Research method of Ver15508 treatment category conversion recombination function impairment

Abstract

The invention discloses a method for researching damage of a Ver15508 treatment category conversion recombination function, and relates to the technical field of immunology. The method specifically comprises the following steps: step 1, preparing experimental materials; step 2, carrying out experiments on effects and influences of VER15508 with different concentrations on CH12F3 cells; 3, carrying out an experiment on the influence of VER15508 with different concentrations on the CH12F3 cell line category conversion function; 4, carrying out an experiment on the influence of VER15508 with different concentrations on AID protein transcribed by the aicda gene of the CH12F3 cell; and 5, carrying out a CD19 + B cell verification experiment. Through experiments of VER15508 with different concentration gradients on CH12F3 cells, it is known that the VER15508 can effectively enhance the category transformation recombination function by promoting AID expression in a B lymphocyte line under the appropriate drug concentration.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a research method for treating impaired class switching recombination function by Ver15508. Background Art

[0002] Class switch recombination (CSR) is an important mechanism of B lymphocytes in the immune response process. It allows B cells to change the type of antibodies they produce according to the needs of the immune response, so that cells that originally produce IgM antibodies can instead produce IgG, IgA and IgE antibodies without changing the specificity of the antibodies. For example, IgG antibodies can pass through the placenta to provide immune protection for newborns; IgA antibodies play an important role in mucosal immunity to prevent the invasion of pathogens. This process is crucial to the diversity and effectiveness of adaptive immune responses.

[0003] AID is a key enzyme in CSR, which is expressed after B cells are stimulated by antigens. AID converts cytosine (C) in DNA into uracil (U) through deamination, triggering the DNA damage and repair process, thereby promoting the occurrence of CSR.

[0004] The CH12F3 cell line is a mouse myeloma cell line developed from a subclone of the IgM CH12.LX lymphoma cell line. It is often used to study the affinity maturation process of B lymphocytes, including somatic hypermutation and class switch recombination. Under appropriate cytokine stimulation, CH12F3 cells can be induced to switch from surface IgM+ cells to surface IgA+ cells with a high switching frequency, making it a useful model for studying the molecular mechanism of class switch recombination.

[0005] VER-155008 is a small molecule inhibitor that is mainly used to inhibit the activity of heat shock protein 70 (Hsp70). Hsp70 is a molecular chaperone that participates in the folding, transport and degradation of proteins and is essential for maintaining the homeostasis of intracellular proteins. VER-155008 affects cell survival and proliferation by inhibiting the function of Hsp70, especially showing significant anti-cancer activity in tumor cells. It also has a significant inhibitory effect on the proliferation of multiple tumor cell lines. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and propose a research method for treating impaired class switching recombination function of Ver15508.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A research method for treating impaired recombination function by class switching of Ver15508, comprising the following steps:

[0009] Step 1: Prepare experimental materials; the experimental materials include CH12F3 cells, organic compound VER155008, and CD19+ B cells;

[0010] Step 2: Experiment on the effects and influence of different concentrations of VER15508 on CH12F3 cells; CH12F3 cells were stained with CFSE fluorescent dye, and then cultured with different concentrations of VER15508, and finally the proportion of living cells and the average fluorescence intensity of CFSE in living cells were analyzed by flow cytometry;

[0011] Step 3: Experiment on the effect of different concentrations of VER15508 on the class switching function of CH12F3 cell line; CH12F3 cells were cultured with different concentrations of VER15508, and then the class switching recombination of CH12F3 cells was induced by CIT, and finally the proportion of IgA+ cells in living cells was analyzed by flow cytometry;

[0012] Step 4: Experiment on the effect of different concentrations of VER15508 on the transcription of AID protein from the aicda gene of CH12F3 cells; CH12F3 cells were cultured with different concentrations of VER15508, and then the expression of AID protein was induced by CIT. A part of the cells were used to extract RNA, and a part of the cells were used to extract cell protein. Finally, RT-qPCR was used to explore the transcription of the AID gene aicda, and Western Blot was used to explore the expression of AID protein;

[0013] Step 5: CD19+ B cell verification experiment; CD19+ B cells were extracted from the spleen of C57BL / 6 mice, and then different concentrations of VER15508, IL4 and LPS were used to induce class conversion recombination from IgM to IgG1 on the surface of B cells. Finally, the proportion of IgG1+ cells in living B220-positive cells was analyzed by flow cytometry.

