CN101570739A - Method for building psoriasis basic research models, and gas-liquid level transmembrane device - Google Patents
Method for building psoriasis basic research models, and gas-liquid level transmembrane device Download PDFInfo
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Abstract
The invention provides a method for building psoriasis basic research models, and a gas-liquid level transmembrane device. The method utilizes a psoriasis streptococcal-induced mechanism and a T cell-mediated excessive proliferation mechanism, innovatively adopts a serum-free medium added with psoriasis peripheral blood T lymphocytes and hemolytic streptococcus antigens, and cultures psoriasis skin lesions in vitro. Characteristic tissue morphology of the psoriasis skin lesions can be maintained for as long as one week. The gas-liquid level transmembrane device used for culturing psoriasis in vitro skin lesions comprises the serum-free medium containing psoriasis peripheral blood T cells and the hemolytic streptococcus antigens in the method for building psoriasis basic research models, as well as sterilizing lens-wiping paper for manufacturing gas-liquid level, wherein tissue blocks and a gas phase are borne on the sterilizing lens-wiping paper; the gas phase is at 37 DEG C with 5 percent of CO2 saturation. The device not only can completely simulate gas-liquid junction environment for human skin growth, but also can reach the requirements of in vitro skin tissue culture. In addition, the device is easy to prepare, saves expenses, and has the advantages of repeatability, controllability and the like.
Description
Technical field
The invention belongs to the field of setting up of experimental model.
Background technology
Along with more and more deeply and extensive studies is explored, be the focus that academia pays close attention to as the psoriatic external model of experiment basis to psoriatic always.On the one hand, people are devoted to the research of psoriatic mouse model: play people as far back as the sixties in last century and just begun to utilize ultraviolet ray, chemicals to bring out and set up the mouse psoriasis model.Yet this model has only been simulated psoriatic certain particular pathologies, and exists the of short duration defective of holding time.
The seventies, Krueger etc. successfully was transplanted to people's psoriatic lesion on the nude mice first, but Baker etc. assess cutify, find that most of psoriatic features can not keep for a long time after transplanting, can provide cytokine because of mouse body itself simultaneously, influence experiment effect.Keep for a long time, can provide cytokine because of mouse body itself simultaneously, influence experiment effect.
People are devoted to study spontaneous animal model in recent years, become mouse, flaky skin sudden change mouse as no chalaza, but cost an arm and a leg, and have artificial orientation's property; Bonder etc. utilizes outgrowth mouse vagina epithelium of oestrogenic hormon phase and Jarrett to utilize mouse tail scale epidermis to carry out the research of curative effect of medication in addition, but it is complete to simulate psoriatic hyperproliferative epidermal and epidermal differentiation, can not react relevant unusual of psoriatic pathological change, particularly T cell comprehensively.
On the other hand, along with the progress of Protocols in Molecular Biology, people attempt to use the external associating cultured method of various kinds of cell and make up the organizational project model, but because of technical sophistication, expense costliness suitability not strong.
Summary of the invention
For these reasons, we are devoted to simulate fully the possible pathogenesis of human body psoriatic, and the vitro culture psoriatic lesion is set up a kind of external realistic model of psoriatic of economical and convenient practicality, is used for psoriatic basis, laboratory and clinical study.
Technical conceive of the present invention is: the mechanism of utilizing psoriatic suis abduction mechanism and the cell-mediated hyper-proliferative of T, 1. the employing of novelty adds the psoriatic periphery blood T lymphocyte and reaches the 2. antigenic serum free medium of Hemolytic streptococcus, vitro culture psoriatic lesion.Can keep psoriatic lesion characteristic tissue morphology and reach week.
The present invention is implemented by following technique means:
One, a kind of establishment method of psoriatic fundamental research model
1. draw materials:
1. skin sample: untreated psoriasis vulgaris skin decreases in half a year;
2. psoriatic peripheral blood: corresponding psoriatic's peripheral blood 2mL, aseptic vacuum anticoagulant blood-collecting pipe is gathered, and the collection of blood sample and all skin samples is all obtained patient's informed consent and is ratified through Ethics Committee;
3. beta hemolytic streptococcus.
