CN107988144A - The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury - Google Patents
The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury Download PDFInfo
- Publication number
- CN107988144A CN107988144A CN201711182612.7A CN201711182612A CN107988144A CN 107988144 A CN107988144 A CN 107988144A CN 201711182612 A CN201711182612 A CN 201711182612A CN 107988144 A CN107988144 A CN 107988144A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- cells
- epithelial cells
- pulmonary
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Dermatology (AREA)
- Vascular Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses pulmonary epithelial cells during a kind of simulation acute lung injury and the method for building up of the external model of capillary endothelial cell barrier function, the described method comprises the following steps：The lower floor that the metainfective mouse C3H/10T1/2 microvascular endothelial cells strains of endotoxin are inoculated in cell using Transwell co-culture systems by step (1) cultivates；Small 1 pulmonary epithelial cells strains of mouse TC are inoculated in the upper strata of cell, cell culture supernatant is collected after co-culturing a period of time；Pulmonary epithelial cells/endothelial cell barrier synthesis and the situation of expression inflammatory factor and inflammatory cell infiltration GAP-associated protein GAP in step (2) detection cell culture supernatant, are created as a pulmonary epithelial cells/microvascular endothelial cells co-culture model for being suitable for LPS-induced acute lung injury research.Barrier cell function when the model that the present invention establishes is close to internal LPS-induced acute lung injury, can provide reliable model basis for clinically acute lung injury research.
Description
Technical field
The present invention relates to cell model establishing techniques field, more particularly to pulmonary epithelial cells during a kind of simulation acute lung injury
With the method for building up of the external model of capillary endothelial cell barrier function.
Background technology
LPS-induced acute lung injury (acute lung injury, ALI) clinically common critical illness.The hair of ALI
Interpretation of the cause, onset and process of an illness system is varied, accounts for the overwhelming majority caused by induced by lipopolysaccharide (LPS), can be divided between LPS coup injuries and LPS
Connect damage (proinflammatory/anti-inflammatory response is unbalance, promotees unbalance, the oxidative stress of solidifying/anticoagulant response, born of the same parents' apoptosis disorder disorder etc.), alveolar
One capillary barrier III, causes the release of capillary permeability increase, neutrophil infiltration and inflammatory mediator out of control, and
A series of symptom is induced in damage location aggregation.ALI is that infection, wound etc. cause body inflammatory reaction out of control as a result, scorching
Sex factor plays an important role in the course of disease occurrence and development of ALI.When interleukin (interleukin, IL-1) is with ALI
The infiltration of inflammatory cell is closely related with activating, and IL-6 is both inflammatory factor, while also has anti-inflammatory effect.But zoopery is shown
Show, in ALI, high-caliber IL6 mainly plays inflammatory factor effect in lung tissue, and it is neutral that it can suppress infiltration in lung tissue
The apoptosis of granulocyte, strengthens the inflammatory reaction of intrapulmonary.
Although inflammatory factor plays an important role in the occurrence and development of AL1, in the pathomechanism of ALI also
There is other influence factors --- pulmonary epithelial cells and capillary endothelial cell barrier.Under pathological state, lung epithelial is thin
Born of the same parents have the function that to prevent rich protein liquid from leaking into alveolar space, and it additionally aids the resorption receipts of pulmonary edema.But ALI and
Pulmonary epithelial cells a large amount of apoptosis during ARDS, epithelial cell/endothelial barrier destroys at this time and function is damaged.Zoopery confirmation, ALI
When by suppressing pulmonary epithelial cells apoptosis can reduce that intrapulmonary inflammatory factor is horizontal, mitigate injury of lungs, to reduce experimental animal dead
Rate.This prompting, the function of lung epithelial/endothelial cell barrier have the function that important in the ALI courses of disease.It is scorching in lung tissue during ALI
Sex factor level be higher than blood plasma, and inflammatory factor is mainly derived from the inflammatory cell that intrapulmonary infiltrates in lung tissue, and pulmonary epithelial cells
Also it is related to the inflammatory reaction of intrapulmonary.Pulmonary epithelial cells is not only involved in secretion inflammatory factor (such as TNF-α, IL-1 and IL6 during ALI
Deng), it can also increase cell related cell adhesion molecule -1 (intercellular adhesion molecule-1, ICAM-1)
Express and promote neutrophil leucocyte intrapulmonary to infiltrate.Above experimental data is prompted, and pulmonary epithelial cells take part in intrapulmonary in the ALI courses of disease
Inflammatory reaction.