[0014] Preferably, in step 2, the concentration of CFSE fluorescent dye is 1 μM, the infection condition of CH12F3 cells is incubation at 37° C. in the dark for 10 minutes, and the concentration of VER15508 is classified into 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM.

[0015] Further: in the step three, 10 ng / μLiL4, 1 μg / mLCD40, and 2 ng / mLTGF-β were added to CIT, and the concentrations of VER15508 were classified as 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM.

[0016] As a preferred embodiment of the present invention: the concentration of VER15508 in step 4 is classified into 0 μM, 2.5 μM, and 5 μM.

[0017] Based on the above scheme: In step 5, the spleen of C57BL / 6 mice was ground, washed with 1 mL of PBS containing 0.5% BSA, and 1 mL of red blood cell lysis buffer was added. After standing at room temperature for 5-10 minutes, the cell suspension was filtered through a mesh and centrifuged at 300 g for 5 minutes.

[0018] Preferably: in step 5, the concentrations of VER15508 are classified into 0 μM and 1.5 μM, the concentration of IL4 is 10 ng / μL, and the concentration of LPS is 80 ng / μL.

[0019] The beneficial effects of the present invention are:

[0020] This research method, through experiments on CH12F3 cells with different concentration gradients of VER15508, found that Ver15508, at appropriate drug concentrations, effectively enhanced the class switch recombination function in B lymphocyte lines by promoting AID expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The chemical structure diagram of VER-155008 of the present invention is shown in FIG.

[0022] Figure 2 This is a graph showing the detection results of CFSE fluorescence intensity of CH12F3 cells under different concentration gradients of VER15508 of the present invention;

[0023] Figure 3 This is a statistical diagram showing the effects of different concentration gradients of VER15508 on CH12F3 cell proliferation in the present invention;

[0024] Figure 4 This is a statistical diagram showing the effect of different concentration gradients of VER15508 on the survival rate of CH12F3 cells in the present invention;

[0025] Figure 5 Schematic diagram of the gating strategy for flow cytometry detection of IgA+ cell ratio on the surface of CH12F3 cells in the present invention;

[0026] Figure 6 This is a graph showing the results of detecting the proportion of IgA+ cells produced by CH12F3 cells under different concentration gradients of VER15508 of the present invention;

[0027] Figure 7 This is a statistical diagram of the proportion of IgA+ cells produced by CH12F3 cells under different concentration gradients of VER15508 of the present invention;

[0028] Figure 8 It is a statistical graph of aicda gene transcription in CH12F3 cells after VER15508 treatment of the present invention;

[0029] Fig. 9 This is a schematic diagram of detecting the expression of AID protein in CH12F3 cells after VER15508 treatment of the present invention;

[0030] Fig.10 Schematic diagram of the gating strategy for flow cytometry detection of IgG1+ cell ratio on the surface of primary spleen B cells in the present invention;

[0031] Fig.11 This is a graph showing the detection result of the ratio of IgG1+ cells on the surface of primary spleen B cells after treatment with 1.5 μM VER15508 of the present invention;

[0032] Fig.12 This is a statistical diagram of IgG1+ cells of primary spleen B cells after treatment with 1.5 μM VER15508 of the present invention;

[0033] Fig.13 This is a statistical graph of the IgG1 antibody content in the culture medium of primary spleen B cells before and after treatment with 1.5 μM VER15508 of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0035] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0036] A method for studying the impairment of recombination function in the treatment of Ver15508 class switch, such as Figures 1 to 13 As shown, the specific steps include:

[0037] Step 1: Prepare experimental materials.

