CN110376366A - A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor - Google Patents
A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor Download PDFInfo
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Abstract
Be applied to treat the experimental method of mastadenitis of cow by GPR109A receptor the invention discloses a kind of niacin, comprising the following steps: the screening of S1, mazoitis milk cow: it is positive milk cow that preliminary screening, which goes out mazoitis, and somatic number is about ten thousand/mL of 80-200；The detection of proinflammatory factor in S2, cow serum and whey: normal milk cow, mazoitis milk cow were collected at the 0th day and the 7th day respectively and feeds the peripheral blood in the mazoitis milk cow of niacin, then extracts the serum in blood.The present invention is by formulating completely new mastadenitis of cow therapeutic strategy; the expression of pro-inflammatory mediator in mazoitis Contents in Cows after nicotinic acid treatment is had detected by the serum and whey that separate milk cow; niacin is comprehensively had evaluated using molecular biology method to the therapeutic effect of mastadenitis of cow; niacin can effectively reduce the expression of pro-inflammatory mediator in cow serum and whey, to reach the effect of protection mammary gland alleviates inflammation.
Description
Technical field
The present invention relates to pharmaceutical technology field, specially a kind of niacin is applied to treatment milk cow's milk by GPR109A receptor
The experimental method of adenositis.
Background technique
Mastadenitis of cow is a kind of inflammatory disease a kind of as caused by multiple pathogenic microorganisms mixed infection breast tissue
Disease.In worldwide, mastadenitis of cow is all the serious problem for hindering cow estate to develop in a healthy way.In nearest decades
Between, mastadenitis of cow problem is not well solved, and still causes huge economic losses to whole world cow estate.Every year
The whole world is up to 35,000,000,000 dollars because of the economic loss caused by mastadenitis of cow.And the main reason for causing mastadenitis of cow, is then
Pathogenic microorganism invades cow mammary gland.Although and milk cow clinic in antibiotic application can kill pathogenic microorganism,
Very good effect is had no for some antibody-resistant bacterium and metainfective inflammatory reaction.After bacterium infection, it can be generated in mammary gland big
Endotoxin is measured, these endotoxic main components are exactly lipopolysaccharides (LPS).It can cause body to generate strong immunogenicity
Reaction.LPS can also cause the immune response of galactophore epithelial cell or macrophage, to secrete a large amount of inflammatory factor, damage
Galactophore epithelial cell leads to mazoitis.It is mainly at present that antibiotic cooperation is answered combining for anti-inflammatory drug for the treatment of mazoitis
With, therefore the research and development of mastitis-resisting drug are of great significance, this is because simple antibiotic application can not cure well
Mastadenitis of cow is needed in the therapeutic process of mastadenitis of cow with the use of anti-inflammatory drug, serious in cow mammary gland to alleviate
Inflammatory reaction.Previous anti-inflammatory drug price is higher and has certain medicament residue, serious in the meeting of Cow product drug residue
The purchase of the produced milk of illness milk cow is influenced, therefore the economic benefit of cattle farm can be seriously affected to cause a large amount of economic damages
It loses.The normal production for so how restoring mazoitis milk cow as early as possible becomes the another difficult point for the treatment of mastadenitis of cow, and of the invention
A kind of middle drug using niacin as novel anti-mastadenitis of cow has the characteristics that noresidue and cheap, in milk cow's milk
Adenositis cure after can fast quick-recovery mazoitis milk cow lactation and production.
GPR109A belongs to g protein coupled receptor family, it can inhibit inflammation by activation downstream associated signal paths
Occurrence and development, thus achieve the effect that it is anti-inflammatory, many studies have shown that GPR109A participates in anti-inflammatory, lipid-loweringing and anticancer in vivo
Function.And ligand of the niacin as GPR109A, anti-inflammatory properties can be played by activation GPR109A, and give in milk cow
Using.
