CN109055496A - Utilize the variation of gene microarray analysis lipopolysaccharides stimulation cow mammary gland epithelial cells express spectra - Google Patents
Utilize the variation of gene microarray analysis lipopolysaccharides stimulation cow mammary gland epithelial cells express spectra Download PDFInfo
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Abstract
The method that the present invention relates to the use of the variation of gene microarray analysis LPS stimulation BEMC express spectra, the described method comprises the following steps: step 1, the foundation of cow mammary gland epithelial cells inflammatory model, Step 2: testing Chinese medicine with cow mammary gland epithelial cells inflammatory model.
Description
Technical field:
The present invention relates to drug screening and its examination of curative effect methods, in particular to stimulate milk with genechip detection lipopolysaccharides
The method of bovine mammary epithelial cell express spectra variation, to detect whether lipopolysaccharides can cause inflammatory reaction, and as inflammation
Disease model is used to effect and mechanism of action of the testing drug to inflammatory model.
Background technique:
Chinese medicine refers to traditional Chinese medical theory for guidance, has unique theoretical system and application form, for preventing and controlling
Disease and natural drug and its processing substitute with rehabilitation and health-care effect are treated, mainly includes botanical medicine, animal drugs, mineral
Medicine.
The therapeutic effect of Chinese medicine is to determine that this method is for new with the symptom variation after taking for foundation for a long time
Indication be obviously not suitable for, be Study of Traditional Chinese Medicine new curative effect and mechanism of action, it is imperative to employ new technology.
Lipopolysaccharides is one of gram-negative bacterial cell wall ingredient, and lipopolysaccharides is virose to host.Rouge is more
Sugar only just releases after bacterial death dissolves or destroys bacterium cell by artificial means, so being called endotoxin.Its toxicity
Ingredient is mainly lipoids A.Endotoxin is located at the outermost layer of cell wall, is covered on the mucopeptide of cell wall.Various bacteriums it is interior
The toxic effect of toxin is weaker, roughly the same, can cause to coagulate in fever, microcirculation disorder, endotoxin shock and disseminated intravascular
Blood etc..Endotoxin is heat-resisting and stablizes, and antigenicity is weak.
Lipopolysaccharides has characteristics that
1. the compound of lipid and polysaccharide；
2. molecular weight is greater than 10000, and structure is multiple for distinctive a kind of chemical component in the outer wall layer of gramnegative bacterium
It is miscellaneous, it is all variant between Different groups, even bacterial strain.By taking salmonella as an example, lipopolysaccharides is by core polysaccharide, O- polysaccharide side
Chain and lipoid A composition.For the main component of gram-negative bacterial cell wall, lipopolysaccharides is endotoxin and important group specificity
Antigen (O antigen).
Lipopolysaccharides consists of three parts: lipoid A, core polysaccharide, O- antigen.Lipid A (Lipid A) is to constitute endotoxin
Active glycolipid has two parts with covalent bonding junction to heteroglycan chain: first is that core polysaccharide, is constant in related bacterial strain
's；Another O specificity chain (O-specific chain) is alterable height.The lipopolysaccharides of Escherichia coli is in Experimental immunization
In be common B cell mitogeneic factor i.e. polyclonal activator (polyclonal activator).
Lipopolysaccharides is the compound containing sugar and lipid, and sugared component is more than rouge in composition, therefore named.Gramnegative bacterium
A kind of main component of outer membrane.Its acyl chain is embedded in the outer membrane of bacterium, and sugar chain is exposed to the surface of bacterium, and has antigen
Property (commonly referred to as O antigen).Lipopolysaccharides is also the main component of bacterial endotoxin.It is released when gramnegative bacterium is burst apart,
It is that very strong fever is former.Its monosaccharide composition is more special, and 3,6 deoxidations including D-Glucose D- galactolipin and D-MANNOSE are spread out
Biological and some octanal sugar.
Outer membrane protein is entrenched on LPS and phospholipid layer outer membrane.In structure, they all have beta-barrel structure, and different is outer
β-bucket of memebrane protein is made of different even number beta-pleated sheets, is differed from 8 to 22；Functionally, they have and mention for outer membrane
For permeability, the effect for maintaining outer membrane structure stable.The outer membrane protein folded has extremely strong stability, under room temperature, 2%
Strong ionic surfactant SDS in, outer membrane protein still has normal foldable structure；And at this temperature, other can
Molten albumen can lose rapidly structure in 0.2% SDS.Therefore usually whether 95C heating will be carried out to sample before sample-adding
The SDS-PAGE of unfolding is known as denatured SDS-PAGE and semi-native SDS-PAGE, and it is outer to be used to research
The folding situation of memebrane protein.