2. material processing and transportation
The untreated psoriasis vulgaris skin of the about 1.5cm * 1.5cm of size is decreased, remove sebum and part corium, put into the 1.5mL centrifuge tube of William ' the s substratum 200ul that contains 10% penicillin, Streptomycin sulphate, be transported to the laboratory in 4 ℃ of 30min; Be transported to the laboratory in 37 ℃ of 30min of blood sample.
3.T lymphocyte separates
With corresponding psoriatic's peripheral blood 2mL, with 1: 1 mixing of PBS volume ratio, carefully be added on the 4mL lymphocyte separation medium, 1500 rev/mins of centrifugal 20min get number second layer ring-type oyster white buffy coat, the PBS washed twice, 24 orifice plates are cultivated, and utilize to adhere to adherent partition method, remove monocyte and part bone-marrow-derived lymphocyte, collect suspension cell, density 10
6Individual/mL is in William ' s culture medium culturing.
4. beta hemolytic streptococcus antigen prepd
With beta hemolytic streptococcus in Colombia's blood agar 37 ℃ cultivated 24 hours.The method of plate culture count counting, density 10
9Individual/m, 12000 rev/mins of nutrient solutions are centrifugal, and bacterial sediment is collected in the rearmounted distilled water, ice bath, and 40W * 8 minutes three times are ultrasonic breaks up the precipitated liquid bacterium, and 25000 rev/mins of centrifugal collections bacterium bacterial sediments that loose are weighed, and-20 ℃ of preservations are standby.
5. simple and easy skin histology gas-liquid culture model preparation
Aseptic six orifice plates, every hole splash into 2mL William ' s substratum, place diameter through sterilising treatment and be the circular lens wiping paper of 2cm thereon.External psoriatic lesion is made simple and easy culture dish such as Fig. 1 by oneself.
6. skin bit is handled
In super clean bench the skin damage evenly is cut into 3mm * 3mm skin bit, with containing 10% pair of anti-PBS washing 3 times, each 2min puts down gently on the loading end of ready culture apparatus, and epidermis is exposed in the air, and corium is in the liquid phase.Add psoriatic periphery blood T lymphocyte, the antigenic William ' s of beta hemolytic streptococcus substratum, in 37 ℃, CO
2Saturation ratio is to cultivate in 5% incubator, the next day change liquid.
Untreated psoriasis vulgaris skin described in the step 2 decreases, and obtains under aseptic technique.
Beneficial effect
Use the model of above method preparation to possess following characteristics:
(1) adopt serum-free culture, can not be subjected to the interference of cytokine and other chemicals in the conventional animal serum product, purifying culture environment;
(2) according to present supposition, add psoriatic self periphery blood T lymphocyte and human disease's property beta hemolytic streptococcus antigen, simulate environment in psoriatic's health relatively onset of psoriasis mechanism.Meanwhile, the success of psoriatic lesion vitro culture under this condition further illustrates the direct relation of psoriatic peripheral blood lymphocyte abnormality proliferation and streptococcal infection and onset of psoriasis again;
(3) incubation period reaches 6 days, has guaranteed skin is decreased the requirement that model self is observed the cycle, provides the competent relatively time to carrying out drug screening and other tests future simultaneously.
Two, the simultaneously claimed air fluid level that is used for the cultivation of the external skin damage of psoriatic of the inventor is striden film device
A kind of air fluid level is striden film device, be that the air fluid level that is used for the cultivation of the external skin damage of psoriatic is striden film device, comprise as one, described in a kind of establishment method of psoriatic fundamental research model contain the psoriatic periphery blood T cell and the antigenic serum free medium of Hemolytic streptococcus, sterilization lens wiping paper are made air fluid level, carrying tissue block and 37 ℃ on the sterilization lens wiping paper, CO
2Saturation ratio is 5% gas phase.
According to above-mentioned psoriatic periphery blood T cell and the antigenic serum free medium of Hemolytic streptococcus of containing, the lens wiping paper of will sterilizing earlier floats on the substratum liquid level, utilize it to make air fluid level again---on the sterilization lens wiping paper, carry tissue block, tissue block corium is in the liquid phase, epidermis is exposed in the gas phase, gaseous environment is 37 ℃, CO
2Saturation ratio is 5%.
