CN109395063A - Application of the G-CSF in treatment ITP patient's dysimmunity - Google Patents
Application of the G-CSF in treatment ITP patient's dysimmunity Download PDFInfo
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- CN109395063A CN109395063A CN201811591053.XA CN201811591053A CN109395063A CN 109395063 A CN109395063 A CN 109395063A CN 201811591053 A CN201811591053 A CN 201811591053A CN 109395063 A CN109395063 A CN 109395063A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
Abstract
This medicine is related to a kind of new medicine use, and in particular to the Th1/Th2 cells ratio that granulocyte colony stimulating factor adjusts primary immune thrombocytopenia patient PBMCs treats primary immune thrombocytopenia.Experiment in vitro proves that colony-stimulating factor can reduce the Th1/Th2 ratio of primary immune thrombocytopenia patient in a manner of dose-dependent, the ratio of T-bet/GATA-3, which reduces, proves that colony-stimulating factor can reduce Th1/Th2 ratio by indirect mode, which shows that colony-stimulating factor acts on the potential treatment of primary immune thrombocytopenia patient for the first time.
Description
Technical field
The present invention relates to the treatments of essential thrombocytopenia, and specifically G-CSF is in treatment ITP Patients Patients
It is applied in dysimmunity.
Background technique
Primary immune thrombocytopenia (ITP) is the autoimmune reduced by blood platelet, with the characteristics of dermatorrhagia
Disease, with complicated Pathologic Characteristics.Macrophage, CD8+T cell, autoantibody can destroy blood platelet, in the hair of ITP
It plays an important role during disease.However helper T lymphocyte (Th) plays important on-off action in the generation of mediate B cell antibody.
In addition, it is the autoimmunity disease mainly showed that ITP, which is with Th1 polarization, the degree that Th1/Th2 ratio increases and platelet count and disease
Sick severity is negatively correlated.Therefore reverse Th1 polarization, restore Th1/Th2 balance may be risen in the treatment of ITP patient it is latent
Important function.
Th cell is played regulatory role in immune system by secrete cytokines.Th1 and Th2 cell is being immunoreacted
Effect it is different.Wherein, Th1 cell Major Secretory cytokines interferon (IFN)-γ and interleukin (IL) -2, in immune disease
It plays an important role in the pathogenic process of disease；And Th2 cell Major Secretory cell factor IL-4 and IL-13, in autoimmune disease
Pathogenic process in shield.In addition, some factors have been demonstrated that the secretion of Th1 and Th2 cell factor, example can be influenced
Such as: regulatory T cells, granulocyte colony stimulating factor (G-CSF), T box (T-bet) and GATA combination egg in T lymphocyte
White 3 (GATA-3).In addition, T-bet and GATA-3 are two important T cell transduced elements, be adjusted Th1 and Th2 cell because
Subbase because expression, T cell differentiation in play an important role.T-bet/GATA-3 is believed to really react Th1/Th2
State.
G-CSF is an important Hemopoietic factor of medullary system, while being the necessary mediation person of immune response again, and connection is inherently exempted from
Epidemic disease reaction and the acquired immune response play an important role in inducing and maintaining T cell tolerance.In health donors, G-CSF can
To reduce cell factor IFN-γ, the generation of IL-4 is improved, reduces Th1/Th2 ratio.Delay and/or treat in addition, G-CSF has
The effect of autoimmunity disease.However, effect of the G-CSF in the dysimmunity of ITP is still unclear.
We assume that G-CSF can reduce the Th1/Th2 ratio that ITP patient increases extremely, in order to confirm this it is assumed that I
Study effect of the G-CSF in ITP patient's Th1/Th2 ratio, our result show that G-CSF change ITP patient Th1/
The potential treatment of Th2 ratio acts on, and illustrates G-CSF in the indirect adjustment mechanism of Th1/Th2 cell.
Summary of the invention
The invention discloses application of the G-CSF in treatment ITP patient's dysimmunity, and G-CSF is by adjusting patient Th1/
The ratio of Th2 realizes the treatment to primary immune thrombocytopenia.
G-CSF can reduce Th1/Th2 cells ratio by reducing T-bet/GATA-3 ratio indirectly.
