CN104762364A - Application method of astragalus polysaccharide in antagonism of dairy cow mammary epithelial cells apoptosis - Google Patents
Application method of astragalus polysaccharide in antagonism of dairy cow mammary epithelial cells apoptosis Download PDFInfo
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Abstract
The invention discloses an application method of astragalus polysaccharide in antagonism of dairy cow mammary epithelial cells apoptosis. The method comprises the following steps: (1) obtaining 2-3 generations of dairy cow mammary epithelial cells; (2) grouping and processing cells; (3) detecting cell survival rate employing a CCK-8 method; (4) detecting a cell cycle employing flow cytometry; (5) detecting the change condition of nucleic acid of apoptotic cells employing a DNA ladder; (6) observing the internal morphological structure change of the cells through a transmission electron microscope; (7) detecting inflammation-associated cytokines content in a cell supernatant employing radioimmunoassay; and (8) detecting inflammation-related enzyme activity in the supernatant employing a biochemical kit method. Through the method disclosed by the invention, the action of the astragalus polysaccharide for antagonism of dairy cow mammary epithelial cells apoptosis is discussed; and the method is efficient, simple, convenient, feasible, and accurate in result.
Description
Technical field
The present invention relates to a kind of application method of astragalus polysaccharides; Particularly relate to the application method of a kind of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis.
Background technology
Mastadenitis of cow has a strong impact on the milk yield of milk cow, reduces milk quality, harm consumer health, and extends the milk cow post-partum estrus phase, increases cow culling rate, thus reduces milk cow production economic benefit.At present, the treatment means about mastadenitis of cow is a lot, as chemical medicinal treatment, recombinant vaccine treatment, cytokine therapy, bacteriocin treatment, hormonotherapy and the treatment of anti-Pathogenic Bacteria of Dairy Cow Mastitis composite yolk antibody etc.These methods have his own strong points, though can not thoroughly cure, can play certain curative effect.But the side effect of these methods but can not be ignored, particularly drug residue, jeopardizes human health indirectly.Therefore, how to utilize biologically active substance to transfer the defense mechanism of mammary tissue self, thus regulate the health level of mammary gland to become the focus of mastadenitis of cow study on prevention.
Mammary epithelial cell forms the chief functional cells of mammary gland, is also the first defensive barrier of host body when resisting pathogenic micro-organism invasion mammary gland.Lipopolysaccharides (Lipopolysaccharide, LPS) is a kind of macromolecular structure composition on Gram-negative bacteria (G-) epicyte.The mammary gland of ox is very responsive to the stimulation of LPS, perfusion LPS in mammary gland, obvious mastitis symptom can be induced, the level of lipopolysaccharide binding protein and soluble cd 14 and the release of inflammatory factor in blood and milk can be increased simultaneously, the inflammatory reaction that its inflammatory reaction brought out and G-bacterium infection mammary gland cause has extremely similar place, therefore has clinically and applies very widely.And studies have reported that and show, stimulate through LPS in vitro, cow mammary gland epithelial cells also can be secreted the relevant medium of inflammation and carry out mediated cell damage.
Herbal medicine and active ingredients thereof early have report in the clinical practice for the treatment of mammitis of cow.Relevant report shows, the control of astragalus polysaccharides to mastadenitis of cow has potential research and development and be worth.Zhong Kai etc. study the impact of astragalus polysaccharides on the experimental goat of escherichia coli endotoxin induced by LPS, milk cow and rat mammary gland inflammation; result shows; animal astragalus polysaccharides is fed in breast regional perfusion astragalus polysaccharides or filling; all can alleviate the destruction of escherichia coli endotoxin lipopolysaccharides to mammary tissue, have certain provide protection to the experimentation on animals mazoitis of induced by LPS.
Summary of the invention
The invention provides the application method of a kind of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis, the inventive method has efficient, easy, the feasible and accurate advantage of result.