Beneficial effect
This device not only can be simulated the gas-liquid boundary environment of human body skin growth fully, also can reach the requirement of vitro skin tissue culture, and this device prepares simplely, reduces expenses, and possesses repeatability, advantages such as controllability.
Description of drawings
The present invention has accompanying drawing 10 width of cloth,
The external psoriatic lesion of Fig. 1 is made simple and easy culture dish synoptic diagram by oneself;
In the drawings: 1,37 ℃, CO
2Saturation ratio is 5% gas phase; 2, the sterilization lens wiping paper is made air fluid level, the carrying tissue block; 3, contain the antigenic serum free medium of psoriatic periphery blood T cell and Hemolytic streptococcus.
Fig. 2 is that external psoriatic lesion is cultivated the naked eyes sight;
External cultivation psoriatic tissue (HE * 100) under Fig. 3 light microscopic;
External cultivation psoriatic lesion tissue under Fig. 4 Electronic Speculum;
Fig. 5 vitro culture psoriatic lesion histogenic immunity group dyeing (* 200);
Fig. 6 SDS-PAGE detects molecular weight of albumen;
Fig. 7 Western blotting half-quantitative detection K17;
Fig. 8 different tissues, the different culture condition homogenate IL-8 of undertissue content curve synoptic diagram;
Fig. 9 different tissues, the different culture condition homogenate INF-γ of undertissue content curve synoptic diagram;
Figure 10 different tissues, the different culture condition homogenate TNF-of undertissue alpha content change curve synoptic diagram.
Embodiment
1. draw materials: 1. skin sample is collected: get untreated psoriasis vulgaris skin damage in half a year; 2. the psoriatic peripheral blood is gathered: get corresponding psoriatic's peripheral blood 2mL, aseptic vacuum anticoagulant blood-collecting pipe is gathered, and the collection of blood sample and all skin samples is all obtained patient's informed consent and ratified through Ethics Committee; 3. beta hemolytic streptococcus
2. material processing and transportation: get skin under the aseptic technique and decrease, the about 1.5cm * 1.5cm of size removes sebum and part corium, puts into the 1.5mL centrifuge tube of William ' the s substratum 200ul that contains 10% penicillin, Streptomycin sulphate, is transported to the laboratory in 4 ℃ of 30min; Be transported to the laboratory in 37 ℃ of 30min of blood sample.
3.T lymphocyte separates: get fresh anticoagulation 2mL, with the PBS1:1 mixing, carefully be added on the 4mL lymphocyte separation medium, 1500 rev/mins of centrifugal 20min get number second layer ring-type oyster white buffy coat, the PBS washed twice, 24 orifice plates are cultivated, and utilize to adhere to adherent partition method, remove monocyte and part bone-marrow-derived lymphocyte, collect suspension cell, density 10
6Individual/mL is in William ' s culture medium culturing.
4. beta hemolytic streptococcus antigen prepd: 37 ℃ of overnight incubation in the Todd-Hewitt substratum.The method of plate culture count counting, density 10
9Individual/m, 12000 rev/mins of nutrient solutions are centrifugal, and bacterial sediment is collected in the rearmounted distilled water, ice bath, and 40W * 8 minutes three times are ultrasonic breaks up the precipitated liquid bacterium, and 25 000 rev/mins of centrifugal collections bacterium bacterial sediments that loose are weighed, and-20 ℃ of preservations are standby.
5. the preparation of simple and easy skin histology gas-liquid culture model: aseptic six orifice plates, every hole splash into 2mL different condition substratum, place diameter through sterilising treatment and be the circular lens wiping paper of 2cm thereon.
6. skin bit is handled: in super clean bench the skin damage evenly is cut into 3mm * 3mm skin bit, with containing 10% pair of anti-PBS washing 3 times, each 2min puts down gently on the loading end of ready culture apparatus, and epidermis is exposed in the air, and corium is in the liquid phase.Add psoriatic periphery blood T lymphocyte, the antigenic William ' s of beta hemolytic streptococcus substratum, in 37 ℃, CO
2Saturation ratio is to cultivate in 5% incubator, the next day change liquid.Get tissue respectively at the 0th day, 3 days, 6 days and carry out paraffin section, HE dyeing, om observation and 3% glutaraldehyde, two fixing, the transmission electron microscope observings of 1% osmic acid.
