CN110687287A - Analysis method of deer fetus oligopeptide for immunoregulation of macrophage - Google Patents
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Abstract
An analysis method for immunoregulation of macrophage by deer fetus oligopeptide belongs to the technical field of biotechnology research and development analysis, and the proliferation index of RAW264.7 cells after the deer fetus oligopeptide is screened out to act is obviously increased compared with blank ratio and is most similar to an LPS positive control group; the method analyzes the influence of different mass concentrations on the phagocytic function, the NO secretion amount and the release of cell factors such as IL-1 beta, IL-6 and TNF-alpha, and the obtained result shows that the NO has the maximum release amount at 25 mu g/mL and is closest to an LPS group; the cell cycle examination is carried out simultaneously in the method, and data results prove that along with the increase of the mass concentration of the deer fetus oligopeptide, the proportion of cells in the G0/G1 phase is in the trend of rising firstly and then falling, the proportion of cells in the S phase and the G2/M phase is in the trend of falling firstly and then rising, and when the proportion of the cells in each phase is closest to that of an LPS group at 25 mu G/mL; G0/G1 is the synthesis phase of RNA and protein, so the expression level of related protein is increased, and the secretion capacity of phagocytosis, NO and cytokines reaches the maximum value in the phase.
Description
Technical Field
The invention belongs to the technical field of research, development and analysis of biotechnology, and particularly relates to an analysis method for immunoregulation of deer fetus oligopeptides on macrophages.
Background
The embryo Cervi is placenta and fetus of Cervus nippon Temminck or Cervus elaphus Linnacus. Deer fetus is a traditional and rare tonic in China, and is known as one of 'three treasures of gynecology' (deer fetus, black-bone chicken and donkey-hide gelatin) in ancient times. Deer fetus, as a Chinese medicine, has a long history of treating diseases, and its pharmacological actions are first listed in compendium of materia Medica. Deer fetus, regulating menstruation, nourishing face and removing toxic materials. The traditional Chinese medicine considers that: deer fetus is mainly used for treating impotence due to kidney deficiency and insufficiency of nourishment, and is suitable for treating kidney deficiency, weakness, blood deficiency, female irregular menstruation, metrorrhagia and the like. The embryo Cervi has various chemical components, mainly including amino acids, vitamins, and peptides. Among them, oligopeptide components are easily absorbed, and have been widely noticed.
The immune system is an important system for the organism to play the role of immune response, can prevent pathogens from invading the organism, and can discover foreign matters, pathogenic microorganisms and the like which cause internal environment disorder. The macrophage is the most important phagocyte of the organism, is the first defense line for pathogenic microorganisms to enter the organism, plays an immune function, has the functions of phagocytizing, expressing corresponding antigen presenting molecules, secreting various cell factors and the like, is widely involved in clearing pathogenic microorganisms and aged cells in the organism, plays an immune regulation role, maintains the homeostasis of the organism internal environment, and plays an important role in natural immunity. Modern researches have shown that placenta contains various active components, such as bioactive peptides, immunoglobulin, amino acids, minerals and other components, and is an ideal immunomodulator, and the action mechanism of the immunomodulator is to participate in macrophage immune response reaction by regulating and controlling phagocytosis and antigen presentation functions of macrophages and expression of related cytokines. Other studies show that bioactive peptide components exhibit important physiological activities in the aspects of immunoregulation, antioxidation and the like. However, the research of the embryo cervi oligopeptide on the immunoregulation of macrophage RAW264.7 in the prior art is not carried out.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the method comprises the steps of extracting deer fetus peptides by an ultrafiltration method, detecting the proliferation activity of each peptide segment of each ultrafiltration component through a thiazole blue MTT (methyl thiazolyl tetrazolium) experiment, detecting the influence of the deer fetus peptides on the immune function by an enzyme-linked immunosorbent assay (ELISA) method through a neutral red phagocytic experiment, and ensuring the accuracy of an experiment result through a cell cycle experiment so as to verify the immune regulation effect of different deer fetus ultrafiltration components.
