CN114854687B - Application of lysophosphatidylcholine in promoting Tfh cells to secrete IL-21 and B cell proliferation - Google Patents
Application of lysophosphatidylcholine in promoting Tfh cells to secrete IL-21 and B cell proliferation Download PDFInfo
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- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 title abstract description 45
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Abstract
The invention discloses an application of lysophosphatidylcholine in promoting Tfh cells to secrete IL-21 and B cell proliferation, wherein the lysophosphatidylcholine is lysoPC 18:0. According to the invention, cell experiments and animal experiments show that LysoPC 18:0 has the effect of obviously promoting the Tfh cells to secrete IL-21 and B cells to proliferate, and can be used for preparing secretion promoters for secreting IL-21 by the Tfh cells in vitro or in vivo and reagents for promoting B cells to proliferate; the detection of the LysoPC 18:0 content instead reflects the function of the Tfh to secrete IL-21, can be used for diagnosing autoimmune diseases related to the Tfh cells, such as lupus erythematosus, scleroderma, sjogren syndrome, vasculitis and the like, and provides a foundation for researching the action mechanism of the Tfh cells secreting the IL-21 and establishing an animal model of the Tfh cells highly secreting the IL-21.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to an application of lysophosphatidylcholine in promoting Tfh cells to secrete IL-21 and B cell proliferation, in particular to an application of lysophosphatidylcholine 18:0 (lysoPC 18:0) in promoting Tfh cells to secrete IL-21 or promoting B cell proliferation.
Background
Follicular helper T (Tfh) cells are a subset of CD4T cells that are predominantly found in germinal centers, the primary CD4T cells that maintain and promote germinal center B cells to proliferate and differentiate into plasma cells and memory B cells. The salient features of Tfh are the expression of molecules such as CD4, CXCR5, PD-1, IL-21, where IL-21 is the most important cytokine for Tfh cells to function, but Tfh cells do not always secrete IL-21.IL-21 is the strongest cytokine promoting plasma cell differentiation and antibody affinity maturation, and is also a cytokine essential for germinal center B cell differentiation. Studies have shown that Tfh cells are elevated in number and percentage in systemic lupus erythematosus patients and are positively correlated with antibody titer, lupus disease activity and circulating plasmablasts, and have also been reported to be involved in the pathogenesis of other autoimmune diseases such as dry rheumatoid arthritis, scleroderma, sjogren's syndrome, vasculitis, and the like. However, the number of Tfh cells in human body is very small, and more peripheral blood is needed to detect the secretion function of Tfh cells IL-21 in human body, so that it is more difficult to separate enough Tfh cells for in vitro research. Therefore, it is particularly important to find new indexes reflecting functional Tfh cells and to establish a Tfh cell expansion system in vitro.
Lysophosphatidylcholine (LysoPC), which is an inflammatory lipid molecule with immune activity, is one of the main components of oxidized low-density lipoprotein, is mainly produced by hydrolyzing phosphatidylcholine with phospholipase A2, and can induce migration of lymphocytes and macrophages and increase secretion of inflammatory cytokines. Studies show that LysoPC can promote adipocytes to secrete IL-1 beta, IL-6 and TNF-alpha, and promote lymphocytes to secrete IFN-gamma and TNF-alpha. There is no report on the effect of LysoPC 18:0 on promoting Tfh cells to secrete IL-21 and the effect on promoting B cell proliferation.
Disclosure of Invention
A first object of the present invention is to provide the use of LysoPC 18:0 for the preparation of a secretion promoter for IL-21 secretion by Tfh cells in vitro or in vivo.
Preferably, the in vitro cell intervention concentration of the LysoPC 18:0 is 5-30 mu M, and the in vivo cell intervention concentration is 5-50mg/Kg.
A second object of the present invention is to provide the use of LysoPC 18:0 for the preparation of an agent for promoting B cell proliferation in vitro or in vivo.
Preferably, the in vitro cell intervention concentration of the LysoPC 18:0 is 5-30 mu M, and the in vivo cell intervention concentration is 5-50mg/Kg.
A third object of the present invention is to provide the use of LysoPC 18:0 in the preparation of a reagent for diagnosing autoimmune diseases associated with Tfh cells
Preferably, the autoimmune disease associated with Tfh cells includes lupus erythematosus, scleroderma, sjogren's syndrome, and vasculitis.
Preferably, the agent is used as an indicator for determining the activity of the autoimmune disease associated with Tfh cells by detecting the level of LysoPC 18:0 in vitro.
