阅读说明：本技术 鹿胎寡肽对巨噬细胞免疫调节的分析方法 (Analysis method of deer fetus oligopeptide for immunoregulation of macrophage ) 是由 张辉 张凯月 吴楠 杜延佳 李志成 李晶峰 边学峰 吕金朋 兰梦 高旭 于 2019-11-12 设计创作，主要内容包括：鹿胎寡肽对巨噬细胞免疫调节的分析方法,属于生物技术研发分析技术领域,筛选出鹿胎寡肽作用后RAW264.7细胞的增殖指数较空白比明显升高,且与LPS阳性对照组最为接近；本发明方法分析了不同质量浓度对吞噬功能、NO分泌量和细胞因子如IL-1β、IL-6、TNF-α的释放的影响,获得结果显示,在25μg/mL时,NO有最大的释放量,且与LPS组最为接近；本发明方法中同时进行细胞周期检查,数据结果证明,随着鹿胎寡肽质量浓度的增长,G0/G1期细胞比例呈先上升后下降趋势,S期与G2/M期的细胞比例呈先下降后上升趋势,在25μg/mL时,细胞各时期比例与LPS组最为接近；G0/G1为RNA和蛋白质的合成期,因此相关蛋白的表达量增加,所以吞噬作用、NO及细胞因子的分泌能力在此时期分泌量达最大值。(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.)

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

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.

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).