Milk cow is one of economic animal important in China's animal husbandry, dairy generally existing output of milk in China's is low,
The problems such as butterfat percnetage and relatively not high content of milk protein, in addition cow mammary gland epithelial cells inflammation is also the weight for causing the output of milk low
Factor is wanted, these are all the principal elements for restricting the development of China's dairy.Chinese herbal medicine is the distinctive natural products in China, and resource is rich
Richness has the characteristics that noresidue, toxic side effect are small and are not likely to produce drug resistance.Many Chinese herbal medicines can significantly improve milk cow production
Milk amount, but its mechanism is unclear, not yet carries out correlative study work both at home and abroad.Genetic chip (gene chip) is the eighties
What mid-term proposed.The sequencing principle of genetic chip is sequencing by hybridization method, i.e., by miscellaneous with the nucleic acid probe of one group of known array
The method for carrying out determining nucleic acid sequence is handed over, the gene order of surveyed DNA fragment can be quickly obtained.Such as in one piece of substrate surface
Secure the probe of eight nucleotide known to sequence.When in solution have fluorescent marker nucleic acid sequence TATGCAATCTAG, with
When the nucleic acid probe of corresponding position generates complementary matching on genetic chip, by determining the strongest probe location of fluorescence intensity, obtain
Obtain the probe sequence of one group of sequence complete complementary.The sequence of target nucleic acid can be recombinated out accordingly.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types:
1) nucleic acid probe or cDNA segment being fixed on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) surface lead to
The target gene of common isotope labelling is hybrid with it, and is detected by radiography technology.The advantages of this method is required
Detection device is consistent with radiography technology used in current molecular biology, relatively mature.But probe is close on chip
Degree is not high, and the demand of sample and reagent is big, and there are more problems for quantitative detection.
2) DNA probe array on a glass is fixed with point sample method, progress is hybridized by the target gene with fluorescent marker
Detection.This method reticular density can be greatly improved, and the binding capacity of each probe on the surface is being marked also than more consistent
Still there is the difficulty being not easily overcome in terms of standardization and mass production.
3) oligonucleotide probe array directly synthesized on the hard surfaces such as glass hybridizes with the target gene of fluorescent marker
It is detected.This method combines microelectronics photoetching technique with DNA chemical synthesising technology, and the probe of genetic chip can be made close
Degree greatly improves, and reduces the dosage of reagent, realizes standardization and mass large-scale production, has highly important development latent
Power.
The present invention uses biochip technology, finds a kind of detection method, using this method, can observe using lipopolysaccharides
The variation of cow mammary gland epithelial cells gene expression profile is stimulated, to judge that machine occurs for the possible inflammation of cow mammary gland epithelial cells
Reason, so that treating cow mammary gland epithelial cells inflammation for Chinese medicine provides model and foundation.
Summary of the invention
The present invention provides a kind of variation using biochip technology detection cow mammary gland epithelial cells gene expression profile
Method the described method comprises the following steps:
Step 1, the foundation of cow mammary gland epithelial cells inflammatory model, the method is as follows:
1), the culture of cow mammary gland epithelial cells separates, purifying；
2), lipopolysaccharides stimulates cow mammary gland epithelial cells
Lipopolysaccharides is dissolved in serum free medium, and the serum free medium containing lipopolysaccharides, control group is added in test group cell
Isometric serum free medium culture is added in cell；
3) total serum IgE of the cow mammary gland epithelial cells of lipopolysaccharides stimulation, is extracted；
4), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA；
5), with genechip detection cow mammary gland epithelial cells inflammatory cell gene variation
CRNA sample and chip hybridization then take out chip, scan in scanner after washing；
6), the expression variation between the sample of judgement addition lipopolysaccharides and the sample that lipopolysaccharides is not added according to testing result；
Step 2: testing Chinese medicine with cow mammary gland epithelial cells inflammatory model, the method is as follows:
1), the culture of cow mammary gland epithelial cells separates, purifying；
2), lipopolysaccharides stimulates cow mammary gland epithelial cells；
3) Chinese medicine, is added；
4) total serum IgE of cow mammary gland epithelial cells, is extracted；
5), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA；
6), genechip detection cow mammary gland epithelial cells inflammatory cell gene variation
CRNA sample and chip hybridization then take out chip, scan in scanner after washing；
7), the expression variation between the sample of judgement addition Chinese medicine and the sample that Chinese medicine is not added according to testing result.