The invention discovers that the LysoPC 18:0 can obviously promote Tfh cells to secrete IL-21 and can promote B cell proliferation, and the effect is achieved in vivo or in vitro, so that the LysoPC 18:0 can be used for preparing secretion promoters for promoting Tfh cells to secrete IL-21 in vitro or in vivo or reagents for promoting B cell proliferation in vitro or in vivo. In addition, the content of the LysoPC 18:0 is higher than that of IL-21, the Tfh cells are sensitive and easy to detect, the high level of the LysoPC 18:0 can reflect the existence of the Tfh cells which secrete IL-21, the detection of the content of the LysoPC 18:0 can be used for reflecting the function of the Tfh cells to secrete IL-21 instead, the detection of the content of the LysoPC 18:0 can be used for diagnosing autoimmune diseases related to the Tfh cells, such as lupus erythematosus, scleroderma, sjogren syndrome, vasculitis and the like, and the detection of the mechanism of action of the Tfh cells which secrete IL-21 and the establishment of an animal model of the Tfh cells which secrete IL-21 in a high way are provided for the subsequent study.
Drawings
FIG. 1 is a flow cytometry test of the effect of LysoPC 18:0 on IL-21 secretion function of Tfh cells induced to differentiate in vitro in the examples, A: flow cytometry results showed that LysoPC 18:0 promoted IL-21 secretion by Tfh cells; b: a statistical plot of the results, comparing the averages of two independent samples using t-test, with x representing P < 0.01.
FIG. 2 is a graph showing the effect of flow-through assay of LysoPC 18:0 on IL-21 secretion by Tfh cells in vivo, A: flow cytometry results showed that LysoPC 18:0 promotes IL-21 secretion by CD4T cells in vivo; b: a results statistical graph, the comparison between the averages of two independent samples adopts t test, which represents P < 0.05 and P < 0.01.
FIG. 3 is a graph showing the effect of flow cytometry on B cell proliferation in vivo of LysoPC 18:0, A: flow cytometry results showed that LysoPC 18:0 promoted B cells in vivo (CD 19 + ) Proliferation; b: a statistical plot of the results, comparing the averages of two independent samples using t-test, with x representing P < 0.01.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
LysoPC 18:0, 1-octadecanoyl-sn-glycero-3-phosphorylcholine, formula: c (C) 26 H 54 NO 7 P has the following structural formula:
the purified LysoPC 18:0 from Sigma was purchased and the commercial LysoPC 18:0 was dissolved in 100mM stock solution using 1% ethanol-distilled water.
Example 1
In the embodiment, experiments on human peripheral blood cells prove that the secretion function of LysoPC 18:0 on Tfh cells IL-21 induced to differentiate in vitro is as follows: human peripheral blood is taken, peripheral mononuclear cells (PBMC) are separated by using a gradient density centrifugation method, then easy Sep Human CD4T Cell Iso Kit of Stemcell company is used for enriching CD4T cells, a storage solution of LysopC 18:0 is added in the Tfh in-vitro induced differentiation process, and then the function of secreting IL-21 by the Tfh cells is measured through a flow method, and the specific steps are as follows:
(1) Gradient Density centrifugation (PBMC) separation of peripheral mononuclear cells
(1) Preparing a commercial LysoPC 18:0 stock solution: 1% ethanol-distilled water was used to dissolve 100mM stock solution.
(2) Peripheral blood PBMCs were isolated by gradient density centrifugation: 3-4mL of lymphocyte separation liquid is added into a new empty 15mL centrifuge tube, and 3 times volume of physiological saline is added into 6mL of anticoagulated peripheral blood, and the mixture is uniformly mixed. Then, the peripheral blood is gently added into a centrifuge tube containing lymphocyte separation liquid, the lymphocyte separation liquid layer is not disturbed, then the slow-rise and slow-fall are arranged, the rotating speed is 3500, and the centrifugation is carried out for 25 minutes.
(3) Delamination was seen after centrifugation: the lower layer of red blood cells and granulocytes, the middle layer of lymphocyte separating liquid and mononuclear cells, the upper layer of physiological saline, carefully sucking the middle mononuclear cell layer, adding into a new centrifuge tube, adding 10mL of physiological saline, and uniformly mixing.
(4) And (3) centrifuging: after centrifugation at 500g for 5 minutes, the supernatant was discarded.
(5) Washing for 2 times: 10mL of physiological saline is added, 500g of the mixture is centrifuged for 5 minutes after being blown and evenly mixed, the supernatant is discarded, and the rest cell mass, namely PBMC, is repeated for subsequent experiments.
(2) Isolation of human peripheral blood CD4T cells
Isolation of Human peripheral blood CD4 using Human CD4T Cell Isolation Kit from Stemcell Co + T cells, in particular, are as follows:
(1) resuspension of cells: adding easy Sep Buffer resuspended cells to PBMC cell pellet, and adjusting cell concentration to 5×10 ^7 Each mL and transferring the cell suspension to a sterile flow tube.