7. immunohistochemical methods detects Keratin sulfate 17: before picked at random is cultivated and cultivate paraffin-embedded tissue and normal epidermis paraffin-embedded tissue after 6 days, cut into slices and carry out (Max Vision
TMQuick Immunohistochemical Staining) immunohistochemical staining.Concrete operations are as follows: the paraffin section de-waxing aquation, and citrate buffer solution High Temperature High Pressure antigen retrieval adds anti-(mouse-anti human keratinous 17 a monoclonal antibodies) incubated at room 60 minutes, and PBS washes 3 times, and each 3 minutes, remove PBS, add Max Vision
TM, to hatch under the room temperature 15 minutes, PBS washes 3 times, removes PBS, adds new system DAB solution 5 minutes, the tap water flushing, Hematorylin is redyed, the tap water flushing, the gradient alcohol dehydration drying, dimethylbenzene is transparent, the neutral gum mounting.The result judges with reference to methods such as Garcia
[4], the visual field counting of 10 same magnifications is got in every section at random, and the sxemiquantitative classification: 1. the cell brownish discoloration is 1, and the cell Vandyke brown is colored as 2; 2. positive painted cell proportion is 1,20% ~ 50% to be 2 less than 20%, is that 3. above two additions equal 2 ~ 3 and are the weak positive (+) greater than 50%, 3 ~ 4 positive (++), and＞4 is strong positive (+++).
8.Western detecting, decreases with the skin of cultivating 6 days before Keratin sulfate 17:II group picked at random is cultivated Western blotting, RIPA lysate homogenate extracts albumen, Bradford method protein quantification, the equivalent application of sample, 1 swimming lane adds normal control, 2,3 swimming lanes add cultivates the front and back sample, other repeats portion, and to be used for confidential reference items quantitative, adding blueness simultaneously dyes low molecular weight protein (LMWP) Marker in advance K17 is carried out sxemiquantitative, the Tricine-SDS-PAGE electrophoretic separation, half-dried commentaries on classics film, 4 ℃ of sealings of 5% skim-milk are spent the night, and add mouse-anti people K17 monoclonal antibody successively and spend the night for 4 ℃, another group adds mouse anti human β-Actin antibody as confidential reference items, two anti-horseradish enzyme labelling goat anti-mouse IgG antibody incubated at room temperature 1 ~ 2 hour, ECL colour developing, X-ray film compressing tablet, develop, photographic fixing.
9.ELISA detect cytokine IL-8, TNF α, INF γ content: before will cultivating, cultivated 3 days, the psoriatic lesion of cultivating 6 days takes out, and it is standby that-20 ℃ of preservation in back are done in the filter paper exhibition, treat that herbarium together after, respectively under 4 ℃ of conditions, utilize pressure-even pulp crusher will organize fully to grind and make homogenate, centrifugal 10 minutes of 10000 rev/mins of refrigerated centrifuges are got supernatant, utilize import ELISA test kit to detect each cytokine absorption photometric value and be converted into concentration, tabulation is analyzed.
Examining report
This detection content is that the model that makes according to the establishment method of the fundamental research of psoriatic described in summary of the invention model carries out.
In following content, W represents simple serum free medium; The serum free medium of the T lymphocyte composition that W+C representative adding psoriatic peripheral blood is separated; The T lymphocyte that W+C+A representative adding psoriatic peripheral blood is separated and the serum free medium of beta hemolytic streptococcus antigenic component; Digitized representation after W, W+C, the W+C+A is cultivated fate.
One. techtology is observed
1. visual inspection
According to the pre-experience that we cultivate skin histology a large amount of early stages, it is glossy that the external skin histology that survives roughly shows as surface wettability, and it is yellowish pink that color is, the nutrient solution clarification; And the many shrinkages of skin histology outward appearance out of order, color is gloomy or to present stratum basale loose, and color is pale.Simple serum-free culture (W) is compared with the psoriatic lesion cultivation that the serum free medium (W+C+A) that adds T lymphocyte that psoriatic's peripheral blood separates and beta hemolytic streptococcus antigenic component is cultivated, in appearance, it is gloomy that epidermis shows slightly, there is shrinkage at the edge, and latter's epidermis is more ruddy; Cultivate to decrease with the skin of cultivating six days in three days and compare, in appearance, it is tender that epidermis shows slightly water, glossyly (sees Fig. 2 a, b, c, d, e; Fig. 2 is that external psoriatic lesion is cultivated naked eyes sight 2a: the vitro tissue culture model; 2b:W3 days, visible skin decreased peripheral shrinkage, colour-darkening; 2c:W+C+A3 days, outward appearance was ruddy; 2d:W6 days, the visible epidermis shrinkage of outward appearance, greyish white; 2e:W+C+A6 days, epidermis was coarse, and dermatoglyph is dark, still kept yellowish pink, the water profit).