Niacin belongs to B family vitamin.Niacin is primarily involved in intracorporal lipid-metabolism, inflammatory reaction and glycometabolism etc..Forefathers
Research shows that niacin can pass through activation GPR109A inhibit colitis development.Also it has been found that niacin can inhibit intravascular
Chrotoplast inflammation.Some researches show that the prolonged applications of traditional anti-inflammatory agent to be easy to generate gastrointestinal reaction, renal toxicity and hepatotoxicity wind agitation
Deng other side effects, expensive due to traditional anti-inflammatory medicament, large-scale anti-inflammatory agent application will also result in a large amount of warps
Ji loss.And the hormonal components that Steroidal anti-inflammatory drug contains can have an impact the milk component of milk cow, to influence ox
The purchase of milk further increases the loss of cattle farm.And niacin itself will not generate medicament residue, and cheap and easy to get.Therefore cigarette
Acid is as a kind of advantage that there is novel mastitis-resisting drug conventional medicament not have, but niacin is for mazoitis at present
Act on not clear, therefore the present invention is quasi- using nicotinic acid treatment and prevention mastadenitis of cow.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of niacin is applied to treatment milk cow by GPR109A receptor
The experimental method of mazoitis confirms that niacin can be by activating GPR109A in ELISA test, and significantly inhibits mammary gland
The protein expression of IL-6 in scorching milk bovine whey and serum, TNF-α and IL-1 β.
(2) technical solution
To achieve the above object, the invention provides the following technical scheme: a kind of niacin is applied to control by GPR109A receptor
Treat the experimental method of mastadenitis of cow, comprising the following steps:
The screening of S1, mazoitis milk cow: it is positive milk cow that preliminary screening, which goes out mazoitis, and somatic number is about 80-200
Ten thousand/mL；
The detection of proinflammatory factor in S2, cow serum and whey: normal milk cow, mammary gland are collected the 0th day and the 7th day respectively
Then peripheral blood in scorching milk cow and the mazoitis milk cow of feeding niacin extracts the serum in blood；
The detection of proinflammatory factor in S3, dairy cow milk: normal milk cow, mazoitis milk cow are collected the 0th day and the 7th day respectively
Milk in the mazoitis milk cow illness breast or Normal breast of feeding niacin, then extracts the whey in milk；
S4, CCK8 experiment
Primary cow mammary gland epithelial cells are inoculated into 96 porocyte culture plates, cell about 0.5 × 10 is contained in every hole4
It is a, 100 μ L of culture medium is added, cell is put into 37 DEG C and 5%CO2Sterile constant-temperature incubator in cultivate 24 hours, then plus
The CCK8 reagent of 10 μ L is added in the niacin or niacin+LPS for entering various concentration into each hole again after 24 hours, continues
It is cultivated 2 hours in culture medium, then takes out 96 orifice plates and be put into microplate reader the measurement absorption photometric at 450 nm wavelength；
S5, niacin test the inhibiting effect of primary cow mammary gland epithelial cells inflammatory reaction
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into the mm Tissue Culture Dish of 60 mm × 15, are divided into 4
Group: normal group, niacin group, LPS (1 μ g/ml) group, LPS+ niacin group use serum-free when cell number reaches the 80% of culture dish
Niacin is added after replacing culture medium culture 3 hours in culture medium, and LPS is added after 1 hour, collects in culture dish after 3 hours
Primary cow mammary gland epithelial cells, the cell gathered extract total serum IgE and reverse transcription with Trizol method and are used for subsequent examination at cDNA
After testing or extract the total protein in cell and being uniformly used for the total protein content in cell using BCA determination of protein concentration method
Continuous test detects IL-6 in primary mammary epithelial cells, TNF-α and IL-1 β using fluorescence quantitative PCR method in follow-up test
Gene expression recycles western blot method to detect the protein expression of COX2 and INOS in primary cow mammary gland epithelial cells；
S6, niacin are to the activation of GPR109A
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into the mm Tissue Culture Dish of 60 mm × 15, are used respectively
Niacin collects cell after handling 0 h, 1 h, 3 h, 6 h and 12 h and extracts the total protein in cell, passes through western blot
Method detection cell in GPR109A expression quantity.
Preferably, the characteristic of the milk cow filtered out is more for sediment of trying to get to the heart of a matter, more sticky, and has a small amount of jelly, inclines
When oblique testing cassete, sediment, which has, obviously to be attached to try to get to the heart of a matter and is difficult to the phenomenon that flowing, and when rotary test disk, the oriented center of sediment is poly-
The tendency of collection.