The experiment in vitro of lung epithelial/endothelial cell barrier needs corresponding cell co-culture model during endotoxin ALI.LPS
It is the primary activation thing of inflammation outburst, lung epithelial/endothelial cell barrier external model is for clinic during ALI caused by establishing endotoxin
The research and development of medicine has great importance.Girth happiness etc. once made using Transwell co-culture systems
The co-culture model of people's typeⅡalveolarcells A549 cell lines and human pulmonary microvascular endothelial cells strain HPMECs, and by it
For studying influence of the flagellin stimulation to pulmonary epithelial cells expression TNF-α, but the research of girth happiness is not directed to clinically
The barrier function of lung epithelial/endothelial cell during most common endotoxin ALI, its co-culture model settling time established are 15
My god, whether induce pulmonary vascular endothelial cell inflammation concern is primarily with the infection of injury of lungs long period pulsellum albumen and can be to
Alveolar epithelial cells transmits Cascaded amplification.
The content of the invention
In view of the deficiencies of the prior art, pulmonary epithelial cells and capillary when the present invention provides a kind of simulation acute lung injury
The method for building up of the external model of barrier function of endothelial cells, the cell model can be as in the ALI caused by research endotoxin
The Pathological Physiology such as epithelium/barrier function of endothelial cells, lung permeability changes, the model basis of microvascular tissue repair mechanism.
Technical scheme is as follows：Pulmonary epithelial cells and capillary endothelial cell during one kind simulation acute lung injury
The method for building up of the external model of barrier function, the described method comprises the following steps：
Step (1) applies Transwell co-culture systems by the metainfective micro- blood of mouse C3H/10T1/2 of endotoxin (LPS)
Endothelial cell strain is inoculated in lower floor's culture of Transwell cells；Small mouse TC-1 pulmonary epithelial cells strains are inoculated in
The upper strata of Transwell cells, upper cell and lower floor's cell culture supernatant are collected after cultivating a period of time；
After step (2) endotoxin infecting mouse pulmonary microvascular endothelial cells, then with epithelial cell co-culture, detection cell training
Support pulmonary epithelial cells/endothelial cell barrier synthesis in supernatant and expression inflammatory factor and inflammatory cell infiltration GAP-associated protein GAP
Situation, all detects that IL-6 and ICAM-1 protein expressions raise, table in epithelial cell and pulmonary microvascular endothelial cells supernatant
It is bright to be created as a pulmonary epithelial cells/microvascular endothelial cells co-culture model for being suitable for ALI researchs.
Further, the culture medium of the microvascular endothelial cells is that with the addition of 10-20% (w/w) hyclone (FBS),
90-95U/mL heparin, 2-4mmol/mL L-Glutamines, 10-15ng/mL endothelial growth factor additives, 1-1.5%
(w/w) the H-DMEM culture mediums of penicillin sodium salt and streptomycin sulphate.
Further, the culture medium of the pulmonary epithelial cells strain is that with the addition of 10-20% (w/w) FBS, 1-1.5% (w/w)
1640 culture mediums of penicillin sodium salt and 1-1.5% streptomycin sulphates.
Further, pulmonary epithelial cells strain is inoculated in again after 4~6h of the microvascular endothelial cells strain culture
The upper strata of Transwell cells.
Further, the inoculum density of the microvascular endothelial cells strain is 3.5 × 104/cm2~4.5 × 104/cm2, connect
Kind volume is 1~2mL；The inoculum density of the pulmonary epithelial cells strain is 1.5 × 104/cm2~2.5 × 104/cm2, inoculum
Product is 0.8~1.5mL.
Further, the concentration of endotoxin (LPS) the infecting mouse C3H/10T1/2 microvascular endothelial cells strains is
0.8~1.2 μ g/mL.