The present invention provides a kind of potential effective therapeutic agent, cell collection for primary immune thrombocytopenia patient
G-CSF can reduce the Th1/Th2 ratio of primary immune thrombocytopenia patient in a manner of dose-dependent,
The ratio of T-bet/GATA-3, which reduces, proves that colony-stimulating factor can reduce Th1/Th2 ratio by indirect mode, this
Application shows that colony-stimulating factor acts on the potential treatment of primary immune thrombocytopenia patient for the first time.
Detailed description of the invention
Fig. 1 be G-CSF to the cytokine release of ITP patient (n=12) and normal healthy controls person (n=12) PBMCs and
The effect of Th1/Th2 cytokine-expressing ratio.After various concentration G-CSF acts on PBMCs for 24 hours, PHA (10ug/mL) training is added
Support 48h.The expression of IFN-γ (A), IL-2 (B), IL-4 (C) and IL-13 (D) in cell culture fluid is detected by ELISA method
Amount.ITP patient (E) and normal healthy controls person (F) Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-13) ratio.Data use
The form of mean ± standard deviation is expressed.
Fig. 2 be G-CSF to the IFN-γ of ITP patient (n=12) and normal healthy controls person (n=12) PBMCs, IL-2, IL-4,
After the effect various concentration G-CSF of IL-13, T-bet and GATA-3mRNA expression acts on PBMCs for 24 hours, PHA culture is added
48h.RT-PCR detects the IFN-γ (A) of PBMCs, IL-2 (B), IL-4 (C), IL-13 (D), T-bet (E) and GATA-3's (F)
MRNA expression.Calculate ITP patient and normal healthy controls person's IFN-γ/IL-4 (G), IL-2/IL-13 (H) and T-bet/GATA-
3 (I) ratios.Data are expressed in the form of mean ± standard deviation.
Specific embodiment
The present invention takes human peripheral blood mononuclear cell (PBMCs) to cultivate, and G-CDF is then added and is cultivated, after
Phase stimulates through PHA, sample to be tested is made in trizol solution effects, is then detected.
Detection specifically includes that
The PBMCs of ELISA detection in vitro culture is secreted into cell factor (IFN-γ, IL-2, IL-4, IL- in supernatant
13) expression.
RT-PCR detect in vitro culture PBMCs based intracellular cvtokine (IFN-γ, IL-2, IL-4, IL-13) and T-bet,
The expression of the mRNA of GATA-3 and β-actin.
Embodiment
Research object includes 12 patients with chronic idiopathic thrombocytopenlc purpura and the normal healthy controls person that 12 ages, genders are consistent.Chronic ITP
The main diagnostic criteria according to the publication of the world ITP working group in 2009 of diagnosis, while excluding the relevant other diseases of ITP and suffering from
Person.Patient does not receive hormone therapy.Male 5, female 7, the median age 49.5 years old (17~79 years old), blood platelet median 10.0
×109/ L (0.4~19.6 × 109/L).In 12 patients of this research, 5 (41.7%) example patient antiplatelet antibodies are positive.
Table 1 shows the clinical characters of ITP patient.
In table 1, M indicates that gender is male；F indicates that gender is female.
The operation that the studies above object is followed the steps below:
1, the separation of PBMCs, culture and the anticoagulant peripheral blood 5ml of experiment process taking heparin sodium, by 1500g at room temperature from
15 minutes acquisition blood plasma of the heart needs to melt in 37 DEG C of water-baths when -80 DEG C of storages, blood plasma use.Pass through lymphocyte point
Cell concentration after giving cell count, is adjusted to 1 × 10 by chaotropic PBMCs6/ ml is placed in 1640 culture medium of RPMI and (contains
10%FCS) cultivate, while be added various concentration G-CSF (0ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml, 100ng/ml,
Xiamen Amoytop Biotech Co., Ltd.), 10ug/ml PHA is added in 5%CO2 incubator, after 24 hours to stimulate
Secrete cytokines.By centrifugation after being co-cultured 72 hours under the conditions of 37 DEG C, supernatant is taken, is placed in the centrifuge tube newly prepared, -80
It is frozen in DEG C refrigerator spare；1ml trizol solution is added in PBMCs, gently blows and beats cell precipitation with suction pipe, it is complete to cell
After dissolution, frozen in -80 DEG C of refrigerators spare.