Preferred the method for the invention, steps are as follows:
Step 1, the foundation of cow mammary gland epithelial cells inflammatory model
1), the culture of cow mammary gland epithelial cells separates, purifying
Cow mammary gland epithelial cells are separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；
Breast tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；By breast tissue
In white acinar tissue shear off and clean up repeatedly, mammary gland alveolus is cut into the tissue block of 1mm3, drawn by no milk residual
Appropriate tissue block is added 0.25% pancreatin of 3 times of volumes, shakes digestion in 37 DEG C of thermostat water baths in 15mL centrifuge tube
30min, 1000r/min are centrifuged 5min, remove supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of constant temperature are then added
Water-bath concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, complete
Cell is resuspended in full culture medium, and cell suspension is moved into Tissue Culture Flask, and 37 DEG C, continue to cultivate in the incubator of 5%CO2,
Whether observation cell is adherent afterwards for 24 hours, if there is other digestion products in culture bottle at this time, the complete medium that can more renew is kept away
Exempt to cause cell contamination, postdigestive cell 2-3d grows up to island shape, grows to 80% to it, passed on；
The purifying of cow mammary gland epithelial cells
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, to cream
Glandular epithelium and fibroblast are separated, and when cell covers with culture bottle, discard cell culture fluid, are washed 2 times with PBS,
0.25% pancreatin of 1mL is added into Tissue Culture Flask, microscopically observation, when fibroblast is rounded part and starts shedding off,
Complete medium is added into culture bottle immediately, terminates digestion, gently blows and beats molten to remove fibroblast and remaining pancreatin
Then, suitable complete medium is then added in liquid, continue to cultivate, and aforesaid operations are repeated when cell covers with bottom of bottle again,
Purify cow mammary gland epithelial cells,
2), lipopolysaccharides stimulates cow mammary gland epithelial cells
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discard hole
Interior culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and test group cell is added containing 50 μ g/mL lipopolysaccharides
Serum free medium, cellular control unit are added isometric serum free medium, and 37 DEG C, continue to train in 5%CO2 cell incubator
12h is supported, is then washed 2 times with PBS, TRIzol is added, keeps cell cracking complete with liquid-transfering gun piping and druming, is transferred in centrifuge tube, -80 DEG C
Refrigerator saves backup,
3) total serum IgE of the cow mammary gland epithelial cells of lipopolysaccharides stimulation, is extracted
4), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA
5), with genechip detection cow mammary gland epithelial cells inflammatory cell gene variation
CRNA sample and chip hybridization then take out chip, scan in scanner after washing；
6), the expression variation between the sample of judgement addition lipopolysaccharides and the sample that lipopolysaccharides is not added according to testing result,
Step 2: testing Chinese medicine with cow mammary gland epithelial cells inflammatory model
) 1, the culture of cow mammary gland epithelial cells separates, purifying
Cow mammary gland epithelial cells are separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；
Breast tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；By breast tissue
In white acinar tissue shear off and clean up repeatedly, mammary gland alveolus is cut into the tissue block of 1mm3, drawn by no milk residual
Appropriate tissue block is added 0.25% pancreatin of 3 times of volumes, shakes digestion in 37 DEG C of thermostat water baths in 15mL centrifuge tube
30min, 1000r/min are centrifuged 5min, remove supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of constant temperature are then added
Water-bath concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, complete
Cell is resuspended in full culture medium, and cell suspension is moved into Tissue Culture Flask, and 37 DEG C, continue to cultivate in the incubator of 5%CO2,
Whether observation cell is adherent afterwards for 24 hours, if there is other digestion products in culture bottle at this time, the complete medium that can more renew is kept away
Exempting to cause cell contamination, postdigestive cell 2-3d grows up to island shape, grows to 80% to it, passed on,
The purifying of cow mammary gland epithelial cells
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, to cream
Glandular epithelium and fibroblast are separated, and when cell covers with culture bottle, discard cell culture fluid, are washed 2 times with PBS,
0.25% pancreatin of 1mL is added into Tissue Culture Flask, microscopically observation, when fibroblast is rounded part and starts shedding off,
Complete medium is added into culture bottle immediately, terminates digestion, gently blows and beats molten to remove fibroblast and remaining pancreatin
Then, suitable complete medium is then added in liquid, continue to cultivate, and aforesaid operations are repeated when cell covers with bottom of bottle again,
Purify cow mammary gland epithelial cells,
2), lipopolysaccharides stimulates cow mammary gland epithelial cells
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discard hole
Interior culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and the nothing for containing 50 μ g/mL LPS is added in test group cell
Blood serum medium, cellular control unit are added isometric serum free medium, and 37 DEG C, continue to cultivate in 5%CO2 cell incubator
Then 12h is washed 2 times with PBS, TRIzol is added, and is kept cell cracking complete with liquid-transfering gun piping and druming, is transferred in centrifuge tube, -80 DEG C of ice
Case saves backup,
3) Chinese medicine grouping, is added,
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discard hole
Interior culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and Chinese medicine group cell is added containing 50 μ g/mL lipopolysaccharides
The serum free medium for containing 50 μ g/mL lipopolysaccharides, the bodies such as cellular control unit addition are added in serum free medium, lipopolysaccharide group cell
Long-pending serum free medium, is then washed 2 times with PBS, is added by 37 DEG C, continue in 5%CO2 cell incubator to cultivate 12h
TRIzol keeps cell cracking complete, is transferred in centrifuge tube with liquid-transfering gun piping and druming, and -80 DEG C of refrigerators save backup,
4) total serum IgE of three other cow mammary gland epithelial cells of group, is extracted
5), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA,；
6), with the other cow mammary gland epithelial cells inflammatory cell genome cRNA sample of three groups of genechip detection
And chip hybridization, chip is then taken out, is scanned in scanner after washing；
7), the expression variation between the sample of judgement addition Chinese medicine and the sample that Chinese medicine is not added according to testing result.
The wherein Chinese medicine are as follows: any Chinese medicine or its extract that may have anti-cow mammary gland epithelial cells inflammation.It is excellent
The Chinese medicine of choosing are as follows: 8-methoxypsoralen.
