CN104152397A - Sensitized animal spleen cell model and use thereof in drug screening - Google Patents
Sensitized animal spleen cell model and use thereof in drug screening Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutical drug screening, and relates to a sensitized animal spleen cell model and use thereof. The sensitized animal spleen cell model is prepared as follows: campylobacter jejuni CJ-S131 is used for inducing mice to generate SLE (systemic lupus erythematosus) like symptoms for preparation of a spleen single cell suspension under sterile conditions; campylobacter jejuni CJ-S131 is added into a culture plate to again stimulate sensitized spleen cells to prepare the sensitized animal spleen cell model, and an anti-SLE drug is used to validate the use of the sensitized animal spleen cell model in preparation of drugs for prevention and treatment of systemic lupus erythematosus (SLE). The invention also provides establishment of an anti-SLE related drug in-vitro screening method by use of the prepared sensitized animal spleen cell model to realize high throughput screening of the anti-SLE drug; and the anti-SLE related drug in-vitro screening method makes up the defects of incapability of high throughput screening of the anti-SLE drug in the prior art, and significantly shortens the cycle of ordinary the SLE model evaluation.
Description
Technical field
The invention belongs to pharmacy drug screening field, relate to cell model and uses thereof, be specifically related to a kind of splenocyte model of sensitized animal and the purposes in drug screening thereof; Described model can be used for setting up in vitro drug screening, especially prevents and treats the high flux screening platform of systemic lupus erythematous (SLE) medicine as screening.
Background technology
Known system lupus erythematosus (SLE) be a kind of refractory, outbreak, serious autoimmune disease that lethality rate is high repeatedly; Although the cause of disease and the pathogenesis of described SLE are illustrated not yet completely, but patient all exists immunoloregulation function seriously to lack of proper care, the high-caliber anti self antibody of body and low-level mononuclear macrophage are removed ability, immunocomplex is increased, and popularity activating complement system, cause inflammation and the injuries of tissues and organs of body, make the identification long-term existence of body to autoantigen simultaneously, cause the protracted course of disease of the state of an illness.
Studies have reported that, the generation of multiple autoantibody can be detected for SLE patient, as antinuclear antibody, anti-double-chain DNA antibody (anti-dsDNA antibody), histonic antibody etc.; And due to its immunomodulatory obstacle, can cause that gamma-globulin raises, and the change of relevant cell factor secretion, as the level of interferon-γ (IFN-γ), interleukin 10 (IL-10) and Interleukin-17 (IL-17) etc. raises.
At present, the animal model adopting in domestic and international anti-SLE drug research has the experimental model of idiopathic SLE model (as MRL/lpr) and campylobacter jejuni induction etc., above-mentioned model validation environmental factors maintaining of immunological tolerance played to same vital effect with inherited genetic factors; But the experimental period of wherein said animal model is long, can not realize medicament high flux screening; Because the heat shock protein(HSP) of the 43KD of campylobacter jejuni CJ-S131 all has the epitope similar to body autoantigen (molecular simulation phenomenon) with LPS, bacterium composition enters in body as cross-reacting antigen, generation stress, cause a series of immune responses, after T cell activation, assist B cell polyclone to activate by alternative pathway, cause the generation of autoantibody, make campylobacter jejuni inducing mouse formation to have typical SLE sample symptom, this model is to study at present SLE pathomechanism and find one of comparatively ideal model of medicine.
Radix bupleuri is traditional Chinese medicine and the conventional Chinese medicine of China, and its nature and flavor hardship is cool, have evacuate bring down a fever, effect that dispersing the stagnated live-QI to relieve the stagnation of QI, liter sun is lifted gas, be used for the treatment of alternate attacks of chills and fever, fullness in the chest and hypochondriac pain, bitter taste deafness, headache and dizziness etc.Herba Sidae Rhombifoliae soup taking radix bupleuri as main ingredient is more gentle, has the merit for the treatment of SHAO YANG disease by mediation, taking the heat of Qing Guo important department as feature, has report for extreme noxious heat SLE, evident in efficacy; Existing document (ZhengWang; Hong Li, et al. Beneficial effect of Bupleurum polysaccharides on autoimmune disease induced by Campylobacter jejuni in BALB/c mice. Journal of Ethnopharmacology. 2009; 124:481-487.) report, Bupleurum chinense polysaccharide has provide protection to SLE sample mouse, can selectivity suppress complement activation, and immune function of mice is had to regulating effect.Prepare the method for preventing and treating systemic lupus erythematous (SLE) medicine in view of all lacking at present in vitro high flux screening both at home and abroad, present inventor intends providing a kind of splenocyte model of sensitized animal, and adopt described Bupleurum chinense polysaccharide to verify the characteristic of this splenocyte model in vitro high-throughput sieve prescription face, further set up in vitro high flux screening and prepare the method for preventing and treating systemic lupus erythematous (SLE) medicine.