The analysis method of deer fetus oligopeptide for macrophage immunoregulation is characterized by comprising the following steps: comprises the following steps which are sequentially carried out,
step one, preparing supernatant
Taking 40g of raw deer fetus, crushing the raw deer fetus into powder with the particle size of 60 meshes, taking distilled water with the weight of 10-30 times of that of the raw deer fetus, adding the distilled water into the crushed raw deer fetus, stirring for 1-3 times at the temperature of 4 ℃, keeping for 6-8 hours each time at the rotating speed of 3600r/min, centrifuging for 10-30 minutes, and taking supernatant;
step two, preparing freeze-dried powder
Filtering the supernatant obtained in the first step by using double-layer oil filter paper, performing microfiltration by using a 0.45-micrometer filter membrane, performing ultrafiltration by using a 1KDa, 3KDa and 10KDa ultrafiltration membrane, performing ultrafiltration separation and purification to obtain a molecular total extract DFP-1, a deer fetus protein DFP-2 with the mass more than 10KDa, a deer fetus polypeptide DFP-3 with the molecular mass of 3KDa to 10KDa, a deer fetus polypeptide DFP-4 with the molecular mass of 1KDa to 3KDa and a deer fetus oligopeptide DFP-5 with the molecular mass less than 1KDa, and freeze-drying to obtain freeze-dried powder for later use;
step three, establishing a protein standard curve
Precisely weighing 1.0mg of bovine serum albumin in a 10mL volumetric flask, dissolving with distilled water and fixing the volume to a scale to obtain a BSA standard solution with the concentration of 0.1 mg/mL;
respectively sucking 0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of BSA standard solution, complementing each tube to 1.0mL by distilled water, adjusting zero by a blank tube, adding 5mL of Coomassie brilliant blue reagent into each tube, uniformly mixing by vortex, reacting for 5min at room temperature, and measuring the absorbance value at 595nm between 5min and 20 min;
taking the protein concentration mu g/mL of the bovine serum albumin standard solution in each tube as an abscissa x, and taking the measured absorbance value as an ordinate Y to perform linear regression to obtain a standard curve regression equation for measuring the protein content;
step four, measuring the sample solution
Taking 1mg of each freeze-dried powder obtained in the step two, adding 1mL of distilled water, preparing 1mg/mL of sample solution, absorbing 1mL of sample solution, adding 5mL of Coomassie brilliant blue reagent, uniformly mixing in a vortex manner, reacting at room temperature for 5min, and measuring the absorbance value at 595nm between 5min and 20 min;
step five, measuring cell proliferation index
Taking RAW264.7 cells in logarithmic growth phase as 105One/well of the cells was plated in 96-well cell culture plates at 150. mu.L/well with 5% CO at 37 ℃%2Culturing for 24h in an incubator, after the cells adhere to the wall, removing supernatant, respectively adding the freeze-dried powder obtained in the step two to prepare 150 mu L of 100 mu g/mL solution, wherein a blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, a positive control group is 150 mu L of 10 mu g/mL Lipopolysaccharide (LPS) and each group has 5 compound wells; at 37 deg.C, 5% CO2Culturing for 12h, 24h and 48h respectively under the condition, discarding supernatant, adding 5mg/mL MTT thiazole blue solution 10 μ L into each well, adding DMSO dimethyl sulfoxide 150 μ L after 4h, shaking for 5min, measuring transmittance OD value at 490nm wavelength to obtain fine powderAn index of cell proliferation;
step six, determining cell proliferation index of deer fetus oligopeptide DFP-5 under drug stimulation
Respectively adding the deer fetus oligopeptide DFP-5 freeze-dried powder with the molecular mass less than 1KDa obtained in the step two into a DMEM high-sugar medium to prepare 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL and 400 mu g/mL of solution 150 mu L, wherein the blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, the positive control group is 150 mu L of LPS bacterial lipopolysaccharide with the molecular mass less than 1KDa, and each group has 5 multiple wells; at 37 deg.C, 5% CO2Culturing for 24h under the condition, discarding culture supernatant, adding 10 μ L of 5mg/mL MTT thiazole blue solution into each hole, adding 150 μ L of DMSO dimethyl sulfoxide after 4h, oscillating for 5min, and measuring transmittance OD value at 490nm wavelength to obtain cell proliferation index;
seventhly, measuring the phagocytosis index of the cells
Taking RAW264.7 cells in logarithmic growth phase as 105150 μ L/well in 96-well plates at 37 ℃ with 5% CO2After incubation in an incubator for 24 hours, discarding the culture solution, giving the deer fetus oligopeptide DFP-5 with the mass concentration of 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL and 200 mu g/mL for drug stimulation, setting 5 multiple holes in each group, setting LPS bacterial lipopolysaccharide groups and blank groups at the same time, incubating for 24 hours, sucking out the supernatant, washing with PBS for 2 times, then adding 200 mu L of cell culture solution, adding 20 mu L of neutral red dye solution, and incubating for 2 hours; removing cell culture fluid containing neutral red dye solution, washing with PBS for 1 time, adding neutral red lysis solution 200 μ L into each well, and lysing for 10min on a shaking bed at room temperature; measuring the transmittance OD value at the wavelength of 540nm to obtain the phagocytosis index of the cells;
