SiRNA for reducing IFNGR1 and application thereof

Abstract

The invention provides siRNA for reducing IFNGR1 and application of the siRNA, and relates to the technical field of biology, the siRNA comprises at least one of IFNGR1-si-1, IFNGR1-si-2 or IFNGR1-si-3, and experiments prove that the knock-down efficiency of the IFNGR1-si-1, the IFNGR1-si-2 and the IFNGR1-si-3 on IFNGR1 genes in mesenchymal stem cells reaches 48% or above. And quantitative expression of the mesenchymal stem cell IFNGR1 can be realized by quantitatively transferring siRNA. A fitting formula is determined by establishing a corresponding relation between the IFNGR1 expression quantity and the total lymphocyte proliferation inhibition rate, and the total lymphocyte proliferation inhibition rate can be calculated by detecting the IFNGR1 expression quantity of the mesenchymal stem cells to be evaluated. Compared with the prior art, the MSC does not need to be evaluated to be co-cultured with the PBMC every time, and a large number of PBMCs are used.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to siRNA for reducing IFNGR1 and application thereof. Background Art

[0002] Mesenchymal stem cells (MSC) are a group of multipotent adult stem cells derived from the mesoderm, which have the potential to self-renew, multidirectional differentiation, promote tissue and organ repair, and immune regulation. MSC can be isolated and cultured from multiple tissues such as bone marrow, umbilical cord, placenta, fat, bone, dental pulp, and endometrium. MSC-like cells can also be differentiated from embryonic stem cells or pluripotent stem cells. MSC has low immunogenicity. Because it does not express co-stimulatory molecules such as CD40, CD80, CD86 and major histocompatibility complex molecules, it is less likely to cause immune rejection. At the same time, because its regulatory effect on immune cells is not restricted by major histocompatibility complex molecules, it can exert immunoregulatory effects on both autologous and allogeneic immune cells. For example, MSC can exert regulatory effects on the proliferation, differentiation, and antibody production of abnormally activated T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, etc. through direct cell-to-cell contact or secretion of cytokines. Many clinical studies have shown that MSC infusion can significantly inhibit the excessive proliferation of T cells in patients and inhibit the activity of cytotoxic CD8+T lymphocytes. Although a large number of scholars have conducted research on the mechanism of action of MSCs, it is still impossible to fully explain the drug mechanism of action (MOA) of MSCs and provide a suitable potency evaluation method.

[0003] At present, the most commonly used method to evaluate the immunoregulatory ability of MSCs is to co-culture MSCs with peripheral blood mononuclear cells (PBMCs) for 3 to 5 days, detect the proliferation ability of immune cells such as lymphocytes in PBMCs, and calculate the inhibitory ability of MSCs on their proliferation to evaluate the immunoregulatory ability of MSCs. However, this method requires the use of a large number of PBMCs.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a siRNA that reduces IFNGR1, so as to at least solve the technical problem in the prior art that there is a lack of a substance that can downregulate the expression of IFNGR1 and can be used to evaluate mammalian cells.

[0006] The second object of the present invention is to provide the use of the above-mentioned siRNA in reducing or inhibiting the expression of IFNGR1 in mammalian cells or preparing products for reducing or inhibiting the expression of IFNGR1 in mammalian cells, reducing or inhibiting the immunosuppressive ability of mammalian cells or preparing products for reducing or inhibiting the immunosuppressive ability of mammalian cells, or evaluating the immunoregulatory ability of mammalian cells.

[0007] A third object of the present invention is to provide a reagent or kit for reducing IFNGR1.

[0008] A fourth object of the present invention is to provide a method for down-regulating the immunoregulatory ability of mesenchymal stem cells.

[0009] A fifth object of the present invention is to provide a mesenchymal stem cell that quantitatively expresses IFNGR1.

[0010] A sixth object of the present invention is to provide a method for evaluating the immunoregulatory ability of mesenchymal stem cells.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides an siRNA for reducing IFNGR1, comprising at least one of IFNGR1-si-1, IFNGR1-si-2 or IFNGR1-si-3;

[0013] The IFNGR1-si-1 comprises a forward sequence IFNGR1-si-1-F as shown in SEQ ID NO.1 and a reverse sequence IFNGR1-si-1-R as shown in SEQ ID NO.2;

[0014] The IFNGR1-si-2 comprises a forward sequence IFNGR1-si-2-F as shown in SEQ ID NO.3 and a reverse sequence IFNGR1-si-2-R as shown in SEQ ID NO.4;

[0015] The IFNGR1-si-3 comprises a forward sequence IFNGR1-si-3-F as shown in SEQ ID NO.5 and a reverse sequence IFNGR1-si-3-R as shown in SEQ ID NO.6.