[0038] The experimental materials include CH12F3 cells, organic compound VER155008, and CD19+ B cells.

[0039] The CH12F3 cell line is a mouse myeloma cell line developed from a subclone of the IgM CH12.LX lymphoma cell line. It is often used to study the affinity maturation process of B lymphocytes, including somatic hypermutation and class switch recombination. Under appropriate cytokine stimulation, CH12F3 cells can be induced to switch from surface IgM+ cells to surface IgA+ cells with a high conversion frequency, making it a useful model for studying the molecular mechanism of class switch recombination.

[0040] VER15508 compound was purchased from (CAS No.: 1134156-31-2), its IC50 for Hsp70, Hsc70 and Grp7 are 0.5μM, 2.6μM and 2.6μM respectively, and its KD for Hsp70 is 0.3μM. Its chemical structure is as follows Figure 1 shown.

[0041] CD19+ B cells The CD19 magnetic beads antibody of Spleen CD19 MicroBead Kit, mouse (Miltenyi Biotech, 130-129-973) was sorted from the spleen of C57BL / 6 mice.

[0042] Step 2: Experiment on the effects and impacts of different concentrations of VER15508 on CH12F3 cells.

[0043] 1. Laying the boards

[0044] (1) Take 1 million CH12F3 cells, wash the CH12F3 cells twice with 1 mL PBS to remove the serum in the cell culture medium, and then count the cells.

[0045] (2) After counting, according to the density of 500,000 cells in 100 μL PBS, CFSE fluorescent dye (purchased from Biolegend) was added at a final concentration of 1 μM and incubated at 37°C in the dark for 10 minutes.

[0046] (3) Add 1 mL of PMRI1640 medium containing 10% FBS to the stained cells to terminate the incubation. Centrifuge at 300 g for 5 minutes and discard the supernatant.

[0047] (4) After repeated washing with PMRI1640, the cell pellet was resuspended in PMRI1640 medium and plated in a 96-well plate at a number of 10,000 cells per well.

[0048] (5) VER15508 compounds at concentrations of 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM were added, respectively, and the cells were placed in a cell culture incubator for 48 h before being taken out.

[0049] 2. Streaming Analysis

[0050] (1) After discarding the culture medium from the cells, wash them once with PBS.

[0051] (2) Resuspend the cell pellet in PBS containing 100 μg / ml PI (propidium iodide) to distinguish between live and dead cells.

[0052] (3) The cells were detected on a flow cytometer (Sony SA3800) within 30 minutes after the addition of PI dye.

[0053] CFSE (Carboxyfluorescein Succinimidyl Ester) is a fluorescent dye that can penetrate cell membranes and covalently bind to intracellular proteins in living cells. After hydrolysis, CFSE releases green fluorescence, and its fluorescence intensity decreases step by step as cell division increases. It is often used to detect cell proliferation.

[0054] The excitation wavelength of CFSE is 494nm, the emission wavelength is 521nm, the excitation wavelength of PI is 488nm, and the emission wavelength is greater than 630nm, producing red fluorescence.

[0055] The experimental results are as follows Figures 2 to 4 As shown, as the concentration of VER15508 added increases, the inhibitory effect on cell proliferation becomes more obvious, and the number of surviving cells decreases; when the concentration of VER15508 is below 2.5 μM, the effect on cell proliferation and survival is small.

[0056] Step 3: Experiment on the effect of different concentrations of VER15508 on the class switching function of CH12F3 cell line.

[0057] 1. Paving

[0058] (1) Cell counting: 10,000 CH12F3 cells were plated in each well of a 96-well plate.

[0059] (2) VER15508 compound was added at concentrations of 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM, respectively.

[0060] (3) CIT containing 10 ng / μLiL4, 1 μg / mL LCD40, and 2 ng / mL TGF-β was added to induce class switch recombination in CH12F3 cells, and the cells were placed in a cell culture incubator for 48 h before being removed.

