CN101633906A - Method for establishing endothelial crossing model for asthmatic serum dendritic cell - Google Patents

Abstract

The invention relates to a method for establishing an endothelial crossing model for asthmatic serum dendritic cells. The method comprises the following steps: adopting Ia centrifugation method to obtain serums, an immunomagnetic bead separation to obtain monocytes, and a trypsin digestion method to obtain human umbilical venous endothelial cells; pretreating the serums, the monocytes and the human umbilical venous endothelial cells with gelatin, adding passage endothelial cell suspension into a culture plate to grow into a single layer, suspending asthmatic patient serums on the monocytes to be added on the endothelial cell single layer, fully washing the endothelial cell single layer, and recovering suspension cells to obtain the endothelial crossing model for the asthmatic serum dendritic cells. The invention overcomes the defect that an experiment for truly simulating differentiation maturation and migration functions of the monocytes in the body of an asthmatic patient can not be carried out because of one experiment platform short. The invention provides an in vitro model depended by non cytokines for objectively simulating the monocytes to migrate and differentiate in the body to become the dendritic cells, and in addition, the asthmatic serums are a true internal environment of the patient.

Description

Set up the method for endothelial crossing model for asthmatic serum dendritic cell

Technical field

The present invention relates to a kind of medical science model, particularly a kind of method of setting up endothelial crossing model for asthmatic serum dendritic cell.

Background technology

Asthma is CD4 ⁺The air flue anaphylactic disease that T is cell-mediated, wherein (eosinophil EOS) soaks into and to be significant pathological characteristics eosinophilic granulocyte, and II type helper T cell (Th2) immunity is hyperfunction then to be most crucial immunological abnormality.(dendritic cell DC) is the startup person and the Sustainer of immunne response to dendritic cell, brings into play keying action in asthma Th2 immunological drift process.

Monocyte is scavenger cell and DC common precursor cell, can directed differentiation be DC under multiple exogenous cytokine induction.In granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-4 (IL-4) classical formalism, monocyte needed 5 days just can be divided into immature DC approximately, continue give ripe inductor lipopolysaccharides (LPS) or tumor necrosis factor alpha (TNF-α) stimulate 2 days after differentiation and maturation.

Before the present invention, though the cytokine environment is given the ability of monocyte directed differentiation, often also changed simultaneously function and the migration potentiality thereof of DC, and with body in actual cytokine concentration greatly differ from each other.Therefore, lack the model of an experiment porch, can't really simulate the experiment of monocyte at intravital differentiation and maturation of asthmatic patient and shift function.

Summary of the invention

Purpose of the present invention just is to overcome above-mentioned defective, design, a kind of method of setting up endothelial crossing model for asthmatic serum dendritic cell for experiment usefulness of development.

Technical scheme of the present invention is:

Set up the method for endothelial crossing model for asthmatic serum dendritic cell, its major technique step is:

(1) adopts centrifuging, obtain serum from the asthmatic patient peripheral blood;

(2) adopt immunomagnetic beads method, obtain monocyte from normal people's peripheral blood;

(3) adopt trypsin digestion, obtain Human umbilical vein endothelial cells from normal puerpera's umbilical cord;

(4) gelatin pre-treatment 24 porocyte culture plates add the endotheliocyte suspension that goes down to posterity culture plate and grow to individual layer;

(5) the resuspended monocyte of asthmatic patient serum, and add on the endothelial cell monolayer;

After (6) 2 hours, the thorough washing endothelial cell monolayer;

After (7) 36 hours-48 hours, the washing endothelial cell monolayer reclaims suspension cell;

(8) obtain endothelial crossing model for asthmatic serum dendritic cell.

Advantage of the present invention and effect be endothelial crossing model be unique so far objective simulation monocyte in vivo migration and differentiation be the external model that the acellular factor of dendritic cell relies on, and asthmatic serum be the patient real in environment.Therefore, purpose of the present invention just is to overcome the defective of above-mentioned classical formalism, endothelial crossing model with best simulation dendritic cell migration and differentiation is a point of penetration, utilize environment in the objective simulation body of the asthmatic patient serum serum, set up a kind of method of endothelial crossing model for asthmatic serum dendritic cell of novelty.

Advantage of the present invention and effect also are to have set up asthmatic patient serum dendritic cell endothelial crossing model.Discovered already by this model that asthmatic patient serum can cause dendritic cell nf κ B (NF-κ B) excessive activation, promote the differentiation of dendritic cells maturation.By this model, not only can further investigate effect and mechanism that asthmatic serum promotes the unusual differentiation of dendritic cell from now on, annotate the immunology pathogenesis of asthma, and can study or screen the novel treating asthma medicine of target dendritic cell, as the experiment porch of preparation and checking anti-asthmatic medicament mechanism of action.

Other advantages of the present invention and effect will go on to say below.

Description of drawings

The structure of Fig. 1---asthmatic serum dendritic cell endotheliocyte crossing model and evaluation synoptic diagram.

Contrary phenotype and the morphological change synoptic diagram that passes through front and back of monocyte endothelium under the environment in Fig. 2---the asthmatic serum.

