CN103860574B - Quercitroside is preparing the application in human gamma delta t cells multiplication agent - Google Patents
Quercitroside is preparing the application in human gamma delta t cells multiplication agent Download PDFInfo
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- CN103860574B CN103860574B CN201410106885.3A CN201410106885A CN103860574B CN 103860574 B CN103860574 B CN 103860574B CN 201410106885 A CN201410106885 A CN 201410106885A CN 103860574 B CN103860574 B CN 103860574B
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Abstract
The invention discloses preparing the application in human gamma delta t cells multiplication agent, relating to medical art.Quercitroside significantly can promote the propagation of human gamma delta t cells, and can promote the expression of its perforin and granzyme B, has certain dose dependent; Gamma delta T cells after Quercitroside process, can promote the expression of activity form p-ERK1/2 and p-Akt, and can promote the expression of anti-apoptotic proteins Bcl-2, has certain dose dependent.Present invention finds the medical usage that Quercitroside is new, can be immunotherapy of tumors medicament research and development and laboratory reference is provided.
Description
Technical field
The present invention relates to medical art, be specifically related to Quercitroside and preparing the application in human gamma delta t cells multiplication agent.
Background technology
In recent years, the Biotherapeutics of tumor has become the focus of therapeutic field of tumor research.Human gamma delta t cells is mainly distributed in people's mucosa and epithelial tissue, there is stronger antitumor action, main with MHC (majorhistocompatibilitycomplex, MHC) non-limiting way identification tumor, by approach killing tumor cells such as granzyme B (granzymeB) and perforins (perforin), and with other inherent immunity cell and adaptive immunity cell interaction, auxiliary play antitumor action.Human gamma delta t cells has become the new candidate effector lymphocyte of tumor patient adoptive immunotherapy one.As can be seen here, effectively increase human peripheral blood cell, and to meet the needs of the research of gamma delta T cells biological function and clinical adoptive immunotherapy, the raising gamma delta T rate of increase and function are very important and necessary.
Herba Polygoni Capitati (PolygonumcapitatumBuch.-Ham.exD.Don) is Polygonaceae (Polygonaceae) Polygonum (Polygonum) plant, perennial herb, for the distinctive Miao Ethnomedicine in Guizhou, all herbal medicine, have clearing away heat-damp and promoting diuresis, removing toxic substances pain relieving and blood dissipating blood stasis, inducing diuresis for treating stranguria syndrome effect Chinese Plants will editorial board of the Chinese Academy of Sciences. Chinese Plants will, 25th volume the 1st fascicle. Beijing: Science Press, 1998:57-58. Nanjing University of Traditional Chinese Medicine, Chinese medicine voluminous dictionary, 2nd edition. Shanghai: Shanghai science tech publishing house, 2006:850-850 ］.Bibliographical information, Herba Polygoni Capitati main chemical compositions volatile oil, fat-soluble, lignanoids, phenolic acids, flavone compound etc., wherein, Quercitroside is its Main Flavonoids compounds ［ Zhao Xinchao, fogbow, Wang Yuanshu, studies etc. Herba Polygoni Capitati total flavone extracting process. time precious traditional Chinese medical science traditional Chinese medicines, 2009,20 (11): 2815-2816 ］.
Quercitroside (quercitroside, quercitrin), i.e. Quercetin-3-O-α-L-rhamnopyranose (5,7,3 ', 4 '-kaempferol-3-O-α-L-rhamnopyranosyloxyhy glucosides); Molecular formula: C21H20O11; Molecular weight: 448.3769; Chemical structural formula is as follows.
Quercitroside has the various biological such as antioxidation, scavenging free radicals, antitumor, antidepressant, antiviral, the liver protecting, protection cardiovascular because of it active; be subject to increasing concern ［ Zhu Xuexin; Jiang Fusheng; Ding Zhishan. the bioactive progress of Quercitroside. Serpentis will; 2012,24 (1): 47-49 ］.And Quercitroside particularly has no pertinent literature report to the impact of human gamma delta t cells to immunocyte.