[0061] 2. Streaming Analysis

[0062] (1) After discarding the culture medium from the cells, wash them once with PBS.

[0063] (2) The cells were stained with 0.5 μg / ml PE-anti Mouse IgA antibody (Invitrogen) on ice in the dark for 45-60 min and washed once with PBS.

[0064] (3) Resuspend the cell pellet in PBS containing 100 μg / ml PI (propidium iodide) to distinguish between live and dead cells.

[0065] (4) After adding PI dye, the cells were detected on a flow cytometer (Sony SA3800) within 30 minutes.

[0066] Figure 5 This is the gating strategy for flow analysis. The results are as follows Figure 6 and Figure 7 As shown, when VER15508 was 2.5 μM, the proportion of IgA+ cells produced by CH12F3 increased, suggesting that its class switch recombination function was enhanced; at concentrations above 2.5 μM, as the concentration increased, the proportion of IgA+ cells produced gradually decreased. Since cell proliferation can affect the rate of class switch recombination, it is considered that the class switch recombination function was impaired due to the inhibition of cell proliferation by VER15508 at high concentrations.

[0067] Step 4: Experiment on the effect of different concentrations of VER15508 on the transcription of AID protein from the aicda gene of CH12F3 cells.

[0068] planking

[0069] (1) Cell counting: 500,000 CH12F3 cells were plated in each well of a 6-well plate.

[0070] (2) VER15508 compound was added at concentrations of 0 μM, 2.5 μM, and 5 μM, respectively.

[0071] (3) CIT was added to induce AID protein expression and incubated in a cell culture incubator for 48 h.

[0072] (4) Collect samples, use a portion of the cells for RNA extraction, and use a portion of the cells for protein extraction.

[0073] Detection

[0074] The transcription of the AID encoding gene aicda was investigated by RT-qPCR, and the expression of AID protein was investigated by Western Blot.

[0075] The experimental results are as follows Figure 8 and Fig. 9As shown, the aicda gene transcription and AID protein expression of CH12F3 cells were increased after treatment with VER15508 at concentrations of 2.5μM and 5μM; this is a possible mechanism by which VER15508 promotes the class switching and recombination function of CH12F3 cells at low concentrations.

[0076] Step 5: CD19+ B cell verification experiment.

[0077] 1. Extraction of primary splenic B cells

[0078] (1) The spleen of a C57BL / 6 mouse was removed, ground, and then washed with 1 mL of PBS containing 0.5% BSA.

[0079] (2) Add 1 mL of red blood cell lysis buffer and leave at room temperature for 5-10 minutes.

[0080] (3) Filter the cell suspension through a mesh and centrifuge at 300 g for 5 min.

[0081] (4) Use CD19+ B cells in the spleen were sorted out using the CD19 magnetic bead antibody of Spleen CD19 MicroBead Kit, mouse (Miltenyi Biotech, 130-129-973).

[0082] 2. Laying the boards

[0083] (1) Count the sorted B cells and plate them into a 96-well plate at a rate of 10,000 cells per well.

[0084] (2) 0 μM and 1.5 μM VER15508 were added respectively.

[0085] (3) Add 10 ng / μL IL4 and 80 ng / μL LPS to each well to induce class switching recombination from IgM to IgG1 on the surface of B cells.

[0086] (4) Place in a cell culture incubator for 5 days.

[0087] 3. Flow cytometry

[0088] (1) After collecting the cell culture medium, wash once with PBS.

[0089] (2) The cells were stained with 0.5 μg / ml PE anti-Mouse IgG1 antibody (Biolegend) and APC anti-MouseCD45R / B220 antibody (Biolegend) on ice in the dark for 45-60 min and washed once with PBS.

[0090] (3) Resuspend the cell pellet in PBS containing 100 μg / ml PI (propidium iodide) to distinguish between live and dead cells.

[0091] (4) After adding PI dye, the cells were detected on a flow cytometer (Sony SA3800) within 30 minutes.