Second aspect of the present invention, lung epithelial is thin when also providing the simulation acute lung injury established by the method for building up
Born of the same parents and the external model of capillary endothelial cell barrier function.
Due in the case of in vivo acute lung injury occur after 48 it is small when it is interior can secrete a large amount of inflammatory factors, it is contemplated that
Replicate a kind of model of closest internal LPS-induced acute lung injury situation, the time point that the present invention is paid close attention to be after damage 48 it is small when
It is interior.During ALI, lung epithelial/endothelial cell barrier can secrete inflammatory factor, expression ICAM-1 promotes inflammatory cell attachment infiltration,
At the same time also between expression cell tight junction protein (such as occludin) to stop inflammatory cell infiltration.It is real in vitro to be consequently adapted to ALI
Synthesis and secretion inflammatory factor should not only be had the function of by testing lung epithelial/endothelial cell co-culture model of research, should be able to also
Tight junction protein between expression cell, and barrier action is presented.The present invention makes mouse in Transwell co-culture systems
LA-4 pulmonary epithelial cellses and the co-culture model of mouse C3H/10T1/2 microvascular endothelial cells, by adjusting cell culture condition
The lung of inflammatory factor (IL6) and inflammatory cell infiltration GAP-associated protein GAP (ICAM-1) can be expressed to establish one under endotoxin stimulation
Epithelium/endothelial cell co-culture model, this model being capable of lung epithelial/endothelial cell barriers during LPS acute lung injury in analogue body
Function.
Compared with prior art, the invention has the advantages that：This model that the present invention establishes is close interior in vivo
Barrier cell function during toxin acute lung injury, can provide a kind of reliable model base for clinically acute lung injury research
Plinth, the present invention establishes one by adjusting cell culture condition can express inflammatory factor (IL6) and inflammatory under endotoxin stimulation
The lung epithelial of cellular infiltration GAP-associated protein GAP (ICAM-1)/endothelial cell co-culture model, this model have synthesis and secretion inflammatory
The function of the factor, should be able to also tight junction protein between expression cell, and barrier action is presented.
Brief description of the drawings
Fig. 1 is IL-6 protein expression result figures；
Fig. 2 is ICAM-1 protein expression result figures.
Embodiment
Technical scheme is described in further details with reference to specific embodiment, but the present invention does not limit to
In following technical scheme.
Embodiment 1
The present embodiment makes mouse TC-1 pulmonary epithelial cellses and mouse C3H/10T1/2 in Transwell co-culture systems
The co-culture model of microvascular endothelial cells, by adjusting cell culture environment so that this pulmonary epithelial cells/microvascular endothelial is thin
Born of the same parents' co-culture model can express inflammatory factor (IL6) and inflammatory cell infiltration GAP-associated protein GAP (ICAM-1) under endotoxin stimulation.
It is as follows to make pulmonary epithelial cells/microvascular endothelial cells co-culture model process：
The mouse C3H/10T1/2 microvascular endothelial cells for infecting 1ug/mL LPS using Transwell co-culture systems
Strain is with 4 × 104/cm2Concentration inoculation 1.5mL in the lower floor of Transwell cells, after cultivating 5h.By small mouse TC-1 lungs
Epithelial cell strain is with 2 × 104/cm2Concentration be inoculated with 1mL in the upper strata of cell.Lower floor's cell culture medium is that with the addition of 10% tire ox
Serum (FBS), 90U/mL heparin, 4mmol/mL L-Glutamines, 10ng/mL endothelial growth factor additives, 1% is blue or green
The H-DMEM culture mediums of mycin sodium salt and streptomycin sulphate.Upper strata culture medium to the addition of 10%FBS, 1% penicillin sodium salt and
1640 culture mediums of streptomycin sulphate.Two kinds of cells, which co-culture, establishes pulmonary epithelial cells/endothelial cell co-culture model, in culture
24h and 48h collects the supernatant of upper cell and lower confluent monolayer cells afterwards.