step eight, determining the influence of embryo cervi oligopeptide DFP-5 on the cytokine release of RAW264.7 cells
Inoculating RAW264.7 cells in logarithmic growth phase into 96-well culture plate, wherein each well volume is 200 μ L, and the density is 105One/well at 37 ℃ with 5% CO2Culturing in incubator for 24h, after cell adherence, discarding supernatant, adding DFP-5 sample solutions of 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, and 200. mu.g/mL 150. mu.L, respectively, and setting blank groupAnd LPS bacterial lipopolysaccharide group, each group has 5 multiple wells, at 37 deg.C and 5% CO2Culturing for 24h under the condition, taking 150 μ L of supernatant, and determining NO secretion, IL-1 β secretion, IL-6 secretion and TFN- α secretion according to the kit instructions;
step nine, determining the influence of deer fetus oligopeptide DFP-5 on RAW264.7 cell cycle
RAW264.7 cell concentration of 105Inoculating 2mL of the cells in a 6-well plate, sucking out the culture solution after 24h, respectively adding deer fetus oligopeptide DFP-5 with mass concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/mL, simultaneously setting LPS bacteria lipopolysaccharide group and blank group, continuing incubation, collecting sample cells with the number of cells of about 6 × 106Washing with cold poly (butylene succinate) PBS for 2 times, blowing off cells to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, resuspending the cells with 500 muL poly (butylene succinate) PBS, dripping 3mL 75% cold ethanol, fixing for 1h at-20 ℃, centrifuging, resuspending the cells with 500 muL cold poly (butylene succinate) PBS, adding RnaseA endonuclease solution 20 muL, bathing for 30min at 37 ℃, centrifuging for 10min, discarding supernatant, adding fluorescent dye PI dye solution 400 muL, uniformly mixing, incubating for 30min at 4 ℃ in the dark, and detecting the result by a flow cytometer;
as a result, to
Wherein x represents mean, s represents standard deviation, and SPSS19.0 statistical software is adopted to carry out pairing t test and one-way variance analysis, and the results show that p is less than 0.01 and p is less than 0.05, wherein p represents significance.
And the preparation method of the Coomassie brilliant blue reagent in the third step and the fourth step comprises the steps of weighing 50mg of Coomassie brilliant blue G-250, dissolving with 25mL of 95% ethanol, adding 50mL of 85% phosphoric acid, shaking up, and metering to 50mL of brown bottle with distilled water to obtain the Coomassie brilliant blue reagent for later use.
Through the design scheme, the invention can bring the following beneficial effects: the method for analyzing immunoregulation of deer fetus oligopeptide on macrophage is characterized in that after the deer fetus oligopeptide is screened out, the proliferation index of RAW264.7 cells is obviously increased compared with blank ratio and is most similar to that of an LPS positive control group;
furthermore, in order to verify the influence of embryo cervi oligopeptide on the immunoregulation effect, the method analyzes the influence of different mass concentrations on the phagocytic function, the NO secretion amount and the release of cytokines such as IL-1 beta, IL-6 and TNF-alpha, and the obtained result shows that the NO has the maximum release amount at 25 mu g/mL and is closest to the LPS group;
after the deer fetus oligopeptide acts for 24 hours, the secretion function of RAW264.7 cytokines is obviously increased, and when the concentration is 25 mu g/mL, the level of each factor of the cells is close to that of an LPS group, so that the deer fetus oligopeptide is proved to have the function of enhancing the secretion of the cytokines;
the cell cycle examination is carried out simultaneously in the method, and data results prove that along with the increase of the mass concentration of the deer fetus oligopeptide, the proportion of cells in the G0/G1 phase is in the trend of rising firstly and then falling, the proportion of cells in the S phase and the G2/M phase is in the trend of falling firstly and then rising, and when the proportion of the cells in each phase is closest to that of an LPS group at 25 mu G/mL; G0/G1 is the synthesis phase of RNA and protein, so the expression level of related protein is increased, and the secretion capacity of phagocytosis, NO and cytokines reaches the maximum value in the phase.
Drawings
The invention is further described with reference to the following figures and detailed description:
FIG. 1 is a graph of bovine albumin in phantom obtained in step three of the present invention.
FIG. 2 is a bar graph showing the effect of the mass concentration of deer fetus oligopeptide DFP-5 on the proliferation capacity of RAW264.7 cells obtained in step six of the present invention.
FIG. 3 is a schematic diagram showing the effect of deer fetus oligopeptide DFP-5 obtained in step seven of the present invention on the phagocytic ability of RAW264.7 cells.
FIG. 4 is a schematic diagram showing the effect of deer fetus oligopeptide DFP-5 obtained in step eight on NO secretion of RAW264.7 cells.
FIG. 5 is a schematic diagram showing the effect of deer fetus oligopeptide DFP-5 obtained in step eight on IL-1 beta secretion of RAW264.7 cells.