Summary of the invention
The invention provides Quercitroside and prepare the application in human gamma delta t cells multiplication agent.
Technical scheme of the present invention is: be separated mononuclearcell by healthy human peripheral blood, adds inducing culture in the RPMI-1640 complete medium containing isopentenylpyrophosphate and recombinant human interleukin--2 and obtains gamma delta T cells.After using the Quercitroside effect gamma delta T cells 48h of variable concentrations, measure variable concentrations Quercitroside group gamma delta T cells multiplication capacity by CCK-8 method; Flow cytometry is adopted respectively to organize the expression of gamma delta T cells perforin and granzyme B; The expression of p-ERK, p-Akt and Bcl-2 albumen is detected by WesternBlot method.
Research shows, Quercitroside significantly can promote the propagation of human gamma delta t cells, and can promote the expression of its perforin and granzyme B, has certain dose dependent; Gamma delta T cells after Quercitroside process, can promote the expression of activity form p-ERK1/2 and p-Akt, and can promote the expression of anti-apoptotic proteins Bcl-2, has certain dose dependent.
Accompanying drawing explanation
Fig. 1 cultivates front and back gamma delta T cells ratio (A: do not cultivate; B: cultivate after 10 days)
The impact (* P<0.05, * * P<0.01, comparedwithcontrolgroup) that the Quercitroside of Fig. 2 variable concentrations is bred gamma delta T cells
After the Quercitroside effect gamma delta T cells 48h of Fig. 3 variable concentrations, on the impact that perforin and granzyme B are expressed
After the Quercitroside effect gamma delta T cells 48h of Fig. 4 variable concentrations, on the impact that p-ERK1/2, p-Akt and Bcl-2 express
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1: the impact experiment that Quercitroside is bred gamma delta T cells
1 materials and methods
1.1 experiment material
The anti-perforin (anti-perforin) of anti-gamma delta T CR, PE labelling of FITC labelling, the particle-resistant enzyme B(anti-granzymeB of PE labelling), the agent of Fix & Perm rupture of membranes is all purchased from ebioscience company; Recombinant human interleukin--2 (rhIL-2), isopentenylpyrophosphate (isopentenylpyrophosphate, IPP) is purchased from Xiamen Amoytop Biotech Co., Ltd.; RPMI-1640 culture medium, calf serum, trypsin are purchased from Gibco company; Lymphocyte separation medium is purchased from Chinese Academy of Sciences hematology institute; CCK-8 test kit, BCIP/NBT alkaline phosphatase colour reagent box and Western and IP lysis liquid kit are purchased from green skies Bioisystech Co., Ltd.People AB type serum is purchased from blood station, Xuzhou City.Phosphoric acid solution is analytical pure, and water is redistilled water.
Quercitroside, by reference literature in Polygonaceae arsesmart Herba Polygoni Capitati (PolygonumcapitatumBuch.-Ham.exD.Don) Yang Yang. Salvia przewalskii and Herba Polygoni Capitati chemical constitution study. The 2nd Army Medical College master thesis. Shanghai: The 2nd Army Medical College, 2009 ］ separation and purification obtains, check through high performance liquid chromatography (HPLC), purity is greater than 98%.
1.2 gamma delta T cells are cultivated and qualification
Get Healthy People periphery anticoagulation 50ml, after being separated acquisition peripheral blood mononuclearcell (PBMCs) with lymph separating medium, reference literature ［ Chen Fuxing, Liu Junquan, Feng Xia, Deng. a kind of new method of amplification in vitro human gamma delta t cells. cell and molecular immunology magazine, 2007,23 (7): 662-664 ］ carry out gamma delta T cells cultivation.PBMC is added in gamma delta T cells RPMI-1640 culture fluid (rhIL-2 containing IPP and 100IU/ml of 10% calf serum, 5%AB serum, 2 μ g/ml), in 37 DEG C, 5%CO
2cultivate 10d in cell culture incubator, use flow cytomery with after anti-TCR-γ δ-FITC labelling.