Preferably, in milk somatic number detection method are as follows: first preparation dyeing liquor, take 40mL tetrachloroethanes and 54mL
Ethyl alcohol is sufficiently mixed uniformly, is then placed into 65 DEG C and is asked progress 3min heating in aqueous solution, then add 0.6g methyl blue, finally
It is placed in temperature to be adjusted in 4 DEG C of refrigerator 2 hours, 6mL glacial acetic acid is then added, is put in and is stored at room temperature；We take one piece again
Clean glass slide instills the fresh milk sample of 10 μ L, is put into drying in insulating box, then immerse in dyeing liquor, eventually forms
Even slide；The sample prepared is placed on progress staining cell counting in tally.
Preferably, in blood serum extracting method are as follows: the blood adopted in step S2 is placed in 4 DEG C of ice in 45 degree of angles
It about 1 hour in case, is then centrifuged 5 minutes with 3000 turns of speed, collects IL- in supernatant and application ELISA method detection serum
6, the protein expression of TNF-α and IL-1 β.
Preferably, in milk whey extracting method are as follows: 3000 turns of speed of the milk adopted in step S3 is centrifuged
20 minutes, collect the protein expression of IL-6 in supernatant and application ELISA method detection serum, TNF-α and IL-1 β.
Preferably, the extracting method of galactophore epithelial cell total protein: Primary breast epithelium is collected using Protein Extraction Reagent
Cell simultaneously extracts total protein, using western blot method detection cow mammary gland epithelial cells in iNOS, COX-2 and
The relative expression quantity of GPR109A.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of niacin is applied to treatment cow mammary gland by GPR109A receptor
Scorching experimental method, have it is following the utility model has the advantages that
1, the present invention is detected by formulating completely new mastadenitis of cow therapeutic strategy by the serum and whey that separate milk cow
After nicotinic acid treatment in mazoitis Contents in Cows pro-inflammatory mediator expression, using molecular biology method comprehensively have evaluated niacin pair
The therapeutic effect of mastadenitis of cow, niacin can effectively reduce the expression of pro-inflammatory mediator in cow serum and whey, to reach
The effect of protecting mammary gland to alleviate inflammation.
2, the present invention confirms the mechanism of action of nicotinic acid treatment mazoitis further through experiment in vitro comprehensively, and this method passes through inspection
The expression quantity for surveying GPR109A, INOS and COX2 albumen in galactophore epithelial cell specifies the anti-inflammatory mechanism of action of niacin.From external
We have found that niacin not only has therapeutic effect to mazoitis but also there are also defencive functions in experimental result, feeding niacin in advance can
The Breast health for maintaining milk cow, reduces mazoitis.
3, the present invention can alleviate mastadenitis of cow by way of oral, facilitate the treatment of mazoitis, it is only necessary to
Niacin will be put into feed upper layer during feeding, it is convenient and efficient, drug resistance and the pollution of other antibiotic will not be generated, and
Stress reaction will not be generated to milk cow, be conducive to the treatment and prevention of mastadenitis of cow.