The application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis, comprises the steps:
(1) cultivate cow mammary gland epithelial cells, obtain 2-3 and be used for follow-up test for logarithmic phase cell;
(2) grouping of cell and process:
1. get described 2-3 and be prepared into 1 × 10 for logarithmic phase cow mammary gland epithelial cells
4the cell suspension of individual/mL, is inoculated in 96 porocyte culture plates by every hole 150 μ L, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 200 μ L, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides;
2. get described 2-3 and be prepared into 5 × 10 for logarithmic phase cell
5-1 × 10
6the cell suspension of individual/mL, is inoculated in 6 porocyte culture plates by every hole 1.5mL, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 tests to test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 2mL, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides.
(3) CCK-8 method detect in described step (2) 1. described in cell survival rate in 5 test group;
(4) in step described in Flow cytometry (2) 2. described in mammary epithelial cell cycle in 5 test group;
(5) agarose gel electrophoresis detect in described step (2) 2. described in mammary epithelial cell DNAladder band in 5 test group;
(6) in step described in transmission electron microscope observing (2) 2. described in the inner Ultrastructural change of mammary epithelial cell in 5 test group;
(7) radioimmunology detect in described step (2) 2. described in inflammation-associated cytokine content in cell conditioned medium liquid in 5 test group;
(8) biochemical reagents box method detect in described step (2) 2. described in 5 test group in cell conditioned medium liquid inflammation relevant enzyme enzyme live.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (1) specifically comprises the steps:
Tissue mass cell culture is adopted to obtain cow mammary gland epithelial cells, with containing substratum based on the DMEM/F12 nutrient solution of 10% volume fraction calf serum, in 37 DEG C, CO
2volume fraction is cultivate in the incubator of 5%, gets 2-3 for logarithmic phase cell for follow-up test after purified.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (3) specifically comprises the steps:
In described step (2) 1. in after 5 test group obtaining continue to cultivate 8h respectively, every hole adds the CCK-8 solution of 10 μ L, continue to hatch 1 ~ 4h, observe substratum colour-change, the absorbance of each hole at 490nm place is measured in full-automatic microplate reader, calculate described 5 test group cell inhibitory rates, cell survival rates respectively, the method of calculation of described cell inhibitory rate are: cell inhibitory rate=1-(test group OD value/control group OD value) × 100%, cell survival rate=1-cell inhibitory rate; The cell survival rate of control group is defined as 100%.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (4) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, carry out trysinization respectively to obtain single cell suspension moving to respectively in centrifuge tube to carry out first time centrifugal, it is the centrifugal 5min of 1500rpm at ambient temperature that described first time is centrifugal, after careful absorption supernatant liquor, adding respectively after 1mL PBS blows and beats cell precipitation is transferred in streaming loading pipe again, recentrifuge also absorbs supernatant, described recentrifuge is identical with described first time centrifugal condition, stay 50 μ L supernatant liquors in order to avoid siphon away cell during described absorption supernatant liquor, obtain cell cycle testing sample, described cell cycle testing sample is carried out Flow cytometry respectively.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (5) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell, uses phenol-chloroform-primary isoamyl alcohol method extracting cell DNA, electrophoresis in 1% sepharose respectively respectively, detects cell DNA integrity.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (6) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell, fixes by the glutaraldehyde that massfraction is 2.5% respectively, fixes after cleaning for several times with 1% osmic acid again; Then dewater respectively, described dehydration is first dewater step by step with the alcohol that massfraction is 50%-70%, then dewater step by step with the acetone that massfraction is 80%-100%; Epon812 resin embedding is used respectively after dehydration, make 1 μm of semithin section, dye with toluidine blue again, 70-80nm ultrathin section(ing) is made after tissue positioned, with acetic acid uranium-lead citrate by described ultrathin section(ing) double staining, under JSM-6700F transmission electron microscope, observe the inner ultrastructural morphology of described 5 test group mammary epithelial cells and apoptosis situation thereof respectively.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (7) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, radioimmunology detects the massfraction of inflammation-associated cytokine TNF-α, IL-1 β, IL-6, IL-2 in described each group of cell conditioned medium liquid, and unit is followed successively by pg/mL, ng/mL, pg/mL, ng/mL respectively.