Under endotoxin (LPS) stimulation, pulmonary epithelial cells/endothelial cell barrier synthesis and expression inflammatory factor and inflammatory are thin
Born of the same parents infiltrate the situation of GAP-associated protein GAP：
24h and 48h collects cell culture supernatant after co-cultivation, using Elisa technology for detection under endotoxin stimulation
Pulmonary epithelial cells/microvascular endothelial cells co-culture model synthesis is related to expression inflammatory factor (IL6) and inflammatory cell infiltration
The level of albumen (ICAM-1).Intrapulmonary inflammatory factor (IL6) and inflammatory cell leaching are detected in the mouse ALI models of LPS inductions
Moisten the expression of GAP-associated protein GAP (ICAM-1).Adjustment cell culture condition establishes a pulmonary epithelial cells for being suitable for ALI researchs
/ microvascular endothelial cells co-culture model.
The present embodiment successfully cultivates Mice lung microvessel endothelial cell, the Mice lung microvessel endothelial cell life of original cuiture
Grow in order, in fusiformis or polygonal；Mouse lung epithelial cells (TC-1) are cultivated in success：Mouse lung epithelial cells strain culture 4h
It is rounded afterwards, it is in cube after culture 12h, with formation paving stone sample after the extension 24h of incubation time.
Inflammatory factor IL6 eggs related to inflammatory cell infiltration in enzyme linked immunosorbent assay (ELISA) (ELISA) detection each group cell
The expression of (ICAM-1) in vain：Compared with co-culturing control group, after LPS stimulates Mice lung microvessel endothelial cell, co-culture
IL-6 and ICAM-1 protein expressions raise (p in mouse lung epithelial and pulmonary microvascular endothelial cells supernatant<0.05), see Fig. 1 and
Fig. 2.
Fig. 1 is IL-6 protein expression situations, and A-H is respectively different treatment groups, and A and B are respectively：Mouse lung epithelial-lung
Microvascular endothelial cells co-cultures the protein expression in the pulmonary microvascular endothelial cells and lung epithelial supernatant of 24h groups；C and D points
It is not：Mouse lung epithelial-pulmonary microvascular endothelial cells is co-cultured in the pulmonary microvascular endothelial cells and lung epithelial supernatant of 48h groups
Protein expression；E and F are respectively：Mouse lung epithelial-pulmonary microvascular endothelial cells of LPS processing co-cultures the micro- blood of lung of 24h groups
Protein expression in endothelial cell and lung epithelial supernatant；G and H are respectively：Mouse lung epithelial-organs except lungs of LPS processing
Endothelial cell co-cultures the protein expression in the pulmonary microvascular endothelial cells and lung epithelial supernatant of 48h groups.With mouse lung epithelial-
Microvascular endothelial cells co-cultures 24h control groups (Figure 1A and B) and compares, LPS infection lung epithelial-pulmonary microvascular endothelial cells 24h
Co-culture system in two kinds of cell supernatant IL-6 protein expression levels significantly raise (P<0.05) (Fig. 1 E and F)；With mouse
Lung epithelial-microvascular endothelial cells co-cultures 48h control groups (Fig. 1 C and D) and compares, LPS infection lung epithelial-organs except lungs endotheliums
Two kinds of cell supernatant IL-6 protein expression levels significantly raise (P in the co-culture system of cell 48h<0.05) (Fig. 1 G and
H)。
Fig. 2 is ICAM-1 protein expression situations, and A-H is respectively different treatment groups in Fig. 2, and A and B are respectively：Mouse lung
Epithelium-pulmonary microvascular endothelial cells co-cultures the protein expression in the pulmonary microvascular endothelial cells and lung epithelial supernatant of 24h groups；
C and D are respectively：Mouse lung epithelial-pulmonary microvascular endothelial cells is co-cultured in the pulmonary microvascular endothelial cells and lung epithelial of 48h groups
Protein expression in clear liquid；E and F are respectively：Mouse lung epithelial-pulmonary microvascular endothelial cells of LPS processing co-cultures 24h groups
Protein expression in pulmonary microvascular endothelial cells and lung epithelial supernatant；G and H are respectively：Mouse lung epithelial-lung of LPS processing
Microvascular endothelial cells co-cultures the protein expression in the pulmonary microvascular endothelial cells and lung epithelial supernatant of 48h groups.Train together
Foster control group compares, and after LPS stimulates Mice lung microvessel endothelial cell, co-cultures mouse lung epithelial and pulmonary microvascular endothelial cells
ICAM-1 protein expressions significantly raise (p in supernatant<0.05).Specifically, with mouse lung epithelial-microvascular endothelial cells
Co-culture 24h control groups (Figure 1A and B) to compare, in the co-culture system of LPS infection lung epithelial-pulmonary microvascular endothelial cells 24h
Two kinds of cell supernatant ICAM-1 protein expression levels significantly raise (P<0.05) (Fig. 1 E and F)；With mouse lung epithelial-capilary
Endothelial cell co-cultures 48h control groups (Fig. 1 C and D) and compares, and LPS infects the common training of lung epithelial-pulmonary microvascular endothelial cells 48h
Two kinds of cell supernatant ICAM-1 protein expression levels significantly raise (P in the system of supporting<0.05) (Fig. 1 G and H).