(2) Antibody incubation: isolation Cocktail was added to the cell suspension at a rate of 50. Mu.L/mL, gently mixed, and incubated at room temperature for 5 minutes.
(3) Adding sorting magnetic beads: after shaking by Rapid sphere vortex for 30 seconds, the cell suspension was added at a ratio of 50. Mu.L/mL, and easy Sep Buffer was added to 2.5mL, and the mixture was gently mixed 2-3 times with a pipette and placed into a magnetic separation rack.
(4) Incubation: incubate for 3 minutes at room temperature.
(5) Sorting: the magnet holder was lifted and the liquid in the flow tube was poured into a new 15mL centrifuge tube. And cell counting was performed.
(6) Cell culture: after centrifugation at 400g for 5 min, the supernatant was decanted and the cell broth was added with a cell resuspension density of 5X 10) 5 And each mL.
(3) In vitro induced differentiation of human Tfh cells
The cells were divided into 2 groups: control group and LysoPC 18:0 group, lysoPC 18:0 group added with 10 mu M final concentration of LysoPC 18:0, control group added with equal amount of 1% ethanol-distilled water, then both groups of cells added with cytokines, induced to differentiate to Tfh cells, induced to differentiate for 3 days, cell changing and 1:2 or 1:3 pore separation treatment, after 5 days, cells were collected for subsequent experiments, and the experiment was repeated 4 times.
The components for inducing the differentiation of the Tfh cells are as follows: culture solution: immunoCurt TM XF T Cell Expansion Medium (from Stemcell, # 10981), beta-mercaptoethanol: 50 mu M, immunoCurt TM Human CD3/CD28/CD 2T Cell Activator (from Stemcell Co., # 10970): 20 μL/mL, activinnA: 50ng/mL, IL-12:5ng/mL, TGF:5ng/mL, IL-23:10ng/mL. Cytokines were all purchased from PeproTech corporation.
(4) Flow cytometry detection of Tfh secreted IL-21 function
A cell stimulator cocktail (available from Invitrogen Co., # 00-4970-03) was added to the culture well and the mixture was stimulated in a cell incubator for 5 hours. The stimulated cells were collected in a flow tube and washed once with 2mL of flow stain, centrifuged at 400g for 5 min, and the supernatant discarded, as follows:
(1) antibody incubation: 0.5 mu L Fixable Viability Dye eFlour 450,2 mu L of anti-human CD4-FITC, anti-human CXCR5-APC and anti-human PD-1-PE flow antibody are added to 100 mu L of flow staining solution to resuspend cell pellet, and the cell pellet is incubated for 30 minutes at normal temperature and in a dark place.
(2) Washing: cells were resuspended by adding 2mL of flow-through staining solution, and after centrifugation at 400g for 5 minutes, the supernatant was discarded.
(3) Fixing: 200. Mu.L of IC Fixation Buffer (from Invitrogen, # 00-8222-49) was added to the flow tube, and the tube was fixed at room temperature for 20 minutes in a dark place.
(4) Rupture of membranes: 3mL of 1X Permeabilization Buffer (available from Invitrogen, inc. # 00-8333-56) was added to the flow tube, 400g was centrifuged for 5 minutes, the supernatant was discarded, and the wash was repeated once.
(5) Antibody incubation: mu.L of anti-human IL-21-percp-cy5 flow antibody was added to 100. Mu.L of 1X Permeabilization Buffer resuspended cell pellet and incubated at room temperature for 30 min in the absence of light. 3mL of 1X Permeabilization Buffer was added to the flow tube, centrifuged at 400g for 5 minutes, the supernatant was discarded, and washing was repeated once.
(6) And (3) resuspension detection: cell pellet was resuspended by adding 500 μl of flow stain and detected using flow cytometer BD Aria III.
The results are shown in FIG. 1, showing that LysoPC 18:0 has the effect of promoting Tfh cells in vitro (CD 4 + CXCR5 + PD-1 + ) Secretion of IL-21.
Example 2
In the embodiment, the promotion function of LysoPC 18:0 on the proliferation of in vivo Tfh cells secreting IL-21 and B cells is verified by intraperitoneal injection of the LysoPC 18:0 into mice or a physiological saline animal experiment, and the process is as follows: the mice are intraperitoneally injected with LysoPC 18:0 or physiological saline, and peripheral blood flow cytometry is collected after 12 hours and 24 hours to detect the function of CD4T cells for secreting IL-21 and the percentage of B cells, and the specific steps are as follows:
10 healthy adult C57/BL6 mice with the age of 8 weeks are selected and randomly divided into 2 groups, 5 groups are respectively injected with 50mg/Kg of LysoPC 18:0 200 mu L or 200 mu L of physiological saline intraperitoneally, each group of mice is euthanized for 12 hours and 24 hours, peripheral anticoagulants of the mice are collected for subsequent experiments, and the experiments are repeated for 3 times.