2.HE dyeing
The tissue of simple serum-free culture (W), along with the prolongation of vitro culture time, pathology table shape differs from the psoriatic performance, double-deck stratum corneum can occur, acantholysis, epidermis dermis separates, and inflammatory cell reduces.And the psoriatic lesion that the serum free medium (W+C+A) of T lymphocyte that adding psoriatic peripheral blood is separated and beta hemolytic streptococcus antigenic component is cultivated is cultivated after 3 days, 6 days, tissue morphology still keeps the histopathology feature of psoriasis vulgaris, stratum corneum thickens, hyperkeratosis companion parakeratosis, between loose parakeratotic cell, be mingled with the clearance; In angleplied laminate or under the angleplied laminate, a large amount of neutrophil infiltration are arranged; Granular layer attenuation or disappearance, acanthosis, epidermal ridge prolong (sees Fig. 3 a, b, c, d; Fig. 3 is an external cultivation psoriatic tissue (HE * 100) under the light microscopic; 3a:W3 days, the stratum corneum hyperkeratosis, inflammatory cell reduces; 3b:W+C+A3 days hyperkeratosiss, granular layer disappears, and acanthosis, epidermis extension downwards are the rostellum shape; 3c:W6 days acantholysis have the crack in the epidermis; 3d:W+C+A6 days hyperkeratosis companion parakeratosis, the visible down a large amount of neutrophil infiltration of angleplied laminate).
3. electron microscopic observation
Before cultivating and add psoriatic lesion that the serum free medium of T lymphocyte that psoriatic's peripheral blood separates and beta hemolytic streptococcus antigenic component cultivates and cultivate and organize transmission electron microscope under observation after 6 days both all present psoriatic ultrastructural characteristics [12], showing as basal layer of epidermis changes little, be mainly the corneocyte apoptosis, a large amount of on every side fat drip; The granular layer cell mitochondrial increases; The broadening of stratum spinosum epidermidis intercellular substance, rrna especially polysome increase, and desmosome reduces, and the tension force silk is thin.No significant difference (is seen Fig. 4 a, b, c, d, e, f before and after cultivating; Fig. 4 organizes 4a and 4d for external cultivation psoriatic lesion under the Electronic Speculum: skin decreases and W+C+A6 days before cultivating, and all presents the keratinocyte layer, the keratinocyte apoptosis, and a large amount of on every side fat drip (* 12000); 4b and 4e: skin decreases and W+C+A6 days before cultivating, and all presents granular layer, and plastosome increases (* 7500); 4c and 4f: skin decreases and W+C+A6 days before cultivating, and all presents intercellular substance broadening, and rrna especially polysome increases, and desmosome reduces, tension force silk thin (* 6000)).
Two. identify the specific expressed of psoriatic lesion Keratin sulfate 17
1. immunohistochemical methods detects K17
K17 was at epidermis during the psoriatic lesion cultivation that the T lymphocyte that cultivation is preceding and adding psoriatic peripheral blood is separated and the serum free medium (W+A+C) of beta hemolytic streptococcus antigenic component are cultivated was organized in 6 days, especially all being strong positive in the acantholysis cell endochylema expresses, the cell color is brown or Vandyke brown (is seen Fig. 5 a, the former is 5a for b, and the latter is 5b).