From Fig. 1 and Fig. 2, endotoxin (LPS) causes Mice lung microvessel endothelial cell inflammatory reaction, and to mouse lung
Epithelial cell transduction cascade amplifies.Illustrate inflammation not only can in same intracellular amplification, but also can adjacent not allogenic cell it
Between carry out Cascaded amplification, illustrate present invention success establish in vitro it is a kind of can LPS Acute Lung Injury situations in analogue body
Under lung epithelial/barrier function of endothelial cells model.
Embodiment 2 changes LPS concentration
Grope to find that cell can in the co-culture system that the LPS of final concentration of 0.8~1.2 μ g/mL induces through preliminary experiment
Survive and upgrowth situation is good.Concentration is less than 0.8 μ g/mL, can cause to cause damage or degree of injury with it is internal acute
Situation gap during injury of lungs is big.Higher than 1.2 μ g/mL, cell death can be caused.
Embodiment 3 changes lower floor's cell culture medium
Lower floor's culture medium of foundation is the culture environment that endothelial cell growth is most suitable for, and changes the component in culture medium, or
A certain component is lacked, or the amount of component and ratio change, and can all cause Endothelial Cell Survival rate low or propagation is slow, or even extremely
Die, be unable to reach two kinds of cells and co-culture required cell density, cell culture the results are shown in Table 1.
Table 1
Under normal conditions, in no addition FBS, cell second day will be dead.Not plus 90-95U/mL heparin feelings
It is little to cell survival and Effects of Density under condition.Penicillin sodium salt and streptomycin sulphate excessive concentration, may cause cell dead
Die, concentration is too low, may cause germ contamination.
Embodiment 4 changes upper cell culture medium
Change the component in the culture medium of upper strata, or a certain component of missing, or the amount of component and ratio change, and can all cause
Endothelial cell survival rate is low or propagation is slow, is unable to reach two kinds of cells and co-cultures required cell density, influences two kinds of cells
Co-cultivation fusion, cell culture the results are shown in Table shown in 2.
Table 2
| Epithelial cell special culture media in this method | Lack FBS | |
| Cell survival/death | Survival | It is dead |
| Cell density | 85-95% | 10-20% |
Penicillin sodium salt and streptomycin sulphate excessive concentration, may cause cell death, and concentration is too low, may cause bacterium
Pollution.
Embodiment 5 replaces levels cell culture order
Levels cell category is different, it is necessary to corresponding specific culture medium.Cell survival rate can be caused by changing culture medium
It is low, do not breed, or it is dead, influence co-culture system foundation.
Embodiment 6, which changes, co-cultures the time
It is not inconsistent with the situation of internal acute lung injury, the acute lung injury inflammation infection caused by LPS usually occurs small 48
When it is interior.So we collect sample detection inflammatory factor expression when co-cultivation 24 and 48 is small.
Embodiment 7 changes the priority inoculation time of upper and lower confluent monolayer cells
The present embodiment finds that in transwell cells the growth of endothelial cell, growth rate usually compare epithelial cell
Slowly.So to be first inoculated with endothelial cell 4-6 it is small when after, when endothelial cell growth breed to certain density when, it is thin to inoculate epithelium
Born of the same parents, to ensure that two kinds of cell co-culture systems are successfully established.