[0016] Furthermore, the 3' ends of the forward sequence and the reverse sequence are connected with 2 additional nucleotides respectively;

[0017] Preferably, the additional nucleotides are deoxythymidine nucleotides.

[0018] In a second aspect, the present invention provides the use of the above siRNA in any of the following:

[0019] A1. Use in reducing or inhibiting the expression of IFNGR1 in mammalian cells or in preparing a product for reducing or inhibiting the expression of IFNGR1 in mammalian cells;

[0020] A2. Use in reducing or inhibiting the immunosuppressive ability of mammalian cells or in preparing products for reducing or inhibiting the immunosuppressive ability of mammalian cells;

[0021] A3. Application in evaluating the immunoregulatory capacity of mammalian cells.

[0022] Further, the cells are mesenchymal stem cells;

[0023] Preferably, the mammal comprises a human.

[0024] In a third aspect, the present invention provides a reagent or kit for reducing IFNGR1, comprising the above-mentioned siRNA.

[0025] In a fourth aspect, the present invention provides a method for down-regulating the immunoregulatory ability of mesenchymal stem cells, comprising transferring the above-mentioned siRNA into mesenchymal stem cells.

[0026] Furthermore, after the mesenchymal stem cells are transferred in, the step also includes culturing for at least 24 hours.

[0027] In a fifth aspect, the present invention provides a mesenchymal stem cell that quantitatively expresses IFNGR1, comprising the above-mentioned siRNA.

[0028] In a sixth aspect, the present invention provides a method for evaluating the immunoregulatory ability of mesenchymal stem cells, comprising detecting the IFNGR1 expression level of the mesenchymal stem cells to be evaluated, substituting the IFNGR1 expression level of the mesenchymal stem cells to be evaluated into a fitting formula to calculate the total lymphocyte proliferation inhibition rate, and evaluating the immunoregulatory ability of the mesenchymal stem cells according to the total lymphocyte proliferation inhibition rate;

[0029] The fitting formula is determined by the IFNGR1 expression levels of several groups of mesenchymal stem cells that quantitatively express IFNGR1 and the total lymphocyte proliferation inhibition rate corresponding to the IFNGR1 expression levels.

[0030] Furthermore, the method for constructing the fitting formula includes the following steps:

[0031] A. preparing several groups of mesenchymal stem cells that quantitatively express IFNGR1, and obtaining the IFNGR1 expression level of each group of mesenchymal stem cells;

[0032] B. Several groups of mesenchymal stem cells quantitatively expressing IFNGR1 were co-cultured with peripheral blood mononuclear cells to obtain the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells;

[0033] C. According to the IFNGR1 expression level of each group of mesenchymal stem cells and the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells, a fitting curve was constructed to obtain a fitting formula;

[0034] Preferably, the IFNGR1 expression levels of the several groups of mesenchymal stem cells that quantitatively express IFNGR1 are all different;

[0035] Preferably, the number of the several groups of mesenchymal stem cells quantitatively expressing IFNGR1 is at least 2;

[0036] Preferably, the method for preparing several groups of mesenchymal stem cells quantitatively expressing IFNGR1 comprises introducing different concentrations of siRNA into mesenchymal stem cells respectively;

[0037] Preferably, the different concentrations of siRNA are 0 to 10 pmol / mL of siRNA, and are not 0;

[0038] Preferably, the culture time of mesenchymal stem cells quantitatively expressing IFNGR1 is 0 to 72 hours, preferably 24 hours;

[0039] Preferably, the co-culture time with peripheral blood mononuclear cells is 72 to 120 hours, preferably 96 hours.

[0040] The present invention provides a siRNA for reducing IFNGR1 and its application. Experiments prove that the knockdown efficiency of IFNGR1 gene in mesenchymal stem cells by IFNGR1-si-1, IFNGR1-si-2 and IFNGR1-si-3 is more than 48%, among which IFNGR1-si-3 is the highest. The quantitative expression of IFNGR1 in mesenchymal stem cells can be achieved by quantitatively transferring siRNA. It can be used to establish the corresponding relationship between the expression level of IFNGR1 in MSC and the inhibitory ability of MSC on the proliferation of immune cells, and is used to evaluate mesenchymal stem cells. On the other hand, a method for evaluating the immunoregulatory ability of mesenchymal stem cells is provided. By quantitatively expressing IFNGR1 in mesenchymal stem cells, the corresponding relationship between the IFNGR1 expression amount and the total lymphocyte proliferation inhibition rate is established to determine the fitting formula. The total lymphocyte proliferation inhibition rate can be calculated by detecting the IFNGR1 expression amount of the mesenchymal stem cells to be evaluated; compared with the prior art, it is not necessary to co-culture MSC with PBMC every time for the evaluation of MSC, and a large number of PBMCs are used. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 A comparison chart of IFNGR expression levels on the surface of MSC cells treated differently provided in Example 2 of the present invention;