Summary of the invention
The object of this invention is to provide a kind of splenocyte model of sensitized animal and the purposes in drug screening thereof.Splenocyte model experiment cycle of this sensitized animal is short, can realize medicament high flux screening.
The splenocyte model of sensitized animal of the present invention is prepared by following method, adopts campylobacter jejuni CJ-S131 induction to set up mouse SLE original mold type, gets model mouse spleen cell, stimulates in vitro with CJ-S131, makes the isolated model of SLE sample performance; This isolated model shows SLE related immune index and raises, and described SLE related immune index comprises: anti-dsDNA antibody, total IgG, IFN-γ and IL-10.
More specifically, the preparation method of the splenocyte model of sensitized animal of the present invention, is characterized in that, it comprises step:
Adopt campylobacter jejuni CJ-S131 inducing mouse to produce SLE sample symptom, process according to a conventional method experiment mice, under aseptic condition, get its spleen, prepare single cell suspension; In culture plate, add campylobacter jejuni CJ-S131, again stimulate the splenocyte of sensitization, make the splenocyte model of sensitized animal.
The present invention further adopts the medicine Bupleurum chinense polysaccharide with anti-SLE effect, verifies the purposes of above-mentioned isolated model in drug screening, especially prevents and treats the purposes in systemic lupus erythematous medicine in preparation.
In the present invention, in the splenocyte model making, add the medicine Bupleurum chinense polysaccharide to be measured of different concns, be placed in 37 DEG C, 5% CO
2incubator is cultivated 48h; After cultivation finishes, draw the cells and supernatant in culture plate, detect the disease-related amynologic index such as anti-dsDNA antibody, total IgG, IFN-γ and IL-10, result shows, in SLE relevant diseases index, in cell culture supernatant, anti-dsDNA antibody, total IgG, relevant inflammatory factors IFN-γ and IL-10 level obviously raise;
Result shows, the splenocyte model that adopts the present invention to make can be used for screening prevents and treats the medicine of systemic lupus erythematous (SLE).
The present invention also provides the method for the anti-SLE related drugs of a kind of vitro Screening, realizes the high flux screening of anti-SLE medicine; Its step comprises:
(1) set up SLE related immune abnormal cells model:
Stimulate again disease model mice spleen cell by campylobacter jejuni CJ-S131, successfully set up the inflammatory model of in vitro SLE sample performance;
(2) adopt SLE like cell model discrimination medicine;
(3) anti-dsDNA TPPA:
(4) total IgG is measured:
(5) IFN-γ measures:
(6) IL-10 measures:
Filter out the medicine of preventing and treating systemic lupus erythematous.
In the present invention, the notable feature of described SLE disease on pathology is the generation of autoantibody in patient body; Described autoantibody comprises anti-double-chain DNA antibody (anti-dsDNA antibody), antinuclear antibody etc.; Experimental result demonstration of the present invention, while stimulation in vitro, model group is compared with Normal group and adjuvant control group, and the level of anti-dsDNA antibody significantly raises, and shows whole animal SLE original mold type modeling success; And splenocyte is after CJ-S131 stimulates again in vitro, the level of the anti-dsDNA antibody of model group further raises, and all has significant difference compared with control group; In addition, in SLE lysis, due to immunomodulatory obstacle, cause that total immunoglobulin IgG albumen raises, and the change of relevant cell factor secretion, as IFN-γ, IL-10, IL-17 etc.; Experimental result of the present invention also shows, before stimulating again in vitro, the level of total IgG, IFN-γ, IL-10 is no significant difference between model group and control group, and splenocyte is after campylobacter jejuni CJ-S131 stimulates again in vitro, the level of the total IgG of model group, IFN-γ, IL-10 all significantly raises compared with control group;
Above-mentioned detected result shows, stimulates disease model mouse spleen cell by campylobacter jejuni CJ-S131 again, has successfully set up the inflammatory model of SLE sample performance; And described Bupleurum chinense polysaccharide has significant protective effect to the in vitro medicaments sifting model of this SLE, show as the obvious effect that improves disease-related immune indexes, confirm the validity of described in vitro sieve prescription method.