The application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, wherein, described step (8) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, the enzyme that biochemical reagents box method detects inflammation relevant enzyme NAGase, AKP, MPO, iNOS in each group of cell conditioned medium liquid is lived, and unit is U/L.
The beneficial effect of the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis is:
The focus of current mazoitis study on prevention how to utilize biologically active substance to transfer the defense mechanism of mammary gland self, regulate the health level of mammary gland, and the unusual effect utilizing Chinese herbal medicine effective ingredients (plant-sourced bioactive ingredients) to prevent and treat mammitis of cow is domestic and international Experts ' Attention day by day.The method of the invention causes in illustrating astragalus polysaccharides to the antagonistic action of lipopolysaccharide-induced cow mammary gland epithelial cells apoptosis and mechanism thereof, contributes to understanding in depth lipopolysaccharides to the mechanism of action of normal cow mammary gland epithelial cells apoptosis damage and astragalus polysaccharides antagonize cellular apoptosis and inner link.Can be the pathogenesis of further investigation mastadenitis of cow, therapeutic trial and pharmacodynamic study and trial model basis and data refer are provided.
And with cow mammary gland epithelial cells for test materials obtains test materials safety, economic and easy to be many than being sampled or massacre macrofauna (as milk cow) by living tissue.In vitro under culture condition drugs or intracellular toxin etc. on the impact of mammary epithelial cell, thus a series of physiological activities of cell under directly can observing normal and pathology situation, and further objective evaluation is made to various medicine or endotoxic effect, solve the many problems being difficult to study in live body situation.
Below in conjunction with accompanying drawing, the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis is described further.
Accompanying drawing explanation
Fig. 1 is the data statistic analysis figure that in the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, CCK-8 method detects each test group mammary epithelial cell survival rate; Wherein, control group is blank group, and model group is LPS model group, and 1mg/mL is the first astragalus polysaccharides model group, and 3mg/mL is the second astragalus polysaccharides model group, and 5mg/mL is the 3rd astragalus polysaccharides model group; * represents: compared with blank group, and LPS model group cell survival rate extremely significantly reduces (P<0.01); ## represents: compared with LPS model group, and each astragalus polysaccharides model group Cell viability all significantly improves (P<0.01) pole.
Fig. 2 is each test group DNA agarose gel electrophoresis figure in the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis; A swimming lane is marker; B swimming lane is blank group; C swimming lane is LPS model group; D swimming lane is the first astragalus polysaccharides model group; E swimming lane is the second astragalus polysaccharides model group; F swimming lane is the 3rd astragalus polysaccharides model group.
Fig. 3 is each test group cell interior ultrastructure figure in the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis; Wherein, A figure is blank group; B figure is LPS model group; C figure is the first astragalus polysaccharides model group; D figure is the second astragalus polysaccharides model group; E figure is the 3rd astragalus polysaccharides model group.
Fig. 4 is the data statistic analysis figure of each test group cell conditioned medium liquid inflammatory cytokine content in the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis; Wherein, A figure is that TNF-alpha content compares; B figure is IL-1 β comparision contents; C figure is IL-6 comparision contents.Wherein, control group is blank group, and model group is LPS model group, and 1mg/mL is the first astragalus polysaccharides model group, and 3mg/mL is the second astragalus polysaccharides model group, and 5mg/mL is the 3rd astragalus polysaccharides model group;
Fig. 5 is the data statistic analysis figure that in the application method of astragalus polysaccharides of the present invention in antagonism cow mammary gland epithelial cells apoptosis, each test group cell conditioned medium liquid inflammation relevant enzyme enzyme is lived; Wherein, A figure is that the work of NAGase enzyme is compared; B figure is that the work of AKP enzyme is compared; C figure is that the work of MPO enzyme is compared; D figure is that the work of iNOS enzyme is compared.Wherein, control group is blank group, and model group is LPS model group, and 1mg/mL is the first astragalus polysaccharides model group, and 3mg/mL is the second astragalus polysaccharides model group, and 5mg/mL is the 3rd astragalus polysaccharides model group.