Embodiment

The method of setting up endothelial crossing model for asthmatic serum dendritic cell is as follows:

(1) adopt immunomagnetic beads method to obtain monocyte from human peripheral;

(2) obtain normal puerpera's fresh umbilical cord (delivery room take out 4 hours in), trypsin digestion (0.25% pancreatin) obtains the Human umbilical vein endothelial cells in normal puerpera's umbilical vein source, and best digestion time is 20 minutes, and primary cultured cell 70% goes down to posterity when merging;

(3) after endothelial cells cultured grew up to individual layer, phase microscope was observed endotheliocyte down and is paving stone sample outward appearance, endotheliocyte endochylema people VIII factor stained positive under the fluorescent microscope;

(4) 0.1% gelatin pre-treatment 24 porocyte culture plates, the endotheliocyte suspension of (s-generation) of will going down to posterity adds culture plate and grows to individual layer, add the monocyte in asthmatic patient serum and normal people's peripheral blood source then, cma staining dynamic observes monocyte and passes through endothelial cell monolayer;

After (5) 2 hours, thorough washing endothelial cell monolayer (be intended to remove the endotheliocyte top monocyte that forward passes through does not take place);

After (6) 36 hours-48 hours, the washing endothelial cell monolayer, (forward has promptly taken place to be passed through with reverse and passes through to reclaim suspension cell, get back to the dendritic cell of the cells of monocytic origin of endotheliocyte top), Jim Sa (Giemsa) dyeing, scanning electron microscope (SEM) and flow cytometry (FCM) are identified dendritic cell.

Hence one can see that, the present invention is the monocyte in human peripheral source that will obtain from human body and Human umbilical vein endothelial cells as intermediate result and the method handled, provides reliable technique platform or method for further analyzing asthma immunology pathogenesis and developing novel treating asthma medicine.

As shown in Figure 1, A-D: phosphate buffered saline buffer (PBS) perfusion human umbilical vein's inwall (endotheliocyte closely sticks), pancreatin pours into 10 minutes (loose sticking), 20 minutes (major part comes off, best digestion time), (excessively digestion) (HE dyeing * 400) in 30 minutes respectively; E-F: endotheliocyte is typical paving stone sample monolayer alignment (phase microscope * 100), the endotheliocyte endochylema VIII factor dyeing green fluorescence (indirect IF staining * 800) of expressing strong; G: monolayer endothelial cell; H: monocyte passed through back 0.5 hour, and more monocyte (red arrow shows silver-colored particle attached cell) is attached on the endotheliocyte gap, and black arrow is shown the silver mirror phenomenon; I: passed through back 1 hour, the silver-colored particle attached cell that is attached on the endotheliocyte gap falls sharply; J: passed through back 2 hours, subcutaneous hypothallus in a large amount of cells (blue arrow does not have silver-colored particle and adheres to) enter, the silver-colored particle attached cell (cma staining * 200) that is passing through is on a small quantity still seen in the endotheliocyte gap; K: dendritic cell is typical dendron shape outward appearance (scanning electron microscope * 7000); L-M: asthma group CD80[(90.8 ± 4.5) %] and CD86[(93.7 ± 4.0) %] all being significantly higher than normal group [(39.0 ± 5.9) % and (36.3 ± 7.1) %], dotted line is a normal group, solid line is an asthma group, n=6.

As shown in Figure 2, a: flow cytometry shows the low CD14 of expression of the monocyte (being dendritic cell) after contrary the passing through, but high expression level CD11, CD80, CD86, CD83 and HLA-DR.B: monocyte (0 hour) passes through in asthmatic serum stimulation subinverse and is divided into typical dendritic cell (Giemsa dyeing, * 400).Selected for use once as representative in five experiments.

We discover recently, in and asthmatic patient serum il-4, can suppress that monocyte prove that to the differentiation of dendritic cells maturation IL-4 is that asthmatic serum promotes the important cytokine that dendritic cell is broken up unusually in the endothelial crossing model.And target (selectivity) suppresses the ripe and stimulation T cell proliferation function of differentiation of dendritic cells that nf κ B can significantly reduce endothelial crossing model asthmatic serum inductive cells of monocytic origin.This provides new experimental model for the immunology pathogenesis of further studying asthma, and provides new intervention target spot for developing and screen New Antiasthma.In addition, this model also provides new thinking and strategy for other immunological disease (as systemic lupus erythematous) of research dendritic cell mediation.

The protection domain of request of the present invention is not limited only to the description of present embodiment.

Claims (1)

1. set up the method for endothelial crossing model for asthmatic serum dendritic cell, its step is:

(1) adopts centrifuging, obtain serum from the asthmatic patient peripheral blood;

(2) adopt immunomagnetic beads method, obtain monocyte from normal people's peripheral blood;

(3) adopt trypsin digestion, obtain Human umbilical vein endothelial cells from normal puerpera's umbilical cord;

(4) gelatin pre-treatment 24 porocyte culture plates add the endotheliocyte suspension that goes down to posterity culture plate and grow to individual layer;

(5) the resuspended monocyte of asthmatic patient serum, and add on the endothelial cell monolayer;

After (6) 2 hours, the thorough washing endothelial cell monolayer;

After (7) 36 hours-48 hours, the washing endothelial cell monolayer reclaims suspension cell;

(8) obtain endothelial crossing model for asthmatic serum dendritic cell.

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