[0043] Figure 2 A comparison chart of the total lymphocyte proliferation inhibition rate of MSCs treated differently provided in Example 3 of the present invention;

[0044] Figure 3 This is the fitting curve of IFNGR1 expression on the surface of MSC cells and total lymphocyte proliferation inhibition rate provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0045] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0046] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0047] In one aspect, the present invention provides an siRNA for reducing IFNGR1, comprising at least one of IFNGR1-si-1, IFNGR1-si-2 or IFNGR1-si-3;

[0048] The IFNGR1-si-1 comprises a forward sequence IFNGR1-si-1-F as shown in SEQ ID NO.1 and a reverse sequence IFNGR1-si-1-R as shown in SEQ ID NO.2;

[0049] The IFNGR1-si-2 comprises a forward sequence IFNGR1-si-2-F as shown in SEQ ID NO.3 and a reverse sequence IFNGR1-si-2-R as shown in SEQ ID NO.4;

[0050] The IFNGR1-si-3 comprises a forward sequence IFNGR1-si-3-F as shown in SEQ ID NO.5 and a reverse sequence IFNGR1-si-3-R as shown in SEQ ID NO.6.

[0051] The present invention designs siRNA that interferes with the expression of IFNGR1 in mesenchymal stem cells. Experiments show that the knockdown efficiency of IFNGR1-si-1, IFNGR1-si-2 and IFNGR1-si-3 for the IFNGR1 gene in mesenchymal stem cells is more than 48%, among which IFNGR1-si-3 is the highest. The quantitative expression of IFNGR1 in mesenchymal stem cells can be achieved by quantitatively transferring siRNA. It can be used to establish the corresponding relationship between the expression level of IFNGR1 in MSC and the inhibitory ability of MSC on the proliferation of immune cells, and is used to evaluate mesenchymal stem cells.

[0052] In some specific embodiments, the 3' ends of the forward sequence and the reverse sequence are respectively connected to 2 additional nucleotides;

[0053] Preferably, the additional nucleotides are deoxythymidine nucleotides.

[0054] According to another aspect of the present invention, there is also provided the use of the above siRNA in any of the following items:

[0055] A1. Use in reducing or inhibiting the expression of IFNGR1 in mammalian cells or in preparing a product for reducing or inhibiting the expression of IFNGR1 in mammalian cells;

[0056] A2. Use in reducing or inhibiting the immunosuppressive ability of mammalian cells or in preparing products for reducing or inhibiting the immunosuppressive ability of mammalian cells;

[0057] A3. Application in evaluating the immunoregulatory capacity of mammalian cells.

[0058] In some specific embodiments, the cells are mesenchymal stem cells.

[0059] In some specific embodiments, the mammal comprises a human.

[0060] According to another aspect of the present invention, a reagent or kit for reducing IFNGR1 is provided, comprising the above-mentioned siRNA.

[0061] According to another aspect of the present invention, a method for down-regulating the immunoregulatory ability of mesenchymal stem cells is provided, comprising transferring the above siRNA into mesenchymal stem cells.

[0062] By transferring siRNA into mesenchymal stem cells to reduce or inhibit the expression of IFNGR1, the purpose of downregulating the immunoregulatory ability of mesenchymal stem cells is achieved. The mesenchymal stem cells with reduced or inhibited IFNGR1 expression prepared by this method can be used in diseases unrelated to MSC immunoregulation, such as tissue repair. The use of MSCs with reduced or inhibited IFNGR1 expression can avoid their immunosuppressive effects. It can be used in the preparation of drugs for treating diseases unrelated to immunoregulation.

[0063] In some specific embodiments, after the mesenchymal stem cells are introduced, the step further includes culturing for at least 24 hours.

[0064] Experiments have shown that the expression of IFNGR1 in mesenchymal stem cells can be quantitatively reduced or inhibited by introducing siRNA of a specific concentration. According to another aspect of the present invention, a mesenchymal stem cell that quantitatively expresses IFNGR1 is provided, comprising the above siRNA.

[0065] According to another aspect of the present invention, a method for evaluating the immunoregulatory ability of mesenchymal stem cells is also provided, comprising detecting the IFNGR1 expression level of the mesenchymal stem cells to be evaluated, substituting the IFNGR1 expression level of the mesenchymal stem cells to be evaluated into a fitting formula to calculate the total lymphocyte proliferation inhibition rate, and evaluating the immunoregulatory ability of the mesenchymal stem cells according to the total lymphocyte proliferation inhibition rate;

[0066] The fitting formula is determined by the IFNGR1 expression levels of several groups of mesenchymal stem cells that quantitatively express IFNGR1 and the total lymphocyte proliferation inhibition rate corresponding to the IFNGR1 expression levels.