Embodiment
Embodiment 1
The application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis, comprises the steps:
(1) cultivate cow mammary gland epithelial cells, obtain 2-3 and be used for follow-up test for logarithmic phase cell;
(2) grouping of cell and process:
1. get described 2-3 and be prepared into 1 × 10 for logarithmic phase cow mammary gland epithelial cells
4the cell suspension of individual/mL, is inoculated in 96 porocyte culture plates by every hole 150 μ L, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 200 μ L, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides;
2. get described 2-3 and be prepared into 5 × 10 for logarithmic phase cow mammary gland epithelial cells
5-1 × 10
6the cell suspension of individual/mL, is inoculated in 6 porocyte culture plates by every hole 1.5mL, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 tests to test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 2mL, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides.
(3) CCK-8 method detect in described step (2) 1. described in cell survival rate in 5 test group;
(4) in step described in Flow cytometry (2) 2. described in mammary epithelial cell cycle in 5 test group;
(5) agarose gel electrophoresis detect in described step (2) 2. described in mammary epithelial cell DNAladder band in 5 test group;
(6) in step described in transmission electron microscope observing (2) 2. described in the inner Ultrastructural change of mammary epithelial cell in 5 test group;
(7) radioimmunology detect in described step (2) 2. described in inflammation-associated cytokine content in cell conditioned medium liquid in 5 test group;
(8) biochemical reagents box method detect in described step (2) 2. described in 5 test group in cell conditioned medium liquid inflammation relevant enzyme enzyme live.
Embodiment 2
The application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis, comprises the steps:
(1) tissue mass cell culture is adopted to obtain cow mammary gland epithelial cells, with containing substratum based on the DMEM/F12 nutrient solution of 10% volume fraction calf serum, in 37 DEG C, CO
2volume fraction is cultivate in the incubator of 5%, gets 2-3 for logarithmic phase cell for follow-up test after purified.
(2) grouping of cell and process:
1. get described 2-3 and be prepared into 1 × 10 for logarithmic phase cow mammary gland epithelial cells
4the cell suspension of individual/mL, is inoculated in 96 porocyte culture plates by every hole 150 μ L, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 200 μ L, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides;
2. get described 2-3 and be prepared into 5 × 10 for logarithmic phase cell
5-1 × 10
6the cell suspension of individual/mL, is inoculated in 6 porocyte culture plates by every hole 1.5mL, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 tests to test group at random: (I) blank group; (II) lipopolysaccharides (Lipopolysaccharide, LPS) model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 2mL, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS; Described first astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration LPS, 5mg/mL mass concentration astragalus polysaccharides.
(3) in described step (2) 1. in after 5 test group obtaining continue to cultivate 8h respectively, every hole adds the CCK-8 solution of 10 μ L, continue to hatch 1 ~ 4h, observe substratum colour-change, the absorbance of each hole at 490nm place is measured in full-automatic microplate reader, calculate described 5 test group cell inhibitory rates, cell survival rates respectively, the method of calculation of described cell inhibitory rate are: cell inhibitory rate=1-(test group OD value/control group OD value) × 100%, cell survival rate=1-cell inhibitory rate; The cell survival rate of control group is defined as 100%.
Result is as shown in Figure 1: compared with blank group, LPS model group cell survival rate extremely significantly reduces (P<0.01); Compared with LPS model group, first, second, and third astragalus polysaccharides model group cell survival rate all significantly improves (P<0.01) pole; Blank group cell survival rate is decided to be 100%, and after statistic data, LPS model group cell survival rate is 50.64%, and show that the ability of LPS model group cell reduction CCK-8 declines, cell viability declines; First, second, and third astragalus polysaccharides model group cell survival rate is respectively: 62.94%, 74.85% and 85.13%, illustrates the mammary epithelial cell apoptosis damage that astragalus polysaccharides can significantly suppress LPS to induce have provide protection to mammary epithelial cell.