Chinese medicine of the present invention, experiment is 8-methoxypsoralen (8-MethylPsoralen, 8-MOP),
It is the coumarin kind compound that separating and extracting comes out from pilose gerbera herb, pilose gerbera herb system composite family pilose gerbera herb, which belongs to, plants
Object GerberaPitose-noldes (L :) Cass., the plant medicinal part are under ground portion or herb, have qi-regulating and blood, clear
Thermal detoxification, it is relieving cough and reducing sputum, control carbuncle swells, acute tonsillitis, lower blood and other effects, modern pharmacological studies have shown that they have antibacterial activity, and poison
Property it is lower, both can be used to cure the disease, be also used as fragrance and come using pertinent literature shows the classes of compounds therefrom separated
Very much, including the compounds such as cumarin, terpene, benzene a pair of horses going side by side pyrans, sterols, flavones.
The related experiment that Chinese medicine is added is as follows:
BMECs is inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discards the culture medium in hole,
PBS is washed 2 times.Chinese medicine is dissolved in serum free medium, lipopolysaccharides (LPS) is dissolved in serum free medium, and Chinese medicine group cell adds
Enter to contain the serum free medium of 25 μ g/mL Chinese medicines, 37 DEG C, cultivate 3h in 5%CO2 cell incubator, is then added and contains 50 μ g/mL
The serum free medium of LPS, 37 DEG C, continue in 5%CO2 cell incubator to cultivate 12h.Lipopolysaccharide group cell, which is added, contains 50 μ g/
The serum free medium of mL LPS.Isometric serum free medium is added in cellular control unit.37 DEG C, 5%CO2 cell incubator
Middle culture 12h.
In method of the invention, wherein the genetic chip containing complementary DNA prepared, belongs to existing skill
Art can be prepared with art methods, for example first extract cow mammary gland epithelial cells DNA, or first extraction LPS is post-stimulatory
Cow mammary gland epithelial cells DNA, is prepared using following methods: light guides polymerization technique, piezoelectricity impact system, micromachine point sample
Method, chemical gunite.
The present invention according to the above technical scheme, in operation by the sample for needing to detect, is set as blank control group
(being added without LPS, be also added without Chinese medicine), LPS stimulate cow mammary gland epithelial cells group (LPS is added), and LPS is stimulated on cow mammary gland
Three groups of chrotoplast+Chinese medicine group (LPS being added, Chinese medicine is also added), measure their cRNA sequence respectively, are carried out according to spectrogram
Comparative analysis, as a result, it has been found that, the inflammation that Chinese medicine is formed after stimulating for LPS is inhibited.Experiment side through the invention
Method determines following index, to judge LPS stimulation cow mammary gland epithelial cells and LPS stimulation cow mammary gland epithelial cells+Chinese medicine group
Between difference, to illustrate the therapeutic effect of Chinese medicine: such as pass through difference expression gene be enriched with functional annotation, differential expression base
Because of the immune related pathways of enrichment, the expression of inflammation relevant molecule.
It is thin by chip analysis 8-methoxypsoralen group cell and lipopolysaccharide group after 8-methoxypsoralen
Born of the same parents' mRNA express spectra, the expression quantity of inflammation-associated cytokine (such as IL-1, IL-6, TNF-), 8-methoxypsoralen group with
Lipopolysaccharide group is lowered compared to obvious, to illustrate to stimulate the inflammation to be formed to have LPS after 8-methoxypsoralen is added
Inhibiting effect.
Chinese medicine is added in the cell culture fluid for causing inflammatory reaction the present invention, is carried out using biochip technology to sample
Genetic chip microarray scanning, to detect variation of the Chinese medicine to inflammatory cell gene expression, to learn the new effect of Chinese medicine
And mechanism of action, the application for next step Study of Traditional Chinese Medicine as drug lay the foundation.
Detailed description of the invention:
Compared with the control group, the scatter plot of difference expression gene, red representative expression quantity raises Fig. 1 LPS processing group, green
Color represents expression quantity downward, and blue represents expression no significant difference.
Fig. 2 LPS processing group is compared with Chinese medicine group, the scatter plot of difference expression gene, red representative expression quantity up-regulation, green
Color represents expression quantity downward, and blue represents expression no significant difference.
Fig. 3 LPS processing group is compared with Chinese medicine group, the scatter plot of difference expression gene, red representative expression quantity up-regulation, green
Color represents expression quantity downward, and blue represents expression no significant difference.
Specific embodiment:
The present invention is further illustrated by the following examples, but not as limitation of the present invention.
Embodiment 1, the foundation of model
It the originally culture of cow mammary gland epithelial cells (BMECs) and isolates and purifies
BMECs's is separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；
Breast tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；By breast tissue
In white acinar tissue shear off and clean up repeatedly, no milk residual.Mammary gland alveolus is cut into the tissue block of 1mm3, is drawn
Appropriate tissue block is added 0.25% pancreatin of 3 times of volumes, shakes digestion in 37 DEG C of thermostat water baths in 15mL centrifuge tube
30min, 1000r/min are centrifuged 5min, remove supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of constant temperature are then added
Water-bath concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, complete
Cell is resuspended in full culture medium, and cell suspension is moved into Tissue Culture Flask, and 37 DEG C, continue to cultivate in the incubator of 5%CO2,
Whether observation cell is adherent afterwards for 24 hours, if there is other digestion products in culture bottle at this time, the complete medium that can more renew is kept away
Exempt to cause cell contamination, postdigestive 2~3d of cell grows up to island shape, grows to 80% to it, passed on.