Detailed description of the invention
Fig. 1 is influence of the niacin to IL-1 β in milk bovine whey；
Fig. 2 is influence of the niacin to IL-6 in milk bovine whey；
Fig. 3 is influence of the niacin to TNF-α in milk bovine whey；
Fig. 4 is influence of the niacin to somatic number in dairy cow milk；
Fig. 5 is influence of the niacin to IL-1 β in cow serum；
Fig. 6 is influence of the niacin to IL-6 in cow serum；
Fig. 7 is influence of the niacin to TNF-α in cow serum；
Fig. 8 is influence of the niacin to IL-1 β gene level in cow mammary gland epithelial cells；
Fig. 9 is influence of the niacin to IL-6 gene level in cow mammary gland epithelial cells；
Figure 10 is influence of the niacin to TNF-α gene level in cow mammary gland epithelial cells；
Figure 11 is the influence that niacin survives to cow mammary gland epithelial cells；
Figure 12 is influence of the niacin to iNOS in cow mammary gland epithelial cells and COX-2；
Figure 13 is influence of the niacin to GPR109A in cow mammary gland epithelial cells.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
It is applied to treat the experimental method of mastadenitis of cow by GPR109A receptor the present invention provides a kind of niacin, packet
Include following steps:
The screening of S1, mazoitis milk cow: it is positive milk cow that preliminary screening, which goes out mazoitis, and somatic number is about 80-200
Ten thousand/mL；
The detection of proinflammatory factor in S2, cow serum and whey: normal milk cow, mammary gland are collected the 0th day and the 7th day respectively
Then peripheral blood in scorching milk cow and the mazoitis milk cow of feeding niacin extracts the serum in blood；
The detection of proinflammatory factor in S3, dairy cow milk: normal milk cow, mazoitis milk cow are collected the 0th day and the 7th day respectively
Milk in the mazoitis milk cow illness breast or Normal breast of feeding niacin, then extracts the whey in milk；
S4, CCK8 experiment
Primary cow mammary gland epithelial cells are inoculated into 96 porocyte culture plates, cell about 0.5 × 10 is contained in every hole4
It is a, 100 μ L of culture medium is added, cell is put into 37 DEG C and 5%CO2Sterile constant-temperature incubator in cultivate 24 hours, then plus
The CCK8 reagent of 10 μ L is added in the niacin or niacin+LPS for entering various concentration into each hole again after 24 hours, continues
It is cultivated 2 hours in culture medium, then takes out 96 orifice plates and be put into microplate reader the measurement absorption photometric at 450 nm wavelength；
S5, niacin test the inhibiting effect of primary cow mammary gland epithelial cells inflammatory reaction
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into the mm Tissue Culture Dish of 60 mm × 15, are divided into 4
Group: normal group, niacin group, LPS (1 μ g/ml) group, LPS+ niacin group use serum-free when cell number reaches the 80% of culture dish
Niacin is added after replacing culture medium culture 3 hours in culture medium, and LPS is added after 1 hour, collects in culture dish after 3 hours
Primary cow mammary gland epithelial cells, the cell gathered extract total serum IgE and reverse transcription with Trizol method and are used for subsequent examination at cDNA
After testing or extract the total protein in cell and being uniformly used for the total protein content in cell using BCA determination of protein concentration method
Continuous test detects IL-6 in primary mammary epithelial cells, TNF-α and IL-1 β using fluorescence quantitative PCR method in follow-up test
Gene expression recycles western blot method to detect the protein expression of COX2 and INOS in primary cow mammary gland epithelial cells；
S6, niacin are to the activation of GPR109A
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into the mm Tissue Culture Dish of 60 mm × 15, are used respectively
Niacin collects cell after handling 0 h, 1 h, 3 h, 6 h and 12 h and extracts the total protein in cell, passes through western blot
Method detection cell in GPR109A expression quantity.
For the above-mentioned detection of cooperation, following substance extracting method is now provided:
Firstly, the characteristic of the milk cow filtered out is more for sediment of trying to get to the heart of a matter, it is more sticky, and have a small amount of jelly, it tilts
When testing cassete, sediment, which has, obviously to be attached to try to get to the heart of a matter and is difficult to the phenomenon that flowing, when rotary test disk, the oriented center aggregation of sediment
Tendency.
The detection method of somatic number in milk are as follows: preparation dyeing liquor first takes 40mL tetrachloroethanes and 54mL ethyl alcohol, fills
Divide and be uniformly mixed, is then placed into 65 DEG C and asks progress 3min heating in aqueous solution, then add 0.6g methyl blue, be finally placed in temperature
Degree is adjusted in 4 DEG C of refrigerator 2 hours, and 6mL glacial acetic acid is then added, is put in and is stored at room temperature；We take one piece of clean load again
Slide instills the fresh milk sample of 10 μ L, is put into drying in insulating box, then immerse in dyeing liquor, eventually forms uniform slide；
The sample prepared is placed on progress staining cell counting in tally.
The extracting method of serum in blood are as follows: be placed in the blood adopted in step S2 about 1 in 4 DEG C of refrigerators in 45 degree of angles
Hour, it is then centrifuged 5 minutes with 3000 turns of speed, collects IL-6, TNF-α in supernatant and application ELISA method detection serum
With the protein expression of IL-1 β.