Claims (7)
1. the external model of pulmonary epithelial cells and capillary endothelial cell barrier function builds during a kind of simulation acute lung injury
Cube method, it is characterised in that the described method comprises the following steps：
Step (1) is thin by the metainfective mouse C3H/10T1/2 microvascular endothelials of endotoxin using Transwell co-culture systems
Born of the same parents' strain is inoculated in lower floor's culture of Transwell cells；Small mouse TC-1 pulmonary epithelial cells strains are inoculated in Transwell cells
Upper strata, collect upper cell and lower floor's cell culture supernatant after cultivating a period of time；
After step (2) endotoxin infecting mouse pulmonary microvascular endothelial cells, then with epithelial cell co-culture, detect cell culture on
Pulmonary epithelial cells/endothelial cell barrier synthesis and the feelings of expression inflammatory factor and inflammatory cell infiltration GAP-associated protein GAP in clear liquid
Condition, all detects that IL-6 and ICAM-1 protein expressions raise in epithelial cell and pulmonary microvascular endothelial cells supernatant, shows
It is created as a pulmonary epithelial cells/microvascular endothelial cells co-culture model for being suitable for ALI researchs.
2. the method for building up of external model as claimed in claim 1, it is characterised in that the culture of the microvascular endothelial cells
Base is that with the addition of 10-20% (w/w) hyclone (FBS), 90-95U/mL heparin, 2-4mmol/mL L-Glutamines, 10-
The H-DMEM cultures of 15ng/mL endothelial growth factor additives, 1-1.5% (w/w) penicillin sodium salts and streptomycin sulphate
Base.
3. the method for building up of external model as claimed in claim 1, it is characterised in that the culture medium of the pulmonary epithelial cells strain
To with the addition of the 1640 of 10-20% (w/w) FBS, 1-1.5% (w/w) penicillin sodium salt and 1-1.5% streptomycin sulphates cultures
Base.
4. the method for building up of external model as claimed in claim 1, it is characterised in that the microvascular endothelial cells strain culture
Pulmonary epithelial cells strain is inoculated in the upper strata of Transwell cells again after 4~6h.
5. the method for building up of external model as claimed in claim 1, it is characterised in that the microvascular endothelial cells strain connects
Kind concentration is 3.5 × 104/cm2~4.5 × 104/cm2, inoculation volume is 1~2mL；The inoculum density of the pulmonary epithelial cells strain
For 1.5 × 104/cm2~2.5 × 104/cm2, inoculation volume is 0.8~1.5mL.
6. the method for building up of external model as claimed in claim 1, it is characterised in that the endotoxin infecting mouse C3H/
The concentration of 10T1/2 microvascular endothelial cells strains is 0.8~1.2 μ g/mL.
7. pulmonary epithelial cells and hair during the simulation acute lung injury that a kind of method for building up by described in claim 1~6 is established
The external model of thin vascular endothelial cell barrier function.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711182612.7A CN107988144A (en) | 2017-11-23 | 2017-11-23 | The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711182612.7A CN107988144A (en) | 2017-11-23 | 2017-11-23 | The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107988144A true CN107988144A (en) | 2018-05-04 |
Family
ID=62032817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711182612.7A Pending CN107988144A (en) | 2017-11-23 | 2017-11-23 | The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107988144A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486746A (en) * | 2018-11-16 | 2019-03-19 | 青岛大学 | A kind of external breath exposure model building method and application |
| CN110656081A (en) * | 2019-10-17 | 2020-01-07 | 大连医科大学 | Construction method of pancreatic ductal epithelial cell and pancreatic acinar cell co-culture system for simulating in-vivo microenvironment |
| CN110804651A (en) * | 2019-11-15 | 2020-02-18 | 南方医科大学珠江医院 | In-vitro experimental method for correlation of CITED2 and CMVA |
| CN113209134A (en) * | 2021-02-25 | 2021-08-06 | 中国人民解放军海军军医大学 | Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by mustard gas |
