new primer pairs are as follows:

[0119] First pair of primers - F: 5'-CTGGAACTGCCTCCTATT-3' (SEQ ID NO: 1);

[0120] First pair of primers - R: 5'-ATGCGAAGAACACTGAAA-3' (SEQ ID NO: 2);

[0121] The second pair of primers - F: 5'-CATCTGCCTGATCTCATAG-3' (SEQ ID NO: 3);

[0122] Second pair of primers - R: 5'-AGCAAAGTGTCCCGTTCT-3' (SEQ ID NO: 4);

[0123] The third pair of primers - F: 5'-AAGTGGGCTTTGCTCTGC-3' (SEQ ID NO: 5);

[0124] The third pair of primers - R: 5'-GGCAAGACCTTACGGACA-3' (SEQ ID NO: 6).

[0125] Table 8 PCR amplification reaction program

[0126]

[0127] The results are shown in Table 9.

[0128] Table 9 Comparison of qPCR test results of public primers and newly designed primers

[0129]

[0130]

[0131] It can be seen that the three pairs of primers designed independently have higher amplification efficiency in qPCR reactions than the known primers, thereby making the detected relative expression level higher.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating the immunomodulatory potential of human mesenchymal stem cells, characterized in that: The following steps are involved: The human mesenchymal stem cells to be tested are divided into a stimulation group and a control group, wherein the human mesenchymal stem cells in the stimulation group are cultured after being treated with IFN-γ, and the human mesenchymal stem cells in the control group are cultured without being treated with IFN-γ; The total genomic RNA of human mesenchymal stem cells in the stimulation group and the control group was extracted, and the expression level of IDO mRNA was determined by RT-qPCR amplification technology to obtain the expression level of IDO in human mesenchymal stem cells in the stimulation group and the control group respectively; The relative expression of IDO mRNA in the stimulated human mesenchymal stem cells was calculated. The relative expression of IDO in the human mesenchymal stem cells was positively correlated with the immunoregulatory potential of the human mesenchymal stem cells.

2. The method according to claim 1, characterized in that: The working concentration of IFN-γ is 18-50 ng / ml.

3. The method according to claim 1, characterized in that: The IFN-γ treatment time is 45 to 52 hours.

4. The method according to claim 1, characterized in that: The qPCR amplification primers include the nucleotide sequences shown in SEQ ID NO:9 to SEQ ID NO:10; The reaction system of the qPCR amplification was 5 μL of 2×TsingKe Master qPCR Mix-SYBR (+UDG), 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer and 4.2 μL of cDNA template.

5. The method according to claim 1, characterized in that: The reaction program of the qPCR amplification is 50°C 2min; 95°C 2min; 95°C 15sec, 55°C 30sec, 72°C 30sec, 40 cycles or 50°C 2min; 95°C 2min; 95°C 15sec, 60°C 30sec, 40 cycles.

6. The method according to claim 1, characterized in that: The culture medium is MSC-T4 complete culture medium.

7. The method according to any one of claims 1 to 6, characterized in that: The relative expression level of IDO mRNA in human mesenchymal stem cells in the stimulation group is calculated according to Formula I: Among them, actin is the internal reference gene.

8. A kit for implementing the method for evaluating the immunomodulatory potential of human mesenchymal stem cells according to any one of claims 1 to 7, characterized in that: Includes primers for detecting IDO mRNA expression in human mesenchymal stem cells; The primers include at least one pair of primers: a first primer pair, a second primer pair and a third primer pair; The nucleotide sequences of the first primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 2; The nucleotide sequences of the second primer pair are shown in SEQ ID NO:3 to SEQ ID NO:4; The nucleotide sequences of the third primer pair are shown in SEQ ID NO:5 to SEQ ID NO:

6.

9. The kit according to claim 8, characterized in that: Includes qPCR assay master mix.

10. The kit according to claim 9, characterized in that: It also includes at least one of the following: an internal reference primer, an RNA extraction reagent and / or a reverse transcription reagent.

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