FIG. 6 is a schematic diagram showing the effect of DFP-5 on IL-6 secretion of RAW264.7 cells obtained in step eight of the present invention.
FIG. 7 is a schematic diagram showing the effect of deer fetus oligopeptide DFP-5 obtained in step eight on the TFN-alpha secretion amount of RAW264.7 cells.
FIG. 8 is a diagram showing the variation of the cell cycle of RAW264.7 obtained in step nine of the present invention.
FIG. 9 is a bar graph showing the effect of RAW264.7 on cell cycle obtained in step nine of the present invention.
Detailed Description
The analysis method of deer fetus oligopeptide for macrophage immunoregulation is characterized by comprising the following steps: comprises the following steps which are sequentially carried out,
step one, preparing supernatant
Taking 40g of raw deer fetus, crushing the raw deer fetus into powder with the particle size of 60 meshes, taking distilled water with the weight of 10-30 times of that of the raw deer fetus, adding the distilled water into the crushed raw deer fetus, stirring for 1-3 times at the temperature of 4 ℃, keeping for 6-8 hours each time at the rotating speed of 3600r/min, centrifuging for 10-30 minutes, and taking supernatant;
step two, preparing freeze-dried powder
Filtering the supernatant obtained in the first step by using double-layer oil filter paper, performing microfiltration by using a 0.45-micrometer filter membrane, performing ultrafiltration by using a 1KDa, 3KDa and 10KDa ultrafiltration membrane, performing ultrafiltration separation and purification to obtain a molecular total extract DFP-1, a deer fetus protein DFP-2 with the mass more than 10KDa, a deer fetus polypeptide DFP-3 with the molecular mass of 3KDa to 10KDa, a deer fetus polypeptide DFP-4 with the molecular mass of 1KDa to 3KDa and a deer fetus oligopeptide DFP-5 with the molecular mass less than 1KDa, and freeze-drying to obtain freeze-dried powder for later use;
step three, establishing a protein standard curve
Precisely weighing 1.0mg of bovine serum albumin in a 10mL volumetric flask, dissolving with distilled water and fixing the volume to a scale to obtain a BSA standard solution with the concentration of 0.1 mg/mL;
respectively sucking 0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of BSA standard solution, complementing each tube to 1.0mL by distilled water, adjusting zero by a blank tube, adding 5mL of Coomassie brilliant blue reagent into each tube, uniformly mixing by vortex, reacting for 5min at room temperature, and measuring the absorbance value at 595nm between 5min and 20 min;
taking the protein concentration mu g/mL of the bovine serum albumin standard solution in each tube as an abscissa x, and taking the measured absorbance value as an ordinate Y to perform linear regression to obtain a standard curve regression equation for measuring the protein content;
the preparation method of the Coomassie brilliant blue reagent comprises the following steps of weighing 50mg of Coomassie brilliant blue G-250, dissolving the Coomassie brilliant blue G-250 with 25mL of 95% ethanol, adding 50mL of 85% phosphoric acid, shaking up, and fixing the volume of the mixture in a50 mL brown bottle with distilled water to obtain the Coomassie brilliant blue reagent for later use;
step four, measuring the sample solution
Taking 1mg of each freeze-dried powder obtained in the step two, adding 1mL of distilled water, preparing 1mg/mL of sample solution, absorbing 1mL of sample solution, adding 5mL of Coomassie brilliant blue reagent, uniformly mixing in a vortex manner, reacting at room temperature for 5min, and measuring the absorbance value at 595nm between 5min and 20 min;
step five, measuring cell proliferation index
Taking RAW264.7 cells in logarithmic growth phase as 105One/well of the cells was plated in 96-well cell culture plates at 150. mu.L/well with 5% CO at 37 ℃%2Culturing for 24h in an incubator, after the cells adhere to the wall, removing supernatant, respectively adding the freeze-dried powder obtained in the step two to prepare 150 mu L of 100 mu g/mL solution, wherein a blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, a positive control group is 150 mu L of 10 mu g/mL Lipopolysaccharide (LPS) and each group has 5 compound wells; at 37 deg.C, 5% CO2Culturing for 12h, 24h and 48h respectively under the condition, discarding the supernatant, adding 10 mu L of MTT thiazole blue solution of 5mg/mL into each hole, adding 150 mu L of DMSO dimethyl sulfoxide after 4h, oscillating for 5min, and measuring the transmittance OD value at the wavelength of 490nm to obtain the cell proliferation index;
step six, determining cell proliferation index of deer fetus oligopeptide DFP-5 under drug stimulation