Do not see K17 to express before normal people's epidermis is cultivated and cultivate after six days as seen through the serum free medium (W+A+C) that adds T lymphocyte that psoriatic's peripheral blood separates and beta hemolytic streptococcus antigenic component, face tissue is brown xanthochromia and (sees Fig. 5 c, d, the former is 5c, and the latter is 5d).Fig. 5 is vitro culture psoriatic lesion histogenic immunity group dyeing (* 200); 5a: K17 expresses at spinose cell's endochylema strong positive before cultivating; K17 was at spinose cell's endochylema high expression level in 5b:W+C+A6 days; 5c: normal people's epidermis spinose cell does not see that K17 expresses; Organized xanthochromia after cultivating in 5d: healthy tissues W+C+A6 days; 5e: rodent cancer blank; 5f: rodent cancer positive control.
With reference to people's such as Garcia decision method, organize positive cell ratio and color after the dyeing of inverted microscope sem observation, press grade scoring, statistics different grades case quantity (seeing Table 1).With before cultivating and add the group coloration result that psoriatic lesion that the serum free medium of T lymphocyte that psoriatic's peripheral blood separates and beta hemolytic streptococcus antigenic component cultivates cultivated 6 days and relatively carry out statistical analysis with normal control respectively, the tool significant difference, P＜0.01; The immunohistochemical methods result of psoriatic group before and after cultivating is compared unknown significance difference, P＞0.05; The immunohistochemical methods result of normal skin before and after cultivating is compared, have significant difference, P＜0.01 (seeing Table 1).
The expression (routine number) of k17 in the psoriatic lesion epidermis of table 1. vitro culture
Annotate: P represents psoriatic lesion; N represents normal skin; * compared P＜0.01 with W+C+A6 days with normal control before cultivating; Before cultivating, the # psoriatic lesion compared P＞0.05 with W+C+A6 days; ﹠amp; The expression normal skin compares P＜0.01 before and after cultivating
2.Western blotting half-quantitative detection K17
Westernblotting detects K17 result before cultivating and after cultivating the back skin in W+C+A6 days and undermining the normal control skin histology and extract albumen, and blueness is dyed low molecular weight protein (LMWP) Marker in advance and K17 molecular weight by estimate is approximately 46kDa (is seen Fig. 6; Fig. 6 SDS-PAGE detects molecular weight of albumen: normal control does not show and the accurate albumen Marker of sighting target of the black stripe correspondence that sample develops roughly infers K17 content), only see before cultivating after the X-ray film imaging and add T lymphocyte that psoriatic's peripheral blood separates and psoriatic lesion cultivation that the serum free medium of beta hemolytic streptococcus antigenic component is cultivated located to occur two consistent obvious bands in 6 days on the sample lane, the brightness indifference of two bands, (see Fig. 7, Fig. 7 Western blotting half-quantitative detection K17 swimming lane 1: the normal skin contrast and normal skin contrast place trace is fuzzy; Swimming lane 2,3: decrease with W+C+A6 days skins of cultivation before cultivating.The top is K17, and the below is β-Actin confidential reference items contrast).Three. cytokine INF-γ, TNF-α, IL-8 content detection
P represents the psoriatic, and N represents normal control, the digitized representation sequence number of P, N back.For example: P1 is the 1st routine psoriatic who randomly draws.
1.ELISA result
Randomly draw three routine psoriatics and two routine normal controls.The psoriatic group, cytokine INF-γ, the equal content of TNF-α, IL-8 significantly increase under the different culture condition, and do not express in the skin histology homogenate before the cultivation of normal control group; After the different group cytokine content of same culture condition are averaged, with normal control not in the cultured skin cytokine content mean value carry out single factor relatively, there is significant difference P＜0.01; Same routine psoriatic lesion, after the T lymphocyte of separating with adding psoriatic peripheral blood before cultivating and the serum free medium of beta hemolytic streptococcus antigenic component are cultivated, cytokine content relatively, there was no significant difference, P＞0.05 proves that content is little, and simple serum-free culture or only add in the psoriatic lesion after T lymphocytes culture medium that psoriatic's peripheral blood separates is cultivated, cytokine content significantly descends, P＜0.05; Normal skin decreases, and after the T lymphocyte that adding psoriatic peripheral blood is separated and the serum free medium of beta hemolytic streptococcus antigenic component are cultivated, INF-γ, TNF-alpha expression occur.