1.3 statistical procedures
Experimental result adopts
± s represents, between group, Average value compare adopts t inspection, with * P<0.05, * * P<0.01 for there being statistical significance.
1.4CCK-8 method detects cell proliferation
Get the gamma delta T cells cultivating 10d, adjustment cell number to 1.0 × 10
5/ ml, gets 180 μ l and is inoculated in 96 orifice plates, adds final concentration and is respectively 0 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml Quercitrosides, and often group establishes 4 multiple holes.After cultivating 48h, every hole adds the CCK-8 liquid of 20 μ l, measures optical density value after continuing to cultivate 4h in 570nm place.
2 results
2.1 human gamma delta t cells culture & identifications
Get the gamma delta T cells basis of microscopic observation cultivating 10d, cell colony large as seen, individual cells is bar shuttle shape.Use flow cytomery, in the new PBMC be separated, the ratio of gamma delta T cells only accounts for 2.96% ± 1.83%, and the ratio of cultivating gamma delta T cells after 10d is up to 88.94% ± 2.36%, and above result prompting gamma delta T cells is cultivated successfully, can be subsequent experimental use.The results are shown in Figure 1.
The impact that 2.2 Quercitrosides are bred gamma delta T cells
After the Quercitroside effect gamma delta T cells 48h that concentration is 0 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, each concentration group is respectively its rate of increase: 100% ± 2.96%, 107.42% ± 5.42%, 120.34% ± 2.25%, 128.94% ± 4.22%, 132.42% ± 3.27%, 118.98% ± 4.08%.Compared with 0 μ g/ml matched group, 5 μ g/ml groups are without obvious facilitation, and other each concentration group rate of increase is significantly higher than matched group (* P<0.05, * * P<0.01).Wherein, the rate of increase of 40 μ g/ml Quercitroside groups reaches peak, and concentration declines to some extent more than 40 μ g/ml hourly growth rates, but still higher than matched group.The results are shown in Figure 2.
Embodiment 2: the expression impact of Quercitroside on perforin on gamma delta T cells and granzyme B is tested
1 materials and methods
Wherein, 1.1 experiment materials; 1.2 gamma delta T cells are cultivated and qualification; 1.3 statistical procedures are all with embodiment 1.
The expression of perforin and granzyme B on 1.4 flow cytomery gamma delta T cells
The successful gamma delta T cells of cultivation is made into the cell suspension that concentration is 1.0 × 105/ml, be inoculated in 6 well culture plates, every hole 3ml, then adds the Quercitroside that final concentration is respectively 0 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, often organizes 3 multiple holes.In 37 DEG C, 5%CO
248h is cultivated in incubator.Collecting cell also washs 2 times with phosphate buffer (PBS), and often pipe adds anti-gamma delta T CR-FITC20 μ l, and lucifuge hatches 30min.Add 100 μ l fixative room temperature lucifuges and hatch 15min, PBS washs 1 time.Often pipe adds 100 μ l rupture of membranes liquid, 100 μ lanti-Perforin antibody and anti-granzymeB-PE antibody, room temperature lucifuge hatches 15min, after PBS washing, be resuspended in the PBS solution of 0.5ml, use the expression of Flow cytometry perforin and granzyme B respectively.
2 Quercitrosides on granzyme B in gamma delta T cells, perforin expression affect result
Compared with 0 μ g/ml matched group, after the Quercitroside effect gamma delta T cells 48h of 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, its perforin and granzyme B positive expression all obviously raise, and when 5 μ g/ml ~ 40 μ g/ml concentration, have certain dose dependent.Wherein, perforin and the granzyme B positive expression rate of 40 μ g/ml Quercitroside groups reach peak, be respectively 71.70% ± 2.31% and 82.16% ± 1.29%, and concentration decline to some extent more than perforin during 40 μ g/ml and granzyme B positive expression rate.The results are shown in Figure 3.