Respectively adding the deer fetus oligopeptide DFP-5 freeze-dried powder with the molecular mass less than 1KDa obtained in the step two into a DMEM high-sugar medium to prepare 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL and 400 mu g/mL of solution 150 mu L, wherein the blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, the positive control group is 150 mu L of LPS bacterial lipopolysaccharide with the molecular mass less than 1KDa, and each group has 5 multiple wells;at 37 deg.C, 5% CO2Culturing for 24h under the condition, discarding culture supernatant, adding 10 μ L of 5mg/mL MTT thiazole blue solution into each hole, adding 150 μ L of DMSO dimethyl sulfoxide after 4h, oscillating for 5min, and measuring transmittance OD value at 490nm wavelength to obtain cell proliferation index;
seventhly, measuring the phagocytosis index of the cells
Taking RAW264.7 cells in logarithmic growth phase as 105150 μ L/well in 96-well plates at 37 ℃ with 5% CO2After incubation in an incubator for 24 hours, discarding the culture solution, giving the deer fetus oligopeptide DFP-5 with the mass concentration of 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL and 200 mu g/mL for drug stimulation, setting 5 multiple holes in each group, setting LPS bacterial lipopolysaccharide groups and blank groups at the same time, incubating for 24 hours, sucking out the supernatant, washing with PBS for 2 times, then adding 200 mu L of cell culture solution, adding 20 mu L of neutral red dye solution, and incubating for 2 hours; removing cell culture fluid containing neutral red dye solution, washing with PBS for 1 time, adding neutral red lysis solution 200 μ L into each well, and lysing for 10min on a shaking bed at room temperature; measuring the transmittance OD value at the wavelength of 540nm to obtain the phagocytosis index of the cells;
step eight, determining the influence of embryo cervi oligopeptide DFP-5 on the cytokine release of RAW264.7 cells
Inoculating RAW264.7 cells in logarithmic growth phase into 96-well culture plate, wherein each well volume is 200 μ L, and the density is 105One/well at 37 ℃ with 5% CO2Culturing in incubator for 24h, after cell adherence, discarding supernatant, adding DFP-5 sample solutions of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL 150 μ L, setting blank group and LPS bacteria lipopolysaccharide group, each group having 5 multiple wells, incubating at 37 deg.C and 5% CO2Culturing for 24h under the condition, taking 150 μ L of supernatant, and determining NO secretion, IL-1 β secretion, IL-6 secretion and TFN- α secretion according to the kit instructions;
the specific operation steps are as follows:
1. sample adding of the standard: setting standard substance holes and sample holes, wherein 50 mu L of standard substances with different concentrations are added into the standard substance holes respectively;
2. and (4) adding auspicious: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. 40 mu L of sample diluent is added into sample holes to be detected on the enzyme-labeled coated plate, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;
3. adding an enzyme: adding 100 mu L of enzyme-labeled reagent into each hole except for blank holes;
4. and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 60 minutes at 37 ℃;
5. preparing liquid: diluting 20 times of the concentrated washing liquid with 20 times of distilled water for later use;
6. washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
7. color development: adding 50 mu L of color-developing agent A into each hole, adding 50 mu L of color-developing agent B, shaking gently, mixing uniformly, and developing for 15 minutes at 37 ℃ in a dark place;
8. and (4) terminating: adding 50 mu L of stop solution into each well to stop the reaction (at the moment, the blue color immediately turns to yellow);
9. and (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The determination should be carried out within 15 minutes after the addition of the stop solution;
step nine, determining the influence of deer fetus oligopeptide DFP-5 on RAW264.7 cell cycle
RAW264.7 cell concentration of 105Inoculating 2mL of the cells in a 6-well plate, sucking out the culture solution after 24h, respectively adding deer fetus oligopeptide DFP-5 with mass concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/mL, simultaneously setting LPS bacteria lipopolysaccharide group and blank group, continuing incubation, collecting sample cells with the number of cells of about 6 × 106Washing with cold poly (butylene succinate) PBS for 2 times, blowing off cells to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, resuspending cells with 500 muL poly (butylene succinate) PBS, dripping 3mL 75% cold ethanol, fixing at-20 ℃ for 1h, centrifuging, resuspending cells with 500 muL cold poly (butylene succinate) PBS, adding RnaseA endonuclease solution 20 muL, bathing in water at 37 ℃ for 30min, centrifuging for 10min, discarding supernatant, adding fluorescent dye PI (dye)Mixing the solution with 400 μ L, incubating at 4 deg.C in dark for 30min, and detecting the result with flow cytometer;
as a result, to
(x represents mean, s represents standard deviation) and the results of the paired t-test and one-way anova using SPSS19.0 statistical software were statistically different for p < 0.01 and p < 0.05(p represents significance).