The extracting method of whey in milk are as follows: 3000 turns of speed of the milk adopted in step S3 is centrifuged 20 minutes,
Collect the protein expression of IL-6 in supernatant and application ELISA method detection serum, TNF-α and IL-1 β.
The extracting method of galactophore epithelial cell total protein: primary mammary epithelial cells are collected using Protein Extraction Reagent and are mentioned
Total protein is taken, using the phase of iNOS, COX-2 and GPR109A in the method detection cow mammary gland epithelial cells of western blot
To expression quantity.
It observes niacin and counts as follows by the effect that GPR109A receptor is applied to treatment mastadenitis of cow:
As can be seen from Figure 1 IL-1 β obtains expression quantity significant decrease in milk bovine whey after feeding niacin 7 days, without raising
Feeding niacin then reduces less, therefore niacin can effectively reduce the expression quantity of IL-1 β in milk bovine whey, to alleviate milk cow's milk
Adenositis.
As can be seen from Figure 2 IL-6 obtains expression quantity significant decrease in milk bovine whey after feeding niacin 7 days, without feeding
Niacin then reduces less, therefore niacin can effectively reduce the expression quantity of IL-6 in milk bovine whey, to alleviate cow mammary gland
It is scorching.
As can be seen from Figure 3 TNF-α obtains expression quantity significant decrease in milk bovine whey after feeding niacin 7 days, without raising
Feeding niacin then reduces less, therefore niacin can effectively reduce the expression quantity of TNF-α in milk bovine whey, to alleviate milk cow's milk
Adenositis.
As can be seen from Figure 4 body cell obtains expression quantity significant decrease in dairy cow milk after feeding niacin 7 days, without raising
Feeding niacin then reduces less, therefore niacin can effectively reduce the expression quantity of somatic number in dairy cow milk, to alleviate milk
Bovine mastitis.
As can be seen from Figure 5 IL-1 β obtains expression quantity significant decrease in cow serum after feeding niacin 7 days, without raising
Feeding niacin then reduces less, therefore niacin can effectively reduce the expression quantity of IL-1 β in cow serum, to alleviate milk cow's milk
Adenositis.
As can be seen from Figure 6 IL-6 obtains expression quantity significant decrease in cow serum after feeding niacin 7 days, without feeding
Niacin then reduces less, therefore niacin can effectively reduce the expression quantity of IL-6 in cow serum, to alleviate cow mammary gland
It is scorching.
As can be seen from Figure 7 TNF-α obtains expression quantity significant decrease in cow serum after feeding niacin 7 days, without raising
Feeding niacin then reduces less, therefore niacin can effectively reduce the expression quantity of TNF-α in cow serum, to alleviate milk cow's milk
Adenositis.
As can be seen from Figure 8 the relative expression quantity of IL-1 β significantly increases in cow mammary gland epithelial cells after LPS stimulation,
And the relative expression quantity of IL-1 β significantly reduces in cow mammary gland epithelial cells after niacin processing, therefore niacin can effectively subtract
The relative expression quantity of IL-1 β in few cow mammary gland epithelial cells inflammatory model, to alleviate the inflammation of cow mammary gland epithelial cells
Reaction.
As can be seen from Figure 9 the relative expression quantity of IL-6 significantly increases in cow mammary gland epithelial cells after LPS stimulation, and
The relative expression quantity of IL-6 significantly reduces in cow mammary gland epithelial cells after niacin processing, therefore niacin can effectively reduce milk
The relative expression quantity of IL-6 in bovine mammary epithelial cell inflammatory model, to alleviate the inflammatory reaction of cow mammary gland epithelial cells.
As can be seen from Figure 10 the relative expression quantity of TNF-α significantly increases in cow mammary gland epithelial cells after LPS stimulation,
And the relative expression quantity of TNF-α significantly reduces in cow mammary gland epithelial cells after niacin processing, therefore niacin can effectively subtract
The relative expression quantity of TNF-α in few cow mammary gland epithelial cells inflammatory model, to alleviate the inflammation of cow mammary gland epithelial cells
Reaction.