The method of the anti-SLE related drugs of vitro Screening of the present invention, has the following advantages:
The splenocyte that the CJ-S131 immunity of learning from else's experience is crossed, adopts CJ-S131 to stimulate in vitro again, sets up in vitro medicine sorting platform, and splenocyte shows the change of SLE related immune index; The high flux screening that can realize antagonism SLE medicine, has shortened experimental period, simplifies experiment condition and step simultaneously.
The present invention finishes overall modeling mid-term at the experimental SLE model of campylobacter jejuni induction, get campylobacter jejuni CJ-S131 has been set up to immunoreactive splenocyte, by stimulating again of in vitro campylobacter jejuni, further set up the sieve prescription method of in vitro SLE sample performance splenocyte; In experiment of the present invention, adopt after in vitro administration, collect above-mentioned model cell culture supernatant, detect the relevant amynologic index of SLE, can observe targetedly drug effect; Observe and adopt Tissue Culture Plate to cultivate the reaction of splenocyte to medicine after immunity, can the anti-SLE medicine of rapid screening, realize high-throughput new medicament screen, greatly shorten the test period.
The method of the anti-SLE related drugs of vitro Screening of the present invention, has made up the defect that the anti-SLE medicine of existing screening can not high-throughput carries out, and has shortened significantly the cycle of common SLE model evaluation simultaneously, and operating process is easy, makes experiment more convenient.
Brief description of the drawings
Fig. 1 has shown SLE sample mouse boosting cell secretion anti-dsDNA TPPA result.
Fig. 2 has shown SLE sample mouse boosting cell secretion total IgG measurement result.
Fig. 3 has shown SLE sample mouse boosting cell secretion of gamma-IFN measurement result.
Fig. 4 has shown SLE sample mouse boosting cell secretion IL-10 measurement result.
Embodiment
Case study on implementation 1 is set up the abnormal cell model of SLE immune function
The animal that experiment adopts comprises: normal control mouse, adjuvant contrast mouse, SLE model mouse (the SLE original mold type of CJ-S131 induction) mouse; Put to death mouse, aseptic technique taking-up spleen is put into the watch-glass of the RPMI-1640 that fills precooling, is prepared into spleen single cell suspension and regulates splenocyte concentration, gets Tissue Culture Plate, adds respectively CJ-S131 and splenocyte suspension; Add after plate, mix gently, be placed in 37 DEG C, 5% CO
2incubator is cultivated 48h.After cultivation finishes, centrifugal, draw the cell conditioned medium liquid in every hole, after packing, put into-80 DEG C of Refrigerator stores, for detection of indexs such as anti-dsDNA antibody, IgG, IFN-γ and IL-10.