Embodiment 3: Quercitroside is tested the impact of Bcl-2, p-ERK1/2 and p-Akt protein expression in gamma delta T cells
1 materials and methods
Wherein, 1.1 experiment materials; 1.2 gamma delta T cells are cultivated and qualification; 1.3 statistical procedures are all with embodiment 1.
1.4Westernblot detects Bcl-2, p-ERK1/2 and p-Akt protein expression
It is 1.0 × 10 that successful for cultivation gamma delta T cells is made into concentration
5the cell suspension of/ml, is inoculated in 6 well culture plates, every hole 3ml, then adds the Quercitroside that final concentration is respectively 0,5,10,20,40,80 μ g/ml, in 37 DEG C, 5%CO
248h is cultivated in incubator.Collecting cell after washing 1 time with PBS, albumen is extracted with the abundant cracking of Western and IP cell pyrolysis liquid 100 μ L, be separated with 10%SDS-PAGE after BCA method test sample product protein concentration, then by protein delivery on pvdf membrane, the defatted milk powder of 5% closes 2h, primary antibodie (1:500) 4 DEG C is closed and is spent the night, and two anti-(1:1000) 37 DEG C hatch 2h.BCIP/NBT alkaline phosphatase colour reagent box is utilized to develop the color, digital camera Taking Pictures recording.Test repetition 3 times.
2Westernblot detects Quercitroside affects result to Bcl-2, p-ERK1/2 and p-Akt protein expression in gamma delta T cells
Compared with 0 μ g/ml matched group, after the Quercitroside effect gamma delta T cells 48h of 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, the expression of its Bcl-2, p-ERK1/2 and p-Akt is all in rising trend, and there is certain dose dependent, and internal reference GAPDH expression does not change.The results are shown in Figure 4.
Claims (2)
1. Quercitroside is preparing the application in human gamma delta t cells multiplication agent.
2. Quercitroside as claimed in claim 1 is preparing the application in human gamma delta t cells multiplication agent, be separated mononuclearcell by healthy human peripheral blood, add inducing culture in the RPMI-1640 complete medium containing isopentenylpyrophosphate and recombinant human interleukin--2 and obtain gamma delta T cells; After using the Quercitroside effect gamma delta T cells 48h of variable concentrations, measure variable concentrations Quercitroside group gamma delta T cells multiplication capacity by CCK-8 method; Flow cytometry is adopted respectively to organize the expression of gamma delta T cells perforin and granzyme B; The expression of p-ERK, p-Akt and Bcl-2 albumen is detected by WesternBlot method.
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| CN106421184A (en) * | 2016-09-27 | 2017-02-22 | 浙江博纳生物科技有限公司 | Application of hemsley rockvine root extract in preparing tumor immunity therapeutic drug and human gamma delta T cell proliferation agent |
| CN106632197B (en) * | 2016-12-15 | 2019-04-02 | 中国科学院昆明植物研究所 | Single plumage fire cylinder tree anticancer activity position LA-D and its pharmaceutical composition is separated with its chemical component and identification method |
| CN115261316B (en) * | 2022-05-17 | 2023-09-01 | 徐州医科大学 | Method for promoting human gamma delta T cell proliferation and enhancing cytotoxic activity thereof |
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| Title |
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| 一种体外扩增人γδT细胞的新方法;陈复兴 等;《细胞与分子免疫学杂志》;20071231;第23卷(第7期);662-664 * |
| 根皮素对人γδT细胞杀伤胃癌SGC-7901细胞的影响及机制探讨;刘刚 等;《中国免疫学杂志》;20111231;第27卷(第7期);602-606、615 * |
| 白藜芦醇对人γδT细胞生长和杀伤功能的影响;陈玲 等;《中草药》;20111031;第42卷(第10期);2056-2059 * |
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