The first embodiment,
Pulverizing 40g embryo Cervi crude product into 60 mesh, adding 10 times of distilled water into pulverized embryo Cervi crude product, stirring at 4 deg.C for 6 hr for 1 time at 3600r/min, centrifuging for 10min, and collecting supernatant. The yield was 0.139%.
Example II,
Pulverizing 40g embryo Cervi crude product into 60 mesh, adding 20 times of distilled water into pulverized embryo Cervi crude product, stirring at 4 deg.C for 7 hr for 2 times, rotating at 3600r/min, centrifuging for 20min, and collecting supernatant. The yield was 0.274%.
Example III,
Pulverizing 40g embryo Cervi crude product into 60 mesh, adding distilled water 25 times of the mass of embryo Cervi crude product into pulverized embryo Cervi crude product, stirring at 4 deg.C for 3 times (8 hr each time) at 3600r/min, centrifuging for 30min, and collecting supernatant. The yield was 0.436%.
EXAMPLE four study of embryo cervi oligopeptide on macrophage RAW264.7 immunomodulation
(1) Deer fetus peptide protein content determination
A standard curve is plotted with bovine serum albumin mass concentration (X) as the abscissa and absorbance OD value (Y) as the ordinate, as shown in FIG. 1. The protein contents of DFP-1, DFP-2, DFP-3, DFP-4 and DFP-5 solutions were calculated by the formula to be 0.4326mg/mL, 0.2914mg/mL, 0.3160mg/mL, 0.3926mg/mL and 0.3460mg/mL, respectively.
(2) Proliferation of RAW264.7 cells by different ultrafiltration fractions
Each ultrafiltration component of DFP has certain promotion effect on proliferation of RAW264.7 cells; after 12h of culture of RAW264.7 cells, the DFP-1, DFP-2 and DFP-3 components have significant difference (p is less than 0.05) in the promotion effect of RAW264.7 cells, and the DFP-4 and DFP-5 components have significant difference (p is less than 0.01) in the promotion effect of RAW264.7 cells. After the RAW264.7 cells are cultured for 24h and 48h, 5 samples have very significant difference compared with a blank group, when the culture time is 24h, the proliferation capacity of each component is strongest, so that the subsequent cell culture time is 24h, and the proliferation effect of the DFP-5 component on the RAW264.7 cells is closest to the positive effect, therefore, DFP-5 is selected for further study.
Table 1 proliferation of RAW264.7 cells by different ultrafiltration fractions (n ═ 5)
Note: represents significant differences between different samples and blanks at the same time (same row in the table). Wherein, represents significant difference (p < 0.05), and represents significant difference (p < 0.01).
(3) Proliferation of RAW264.7 cells by different mass concentrations of DFP-5
Both LPS and DFP-5 were able to promote proliferation of RAW264.7 cells, and the proliferation index of RAW264.7 cells tended to increase first and then decrease in the mass concentration range of 6.25. mu.g/mL-400. mu.g/mL. When the mass concentration of DFP-5 is 12.5 mug/mL and 200 mug/mL, the influence on the proliferation of RAW264.7 cells is remarkable (p is less than 0.05); when the mass concentration of DFP-5 was 25. mu.g/mL, 50. mu.g/mL, or 100. mu.g/mL, the effect on proliferation of RAW264.7 cells was very significant (p < 0.01), and the proliferation index was at most 1.95. + -. 0.09 at the mass concentration of 25. mu.g/mL. As shown in fig. 2, in the graph, the difference was significant (p < 0.05), and the difference was very significant (p < 0.01), compared to the blank group. As the mass concentration of DFP-5 continues to increase, the proliferation index begins to drop sharply, and when the mass concentration increases to 400. mu.g/mL, the influence of DFP-5 on the proliferation of RAW264.7 cells is not statistically significant compared with that of a blank control group, so that DFP-5 of 12.5. mu.g/mL-200. mu.g/mL is selected for further study of the immunoregulatory mechanism.
(4) Results of neutral Red phagocytosis experiment
The LPS group and DFP-5 can remarkably enhance the phagocytosis capacity of RAW264.7 cells, and the stimulation effect on the phagocytosis capacity of the RAW264.7 cells tends to be increased and then decreased along with the increase of mass concentration. As shown in fig. 3, in the graph, the difference was significant (p < 0.05), and the difference was very significant (p < 0.01), compared to the blank group. When the mass concentration is increased to 25 mug/mL, the phagocytosis index reaches 1.87 +/-0.12, and the DFP-5 can effectively activate the RAW264.7 cells to play a phagocytosis role.