Table 2. tissue homogenate IL-8 (pg/mL) detected result (x ± s)
Annotate: a represents that similar skin decreases not cultivation and cultivates the back relatively; B represents that psoriatic lesion and normal control skin decrease relatively; * represent P＜0.01; # represents P＞0.05
Table 3 tissue homogenate INF-γ (pg/mL) detected result (x ± s)
Annotate: a represents that similar skin decreases not cultivation and cultivates the back relatively; B represents that psoriatic lesion and normal control skin decrease relatively; * represent P＜0.01; # represents P＞0.05
Table 4 tissue homogenate TNF-α (pg/mL) detected result (x ± s)
Annotate: a represents that similar skin decreases not cultivation and cultivates the back relatively; B represents that psoriatic lesion and normal control skin decrease relatively; * represent P＜0.01; # represents P＞0.05
2. utilize EXCEL software to make the cytokine content changing trend diagram of different condition, different cases
From Fig. 8,9,10, we can find out very intuitively that the curve of the cytokine content of psoriatic lesion will be apparently higher than the curve of normal skin; And the cytokine content graphic representation of same case different condition in the time of three days, six days is " W " type trend, explanation is adding the state that T lymphocyte that psoriatic's peripheral blood separates and the psoriatic lesion under the streptococcal antigen culture medium culturing condition have remained the cytokine high expression level, and simple serum-free culture and only adding under the T lymphocyte culture condition, cytokine content significantly reduces.In addition from Fig. 8,9 blue line we as can be seen normal skin cytokine INF-γ, TNF-alpha content have had the fluctuation that makes progress under T lymphocyte that psoriatic's peripheral blood separates and the streptococcal antigen culture medium culturing condition adding, but normal skin is whatsoever cultivated under the condition among Figure 10, and IL-8 is negative all the time to express.
Conclusion: modern technique and method that the present invention adopts modern organ culture institute to use, simulate psoriatic pathogenesis in the human body fully, establish culture condition and utilize technology such as histopathology, immunohistochemical methods that experimental result is identified through experiment screening repeatedly, draw the method for vitro culture psoriatic lesion at last, verified the feasibility of used experimental model and detection index.The method of the vitro culture psoriatic lesion that research obtains, operation is simple, with low cost, avoided the limitation and the unstable of psoriatic animal model in the past.Further detect the index of correlation of Keratin sulfate and cytokine, be used for psoriatic study of pathogenesis and clinical detection.Further exploitation can become a kind of new mode of external psoriasis model, is used for psoriatic infrastest and clinical study.
Claims (3)
1, a kind of establishment method of psoriatic fundamental research model is characterized in that it being to set up according to following step:
(1) draw materials:
1. skin sample: untreated psoriasis vulgaris skin decreases in half a year;
2. psoriatic peripheral blood: corresponding psoriatic's peripheral blood 2mL;
3. beta hemolytic streptococcus;
(2) material processing and transportation
The untreated psoriasis vulgaris skin of the about 1.5cm * 1.5cm of size is decreased, remove sebum and part corium, put into and contain the 1.5mL centrifuge tube of William ' s substratum of reagent 200ul that volume ratio content accounts for the penicillin+Streptomycin sulphate of cumulative volume 10%, be transported to the laboratory in 4 ℃, 30min; Be transported to the laboratory in 37 ℃ of blood samples, the 30min;
(3) the T lymphocyte separates
With corresponding psoriatic's peripheral blood 2mL, with 1: 1 volume ratio mixing of PBS, carefully be added on the 4mL lymphocyte separation medium, 1500 rev/mins of centrifugal 20min get number second layer ring-type oyster white buffy coat, the PBS washed twice, 24 orifice plates are cultivated, and utilize to adhere to adherent partition method, remove monocyte and part bone-marrow-derived lymphocyte, collect suspension cell, density 10
6Individual/mL is in William ' s culture medium culturing;
(4) beta hemolytic streptococcus antigen prepd
With beta hemolytic streptococcus in Colombia's blood agar 37 ℃ cultivated the method for plate culture count counting, density 10 24 hours
9Individual/m, 12000 rev/mins of nutrient solutions are centrifugal, and bacterial sediment is collected in the rearmounted distilled water, ice bath, and 40W * 8 minutes three times are ultrasonic breaks up the precipitated liquid bacterium, and 25000 rev/mins of centrifugal collections bacterium bacterial sediments that loose are weighed, and-20 ℃ of preservations are standby;
(5) simple and easy skin histology gas-liquid culture model preparation
Aseptic six orifice plates, every hole splash into 2mL William ' s substratum, include psoriatic periphery blood T cell density about 10
5Individual/ml and beta hemolytic streptococcus antigen density about 10
6Individual/ml.Placement is that the circular lens wiping paper of 2cm is floating thereon through the diameter of sterilising treatment;
(6) skin bit is handled
In super clean bench, the skin damage evenly is cut into 3mm * 3mm skin bit, with containing 10% pair of anti-PBS washing 3 times, each 2min, put down gently on the loading end of ready culture apparatus, epidermis is exposed in the air, corium is in the liquid phase, adds prepared psoriatic periphery blood T lymphocyte in the step (3), makes its density in 2mL William ' s substratum reach 10
5Individual/ml; Prepared beta hemolytic streptococcus antigen in the step (4) makes its density in nutrient solution reach 10
6Individual/ml, in 37 ℃, CO
2Saturation ratio is to cultivate in 5% incubator, changes liquid in 48～72 hours.