As can be seen from Figure 11 influence is had no on the survival of cow mammary gland epithelial cells after the processing of various concentration niacin, because
The dosage of this niacin is in safe range.
As can be seen from Figure 12 the relative expression quantity of iNOS and COX-2 is aobvious in cow mammary gland epithelial cells after LPS stimulation
It writes and increases, and the relative expression quantity of iNOS and COX-2 significantly reduces in cow mammary gland epithelial cells after niacin processing, this illustrates cigarette
Acid can effectively reduce the relative expression quantity of iNOS and COX-2 in cow mammary gland epithelial cells inflammatory model, to alleviate milk
The inflammatory reaction of bovine mammary epithelial cell.
As can be seen from Figure 13 as time increases, niacin can significantly increase the expression of GPR109A to activate
GPR109A plays anti-inflammatory properties.
In conclusion should be the experiment proves that niacin can play the function for the treatment of and protection to mastadenitis of cow, from animal
In test we have found that after feeding niacin 7 days, pro-inflammatory mediator in the whey of milk cow, serum or milk (IL-1 β, IL-6 and
TNF-α) there is significant downward, it is also significantly reduced compared with the same period does not feed the milk cow of niacin, this illustrates that niacin can
Alleviate mastadenitis of cow well.We demonstrate niacin again in cell experiment to reduce cell by activation GPR109A
The expressing quantity of middle iNOS and COX-2 and reduce LPS induction galactophore epithelial cell inflammatory response model in IL-1 β, IL-6
With the expression of TNF-α, and the niacin of various concentration does not have an impact cow mammary gland epithelial cells.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of niacin by GPR109A receptor be applied to treatment mastadenitis of cow experimental method, which is characterized in that including with
Lower step:
The screening of S1, mazoitis milk cow: it is positive milk cow that preliminary screening, which goes out mazoitis, somatic number be about 80-200 ten thousand/
mL；
The detection of proinflammatory factor in S2, cow serum and whey: normal milk cow, mazoitis milk are collected the 0th day and the 7th day respectively
Then peripheral blood in ox and the mazoitis milk cow of feeding niacin extracts the serum in blood；
The detection of proinflammatory factor in S3, dairy cow milk: normal milk cow, mazoitis milk cow and feeding are collected the 0th day and the 7th day respectively
Then the mazoitis milk cow illness breast or the milk in Normal breast for feeding niacin extract the whey in milk；
S4, CCK8 experiment
Primary cow mammary gland epithelial cells are inoculated into 96 porocyte culture plates, cell about 0.5 × 10 is contained in every hole4It is a, it is added
100 μ L of culture medium, is put into 37 DEG C and 5%CO for cell2Sterile constant-temperature incubator in cultivate 24 hours, be then added different dense
The CCK8 reagent of 10 μ L is added in the niacin or niacin+LPS of degree into each hole again after 24 hours, continues in the medium
Culture 2 hours then takes out 96 orifice plates and is put into microplate reader the measurement absorption photometric at 450nm wavelength；
S5, niacin test the inhibiting effect of primary cow mammary gland epithelial cells inflammatory reaction
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into 60mm × 15mm Tissue Culture Dish, are divided into 4 groups: normal
Group, niacin group, LPS (1 μ g/ml) group, LPS+ niacin group use serum free medium when cell number reaches the 80% of culture dish
Instead of niacin is added after culture medium culture 3 hours, LPS is added after 1 hour, the primary milk in culture dish is collected after 3 hours
Bovine mammary epithelial cell, the cell gathered with Trizol method extract total serum IgE and reverse transcription at cDNA for follow-up test or
It extracts the total protein in cell and the total protein content in cell is uniformly used for subsequent examination by application BCA determination of protein concentration method
It tests, detects the gene of IL-6 in primary mammary epithelial cells, TNF-α and IL-1 β using fluorescence quantitative PCR method in follow-up test
Expression recycles western blot method to detect the protein expression of COX2 and INOS in primary cow mammary gland epithelial cells；
S6, niacin are to the activation of GPR109A
By 0.3 × 105A primary cow mammary gland epithelial cells are inoculated into 60mm × 15mm Tissue Culture Dish, respectively at niacin
Cell is collected after reason 0h, 1h, 3h, 6h and 12h and extracts the total protein in cell, is detected by the method for western blot thin
The expression quantity of GPR109A in born of the same parents.