The purifying of BMECs
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, to cream
Glandular epithelium and fibroblast are separated.When cell covers with culture bottle, cell culture fluid is discarded, is washed 2 times with PBS,
0.25% pancreatin of 1mL is added into Tissue Culture Flask, microscopically observation.When fibroblast is rounded part and starts shedding off,
Complete medium is added into culture bottle immediately, terminates digestion, gently blows and beats molten to remove fibroblast and remaining pancreatin
Then, suitable complete medium is then added in liquid, continue to cultivate.Aforesaid operations are repeated when cell covers with bottom of bottle again,
Purify BMECs.
The passage of BMECs
The growth conditions for observing cell are inhaled when cell covers with bottom of bottle and abandon cell culture fluid, and PBS is washed 2 times, and 1mL is added
The digestion of 0.25% pancreatin, microscopically observation.It is loose when being connected between most cells, when cell starts to be rounded, training completely is added
It supports base and terminates digestion, blowing and beating repeatedly, which makes cell fall off from bottom of bottle, is made single cell suspension, new training is inoculated according to the ratio of 1:2
It supports in bottle, continues to cultivate.
The identification of BMECs
Cell density is adjusted to 1 × 105/mL, is added in 12 porocyte culture plates and cultivates, abandoning when cell grows to 80%
Remove culture medium；10% neutral formalin fixes 20min, and PBS is washed 3 times；0.5%TritonX-100 acts on 20min, and PBS is washed 3 times；
3%H2O2 is incubated for 10min, blocks endogenous peroxydase, and PBS is washed 3 times；- 18 primary antibody of mouse anti-keratin, 4 DEG C of mistakes are added dropwise
Night；The rabbit anti-mouse secondary antibody of biotin labeling is added dropwise, 37 DEG C of incubation 30min, PBS are washed 3 times；The strepto- albumen of HRP label is added dropwise
Element, 37 DEG C of incubation 30min, PBS are washed 3 times；DAB develops the color (being protected from light), under the microscope to light brown, terminates in time after developing the color sufficiently,
Microscopy is taken pictures.As a result with analysis
The adherent and growth that extends outwardly cell island can be seen after cultivating 48h, with the extension of incubation time, cell
Island area is also constantly expanding, and gradually converges in flakes, and at paving stone shape, covering with bottom of bottle 80% to cell can be passed on.Cream
Glandular epithelium has the brown color of specificity or the calmness of sepia, after purification after -18 immunohistochemical staining of keratin
Cow mammary gland epithelial cells purity is higher, can provide basis for follow-up test.
This test purifies galactophore epithelial cell using digestion method when difference, according to epithelial cell and fibroblast to same
The different sensibility of enzyme purify, substantially available purer epithelial cell by 2-3 times.
The processing of BMECs
Cell packet transaction
BMECs is inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discards the culture medium in hole,
PBS is washed 2 times.Lipopolysaccharides (LPS) is dissolved in serum free medium, and test group cell is added the serum-free containing 50 μ g/mL LPS and trains
Base is supported, isometric serum free medium is added in cellular control unit.37 DEG C, continue in 5%CO2 cell incubator to cultivate 12h.So
It is washed 2 times with PBS afterwards, TRIzol is added, kept cell cracking complete with liquid-transfering gun piping and druming, be transferred in centrifuge tube, -80 DEG C of refrigerators save
It is spare.
Cell total rna extracts
RNA is quantitative
Reverse transcription is at cDNA
Genechip detection
As a result with analysis
Compared with the control group, the scatter plot of difference expression gene is as shown below for LPS processing group, red representative expression quantity
Up-regulation, green represent expression quantity downward, and blue represents expression no significant difference.
LPS processing group compared with the control group, difference expression gene totally 1435, wherein raising 1301, lowers 134.
These differential genes are mainly related with the functions such as immune response, congenital immunity, inflammatory reaction, cytokine modulating, difference base
Because being primarily involved in virus infection, disease of immune system, NF- κ B signal access, Toll-like receptor access, antigen processes the phases such as submission
In OFF signal access.These all illustrate, after LPS stimulates BMECs, so that changed with immune-related gene expression, from
And the immune function of cell is changed, so that cell is generated inflammatory reaction, leads to cell damage.
Differential gene GO enrichment analysis (first 10)
Differential gene Pathway enrichment analysis (first 10)
Embodiment 2, with model inspection Chinese medicine
It the originally culture of cow mammary gland epithelial cells (BMECs) and isolates and purifies
BMECs's is separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；
Breast tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；By breast tissue
In white acinar tissue shear off and clean up repeatedly, no milk residual.Mammary gland alveolus is cut into the tissue block of 1mm3, is drawn
Appropriate tissue block is added 0.25% pancreatin of 3 times of volumes, shakes digestion in 37 DEG C of thermostat water baths in 15mL centrifuge tube
30min, 1000r/min are centrifuged 5min, remove supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of constant temperature are then added
Water-bath concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, complete
Cell is resuspended in full culture medium, and cell suspension is moved into Tissue Culture Flask, and 37 DEG C, continue to cultivate in the incubator of 5%CO2,
Whether observation cell is adherent afterwards for 24 hours, if there is other digestion products in culture bottle at this time, the complete medium that can more renew is kept away
Exempt to cause cell contamination, postdigestive 2~3d of cell grows up to island shape, grows to 80% to it, passed on.