(5) Effect of DFP-5 on NO secretion amount in RAW264.7 cells
The stimulation effect of DFP-5 on the NO secretion of RAW264.7 cells is related to the dose, and when the mass concentration of DFP-5 is 25 mu g/mL, the NO secretion of cells reaches the maximum value, namely 21.57 +/-1.8 mu mol/L; as shown in fig. 4, in the graph, the difference was significant (p < 0.05), and the difference was very significant (p < 0.01), compared to the blank group. Each mass concentration of DFP-5 has a very significant difference (p < 0.01) in the amount of NO secreted by the cell active substance, but is weaker than that of LPS group.
(6) Effect of DFP-5 on cytokine Release from RAW264.7 cells
The effect of DFP-5 on the amount of cytokine secretion in RAW264.7 cells was dose-dependent. As shown in fig. 5 to 7, in the graphs, the difference was significant (p < 0.05) and the difference was very significant (p < 0.01) compared to the blank group. After being treated by DFP-5 with 25 mug/mL, the secretion of the cytokines IL-1 beta, IL-6 and TNF-alpha has very significant effect (p is less than 0.01) compared with the blank group, and the secretion of each factor is the highest and approaches to the positive control group.
(7) Effect of DFP-5 on RAW264.7 cell cycle
As shown in FIGS. 8 and 9, the proportion of cells in G0/G1 phase tended to increase first and then decrease after DFP-5 treatment for 24h, and the proportion of cells in S phase and G2/M phase to the total cell count tended to decrease first and then increase, compared with the blank group. When the DFP-5 mass concentration reaches 25. mu.g/mL, the trend of the change at each period is closest to that of the LPS group.
Claims (2)
1. The analysis method of deer fetus oligopeptide for macrophage immunoregulation is characterized by comprising the following steps: comprises the following steps which are sequentially carried out,
step one, preparing supernatant
Taking 40g of raw deer fetus, crushing the raw deer fetus into powder with the particle size of 60 meshes, taking distilled water with the weight of 10-30 times of that of the raw deer fetus, adding the distilled water into the crushed raw deer fetus, stirring for 1-3 times at the temperature of 4 ℃, keeping for 6-8 hours each time at the rotating speed of 3600r/min, centrifuging for 10-30 minutes, and taking supernatant;
step two, preparing freeze-dried powder
Filtering the supernatant obtained in the first step by using double-layer oil filter paper, performing microfiltration by using a 0.45-micrometer filter membrane, performing ultrafiltration by using a 1KDa, 3KDa and 10KDa ultrafiltration membrane, performing ultrafiltration separation and purification to obtain a molecular total extract DFP-1, a deer fetus protein DFP-2 with the mass more than 10KDa, a deer fetus polypeptide DFP-3 with the molecular mass of 3KDa to 10KDa, a deer fetus polypeptide DFP-4 with the molecular mass of 1KDa to 3KDa and a deer fetus oligopeptide DFP-5 with the molecular mass less than 1KDa, and freeze-drying to obtain freeze-dried powder for later use;
step three, establishing a protein standard curve
Precisely weighing 1.0mg of bovine serum albumin in a 10mL volumetric flask, dissolving with distilled water and fixing the volume to a scale to obtain a BSA standard solution with the concentration of 0.1 mg/mL;
respectively sucking 0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of BSA standard solution, complementing each tube to 1.0mL by distilled water, adjusting zero by a blank tube, adding 5mL of Coomassie brilliant blue reagent into each tube, uniformly mixing by vortex, reacting for 5min at room temperature, and measuring the absorbance value at 595nm between 5min and 20 min;
taking the protein concentration mu g/mL of the bovine serum albumin standard solution in each tube as an abscissa x, and taking the measured absorbance value as an ordinate Y to perform linear regression to obtain a standard curve regression equation for measuring the protein content;
step four, measuring the sample solution
Taking 1mg of each freeze-dried powder obtained in the step two, adding 1mL of distilled water, preparing 1mg/mL of sample solution, absorbing 1mL of sample solution, adding 5mL of Coomassie brilliant blue reagent, uniformly mixing in a vortex manner, reacting at room temperature for 5min, and measuring the absorbance value at 595nm between 5min and 20 min;
step five, measuring cell proliferation index
Taking RAW264.7 cells in logarithmic growth phase as 105One/well of the cells was plated in 96-well cell culture plates at 150. mu.L/well with 5% CO at 37 ℃%2Culturing for 24h in an incubator, after the cells adhere to the wall, removing supernatant, respectively adding the freeze-dried powder obtained in the step two to prepare 150 mu L of 100 mu g/mL solution, wherein a blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, a positive control group is 150 mu L of 10 mu g/mL Lipopolysaccharide (LPS) and each group has 5 compound wells; at 37 deg.C, 5% CO2Culturing for 12h, 24h and 48h respectively under the condition, discarding the supernatant, adding 10 mu L of MTT thiazole blue solution of 5mg/mL into each hole, adding 150 mu L of DMSO dimethyl sulfoxide after 4h, oscillating for 5min, and measuring the transmittance OD value at the wavelength of 490nm to obtain the cell proliferation index;