2, ELISA detects cell factor
ELISA kit detects cell factor IFN-γ, IL-2, IL-4, IL-13 after blood plasma G-CSF and PBMCs culture
Variation, each experiment are repeated twice.Without significant cross reaction between different recombinant cytokines.Detecting the minimum rate of accumulation is respectively
39.1pg/mL (G-CSF), 0.99pg/mL (IFN-γ), 9.2pg/Ml (IL-2), 1.3pg/mL (IL-4), 0.7pg/ml (IL-
13)。
3, IFN-γ in RT-PCR method detection PBMCs, IL-2, IL-4, the transcription of IL-13, T-bet and GATA-3
After G-CSF is handled 72 hours, total serum IgE is extracted from 1 × 106PBMCs using TRIzol, passes through ImProm-IITMIt is inverse
The temperature that transcriptase reverse transcription 500ng RNA. is used is: 20 DEG C of 5min, 42 DEG C of 30min, 70 DEG C of 15min, 4 DEG C of maintenances.cDNA
Be stored in -20 DEG C it is spare.The loop parameter setting of PCR reaction: 95 DEG C, 95 DEG C of 15s, 60 DEG C of 30s, 72 DEG C of 30s, connect after ten minutes
Continuous 40 circulations.All reactions are in triplicate.
Pass through 2-ΔΔCtMethod calculates target gene (IFN-γ, IL-2, IL-4, IL-13, T-bet and GATA-3) and pipe
The opposite variation that family's gene (β-actin) is expressed.Wherein β-actin, IFN-γ, IL-2, IL-4, IL-13, T-bet and GATA-
3 primers sequence such as table 2:
Above-mentioned testing result and data are analyzed and summarized:
Effect of the 1.G-CSF to Th1 and Th2 cell cytokine secretion
Under PHA effect, G-CSF and the PBMCs co-incubation of various concentration detect PBMCs secretion by ELISA method
Cell factor amount, comprising: IFN-γ, IL-2, IL-4 and IL-13.Such as Figure 1A and B, when no G-CSF is handled, ITP patient IFN-
The expression of γ and IL-2 is significantly higher than normal healthy controls person (IFN-γ, P < 0.001；IL-2, P < 0.001), and IL-4 (figure C)
And IL-13 (figure D) expression is lower than normal healthy controls person (IL-4, P < 0.01；IL-13, P < 0.001).G-CSF is with dose-dependant
Property mode reduce ITP patient and normal healthy controls person Th1 (IFN-γ+IL-2)/Th2 (IL-4+IL-13) ratio (figure E and
F)；And IFN-γ and IL-2 are gradually decreased, IL-4 and IL-13 are gradually increased.In ITP patient, 100ng/mL there are the case where
Under, IFN-γ and IL-2 reduce by 34.0% ± 1.2% (P < 0.05) and 22.4% ± 3.5% (P < 0.05) respectively；IL-4 and IL-
13 are respectively increased 111.7% ± 75.8% (P < 0.05) and 66.9% ± 17.5% (P < 0.05).Finally, Th1 (IFN-γ+IL-
2) ratio of/Th2 (IL-4+IL-13) reduces by 58.9% ± 2.8% (P < 0.05).In normal healthy controls person, in 100ng/mLG-
In the presence of CSF, IFN-γ and IL-2 reduce respectively 43.0% ± 6.7% (P < 0.05) and 27.9% ± 7.0% (P <
0.05)；39.9% ± 17.2% (P < 0.05) and 18.0% ± 5.6% (P < 0.05) is respectively increased in IL-4 and IL-13.Finally,
The ratio of Th1 (IFN-γ+IL-2)/Th2 (IL-4+IL-13) reduces by 44.6% ± 6.5% (P < 0.05).
Blood plasma G-CSF concentration is lower than the minimum detection value of ELISA kit.