2, the establishment method of psoriatic fundamental research model according to claim 1 is characterized in that: the untreated psoriasis vulgaris skin described in the step (2) decreases, and obtains under aseptic technique.
3, a kind of air fluid level is striden film device, be that the air fluid level that is used for the cultivation of the external skin damage of psoriatic is striden film device, comprise according to described psoriatic periphery blood T cell and the antigenic serum free medium of Hemolytic streptococcus of containing of claim 1, the lens wiping paper of will sterilizing earlier floats on the substratum liquid level, utilize it to make air fluid level again--carry tissue block on the sterilization lens wiping paper, tissue block corium is in the liquid phase, and epidermis is exposed in the gas phase, gaseous environment is 37 ℃, CO
2Saturation ratio is 5%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010823A (en) * | 2010-10-28 | 2011-04-13 | 天津开发区合普工贸有限公司 | Cell culture solution flow supply device |
| CN102010821A (en) * | 2010-09-02 | 2011-04-13 | 天津开发区合普工贸有限公司 | Cell gaseous exposure experiment control device |
| RU2575338C1 (en) * | 2014-12-31 | 2016-02-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Чувашский государственный университет имени И.Н. Ульянова" | Method for simulating psoriasis in experimental animals |
| CN106754698A (en) * | 2016-12-05 | 2017-05-31 | 刘晓明 | A kind of method of separation and the activation for human peripheral T cell |
| CN112870231A (en) * | 2021-02-06 | 2021-06-01 | 澎立生物医药技术（上海）有限公司 | Construction method of psoriasis animal model |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6028245A (en) * | 1997-07-03 | 2000-02-22 | Rhone-Poulenc Rorer Sa | Transgenic animals overexpressing MDM2 |
| EP1557085A1 (en) * | 2004-01-21 | 2005-07-27 | Boehringer Ingelheim International GmbH | Mouse model for human psoriasis |
| JP2009112203A (en) * | 2007-11-02 | 2009-05-28 | Kobe Univ | METHOD FOR SCREENING NEW ANTIINFLAMMATORY DRUG USING PHOSPHOLIPASE Cepsilon AS MOLECULAR TARGET, AND ANIMAL MODEL FOR PSORIASIS VULGARIS-LIKE CHRONIC DERMATITIS |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010821A (en) * | 2010-09-02 | 2011-04-13 | 天津开发区合普工贸有限公司 | Cell gaseous exposure experiment control device |
| CN102010823A (en) * | 2010-10-28 | 2011-04-13 | 天津开发区合普工贸有限公司 | Cell culture solution flow supply device |
| RU2575338C1 (en) * | 2014-12-31 | 2016-02-20 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Чувашский государственный университет имени И.Н. Ульянова" | Method for simulating psoriasis in experimental animals |
| CN106754698A (en) * | 2016-12-05 | 2017-05-31 | 刘晓明 | A kind of method of separation and the activation for human peripheral T cell |
| CN112870231A (en) * | 2021-02-06 | 2021-06-01 | 澎立生物医药技术（上海）有限公司 | Construction method of psoriasis animal model |
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| CN101570739B (en) | 2012-10-03 |
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