[0067] By quantitatively expressing IFNGR1 in mesenchymal stem cells, the corresponding relationship between IFNGR1 expression and total lymphocyte proliferation inhibition rate was established and a fitting formula was determined. The total lymphocyte proliferation inhibition rate can be calculated by detecting the IFNGR1 expression level of the mesenchymal stem cells to be evaluated. Compared with the existing technology, there is no need to co-culture MSCs with PBMCs each time for evaluation, and use a large number of PBMCs.

[0068] In some specific embodiments, the method for constructing the fitting formula comprises the following steps:

[0069] A. preparing several groups of mesenchymal stem cells that quantitatively express IFNGR1, and obtaining the IFNGR1 expression level of each group of mesenchymal stem cells;

[0070] B. Several groups of mesenchymal stem cells quantitatively expressing IFNGR1 were co-cultured with peripheral blood mononuclear cells to obtain the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells;

[0071] C. According to the IFNGR1 expression level of each group of mesenchymal stem cells and the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells, a fitting curve was constructed to obtain a fitting formula;

[0072] In some specific embodiments, the IFNGR1 expression levels of the several groups of mesenchymal stem cells that quantitatively express IFNGR1 are all different. Through several groups of mesenchymal stem cells that quantitatively express IFNGR1 with different IFNGR1 expression levels, several groups of corresponding IFNGR1 expression levels and total lymphocyte proliferation inhibition rates can be obtained for constructing a fitting curve.

[0073] In some specific embodiments, the number of the groups of mesenchymal stem cells quantitatively expressing IFNGR1 is at least 2. The number can be adjusted according to the range of immunoregulatory ability of the mesenchymal stem cells to be evaluated, and can also be set to 3, 4, 5, 6, 7, 8, 9 or 10, or more than 10, to meet the needs of constructing the fitting curve.

[0074] In some specific embodiments, the method for preparing several groups of mesenchymal stem cells that quantitatively express IFNGR1 includes respectively introducing siRNA of different concentrations into mesenchymal stem cells, so as to obtain several groups of mesenchymal stem cells with different IFNGR1 expression levels.

[0075] In some specific embodiments, the different concentrations of siRNA are 0 to 10 pmol / mL of siRNA, and are not zero.

[0076] In some specific embodiments, the culture time of mesenchymal stem cells quantitatively expressing IFNGR1 is 0 to 72 hours, preferably 24 hours.

[0077] In some specific embodiments, the co-culture time with peripheral blood mononuclear cells is 72 to 120 hours, preferably 96 hours.

[0078] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] 1. Cells

[0080] Mesenchymal stem cells (MSC): isolated from the umbilical cord of full-term newborns without congenital diseases (the mothers of the newborns were from the General Hospital of the Chinese People's Liberation Army, without hepatitis, syphilis, AIDS and other infectious diseases, and the mothers and their families gave informed consent for the use of the umbilical cord for experimental research).

[0081] MSC isolation and culture method: take the umbilical cord from the body and sterilize it to obtain the sterilized umbilical cord; then wash the sterilized umbilical cord and cut it into 1-2 cm small segments to obtain umbilical cord small segments; then wash the umbilical cord small segments and cut them into 1-2 mm3 tissue blocks, use fetal bovine serum complete culture medium (90% DMEM / F12 culture medium + 10% FBS), place in 5% CO 2 The isolated umbilical cord tissue blocks were cultured under saturated humidity and 37°C; after the mesenchymal stem cells crawled out, they were subcultured to P4 using a mesenchymal stem cell serum-free medium (500 mL of mesenchymal stem cell serum-free medium base + 5 mL of mesenchymal stem cell serum-free medium additive).

[0082] Peripheral blood mononuclear cells (PBMC): isolated from platelet-rich buffy coat (platelet-rich buffy coat was derived from healthy adults from Tianjin Blood Center), batch number 20240201-02.

[0083] PBMC separation method: slowly add platelet-rich buffy coat to the surface of human lymphocyte separation solution, centrifuge at 300g for 20 minutes to separate the layers. Carefully aspirate the middle buffy coat layer with a pipette, and wash twice with physiological saline at 300g for 5 minutes.

[0084] 2. Reagents

[0085] Opti-Mem (reduced serum medium): purchased from Gibco, product number 51985-034.

[0086] Lipofectamine RNAiMAX: purchased from Invitrogen, catalog number 13778150.