The purifying of BMECs
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, to cream
Glandular epithelium and fibroblast are separated.When cell covers with culture bottle, cell culture fluid is discarded, is washed 2 times with PBS,
0.25% pancreatin of 1mL is added into Tissue Culture Flask, microscopically observation.When fibroblast is rounded part and starts shedding off,
Complete medium is added into culture bottle immediately, terminates digestion, gently blows and beats molten to remove fibroblast and remaining pancreatin
Then, suitable complete medium is then added in liquid, continue to cultivate.Aforesaid operations are repeated when cell covers with bottom of bottle again,
Purify BMECs.
The passage of BMECs
The growth conditions for observing cell are inhaled when cell covers with bottom of bottle and abandon cell culture fluid, and PBS is washed 2 times, and 1mL is added
The digestion of 0.25% pancreatin, microscopically observation.It is loose when being connected between most cells, when cell starts to be rounded, training completely is added
It supports base and terminates digestion, blowing and beating repeatedly, which makes cell fall off from bottom of bottle, is made single cell suspension, new training is inoculated according to the ratio of 1:2
It supports in bottle, continues to cultivate.
The identification of BMECs
Cell density is adjusted to 1 × 105/mL, is added in 12 porocyte culture plates and cultivates, abandoning when cell grows to 80%
Remove culture medium；10% neutral formalin fixes 20min, and PBS is washed 3 times；0.5%TritonX-100 acts on 20min, and PBS is washed 3 times；
3%H2O2 is incubated for 10min, blocks endogenous peroxydase, and PBS is washed 3 times；- 18 primary antibody of mouse anti-keratin, 4 DEG C of mistakes are added dropwise
Night；The rabbit anti-mouse secondary antibody of biotin labeling is added dropwise, 37 DEG C of incubation 30min, PBS are washed 3 times；The strepto- albumen of HRP label is added dropwise
Element, 37 DEG C of incubation 30min, PBS are washed 3 times；DAB develops the color (being protected from light), under the microscope to light brown, terminates in time after developing the color sufficiently,
Microscopy is taken pictures.As a result with analysis
The adherent and growth that extends outwardly cell island can be seen after cultivating 48h, with the extension of incubation time, cell
Island area is also constantly expanding, and gradually converges in flakes, and at paving stone shape, covering with bottom of bottle 80% to cell can be passed on.Cream
Glandular epithelium has the brown color of specificity or the calmness of sepia, after purification after -18 immunohistochemical staining of keratin
Cow mammary gland epithelial cells purity is higher, can provide basis for follow-up test.
This test purifies galactophore epithelial cell using digestion method when difference, according to epithelial cell and fibroblast to same
The different sensibility of enzyme purify, substantially available purer epithelial cell by 2-3 times.
The processing of BMECs
Cell packet transaction
BMECs is inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, discards the culture medium in hole,
PBS is washed 2 times.Chinese medicine and lipopolysaccharides (LPS) are dissolved in serum free medium, and the nothing for containing 50 μ g/mL LPS is added in Chinese medicine group cell
Isometric serum free medium is added in blood serum medium, lipopolysaccharide group cell.37 DEG C, continue to train in 5%CO2 cell incubator
Support 12h.Then it is washed 2 times with PBS, TRIzol is added, kept cell cracking complete with liquid-transfering gun piping and druming, be transferred in centrifuge tube, -80 DEG C
Refrigerator saves backup.
Cell total rna extracts
RNA is quantitative
Reverse transcription is at cDNA
As a result genechip detection is shown in Fig. 2, Fig. 3
The wherein Chinese medicine, this experiment is 8-methoxypsoralen
According to the above technical scheme, in operation by the sample for needing to detect, it is set as blank control group and (is added without
LPS is also added without Chinese medicine), LPS is stimulated cow mammary gland epithelial cells group (LPS is added), LPS stimulation cow mammary gland epithelial cells+
Three groups of Chinese medicine group (be added LPS, Chinese medicine is also added), measure their cRNA sequence respectively, are compared point according to spectrogram
Analysis, as a result, it has been found that, the inflammation that Chinese medicine is formed after stimulating for LPS is inhibited.
Experimental method through the invention determines following index, to judge LPS stimulation cow mammary gland epithelial cells and LPS
The difference between cow mammary gland epithelial cells+Chinese medicine group is stimulated, to illustrate the therapeutic effect of Chinese medicine: as passed through differential expression base
Because of the functional annotation of enrichment, the immune related pathways of difference expression gene enrichment, the expression of inflammation relevant molecule.