2, G-CSF is to IFN-γ on PBMCs, IL-2, IL-4, the expressional function of the mRNA of IL-13, T-bet and GATA-3
Analysis
As shown in Fig. 2, under various concentration G-CSF effect, we by the RT-PCR detection IFN-γ of PBMCs, IL-2,
The expression of the mRNA of IL-4, IL-13, T-bet and GATA-3.When no G-CSF is acted on, IFN-γ/IL-4, IL-2/ of ITP patient
The expression ratio of IL-13 and T-bet/GATA-3 4.23 times higher than the expression ratio of normal healthy controls person, 3.97 times and 3.26 respectively
Again (P < 0.05).With increasing for G-CSF concentration, in ITP patient and normal healthy controls person, IFN-γ, IL-2 and T-bet
The expression of mRNA reduces；The expression of the mRNA of IL-4, IL-13 and GATA-3 are increased；IFN-γ/IL-4, IL-2/IL-13 and T-
The expression ratio of bet/GATA-3 reduces.In 100ng/mL G-CSF effect, IFN-γ, IL-2 and the T- of ITP Patient cells
The expression of the mRNA of bet reduce by 38.9% ± 13.8%, 30.9% ± 9.8% and 22.1% ± 7.1% respectively (P <
0.05)；The expression of these mRNA of normal healthy controls person reduces by 27.7% ± 2.5%, 24.7% ± 5.5% He respectively
15.8% ± 5.5% (P < 0.05).In addition, the expression of the mRNA of IL-4, IL-13, GATA-3 of ITP Patient cells are distinguished
Improve 54.4% ± 12.2%, 26.7% ± 9.5% and 70.2% ± 24.8% (P < 0.05)；These mRNA of normal healthy controls person
Expression 46.7% ± 17.5%, 24.0% ± 9.2% and 31.7% ± 11.4% (P < 0.05) is respectively increased.Finally,
ITP patient's IFN-γ/IL-4, IL-2/IL-13 and T-bet/GATA-3 ratio reduces by 60.4% ± 13.7%, 45.5% respectively
± 8.2% and 53.3% ± 9.8% (P < 0.05), these ratios of normal healthy controls person reduce by 50.7% ± 5.3% respectively,
39.5% ± 10.2% and 35.5% ± 8.7% (P < 0.05).
The experiment confirms that G-CSF can be in the Th1/Th2 cell for reducing ITP patient in a manner of relying property of dose-dependant in vitro
Ratio.T-bet/GATA ratio, which reduces, confirms that G-CSF has indirect immune regulation mechanism to Th1/Th2.Evidence is bright at present, G-
CSF has potential therapeutic effect to ITP patient.
Claims (3)
1. granulocyte colony stimulating factor is treating the application in primary immune thrombocytopenia.
2. the Th1/Th2 cells ratio that granulocyte colony stimulating factor adjusts primary immune thrombocytopenia patient PBMCs
Treat primary immune thrombocytopenia.
3. colony-stimulating factor can reduce primary immune thrombocytopenia patient's in a manner of dose-dependent
Th1/Th2 ratio, the ratio of T-bet/GATA-3, which reduces, proves that colony-stimulating factor can be reduced by indirect mode
Th1/Th2 ratio.
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| CN201811591053.XA CN109395063A (en) | 2018-12-22 | 2018-12-22 | Application of the G-CSF in treatment ITP patient's dysimmunity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113156141A (en) * | 2021-03-23 | 2021-07-23 | 复旦大学附属中山医院 | Multi-cytokine detection chip and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159462A (en) * | 1996-08-16 | 2000-12-12 | Genentech, Inc. | Uses of Wnt polypeptides |
| CN1756561A (en) * | 2002-12-31 | 2006-04-05 | 免疫医疗公司 | Immunotherapy of B cell malignancies and autoimmune diseases using unconjugated antibodies and conjugated antibodies and antibody combinations and fusion proteins |
-
2018
- 2018-12-22 CN CN201811591053.XA patent/CN109395063A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159462A (en) * | 1996-08-16 | 2000-12-12 | Genentech, Inc. | Uses of Wnt polypeptides |
| CN1756561A (en) * | 2002-12-31 | 2006-04-05 | 免疫医疗公司 | Immunotherapy of B cell malignancies and autoimmune diseases using unconjugated antibodies and conjugated antibodies and antibody combinations and fusion proteins |
Non-Patent Citations (2)
| Title |
|---|
| FEI GE等: "Granulocyte colony-stimulating factor decreases the Th1/Th2 ratio in peripheral blood mononuclear cells from patients with chronic immune thrombocytopenic purpura in vitro", 《THROMBOSIS RESEARCH》 * |
| 黄玉等: "成人难治及复发ITP的个体化治疗及研究进展", 《中国实验血液学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113156141A (en) * | 2021-03-23 | 2021-07-23 | 复旦大学附属中山医院 | Multi-cytokine detection chip and application thereof |
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Application publication date: 20190301 |