[0087] Mesenchymal stem cell serum-free culture medium base: purchased from Youkang Hengye Biotechnology (Beijing) Co., Ltd., catalog number NC0103.

[0088] Mesenchymal stem cell serum-free culture medium additive: purchased from Youkang Hengye Biotechnology (Beijing) Co., Ltd., product number NC0103.S.

[0089] Control-siRNA: purchased from Beijing Qingke Biotechnology Co., Ltd., its sequence is:

[0090] control-siRNA-F: 5′-UUCUGGGAACACGAGUGCUTT-3′ (SEQ ID NO.7);

[0091] control-siRNA-R: 5′-AGCAUCGUGUUCCCAGAATT-3′ (SEQ ID NO. 8).

[0092] Anti-human CD3 mAb: purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd., with the catalog number GMP-TL101-0500.

[0093] Anti-human CD28 mAb: purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd., with the catalog number GMP-TL102-0500.

[0094] 1640 culture medium: purchased from Gibco, catalog number 11875093.

[0095] DMEM / F12 culture medium: purchased from Sigma-Aldrich, catalog number 01-072-1A.

[0096] Human lymphocyte separation medium: purchased from Beijing Oriental Huahui Biotechnology Co., Ltd., the product number is 25710.

[0097] Sodium chloride injection: purchased from Shijiazhuang Siyao Co., Ltd., the drug approval number is National Medicine Standard H13023201.

[0098] RNA extraction kit: Takara, catalog number 9767.

[0099] Fetal bovine serum: purchased from Ekosai Biotechnology, product number FND500.

[0100] CFSE: purchased from Becton, Dickinson and Company, catalog number 565082.

[0101] IFNGR1 flow cytometry antibody: purchased from Becton, Dickinson and Company, catalog number 558934.

[0102] 3. Instruments

[0103] Fluorescence quantitative PCR instrument: Roche, model LC96.

[0104] Dual fluorescence activity analyzer: Nexcelom, model Auto 2000.

[0105] Flow cytometer: Becton, Dickinson and Company, model: FACS Calibur.

[0106] 4. Primer sequences for detecting IFNGR1 gene

[0107] IFNGR1 forward primer: 5′-AGTGCTTAGCCTGGTATTCATCTG-3′ (SEQ ID NO. 9);

[0108] IFNGR1 reverse primer: 5′-GGCTGGTATGACGTGATGAGTG-3′ (SEQ ID NO. 10).

[0109] Example 1. siRNA interfering with IFNGR1 expression in stem cells

[0110] The siRNA sequences designed by the present invention for interfering with the expression of IFNGR1 are shown in Table 1.

[0111] Table 1 siRNA sequences

[0112]

[0113] 1. Resuscitate mesenchymal stem cells and add 3×10 4 / cm 2 The cells were seeded at a density of 100 μg / mL in a 48-well plate, with three replicate wells set for each siRNA (each siRNA in Table 1), and cultured in Yokohama serum-free complete medium (500 mL of mesenchymal stem cell serum-free medium base + 5 mL of mesenchymal stem cell serum-free medium supplement) at 37°C, 5% CO2 overnight for 16 h.

[0114] 2. After completing step 1, prepare transfection solution A according to the final concentration of 10pmol / mL siRNA per well, and calculate the amount of siRNA and 12.5μL Opti-Mem per well. Prepare transfection solution B according to the amount of 0.75μL Lipofectamine RNAiMAX per well and 12.5μL Opti-Mem per well. Mix well and let stand at room temperature for 20min. After mixing transfection solution A and transfection solution B, let stand at room temperature for 5min. Add dropwise to a 48-well plate and culture at 37℃, 5% CO2 for 24h. At the same time, set up a control group (10pmol / mL control-siRNA) and operate in the same way.

[0115] 3. After completing step 2, replace the culture medium in the 48-well plate with fresh culture medium at 37°C and 5% CO 2 Culture for 24 hours.

[0116] 4. After completing step 3, discard the culture medium in the well plate, add PBS buffer to rinse the cells once, and after drying, add 120 μL RNA extraction lysis buffer to each well to lyse the cells, and mix the lysis buffers in duplicate wells.

[0117] 5. After completing step 4, use an RNA extraction kit to extract total RNA. Use a reverse transcription kit to reverse the RNA and finally obtain cDNA.

[0118] 6. After completing step 5, use cDNA as a template and the primer sequence used to detect the IFNGR1 gene for fluorescence quantitative PCR, and use β-actin mRNA as an internal reference gene to detect the expression of the IFNGR1 gene. Use a QPCR instrument to detect the CT value of the target gene and the internal reference gene, and calculate the relative expression of mRNA according to the following formula: relative expression = 2 -Δct =2 -(目的基因的CT值-内参基因的CT值) , and the knockdown efficiency of siRNA was calculated according to the following formula: Knockdown efficiency = (1-experimental group 2 -Δct / Control group 2 -Δct )×100%, where the experimental group was siRNA interfering with IFNGR1 gene expression, and the control group was control-siRNA.