It is thin by chip analysis 8-methoxypsoralen group cell and lipopolysaccharide group after 8-methoxypsoralen
Born of the same parents' mRNA express spectra, the expression quantity of inflammation-associated cytokine (such as IL-1, IL-6, TNF-), 8-methoxypsoralen group with
Lipopolysaccharide group is lowered compared to obvious, to illustrate to stimulate the inflammation to be formed to have LPS after 8-methoxypsoralen is added
Inhibiting effect.
Claims (4)
1. a kind of method of the variation using biochip technology detection cow mammary gland epithelial cells gene expression profile, the method
The following steps are included:
Step 1, the foundation of cow mammary gland epithelial cells inflammatory model, the method is as follows:
1), the culture of cow mammary gland epithelial cells separates, purifying；
2), lipopolysaccharides stimulation cow mammary gland epithelial cells lipopolysaccharides is dissolved in serum free medium, and test group cell, which is added, contains rouge
Isometric serum free medium culture is added in the serum free medium of polysaccharide, cellular control unit；
3) total serum IgE of the cow mammary gland epithelial cells of lipopolysaccharides stimulation, is extracted；
4), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA；
5), with genechip detection cow mammary gland epithelial cells inflammatory cell gene variation cRNA sample and chip hybridization, then
Chip is taken out, is scanned in scanner after washing；
6), the expression variation between the sample of judgement addition lipopolysaccharides and the sample that lipopolysaccharides is not added according to testing result；
Step 2: testing Chinese medicine with cow mammary gland epithelial cells inflammatory model, the method is as follows:
1), the culture of cow mammary gland epithelial cells separates, purifying；
2), lipopolysaccharides stimulates cow mammary gland epithelial cells；
3) Chinese medicine, is added；
4) total serum IgE of cow mammary gland epithelial cells, is extracted；
5), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA；
6), genechip detection cow mammary gland epithelial cells inflammatory cell gene variation cRNA sample and chip hybridization, then take
Chip out scans in scanner after washing；
7), the expression variation between the sample of judgement addition Chinese medicine and the sample that Chinese medicine is not added according to testing result.
2. the method according to claim 1, wherein the method comprises the following steps:
Step 1, the foundation of cow mammary gland epithelial cells inflammatory model
1), the culture of cow mammary gland epithelial cells separates, purifying
Cow mammary gland epithelial cells are separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；Mammary gland
Tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；It will be in breast tissue
White acinar tissue shears off to be cleaned up repeatedly, and mammary gland alveolus is cut into the tissue block of 1mm3, drawn appropriate by no milk residual
0.25% pancreatin of 3 times of volumes is added in 15mL centrifuge tube in tissue block, concussion digestion 30min in 37 DEG C of thermostat water baths,
1000r/min is centrifuged 5min, removes supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of thermostat water baths are then added
Concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, cultivate completely
Base weight hangs cell, and cell suspension is moved into Tissue Culture Flask, 37 DEG C, continue to cultivate in the incubator of 5%CO2, observes afterwards for 24 hours
Whether cell is adherent, if there is other digestion products in culture bottle at this time, the complete medium that can more renew avoids cell
Pollution, postdigestive cell 2-3d grow up to island shape, grow to 80% to it, passed on；
The purifying of cow mammary gland epithelial cells
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, on mammary gland
Chrotoplast and fibroblast are separated, and when cell covers with culture bottle, discard cell culture fluid, are washed 2 times with PBS, are added
0.25% pancreatin of 1mL is into Tissue Culture Flask, microscopically observation, when fibroblast is rounded part and starts shedding off, immediately
Complete medium is added into culture bottle, terminates digestion, gently blows and beats right to remove fibroblast and remaining trypsin solution
Afterwards, suitable complete medium is then added, continues to cultivate, aforesaid operations, purifying are repeated when cell covers with bottom of bottle again
Cow mammary gland epithelial cells,
2), lipopolysaccharides stimulates cow mammary gland epithelial cells
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, are discarded in hole
Culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and the addition of test group cell is containing 50 μ g/mL lipopolysaccharides without blood
Clear culture medium, cellular control unit are added isometric serum free medium, and 37 DEG C, continue to cultivate in 5%CO2 cell incubator
Then 12h is washed 2 times with PBS, TRIzol is added, and is kept cell cracking complete with liquid-transfering gun piping and druming, is transferred in centrifuge tube, -80 DEG C of ice
Case saves backup,
3) total serum IgE of the cow mammary gland epithelial cells of lipopolysaccharides stimulation, is extracted
4), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA
5), with genechip detection cow mammary gland epithelial cells inflammatory cell gene variation cRNA sample and chip hybridization, then
Chip is taken out, is scanned in scanner after washing；
6), the expression variation between the sample of judgement addition lipopolysaccharides and the sample that lipopolysaccharides is not added according to testing result,
Step 2: testing Chinese medicine with cow mammary gland epithelial cells inflammatory model