(4) in described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, carry out trysinization respectively to obtain single cell suspension moving to respectively in centrifuge tube to carry out first time centrifugal, it is the centrifugal 5min of 1500rpm at ambient temperature that described first time is centrifugal, after careful absorption supernatant liquor, adding respectively after 1mL PBS blows and beats cell precipitation is transferred in streaming loading pipe again, recentrifuge also absorbs supernatant, described recentrifuge is identical with described first time centrifugal condition, stay 50 μ L supernatant liquors in order to avoid siphon away cell during described absorption supernatant liquor, obtain cell cycle testing sample, described cell cycle testing sample is carried out Flow cytometry respectively.
Result is as shown in table 1: LPS model group compares with control group, G
0/ G
1phase mammary epithelial cell extremely significantly increases (P<0.01), S phase, G
2/ M phase cell count, proliferation index (PI) all extremely significantly reduce (P<0.01), and have occurred apoptotic peak; Compared with LPS model group, first astragalus polysaccharides model group each cell cycle inner cell ratio and proliferation index, all without noticeable change, also have the appearance of apoptotic peak; Compared with LPS model group, the second astragalus polysaccharides model group G
0/ G
1phase mammary epithelial cell extremely significantly reduces (P<0.01), and S phase cell proportion, proliferation index all extremely significantly increase (P<0.01); Compared with LPS model group, the 3rd astragalus polysaccharides model group G
0/ G
1phase mammary epithelial cell extremely significantly reduces (P<0.01), G
2/ M phase cell proportion significantly increases (P<0.05), S phase cell proportion, proliferation index all extremely significantly increase (P<0.01).In cell cycle, G
0/ G
1phase cell quantity per-cent reduces, and the increase of S phase cell quantity per-cent reflects DNA propagation in cell.Result is pointed out, and makes G after the process of 3-5mg/mL astragalus polysaccharides
0/ G
1the cell quantity per-cent of phase reduces; S phase cell quantity per-cent and proliferation index increase; illustrate that astragalus polysaccharides can impel the mammary epithelial cell of apoptosis to rest on the S phase, cell DNA synthesis increases; thus the mammary epithelial cell apoptosis that can significantly suppress LPS to induce damage, there is provide protection to mammary epithelial cell.
Table 1 astragalus polysaccharides is on the impact in LPS inducing cows mammary epithelial cell apoptosis cycle
Note: LPS model group is compared with control group: * p<0.05, * * p<0.01, astragalus polysaccharides model group is compared with LPS model group: #p<0.05, ##p<0.01.
(5) in described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell respectively, use phenol-chloroform-primary isoamyl alcohol method extracting cell DNA respectively, electrophoresis in 1% sepharose, detect cell DNA integrity.
Result is as shown in Figure 2: compared with blank group cell, and LPS model group cell occurs that DNA break degree is comparatively serious, presents the DNA fragmentation of a series of different lengths in electrophorogram; Second and third astragalus polysaccharides model group cellular DNA fragments situation and blank group close; And the first concentration astragalus polysaccharides model group is similar to model group cellular DNA fragments situation, all there is DNA ladder shape band.Apoptotic generation, can cause its endogenous endonuclease and be activated, cause DNA in cell be cut into 180 ~ 200bp length not wait multiple DNA fragmentations, length not wait DNA fragmentation can present different bands by 1% agarose gel electrophoresis; Test-results shows, astragalus polysaccharides can reduce the DNA break degree of LPS inducing mammary epithelial cell, has antagonistic action to apoptosis, and in dose-dependent relationship within the scope of astragalus polysaccharides mass concentration described in present method.