| CN114561338A (en) * | 2022-03-22 | 2022-05-31 | 闻庆平 | Lung qi and blood barrier damage model, and establishing method and application thereof |
| CN114908035A (en) * | 2022-05-06 | 2022-08-16 | 中国疾病预防控制中心营养与健康所 | Kidney podocyte and glomerular endothelial cell co-culture model and application thereof |
-
2017
- 2017-11-23 CN CN201711182612.7A patent/CN107988144A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486746A (en) * | 2018-11-16 | 2019-03-19 | 青岛大学 | A kind of external breath exposure model building method and application |
| CN110656081A (en) * | 2019-10-17 | 2020-01-07 | 大连医科大学 | Construction method of pancreatic ductal epithelial cell and pancreatic acinar cell co-culture system for simulating in-vivo microenvironment |
| CN110804651A (en) * | 2019-11-15 | 2020-02-18 | 南方医科大学珠江医院 | In-vitro experimental method for correlation of CITED2 and CMVA |
| CN113209134A (en) * | 2021-02-25 | 2021-08-06 | 中国人民解放军海军军医大学 | Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by mustard gas |
| CN114561338A (en) * | 2022-03-22 | 2022-05-31 | 闻庆平 | Lung qi and blood barrier damage model, and establishing method and application thereof |
| CN114561338B (en) * | 2022-03-22 | 2024-04-19 | 闻庆平 | Lung qi and blood barrier injury model, and establishment method and application thereof |
| CN114908035A (en) * | 2022-05-06 | 2022-08-16 | 中国疾病预防控制中心营养与健康所 | Kidney podocyte and glomerular endothelial cell co-culture model and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988144A (en) | The method for building up of the external model of pulmonary epithelial cells and capillary endothelial cell barrier function when simulating acute lung injury | |
| JP2016039806A (en) | Cytokine-producing cell sheet and method for using same | |
| CN114574390B (en) | Bifidobacterium longum subspecies infantis for relieving colonitis and application thereof | |
| CN105087487A (en) | Efficient CIK amplifying method | |
| CN102220281B (en) | Co-culture system for liver cells and Kupffer cells and application thereof | |
| CN105296426B (en) | A kind of method for inducing and cultivating of NK cell | |
| CN105331580A (en) | Method for enhancing mesenchymal stem cell chemotactic capacity and chemotactic factor CCL5 expression | |
| CN105462923A (en) | Efficient in-vitro amplification method of human natural killer cells | |
| CN108004209A (en) | A kind of bleeding of the umbilicus regulatory T cells amplification in vitro method | |
| Viola et al. | Microphysiological systems modeling acute respiratory distress syndrome that capture mechanical force-induced injury-inflammation-repair | |
| CN103642752A (en) | Preparation method of human CD3+CD8+CIK (cytokine induced killer) cells | |
| CN104711224A (en) | In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof | |
| Sakai et al. | Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities | |
| Hentschel et al. | Intestinal organoids in coculture: redefining the boundaries of gut mucosa ex vivo modeling | |
| CN114032208B (en) | In-vitro cytokine storm model and construction method and application thereof | |
| Bouchelouche et al. | Monocyte chemoattractant protein-1 production by human detrusor smooth muscle cells | |
| CN1844373A (en) | Method for in vitro preparation of hepatocyte induced by umbilical cord blood hematopoietic stem cell | |
| Ren et al. | Evaluation of nutritional and physical stress conditions during vegetative growth on conidial production and germination in Ophiocordyceps sinensis | |
| CN103131667B (en) | Pancreatic cancer cell line with lymphatic channel high migration activity | |
| CN103952377B (en) | A kind of clone for sheep infective pustule virus separation and Culture and propagation and its preparation method and application | |
| CN108703019A (en) | A kind of the sealing cultivation case and its cultural method of ganoderma lucidum | |
| CN104357542A (en) | Method for determining activity of trichophyton rubrum keratinase | |
| CN105112354B (en) | Retinal pigment epithelium epithelial-mesenchymal transformation model and its application | |
| CN103558392B (en) | A kind of effect of the antiatherosclerotic based on inflammatory reaction evaluation method | |
| CN106701680A (en) | Combination factor for promoting maturity of dendritic cells and method for cultivating dendritic cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180504 |