1), the culture of cow mammary gland epithelial cells separates, purifying
Cow mammary gland epithelial cells are separately cultured
Lactation period and the milk cow without recessive mastitis for milk cows are selected, sterile surgical method obtains cow mammary gland tissue；Mammary gland
Tissue is impregnated 5 minutes with alcohol, tissue is trimmed, distilled water repeated flushing, until liquid is limpid；It will be in breast tissue
White acinar tissue shears off to be cleaned up repeatedly, and mammary gland alveolus is cut into the tissue block of 1mm3, drawn appropriate by no milk residual
0.25% pancreatin of 3 times of volumes is added in 15mL centrifuge tube in tissue block, concussion digestion 30min in 37 DEG C of thermostat water baths,
1000r/min is centrifuged 5min, removes supernatant, and the II Collagenase Type (0.2%) of 3 times of volumes, 37 DEG C of thermostat water baths are then added
Concussion digestion 1.5h, sufficiently piping and druming cell mass, the sieving of 80 mesh collect filtrate in centrifuge tube, and supernatant is abandoned in centrifugation, cultivate completely
Base weight hangs cell, and cell suspension is moved into Tissue Culture Flask, 37 DEG C, continue to cultivate in the incubator of 5%CO2, observes afterwards for 24 hours
Whether cell is adherent, if there is other digestion products in culture bottle at this time, the complete medium that can more renew avoids cell
Pollution, postdigestive cell 2-3d grow up to island shape, grow to 80% to it, passed on,
The purifying of cow mammary gland epithelial cells
It is different from susceptibility of the fibroblast to pancreatin digestive juice according to epithelial cell, using two step digestion methods, on mammary gland
Chrotoplast and fibroblast are separated, and when cell covers with culture bottle, discard cell culture fluid, are washed 2 times with PBS, are added
0.25% pancreatin of 1mL is into Tissue Culture Flask, microscopically observation, when fibroblast is rounded part and starts shedding off, immediately
Complete medium is added into culture bottle, terminates digestion, gently blows and beats right to remove fibroblast and remaining trypsin solution
Afterwards, suitable complete medium is then added, continues to cultivate, aforesaid operations, purifying are repeated when cell covers with bottom of bottle again
Cow mammary gland epithelial cells,
2), lipopolysaccharides stimulates cow mammary gland epithelial cells
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, are discarded in hole
Culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and the serum-free for containing 50 μ g/mL LPS is added in test group cell
Culture medium, cellular control unit are added isometric serum free medium, and 37 DEG C, continue to cultivate 12h in 5%CO2 cell incubator,
Then it is washed 2 times with PBS, TRIzol is added, kept cell cracking complete with liquid-transfering gun piping and druming, be transferred in centrifuge tube, -80 DEG C of refrigerators are protected
Deposit it is spare,
3) Chinese medicine grouping, is added,
Cow mammary gland epithelial cells are inoculated into tissue culture plate, when every hole cell confluency is to 90% or so, are discarded in hole
Culture medium, PBS are washed 2 times, and lipopolysaccharides is dissolved in serum free medium, and the addition of Chinese medicine group cell is containing 50 μ g/mL lipopolysaccharides without blood
The serum free medium for containing 50 μ g/mL lipopolysaccharides is added in clear culture medium, lipopolysaccharide group cell, and cellular control unit is added isometric
Serum free medium, is then washed 2 times with PBS, TRIzol is added, used by 37 DEG C, continue in 5%CO2 cell incubator to cultivate 12h
Liquid-transfering gun piping and druming keeps cell cracking complete, is transferred in centrifuge tube, and -80 DEG C of refrigerators save backup,
4) total serum IgE of three other cow mammary gland epithelial cells of group, is extracted
5), total serum IgE reverse transcription is then converted to cRNA at cDNA, purifies cRNA,；
6), with the other cow mammary gland epithelial cells inflammatory cell genome cRNA sample of three groups of genechip detection and chip
Hybridization, then takes out chip, scans in scanner after washing；
7), the expression variation between the sample of judgement addition Chinese medicine and the sample that Chinese medicine is not added according to testing result.
3. according to the method described in claim 2, it is characterized in that, the Chinese medicine are as follows: any to have on anti-cow mammary gland
The Chinese medicine of chrotoplast inflammation or its extract.
4. according to the method described in claim 3, it is characterized in that, the Chinese medicine are as follows: 8-methoxypsoralen.
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| CN110376366A (en) * | 2019-07-19 | 2019-10-25 | 吉林大学 | A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor |
| CN111944756A (en) * | 2020-08-06 | 2020-11-17 | 五邑大学 | Method for establishing RAW264.7 cell inflammation model |
| CN114377019A (en) * | 2022-01-25 | 2022-04-22 | 吉林大学 | Application of beta-sitosterol in relieving mastitis of dairy cows |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110376366A (en) * | 2019-07-19 | 2019-10-25 | 吉林大学 | A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor |
| CN110376366B (en) * | 2019-07-19 | 2020-10-02 | 吉林大学 | Experimental method for applying nicotinic acid to treatment of cow mastitis through GPR109A receptor |
| CN111944756A (en) * | 2020-08-06 | 2020-11-17 | 五邑大学 | Method for establishing RAW264.7 cell inflammation model |
| CN114377019A (en) * | 2022-01-25 | 2022-04-22 | 吉林大学 | Application of beta-sitosterol in relieving mastitis of dairy cows |
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