[0119] The results are shown in Table 2. The results showed that after siRNA was transfected into cells, compared with control-siRNA, IFNGR1-siRNA-1, IFNGR1-siRNA-2 and IFNGR1-siRNA-3 could effectively interfere with the expression of IFNGR1, among which IFNGR1-siRNA-3 had the highest knockdown efficiency (89.84%), and IFNGR1-siRNA-3 was selected as a representative for use in the following examples.

[0120] Table 2 Knockdown efficiency

[0121] Group <![CDATA[Experimental Group 2 -Δct > <![CDATA[Control group 2 -Δct > Knockdown efficiency IFNGR1-si-1 0.001376981 0.002656017 48.16 IFNGR1-si-2 0.000514283 0.002656017 80.64 IFNGR1-si-3 0.000269945 0.002656017 89.84

[0122] Example 2: Effect of IFNGR1-siRNA-3 on the expression of IFNGR1 in mesenchymal stem cells

[0123] 1. Resuscitate mesenchymal stem cells and add 3×10 4 / cm 2 The cells were seeded at a density of 1.50 μg / mL in a 48-well plate and cultured in serum-free complete medium at 37°C with 5% CO. 2 Overnight culture for 16 h.

[0124] 2. After completing step 1, add 2.44×10 -3 pmol / mL siRNA, 9.77×10 - 3 pmol / mL of siRNA, 3.91×10 -2Calculate the amount of siRNA with pmol / mL, 0.156pmol / mL, 0.625pmol / mL, 2.50pmol / mL or 10pmol / mL siRNA and prepare transfection solution A with 12.5μL Opti-Mem per well. Prepare transfection solution B with 0.75μL Lipofectamine RNAiMAX per well and 12.5μL Opti-Mem per well. Mix well and let stand at room temperature for 20 minutes. Mix transfection solution A and transfection solution B and let stand at room temperature for 5 minutes. Add dropwise to a 48-well plate and incubate at 37°C with 5% CO 2 The cells were cultured for 24 h, and a control group (control-siRNA) was set up at the same time. The same operation was performed, and three parallel experiments were set up in each experimental group and control group.

[0125] 3. After completing step 2, replace the culture medium in the 48-well plate with fresh culture medium at 37°C and 5% CO 2 Culture for 48h.

[0126] 4. After completing step 3, tryple digestion and collect MSCs in the well plate, 4×10 5 Cells / 500μL / tube were resuspended in saline, 5μL IFNGR1 flow cytometry antibody was added and incubated at room temperature for 30min. The same operation was performed with isotype control antibody. After incubation, centrifugation was performed at 300g for 5min, and the cells were washed twice with saline. After resuspending, the expression level of IFNGR1 was determined by flow cytometry. The results are shown in Figure 1 As shown in the figure, the value of CD119 represents the expression of IFNGR, and a to h are the control group and the final concentration of siRNA added to each well is 2.44×10 -3 pmol / mL siRNA, 9.77×10 -3 pmol / mL of siRNA, 3.91×10 -2 There were experimental groups with 10 pmol / mL siRNA, 0.156 pmol / mL siRNA, 0.625 pmol / mL siRNA, 2.50 pmol / mL siRNA or 10 pmol / mL siRNA.

[0127] The results are shown in Table 3. The results showed that the expression of IFNGR1 in cells could be reduced by transfection with IFNGR1-si-3, and the level of IFNGR1 expression in MSCs could be adjusted by changing the amount of IFNGR1-si3 used in transfection, and the relationship was inversely proportional.

[0128] Table 3

[0129] siRNA concentration (pmol) IFNGR1 expression (%) 0 (control group) 44.5 <![CDATA[2.44×10 -4 ]]> 30.5 <![CDATA[9.77×10 -3 ]]> 33.1 <![CDATA[3.91×10 -2 ]]> 26 0.156 18.3 0.625 15.6 2.50 11.6 10 8.29

[0130] Example 3: Effect of IFNGR1 siRNA on the ability of MSC to inhibit total lymphocyte proliferation

[0131] 1. Resuscitate mesenchymal stem cells and add 3×10 4 / cm 2 The cells were seeded at a density of 1.54 μg / ml in 48-well plates using serum-free complete medium and cultured at 37°C with 5% CO. 2 Overnight culture for 16 h.