[0092] The gating strategy for streaming analysis is as follows Fig.10 The specific results are shown in Fig.11 and Fig.12 As shown, when 1.5 μM VER15508 was added, the proportion of IgG1+ cells converted by primary splenic B cells increased, suggesting that their class switching recombination function was enhanced; this was consistent with the results of the CH12F3 cell line.

[0093] The cell culture medium was collected during flow cytometry sampling, and the IgG1 antibody content in the cell culture medium was detected by ELISA. The SBA Clonotyping System-HRP kit (Southern Biotech, 5300-05) was used for detection. It was found that the absorbance of the sample of the primary spleen B cell culture medium treated with 1.5 μM VER15508 increased significantly, indicating that the IgG1 antibody content in the culture medium increased, which was consistent with the results of flow cytometry analysis. The results are as follows: Fig.13 shown.

[0094] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A research method for treating impaired class switching recombination function by Ver15508, characterized in that: The specific steps include: Step 1: Prepare experimental materials; the experimental materials include CH12F3 cells, organic compound VER155008, and CD19+ B cells; Step 2: Experiment on the effects and influence of different concentrations of VER15508 on CH12F3 cells; CH12F3 cells were stained with CFSE fluorescent dye, and then cultured with different concentrations of VER15508, and finally the proportion of living cells and the average fluorescence intensity of CFSE in living cells were analyzed by flow cytometry; Step 3: Experiment on the effect of different concentrations of VER15508 on the class switching function of CH12F3 cell line; CH12F3 cells were cultured with different concentrations of VER15508, and then the class switching recombination of CH12F3 cells was induced by CIT, and finally the proportion of IgA+ cells expressing in living cells was analyzed by flow cytometry; Step 4: Experiment on the effect of different concentrations of VER15508 on the transcription of AID protein from the aicda gene of CH12F3 cells; CH12F3 cells were cultured with different concentrations of VER15508, and then the expression of AID protein was induced by CIT. A part of the cells were used to extract RNA, and a part of the cells were used to extract cell protein. Finally, RT-qPCR was used to explore the transcription of the AID gene aicda, and Western Blot was used to explore the expression of AID protein; Step 5: CD19+ B cell verification experiment; CD19+ B cells were extracted from the spleen of C57BL / 6 mice, and then different concentrations of VER15508, IL4 and LPS were used to induce class conversion recombination from IgM to IgG1 on the surface of B cells. Finally, the proportion of IgG1+ cells in living B220-positive cells was analyzed by flow cytometry.

2. A research method for treating impaired class switching recombination function by Ver15508 according to claim 1, characterized in that: In the step 2, the concentration of CFSE fluorescent dye is 1 μM, the infection condition of CH12F3 cells is incubation at 37° C. in the dark for 10 minutes, and the concentration of VER15508 is classified into 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM.

3. A research method for treating impaired class switching recombination function by Ver15508 according to claim 1, characterized in that: In the step three, 10 ng / μLiL4, 1 μg / mLCD40, and 2 ng / mLTGF-β were added to CIT, and the concentrations of VER15508 were classified as 0 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM.

4. A research method for treating impaired class switching recombination function by Ver15508 according to claim 1, characterized in that: The concentrations of VER15508 in step 4 are classified into 0 μM, 2.5 μM, and 5 μM.

5. A research method for treating impaired class switching recombination function by Ver15508 according to claim 1, characterized in that: In the step 5, the spleen of the C57BL / 6 mouse is ground and washed with 1 mL of PBS containing 0.5% BSA, and then 1 mL of red blood cell lysis buffer is added. After standing at room temperature for 5-10 minutes, the cell suspension is filtered through a mesh and centrifuged at 300 g for 5 minutes.

6. A research method for treating impaired class switching recombination function by Ver15508 according to claim 1, characterized in that: In the step 5, the concentrations of VER15508 were classified into 0 μM and 1.5 μM, the concentration of IL4 was 10 ng / μL, and the concentration of LPS was 80 ng / μL.

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