The IL-21 secretion function and the B cell quantity of Tfh cells in the peripheral blood of the mice are detected by adopting a flow cytometry, and the method is concretely as follows:
(1) split red: 5mL of the split red solution was added and incubated at room temperature for 10mL.
(2) Washing: physiological saline 20mL was added and 400g centrifuged for 5 minutes and the mixture was washed 2 more times.
(3) Stimulation: resuspension of cells 1.5X10A with RPMI1640 cell culture 6 The cell stimulator cocktail was stimulated in a cell incubator for 5 hours at each mL.
(4) Flow-through dyeing: cells were collected in flow tubes and washed once with 2mL of flow staining solution, then flow antibody was added, 0.5. Mu. L Fixable Viability Dye eFlour 450,2. Mu.L of anti-mouse CD4-FITC, anti-mouse CXCR5-APC, anti-mouse PD-1-PE, anti-mouse CD19-percp-cy5 flow antibody was resuspended in 100. Mu.L of flow staining solution for cell precipitation and incubated at room temperature for 30 minutes in the absence of light.
(5) Washing: cells were resuspended by adding 2mL of flow-through staining solution, and after centrifugation at 400g for 5 minutes, the supernatant was discarded.
(6) Fixing: 200. Mu.L of IC Fixation Buffer was added to the flow tube and the tube was fixed at room temperature for 20 minutes in the absence of light.
(7) Rupture of membranes: 3mL of 1X Permeabilization Buffer was added to the flow tube, and 400g was centrifuged for 5 minutes, and the supernatant was discarded. The wash was repeated once.
(8) Antibody incubation: mu.L of anti-mouse IL-21-PE flow antibody was added to 100. Mu.L of 1X Permeabilization Buffer to resuspend the cell pellet and incubated at room temperature for 30 minutes in the absence of light.
3mL of 1X Permeabilization Buffer was added to the flow tube, and 400g was centrifuged for 5 minutes, and the supernatant was discarded. The wash was repeated once, and the cell pellet was resuspended in 500 μl of flow stain and detected using a flow cytometer BD Aria III.
The results are shown in FIGS. 2 and 3, which show that LysoPC 18:0 promotes IL-21 secretion by Tfh cells in vivo (FIG. 2), while LysoPC 18:0 promotes B cell secretion in vivo (CD 19 + ) Proliferation (FIG. 3).
In conclusion, the in vitro cell intervention concentration of the LysoPC 18:0 is 5-30 mu M, and the in vivo intervention concentration is 5-50mg/Kg, so that the secretion of IL-21 by Tfh cells can be obviously promoted, and meanwhile, the proliferation of B cells can be obviously promoted, and the effect is achieved in vivo or in vitro. In addition, the content of the LysoPC 18:0 is higher than that of IL-21, is sensitive and easy to detect than Tfh, can be detected by a conventional mode known to relevant technicians such as high performance liquid chromatography-tandem mass spectrometry, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, high-throughput electrospray tandem mass spectrometry and the like, and can be used for replacing and reflecting the IL-21 secretion function of Tfh by detecting the content of the LysoPC 18:0 and diagnosing autoimmune diseases related to Tfh cells such as lupus erythematosus, scleroderma, sjogren syndrome, vasculitis and the like.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (2)
- Use of lysopc 18:0 for the preparation of a secretion promoter for secretion of IL-21 by Tfh cells in vitro.
- 2. The use according to claim 1, wherein the in vitro cell intervention concentration of LysoPC 18:0 is 5-30 μm.
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| WO2021142835A1 (en) * | 2020-01-19 | 2021-07-22 | 北京卡替医疗技术有限公司 | Strengthened receptor for improving immune cell function |
| CN113811602A (en) * | 2019-02-11 | 2021-12-17 | 北京卡替医疗技术有限公司 | Adjuvant capable of boosting immune cells to expand in vivo |
| CN114002421A (en) * | 2021-12-30 | 2022-02-01 | 佛山市第三人民医院(佛山市精神卫生中心) | Application of exosome metabolite as bipolar affective disorder marker |
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| CN113811602A (en) * | 2019-02-11 | 2021-12-17 | 北京卡替医疗技术有限公司 | Adjuvant capable of boosting immune cells to expand in vivo |
| WO2021142835A1 (en) * | 2020-01-19 | 2021-07-22 | 北京卡替医疗技术有限公司 | Strengthened receptor for improving immune cell function |
| CN114002421A (en) * | 2021-12-30 | 2022-02-01 | 佛山市第三人民医院(佛山市精神卫生中心) | Application of exosome metabolite as bipolar affective disorder marker |
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