[0132] 2. After completing step 1, add 2.44×10 -3 pmol / mL siRNA, 9.77×10 - 3 pmol / mL of siRNA, 3.91×10 -2 Calculate the amount of siRNA with pmol / mL, 0.156pmol / mL, 0.625pmol / mL, 2.50pmol / mL or 10pmol / mL siRNA and prepare transfection solution A with 12.5μL Opti-Mem per well. Prepare transfection solution B with 0.75μL Lipofectamine RNAiMAX per well and 12.5μL Opti-Mem per well. Mix well and let stand at room temperature for 20 minutes. Mix transfection solution A and transfection solution B and let stand at room temperature for 5 minutes. Add dropwise to a 48-well plate and incubate at 37°C with 5% CO 2 The cells were cultured for 24 hours. A control group (10 pmol / mL control-siRNA) was set up and operated in the same way. Three parallel groups were set up in each experimental group and control group.

[0133] 3. After completing step 2, replace the culture medium in the 48-well plate with fresh culture medium at 37°C and 5% CO 2 Culture for 24 hours.

[0134] 4. After completing step 3, resuscitate PBMC and dilute to 1×10 7 / mL, add CFSE to make the final concentration of 10μM / mL, dye in a 37℃ water bath for 15min, then centrifuge and wash twice, according to 1.2×10 6 / mL density, use 1640 medium, add antiCD3 and antiCD28 antibodies at a final concentration of 500ng / mL, prepare PBMC solution, and set up a negative control group without antiCD3 / CD28. Aspirate the medium in the 48-well plate where MSCs are cultured, add 500μLPBMC, and culture for 96h. At the same time, add 500μL PBMC activated by antiCD3 / CD28 to the 48-well plate without MSC as the positive group, and add PBMC without antiCD3 / CD28 as the negative group.

[0135] 5. After 96 hours, PBMCs were collected from 48-well plates and the proliferation capacity of total lymphocytes was determined by flow cytometry. The generation of total lymphocytes was analyzed by FlowJo. The results are as follows: Figure 2 As shown in the figure, the CFSE subset value is the proportion of progeny cells, a~j are the negative group, positive group, control group, and the total amount of siRNA added to each well is 2.44×10 -3 pmol / mL siRNA, 9.77×10 -3 pmol / mL of siRNA, 3.91×10 -2 The experimental groups were siRNA of 10 pmol / mL, 0.156 pmol / mL, 0.625 pmol / mL, 2.50 pmol / mL or 10 pmol / mL. The daughter cell ratio was obtained, and the average value was taken to calculate the total lymphocyte proliferation inhibition rate.

[0136] Total lymphocyte proliferation inhibition rate = [(progeny cell percentage of positive control group - progeny cell percentage of sample group) / (progeny cell percentage of positive control group - progeny cell percentage of negative control group)] × 100%.

[0137] Table 4

[0138] Final siRNA concentration (pmol / mL) Total lymphocyte proliferation inhibition rate (%) 0 (control group) 40.6 <![CDATA[2.44×10 -4 ]]> 45.55 <![CDATA[9.77×10 -3 ]]> 43.85 <![CDATA[3.91×10 -2 ]]> 44.08 0.156 30.33 0.625 14.87 2.50 10.82 10 11.7

[0139] The results are shown in Table 4. The results showed that IFNGR1-si3 could reduce the level of MSC lymphocyte proliferation ability, and by changing the amount of IFNGR1-si3 used in transfection, the level of MSC lymphocyte proliferation inhibition ability could be changed, and the relationship was inversely proportional.

[0140] Combined with the results of Table 3 in Example 2, Origin was used to perform fitting analysis on the expression of IFNGR1 on the surface of MSC cells and the inhibition rate of total lymphocyte proliferation. Figure 3As shown, the fitting formula is: y = 43.54242 + (11.26812 - 43.54242) / (1 + (x / 17.86594) ^ 15.4775); its R 2 =0.98574, with a very high degree of fit, and the total lymphocyte proliferation inhibition rate can be estimated by the expression level of IFNGR1 in MSCs.

[0141] Example 4 Evaluation of the immunomodulatory ability of mesenchymal stem cells

[0142] In this Example 4, four different batches of MSCs were selected, and the total lymphocyte proliferation inhibition rate of each batch of MSCs was obtained using the following two methods respectively.

[0143] 1. According to the method provided in Example 2, 10 pmol / mL control-siRNA was used to treat each batch of MSCs, and the IFNGR1 expression level of each batch of MSCs was collected, and the total lymphocyte proliferation inhibition rate corresponding to each batch of MSCs was calculated according to the fitting formula provided in Example 3;

[0144] 2. According to the method provided in steps 1 to 5 of Example 3, each batch of MSCs was treated with 10 pmol / mL control-siRNA, and the actual value of the total lymphocyte proliferation inhibition rate of each batch of MSCs was obtained.