(6) in described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell, fixes by the glutaraldehyde that massfraction is 2.5% respectively, fixes after cleaning for several times with 1% osmic acid again; Then dewater respectively, described dehydration is first dewater step by step with the alcohol that massfraction is 50%-70%, then dewater step by step with the acetone that massfraction is 80%-100%; Epon812 resin embedding is used respectively after dehydration, make 1 μm of semithin section, dye with toluidine blue again, 70-80nm ultrathin section(ing) is made after tissue positioned, with acetic acid uranium-lead citrate by described ultrathin section(ing) double staining, under JSM-6700F transmission electron microscope, observe the inner ultrastructural morphology of described 5 test group mammary epithelial cells and apoptosis situation thereof respectively.
Result as shown in Figure 3, can be observed under transmission electron microscope, control group mammary epithelial cell cytolemma complete display, and be positioned at cell central authorities, in core, chromatin is uniformly distributed; LPS model group occurs that nucleus is cracked, Chromatin condensation, and cavity increases, cytoplasmic condensation, occurs the apoptotic body differed in size; First astragalus polysaccharides model group, karyopyknosis, Chromatin condensation; Second and third astragalus polysaccharides model group, karyomorphism is tending towards normal, and chromatin is uniformly distributed, nuclear membrane is high-visible.Result shows, astragalus polysaccharides can reduce the apoptosis damage of LPS inducing mammary epithelial cell, has antagonistic action to apoptosis, and in dose-dependent relationship within the scope of astragalus polysaccharides mass concentration described in present method.
(7) in described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, radioimmunology detects the massfraction of inflammation-associated cytokine TNF-α, IL-1 β, IL-6, IL-2 in described each group of cell conditioned medium liquid, and unit is followed successively by pg/mL, ng/mL, pg/mL, ng/mL respectively.
Result is as shown in Figure 4: compared with blank group, in LPS model group cell conditioned medium liquid, the secretion of cytokine TNF-α, IL-1 β, IL-6 extremely significantly rises (P<0.01); Compared with LPS model group, in the first astragalus polysaccharides model group cell conditioned medium liquid, the secretion level of TNF-α, IL-1 β, IL-6 obviously reduces (P<0.05); Compared with LPS model group, in second and third astragalus polysaccharides model group cell conditioned medium liquid, the secretion level of TNF-α, IL-1 β, IL-6 extremely significantly reduces (P<0.01); Result shows that astragalus polysaccharides by reducing excessive secretion and the release of inflammatory cytokine, can alleviate the damage of LPS to mammary epithelial cell, and in dose-dependent relationship within the scope of astragalus polysaccharides mass concentration described in present method.
(8) in described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, the enzyme that biochemical reagents box method detects inflammation relevant enzyme NAGase, AKP, MPO, iNOS in each group of cell conditioned medium liquid is lived, and unit is U/L.
Result is as shown in Figure 5: compared with Normal group, in LPS model group cell conditioned medium liquid, the activity of NAGase, AKP, MPO, iNOS all extremely significantly raises (P<0.01); Compared with LPS model group, the first astragalus polysaccharides model group pole significantly reduces MPO enzyme and lives (P<0.01); Compared with LPS model group, the second astragalus polysaccharides model group pole significantly reduces NAGase, MPO, iNOS enzyme and lives (P<0.01), significantly reduces AKP enzyme and lives (P<0.05); Compared with LPS model group, the 3rd astragalus polysaccharides model group pole significantly reduces NAGase, AKP, MPO, iNOS enzyme and lives (P<0.01); The classical index that NAGase, AKP, MPO, iNOS are all prompting inflammation in body, mammary gland is damaged degree, in the mazoitis of pathogenic bacterium induction, the excessive secretion of inflammatory cytokine and inflammation relevant enzyme is the principal element causing mammary epithelial cell to damage.Compared with model group, second and third astragalus polysaccharides model group obviously can reduce the activity of these four kinds of enzymes, first astragalus polysaccharides model group change is not obvious, illustrate that astragalus polysaccharides can by reducing excessive secretion and the release of inflammatory relevant enzyme, alleviate the damage of LPS to mammary epithelial cell, and in dose-dependent relationship within the scope of astragalus polysaccharides mass concentration described in present method.