The anti-SLE related drugs of embodiment 2 vitro Screening, realizes the high flux screening of anti-SLE medicine
(1) set up SLE related immune abnormal cells model:
Experiment adopts: normal control mouse, adjuvant contrast mouse, SLE model mouse (the SLE original mold type of CJ-S131 induction) mouse; Conventional processing experiment mice, aseptic technique taking-up spleen is put into the watch-glass of the RPMI-1640 that fills precooling; Make spleen single cell suspension and regulate splenocyte concentration, getting Tissue Culture Plate, adding respectively CJ-S131 and splenocyte suspension; After splice, mix, be placed in 37 DEG C, 5% CO
2incubator is cultivated 48h; After cultivation finishes, centrifugal, draw the cell conditioned medium liquid in every hole, after packing, put into-80 DEG C of Refrigerator stores, for detection of indexs such as anti-dsDNA antibody, IgG, IFN-γ and IL-10; Detected result is, in vitro splenocyte is after CJ-S131 stimulates again, and the level of the anti-dsDNA antibody of model group further raises, and all has significant difference compared with control group; Before stimulating again in vitro, the level of total IgG, IFN-γ, IL-10 is no significant difference between model group and control group, and splenocyte is after campylobacter jejuni CJ-S131 stimulates again in vitro, the level of the total IgG of model group, IFN-γ, IL-10 all significantly raises compared with control group;
State detected result and show, stimulate again disease model mice spleen cell by campylobacter jejuni CJ-S131, successfully set up the inflammatory model of in vitro SLE sample performance;
(2) adopt SLE like cell model discrimination medicine
Experiment adopts: SLE model mouse (the SLE original mold type of CJ-S131 induction) mouse; Conventional processing mouse, aseptic technique taking-up spleen is put into the watch-glass of the RPMI-1640 that fills precooling, make spleen single cell suspension and regulate splenocyte concentration, get Tissue Culture Plate, add respectively medicine to be measured, CJ-S131 and splenocyte suspension, add after plate, mix gently, be placed in 37 DEG C, 5% CO
2incubator is cultivated after 48h, centrifugal, draws the cell conditioned medium liquid in every hole, puts into-80 DEG C of Refrigerator stores, for detection of indexs such as anti-dsDNA antibody, IgG, IFN-γ and IL-10 after packing;
(3) anti-dsDNA TPPA:
50 μ g/ml dsDNA coated elisa plates, 4 DEG C are spent the night; PBS-1%BSA sealing, 100 μ l/ holes, hatch 2h for 37 DEG C; PBS-T washing, adds cells and supernatant stoste to be measured, 100ul/ hole, and 35C is hatched 2h; Add the goat anti-mouse igg-HRP of 1:1000 dilution, hatch 2h for 37 DEG C; Add goat anti-rabbit igg-HRP enzyme labelled antibody of 1/1000 dilution, hatch 1h for 37 DEG C; Add OPD substrate solution, 37 DEG C of lucifuges are hatched 20min; Add 2M H
2sO4 termination reaction, with microplate reader 492nm mensuration OD;
Result demonstration, while stimulation, model group is compared with Normal group and adjuvant control group, and the level of anti-dsDNA antibody significantly raises; And in vitro CJ-S131 stimulates after splenocyte again, the level of the anti-dsDNA antibody of model group further raises, and all has significant difference compared with control group; Medicine Bupleurum chinense polysaccharide to be measured (20,40,80ug/ml) can reduce model group is significantly stimulated the high level of the anti-dsDNA antibody causing again by CJ-S131;
(4) total IgG is measured:
10 μ g/ml goat anti-mouse igg coated elisa plates, 4 DEG C are spent the night; Prepare pure mouse IgG typical curve; Take out enzyme plate, its supernatant that inclines, PBS-1%BSA sealing, 100 μ l/ holes, hatch 2h for 37 DEG C; Add respectively pure mouse IgG or the cells and supernatant stoste to be measured of each concentration, 100 μ l/ holes, 35C is hatched 2h, PBS-T washing 5 times, 250 μ l/ holes; Goat anti-mouse igg-the HRP that adds 1:1000 dilution, 100ul/ hole, hatches 2h for 37 DEG C; PBS-T washing 5 times, 300 μ l/ holes; Goat anti-mouse igg-HRP the enzyme labelled antibody that adds 1:5000 dilution, 100 μ l/ holes, hatch 2h for 37 DEG C; PBS-T washing 4 times, 300 μ l/ holes; Add OPD substrate solution, 100 μ l/ holes, 37 DEG C of lucifuges are hatched 20min; Add 2M H
2sO
4termination reaction, 50 μ l/ holes, with microplate reader 492nm mensuration OD;
Result demonstration, while stimulation, total IgG level is no significant difference between model group and control group, and Bupleurum chinense polysaccharide has no significant effect the total IgG level of testing each group; And after campylobacter jejuni CJ-S131 stimulates again in vitro, the level of the total IgG of model group all significantly raises compared with control group, the each concentration of medicine Bupleurum chinense polysaccharide to be measured all can reduce total IgG level significantly, and wherein model group total IgG is almost down to normal level;
(5) IFN-γ measures:
Described cells and supernatant is pressed the method mensuration that IFN-γ test kit specification sheets is recorded; Result demonstration, while stimulation, the level of IFN-γ is no significant difference between model group and control group, and medicine Bupleurum chinense polysaccharide to be measured is also without obvious effect; And after in vitro CJ-S131 stimulates again, the level of the IFN-γ of model group all significantly raises compared with control group, and the each concentration of Bupleurum chinense polysaccharide all can reduce IFN-γ level significantly, and the amplitude maximum that wherein model group IFN-γ level reduces, is almost down to normal level;
(6) IL-10 measures:
Described cells and supernatant is pressed the method mensuration that IFN-γ test kit specification sheets is recorded; Result demonstration, while stimulation, the level of IL-10 is no significant difference between model group and control group, and medicine Bupleurum chinense polysaccharide to be measured is also without obvious effect; After in vitro campylobacter jejuni CJ-S131 stimulates again, the level of the IL-10 of model group all significantly raises compared with control group, and the each concentration of medicine Bupleurum chinense polysaccharide to be measured all can reduce IL-10 level significantly, filters out the medicine of preventing and treating systemic lupus erythematous.