[0145] The specific results are shown in Table 5. The total lymphocyte proliferation inhibition rate calculated by the fitting formula using the IFNGR1 expression level was within ±1.8% compared with the actual total lymphocyte proliferation inhibition rate obtained by co-culture, indicating that the method for evaluating the immunoregulatory ability of mesenchymal stem cells provided by the present invention can completely replace the traditional method.

[0146]

[0147]

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A siRNA for reducing IFNGR1, characterized in that comprising at least one of IFNGR1-si-1, IFNGR1-si-2 or IFNGR1-si-3; The IFNGR1-si-1 comprises a forward sequence IFNGR1-si-1-F as shown in SEQ ID NO.1 and a reverse sequence IFNGR1-si-1-R as shown in SEQ ID NO.2; The IFNGR1-si-2 comprises a forward sequence IFNGR1-si-2-F as shown in SEQ ID NO.3 and a reverse sequence IFNGR1-si-2-R as shown in SEQ ID NO.4; The IFNGR1-si-3 comprises a forward sequence IFNGR1-si-3-F as shown in SEQ ID NO.5 and a reverse sequence IFNGR1-si-3-R as shown in SEQ ID NO.

6.

2. The siRNA according to claim 1, characterized in that The 3' ends of the forward sequence and the reverse sequence are respectively connected with 2 additional nucleotides; Preferably, the additional nucleotides are deoxythymidine nucleotides.

3. Use of the siRNA according to claim 1 or 2 in any of the following: A1. Use in reducing or inhibiting the expression of IFNGR1 in mammalian cells or in preparing a product for reducing or inhibiting the expression of IFNGR1 in mammalian cells; A2. Use in reducing or inhibiting the immunosuppressive ability of mammalian cells or in preparing products for reducing or inhibiting the immunosuppressive ability of mammalian cells; A3. Application in evaluating the immunoregulatory capacity of mammalian cells.

4. The use according to claim 3, characterized in that: The cells are mesenchymal stem cells; Preferably, the mammal comprises a human.

5. A reagent or kit for reducing IFNGR1, characterized in that: Comprising the siRNA according to claim 1 or 2.

6. A method for downregulating the immunomodulatory ability of mesenchymal stem cells, characterized in that: The method comprises transferring the siRNA described in claim 1 or 2 into mesenchymal stem cells.

7. The method according to claim 6, characterized in that After the mesenchymal stem cells are transferred, the culture is also continued for at least 24 hours.

8. A mesenchymal stem cell that quantitatively expresses IFNGR1, characterized in that: Containing the siRNA according to claim 1 or 2.

9. A method for evaluating the immunoregulatory ability of mesenchymal stem cells, characterized in that: The method comprises detecting the IFNGR1 expression of the mesenchymal stem cells to be evaluated, substituting the IFNGR1 expression of the mesenchymal stem cells to be evaluated into a fitting formula to calculate the total lymphocyte proliferation inhibition rate, and evaluating the immunoregulatory ability of the mesenchymal stem cells according to the total lymphocyte proliferation inhibition rate; The fitting formula is determined by several groups of IFNGR1 expression levels of mesenchymal stem cells that quantitatively express IFNGR1 as described in claim 8 and the total lymphocyte proliferation inhibition rate corresponding to the IFNGR1 expression levels.

10. The method according to claim 9, characterized in that The method for constructing the fitting formula comprises the following steps: A. preparing several groups of mesenchymal stem cells that quantitatively express IFNGR1, and obtaining the IFNGR1 expression level of each group of mesenchymal stem cells; B. Several groups of mesenchymal stem cells quantitatively expressing IFNGR1 were co-cultured with peripheral blood mononuclear cells to obtain the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells; C. According to the IFNGR1 expression level of each group of mesenchymal stem cells and the total lymphocyte proliferation inhibition rate corresponding to each group of mesenchymal stem cells, a fitting curve was constructed to obtain a fitting formula; Preferably, the IFNGR1 expression levels of the several groups of mesenchymal stem cells that quantitatively express IFNGR1 are all different; Preferably, the number of the several groups of mesenchymal stem cells quantitatively expressing IFNGR1 is at least 2; Preferably, the method for preparing several groups of mesenchymal stem cells quantitatively expressing IFNGR1 comprises introducing different concentrations of siRNA into mesenchymal stem cells respectively; Preferably, the different concentrations of siRNA are 0 to 10 pmol / mL of siRNA, and are not 0; Preferably, the culture time of mesenchymal stem cells quantitatively expressing IFNGR1 is 0 to 72 hours, preferably 24 hours; Preferably, the co-culture time with peripheral blood mononuclear cells is 72 to 120 hours, preferably 96 hours.

Images