step six, determining cell proliferation index of deer fetus oligopeptide DFP-5 under drug stimulation
Respectively adding the deer fetus oligopeptide DFP-5 freeze-dried powder with the molecular mass less than 1KDa obtained in the step two into a DMEM high-sugar medium to prepare 6.25 mu g/mL, 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL and 400 mu g/mL of solution 150 mu L, wherein the blank control group is 150 mu L of DMEM high-sugar medium containing 10% fetal calf serum, the positive control group is 150 mu L of LPS bacterial lipopolysaccharide with the molecular mass less than 1KDa, and each group has 5 multiple wells; at 37 deg.C, 5% CO2Culturing for 24h under the condition, discarding culture supernatant, adding 10 μ L of 5mg/mL MTT thiazole blue solution into each hole, adding 150 μ L of DMSO dimethyl sulfoxide after 4h, oscillating for 5min, and measuring transmittance OD value at 490nm wavelength to obtain cell proliferation index;
seventhly, measuring the phagocytosis index of the cells
Taking RAW264.7 cells in logarithmic growth phase as 105150 μ L/well in 96-well plates at 37 ℃ with 5% CO2After incubation in an incubator for 24h, the culture solution is discarded, 5 compound wells are set in each group by giving the deer fetus oligopeptide DFP-5 with the mass concentration of 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL and 200 mu g/mL drug stimulation, the LPS bacterial lipopolysaccharide group and the blank group are incubated for 24h, the supernatant is sucked out, poly (butylene succinate) PBS is washed for 2 times, and then 200 mu L fine particles are addedAdding 20 mu L of neutral red dye solution into the cell culture solution, and incubating for 2 h; removing cell culture fluid containing neutral red dye solution, washing with PBS for 1 time, adding neutral red lysis solution 200 μ L into each well, and lysing for 10min on a shaking bed at room temperature; measuring the transmittance OD value at the wavelength of 540nm to obtain the phagocytosis index of the cells;
step eight, determining the influence of embryo cervi oligopeptide DFP-5 on the cytokine release of RAW264.7 cells
Inoculating RAW264.7 cells in logarithmic growth phase into 96-well culture plate, wherein each well volume is 200 μ L, and the density is 105One/well at 37 ℃ with 5% CO2Culturing in incubator for 24h, after cell adherence, discarding supernatant, adding DFP-5 sample solutions of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL 150 μ L, setting blank group and LPS bacteria lipopolysaccharide group, each group having 5 multiple wells, incubating at 37 deg.C and 5% CO2Culturing for 24h under the condition, taking 150 μ L of supernatant, and determining NO secretion, IL-1 β secretion, IL-6 secretion and TFN- α secretion according to the kit instructions;
step nine, determining the influence of deer fetus oligopeptide DFP-5 on RAW264.7 cell cycle
RAW264.7 cell concentration of 105Inoculating 2mL of the cells in a 6-well plate, sucking out the culture solution after 24h, respectively adding deer fetus oligopeptide DFP-5 with mass concentration of 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/mL, simultaneously setting LPS bacteria lipopolysaccharide group and blank group, continuing incubation, collecting sample cells with the number of cells of about 6 × 106Washing with cold poly (butylene succinate) PBS for 2 times, blowing off cells to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, resuspending the cells with 500 muL poly (butylene succinate) PBS, dripping 3mL 75% cold ethanol, fixing for 1h at-20 ℃, centrifuging, resuspending the cells with 500 muL cold poly (butylene succinate) PBS, adding RnaseA endonuclease solution 20 muL, bathing for 30min at 37 ℃, centrifuging for 10min, discarding supernatant, adding fluorescent dye PI dye solution 400 muL, uniformly mixing, incubating for 30min at 4 ℃ in the dark, and detecting the result by a flow cytometer;
as a result, to
Wherein x represents mean, s represents standard deviation, and SPSS19.0 statistical software is adopted to carry out pairing t test and one-way variance analysis, and the results show that p is less than 0.01 and p is less than 0.05, wherein p represents significance.
2. The method of claim 1, wherein the deer fetus oligopeptide is used for the analysis of macrophage immunoregulation: and the preparation method of the Coomassie brilliant blue reagent in the third step and the fourth step comprises the steps of weighing 50mg of Coomassie brilliant blue G-250, dissolving with 25mL of 95% ethanol, adding 50mL of 85% phosphoric acid, shaking up, and metering to 50mL of brown bottle with distilled water to obtain the Coomassie brilliant blue reagent for later use.
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