Case study on implementation 3 is measured the in vitro anti-SLE activity of root of Smallleaf Thorowax polysaccharide
The animal that experiment adopts comprises: SLE model mouse (the SLE original mold type of CJ-S131 induction).Put to death mouse, aseptic technique taking-up spleen is put into the watch-glass of the RPMI-1640 that fills precooling.Be prepared into spleen single cell suspension and regulate splenocyte concentration, getting Tissue Culture Plate, adding respectively medicine to be measured, CJ-S131 and splenocyte suspension; Add after plate, mix gently, be placed in 37 DEG C, 5% CO
2incubator is cultivated after 48h, centrifugal, draws the cell conditioned medium liquid in every hole, puts into-80 DEG C of Refrigerator stores after packing, to be measured
.for detection of indexs such as anti-dsDNA antibody, IgG, IFN-γ and IL-10
;
Adopt Bupleurum chinense polysaccharide to verify that as positive drug this in vitro medicaments sifting model prevents and treats the purposes in systemic lupus erythematous medicine in preparation, experimental result shows, Bupleurum chinense polysaccharide (20,40,80 μ g/ml) can reduce model group is significantly stimulated the high level expression of the anti-dsDNA antibody causing again by CJ-S131; Total IgG measurement result shows, before stimulating again in vitro, Bupleurum chinense polysaccharide has no significant effect the total IgG level of model group and control group, and after stimulating again in vitro, the each concentration of Bupleurum chinense polysaccharide all can reduce total IgG level significantly, the amplitude maximum that wherein model group total IgG level reduces, almost makes total IgG be down to normal level; By detecting SLE relevant cytokine IFN-γ and IL-10, find the secretion level of the inflammation-inhibiting factor that the each concentration of Bupleurum chinense polysaccharide also can significance, make IFN-γ and IL-10 level almost be down to normal level (detected result is as shown in Figure 1,2,3, 4).
The administration of the known root of Smallleaf Thorowax polysaccharide entirety of prior art is evident in efficacy to SLE animal model; On this basis, experimental result of the present invention shows, on this in vitro medicaments sifting model, observe equally the rising that this medicine can suppress to significance SLE related immune index: comprise anti self antibody (anti-dsDNA antibody), total IgG and relevant inflammatory factors (IFN-γ and IL-10); Result shows, described in vitro drug screening method can be used as screening prevents and treats the high flux screening platform of the medicine of systemic lupus erythematous.
Claims (4)
1. the splenocyte model of a sensitized animal, it is characterized in that, the splenocyte model of described sensitized animal, by adopting campylobacter jejuni CJ-S131 induction to set up mouse SLE original mold type, is got model mouse spleen cell, stimulate in vitro with CJ-S131, make the isolated model of SLE sample performance; This isolated model shows SLE related immune index and raises, and described SLE related immune index comprises: anti-dsDNA antibody, total IgG, IFN-γ and IL-10.