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Claims (8)
1. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis, is characterized in that, comprise the steps:
(1) cultivate cow mammary gland epithelial cells, obtain 2-3 and be used for follow-up test for logarithmic phase cell;
(2) grouping of cell and process:
1. get described 2-3 and be prepared into 1 × 10 for logarithmic phase cow mammary gland epithelial cells
4the cell suspension of individual/mL, is inoculated in 96 porocyte culture plates by every hole 150 μ L, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 test group at random: (I) blank group; (II) lipopolysaccharides model group, also referred to as Lipopolysaccharide model group, i.e. LPS model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 200 μ L, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described lipopolysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration lipopolysaccharides; Described first astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration lipopolysaccharides, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration lipopolysaccharides, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 200 μ L containing 100 μ g/mL mass concentration lipopolysaccharides, 5mg/mL mass concentration astragalus polysaccharides;
2. get described 2-3 and be prepared into 5 × 10 for logarithmic phase cow mammary gland epithelial cells
5-1 × 10
6the cell suspension of individual/mL, is inoculated in 6 porocyte culture plates by every hole 1.5mL, under 37 DEG C of conditions, and CO
2volume fraction is in the incubator of 5% after cellar culture 24h, is divided into following 5 test group at random: (I) blank group; (II) lipopolysaccharides model group, i.e. LPS model group; (III) the first astragalus polysaccharides model group; (IV) the second astragalus polysaccharides model group; (V) the 3rd astragalus polysaccharides model group; Often organize 3 plates to repeat; After supernatant is abandoned in suction, described blank group adds basic culture solution 2mL, and described basic culture solution is the DMEM/F12 nutrient solution containing 10% volume fraction calf serum; Described LPS model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration lipopolysaccharides; Described first astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration lipopolysaccharides, 1mg/mL mass concentration astragalus polysaccharides; Described second astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration lipopolysaccharides, 3mg/mL mass concentration astragalus polysaccharides; Described 3rd astragalus polysaccharides model group adds the nutrient solution 2mL containing 100 μ g/mL mass concentration lipopolysaccharides, 5mg/mL mass concentration astragalus polysaccharides.
(3) CCK-8 method detect in described step (2) 1. described in cell survival rate in 5 test group;
(4) in step described in Flow cytometry (2) 2. described in mammary epithelial cell cycle in 5 test group;
(5) agarose gel electrophoresis detect in described step (2) 2. described in mammary epithelial cell DNAladder band in 5 test group;
(6) in step described in transmission electron microscope observing (2) 2. described in the inner Ultrastructural change of mammary epithelial cell in 5 test group;
(7) radioimmunology detect in described step (2) 2. described in inflammation-associated cytokine content in cell conditioned medium liquid in 5 test group;
(8) biochemical reagents box method detect in described step (2) 2. described in 5 test group in cell conditioned medium liquid inflammation relevant enzyme enzyme live.
2. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (1) specifically comprises the steps:
Tissue mass cell culture is adopted to obtain cow mammary gland epithelial cells, with containing substratum based on the DMEM/F12 nutrient solution of 10% volume fraction calf serum, in 37 DEG C, CO
2volume fraction is cultivate in the incubator of 5%, gets 2-3 for logarithmic phase cell for follow-up test after purified.
3. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (3) specifically comprises the steps:
In described step (2) 1. in after 5 test group obtaining continue to cultivate 8h respectively, every hole adds the CCK-8 solution of 10 μ L, continue to hatch 1 ~ 4h, observe substratum colour-change, the absorbance of each hole at 490nm place is measured in full-automatic microplate reader, calculate described 5 test group cell inhibitory rates, cell survival rates respectively, the method of calculation of described cell inhibitory rate are: cell inhibitory rate=1-(test group OD value/control group OD value) × 100%, cell survival rate=1-cell inhibitory rate; The cell survival rate of control group is defined as 100%.
4. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (4) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, carry out trysinization respectively to obtain single cell suspension moving to respectively in centrifuge tube to carry out first time centrifugal, it is the centrifugal 5min of 1500rpm at ambient temperature that described first time is centrifugal, after careful absorption supernatant liquor, adding respectively after 1mL PBS blows and beats cell precipitation is transferred in streaming loading pipe again, recentrifuge also absorbs supernatant, described recentrifuge is identical with described first time centrifugal condition, stay 50 μ L supernatant liquors in order to avoid siphon away cell during described absorption supernatant liquor, obtain cell cycle testing sample, described cell cycle testing sample is carried out Flow cytometry respectively.
5. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (5) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell, uses phenol-chloroform-primary isoamyl alcohol method extracting cell DNA, electrophoresis in 1% sepharose respectively respectively, detects cell DNA integrity.
6. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (6) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collected by trypsinisation cell, fixes by the glutaraldehyde that massfraction is 2.5% respectively, fixes after cleaning for several times with 1% osmic acid again; Then dewater respectively, described dehydration is first dewater step by step with the alcohol that massfraction is 50%-70%, then dewater step by step with the acetone that massfraction is 80%-100%; Epon812 resin embedding is used respectively after dehydration, make 1 μm of semithin section, dye with toluidine blue again, 70-80nm ultrathin section(ing) is made after tissue positioned, with acetic acid uranium-lead citrate by described ultrathin section(ing) double staining, under JSM-6700F transmission electron microscope, observe the inner ultrastructural morphology of described 5 test group mammary epithelial cells and apoptosis situation thereof respectively.
7. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (7) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, radioimmunology detects the massfraction of inflammation-associated cytokine TNF-α, IL-1 β, IL-6, IL-2 in described each group of cell conditioned medium liquid, and unit is followed successively by pg/mL, ng/mL, pg/mL, ng/mL respectively.
8. the application method of astragalus polysaccharides in antagonism cow mammary gland epithelial cells apoptosis according to claim 1, it is characterized in that, described step (8) specifically comprises the steps:
In described step (2) 2. in obtain described 5 test group and continue respectively to cultivate after 8h, collect each group of cell conditioned medium liquid respectively, the enzyme that biochemical reagents box method detects inflammation relevant enzyme NAGase, AKP, MPO, iNOS in each group of cell conditioned medium liquid is lived, and unit is U/L.
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| CN110894486A (en) * | 2019-11-08 | 2020-03-20 | 江苏省农业科学院 | Application of dihydromyricetin in antagonizing heat stress injury of mammary epithelial cells of dairy cows |
| CN110907372A (en) * | 2019-12-02 | 2020-03-24 | 上海海洋大学 | In-vitro evaluation model and method for anti-inflammatory performance of toothpaste containing bletilla striata extract |
| 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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| CN108841784A (en) * | 2018-07-17 | 2018-11-20 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of nucleus pulposus cell anti-aging anti-apoptotic culture medium containing Astragaloside IV |
| CN110373376A (en) * | 2019-04-08 | 2019-10-25 | 北京农学院 | Cow mammary gland epithelial cells damage model and its construction method and application |
| CN110894486A (en) * | 2019-11-08 | 2020-03-20 | 江苏省农业科学院 | Application of dihydromyricetin in antagonizing heat stress injury of mammary epithelial cells of dairy cows |
| CN110894486B (en) * | 2019-11-08 | 2023-02-24 | 江苏省农业科学院 | Application of dihydromyricetin in antagonizing heat stress injury of mammary epithelial cells of dairy cows |
| CN110907372A (en) * | 2019-12-02 | 2020-03-24 | 上海海洋大学 | In-vitro evaluation model and method for anti-inflammatory performance of toothpaste containing bletilla striata extract |
| CN110907372B (en) * | 2019-12-02 | 2023-02-14 | 上海海洋大学 | In-vitro evaluation model and method for anti-inflammatory performance of toothpaste containing bletilla striata extract |
| 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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