2. the experiment side that a kind of niacin according to claim 1 is applied to treatment mastadenitis of cow by GPR109A receptor
Method, it is characterised in that: the characteristic of the milk cow filtered out is more for sediment of trying to get to the heart of a matter, more sticky, and has a small amount of jelly, tilts
When testing cassete, sediment, which has, obviously to be attached to try to get to the heart of a matter and is difficult to the phenomenon that flowing, when rotary test disk, the oriented center aggregation of sediment
Tendency.
3. the experiment side that a kind of niacin according to claim 1 is applied to treatment mastadenitis of cow by GPR109A receptor
Method, it is characterised in that: the detection method of somatic number in milk are as follows: preparation dyeing liquor first takes 40mL tetrachloroethanes and 54mL
Ethyl alcohol is sufficiently mixed uniformly, is then placed into 65 DEG C and is asked progress 3min heating in aqueous solution, then add 0.6g methyl blue, finally
It is placed in temperature to be adjusted in 4 DEG C of refrigerator 2 hours, 6mL glacial acetic acid is then added, is put in and is stored at room temperature；We take one piece again
Clean glass slide instills the fresh milk sample of 10 μ L, is put into drying in insulating box, then immerse in dyeing liquor, eventually forms
Even slide；The sample prepared is placed on progress staining cell counting in tally.
4. the experiment side that a kind of niacin according to claim 1 is applied to treatment mastadenitis of cow by GPR109A receptor
Method, it is characterised in that: the extracting method of serum in blood are as follows: the blood adopted in step S2 is placed in 4 DEG C of ice in 45 degree of angles
It about 1 hour in case, is then centrifuged 5 minutes with 3000 turns of speed, collects IL- in supernatant and application ELISA method detection serum
6, the protein expression of TNF-α and IL-1 β.
5. the experiment side that a kind of niacin according to claim 1 is applied to treatment mastadenitis of cow by GPR109A receptor
Method, it is characterised in that: the extracting method of whey in milk are as follows: 3000 turns of speed of the milk adopted in step S3 is centrifuged 20
Minute, collect the protein expression of IL-6 in supernatant and application ELISA method detection serum, TNF-α and IL-1 β.
6. the experiment side that a kind of niacin according to claim 1 is applied to treatment mastadenitis of cow by GPR109A receptor
Method, it is characterised in that: the extracting method of galactophore epithelial cell total protein: it is thin that Primary breast epithelium is collected using Protein Extraction Reagent
Born of the same parents simultaneously extract total protein, using western blot method detection cow mammary gland epithelial cells in iNOS, COX-2 and
The relative expression quantity of GPR109A.
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| CN112680418A (en) * | 2020-10-30 | 2021-04-20 | 吉林大学 | Cell model for screening GPR109A receptor agonist and screening method |
| CN113564221A (en) * | 2021-07-27 | 2021-10-29 | 吉林大学 | Application of nicotinic acid in preparation of medicine for relieving cow's milk gland fibrosis through GPR109A receptor |
| CN114377019A (en) * | 2022-01-25 | 2022-04-22 | 吉林大学 | Application of beta-sitosterol in relieving mastitis of dairy cows |
| CN114606232A (en) * | 2021-11-04 | 2022-06-10 | 扬州大学 | lncRNA related to mastitis of dairy cattle and application thereof |
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| CN111424008A (en) * | 2020-03-31 | 2020-07-17 | 吉林大学 | Experimental method for promoting animal mammary gland development by nicotinic acid |
| CN112680418A (en) * | 2020-10-30 | 2021-04-20 | 吉林大学 | Cell model for screening GPR109A receptor agonist and screening method |
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| CN114606232A (en) * | 2021-11-04 | 2022-06-10 | 扬州大学 | lncRNA related to mastitis of dairy cattle and application thereof |
| CN114606232B (en) * | 2021-11-04 | 2023-12-08 | 扬州大学 | Cow mastitis related lncRNA and application thereof |
| 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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