2. the preparation method of the splenocyte model of sensitized animal claimed in claim 1, is characterized in that, it comprises step:
Adopt campylobacter jejuni CJ-S131 inducing mouse to produce SLE sample symptom, process according to a conventional method experiment mice, under aseptic condition, prepare its spleen single cell suspension; In culture plate, add campylobacter jejuni CJ-S131, again stimulate the splenocyte of sensitization, make the splenocyte model of sensitized animal.
3. the splenocyte model of sensitized animal claimed in claim 1 is prevented and treated the purposes in systemic lupus erythematous medicine in screening.
4. by purposes described in claim 3, it is characterized in that, described screening method comprises step:
(1) set up SLE related immune abnormal cells model:
Adopt laboratory animal: the SLE sample model mice of normal control mouse, adjuvant contrast mouse, CJ-S131 induction; Ordinary method is processed experiment mice, the watch-glass of the RPMI-1640 that fills precooling is put into the experiment mice spleen of acquisition in aseptic technique, make spleen single cell suspension and regulate splenocyte concentration, get Tissue Culture Plate, add respectively CJ-S131 and splenocyte suspension, after splice, mix, be placed in 37 DEG C, 5% CO
2incubator is cultivated 48h; After cultivation finishes, centrifugal, draw the cell conditioned medium liquid in every hole, packing, puts into-80 DEG C of Refrigerator stores, for subsequent use;
(2) adopt SLE like cell model discrimination medicine
Adopt the SLE sample model mice of laboratory animal: CJ-S131 induction; Ordinary method is processed experiment mice, the watch-glass of the RPMI-1640 that fills precooling is put into the experiment mice spleen of acquisition in aseptic technique, make spleen single cell suspension and regulate splenocyte concentration, get Tissue Culture Plate, add respectively medicine to be measured, CJ-S131 and splenocyte suspension, after splice, mix, be placed in 37 DEG C, 5% CO
2incubator is cultivated after 48h, centrifugal, draws the cell conditioned medium liquid in every hole, puts into-80 DEG C of Refrigerator stores after packing, detects anti-dsDNA antibody, IgG, IFN-γ and IL-10 index;
(3) anti-dsDNA TPPA:
50 μ g/ml dsDNA coated elisa plates, 4 DEG C are spent the night; PBS-1%BSA sealing, 100 μ l/ holes, hatch 2h for 37 DEG C; PBS-T washing, adds cells and supernatant stoste to be measured, 100ul/ hole, and 35C is hatched 2h; Add the goat anti-mouse igg-HRP of 1:1000 dilution, hatch 2h for 37 DEG C; Add goat anti-rabbit igg-HRP enzyme labelled antibody of 1/1000 dilution, hatch 1h for 37 DEG C; Add OPD substrate solution, 37 DEG C of lucifuges are hatched 20min; Add 2M H
2sO4 termination reaction, with microplate reader 492nm mensuration OD;
(4) total IgG is measured:
10 μ g/ml goat anti-mouse igg coated elisa plates, 4 DEG C are spent the night; Prepare pure mouse IgG typical curve; Take out enzyme plate, its supernatant that inclines, PBS-1%BSA sealing, 100 μ l/ holes, hatch 2h for 37 DEG C; Add respectively pure mouse IgG or the cells and supernatant stoste to be measured of each concentration, 100 μ l/ holes, 35C is hatched 2h, PBS-T washing 5 times, 250 μ l/ holes; Goat anti-mouse igg-the HRP that adds 1:1000 dilution, 100ul/ hole, hatches 2h for 37 DEG C; PBS-T washing 5 times, 300 μ l/ holes; Goat anti-mouse igg-HRP the enzyme labelled antibody that adds 1:5000 dilution, 100 μ l/ holes, hatch 2h for 37 DEG C; PBS-T washing 4 times, 300 μ l/ holes; Add OPD substrate solution, 100 μ l/ holes, 37 DEG C of lucifuges are hatched 20min; Add 2M H
2sO
4termination reaction, 50 μ l/ holes, microplate reader 492nm measures OD;
(5) measure cells and supernatant IFN-γ;
(6) measure cells and supernatant IL-10; Screening obtains preventing and treating the medicine of systemic lupus erythematous.
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Application publication date: 20141119 |