CN117603910A - Method for obtaining high-efficiency leukocyte extract by eliminating blood cell stress state of slaughtered animals - Google Patents
Method for obtaining high-efficiency leukocyte extract by eliminating blood cell stress state of slaughtered animals Download PDFInfo
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- CN117603910A CN117603910A CN202311585729.5A CN202311585729A CN117603910A CN 117603910 A CN117603910 A CN 117603910A CN 202311585729 A CN202311585729 A CN 202311585729A CN 117603910 A CN117603910 A CN 117603910A
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Abstract
The invention discloses a method for eliminating stress state treatment fluid of slaughter animal blood cells and obtaining high-efficiency leukocyte extract, which adopts taurine, vitamin C sodium phosphate, ectoin, proline and sodium butyrate as main raw materials, and is accompanied by physical sub-low temperature treatment, so that the influence of slaughter stress stimulation of cultured animals on the leukocytes of the slaughter animal is effectively relieved, the improvement of key indexes (such as cell activation rate, morphology and apoptosis state) after the leukocyte stress is realized, and the active protein indexes of the leukocyte extract are further improved, in particular the key indexes (such as inflammatory factors, heat stress indexes and the like) as cosmetic raw materials; all components are listed in the catalogue of raw material medicines in pharmacopoeia, so that the biological safety and compatibility are further improved, all components are listed in the quality control of the 'registration and recording data management of new raw materials for cosmetics', and the stability among batches is high and the safety is high.
Description
Technical Field
The invention belongs to the technical field of cells and bioengineering, and particularly relates to a method for obtaining a high-efficiency white blood cell extract by eliminating a blood cell stress state of slaughtered animals.
Background
Whole blood fractions include erythrocytes, leukocytes and platelets, which can be finely isolated using modern biological techniques. Leukocytes are heterogeneous cell populations comprising monocytes, lymphocytes, granulocytes and micro-stem cells, and as the most important immune barrier of the body, they secrete a variety of cytokines including Growth Factors (GF), interleukins (IL), colony stimulating factors (colony stimulating factor, CSF), interferons (IFN) and transforming growth factors that regulate the growth and metabolic functions of specific cells. The purified white blood cells are further crushed to release the similar substances, namely white blood cell extract, contain a large amount of cytokines, can directionally promote the growth of various cells, promote the formation of intercellular matrixes, promote the repair of tissue injury, and play an important role in the processes of cell interaction, immunoregulation, hematopoiesis and inflammation.
The leukocyte extract (LEUKOCYTE EXTRACT; CAS 225234-41-3) is a cosmetic material in the national drug administration (New Material registration and recording data management for cosmetics). Since the biological functions of leukocytes are mainly developed around inflammatory controls, various cytokines (such as PDGF, VEGF, HGF, PDGF, bFGF, TGF- β, EGF, etc.), lipids, nucleic acids, etc., have effects of promoting proliferation and division, promoting angiogenesis, repairing mucosa, regenerating cells, etc., on fibroblasts, epithelial cells, endothelial cells, etc. Can effectively activate aging cells, promote cell regeneration, repair inflammatory injury, improve skin immunity, reduce pigmentation, etc.; these are all the scientific bases for their use as active ingredients in biological cosmetics.
Stress is a series of nonspecific reactions generated by animals after the animals are subjected to strong stimulation factors such as biology, physics, chemistry, psychology and the like in the external or internal environment, and comprises physical stress, chemical stress, feeding stress, production stress, trauma stress and transportation stress, and the stress is treated. The stress reaction generation mechanism is complex, the neuroendocrine system can mobilize almost all organs and tissues to deal with the stimulation, and the physiological and biochemical processes in the body are kept to be coordinated and balanced through complex neurohumoral regulation, so that a new steady state is established.
Stress directly affects animal physiology, resulting in mental harm, cardiac burden, digestive system effects, respiratory system effects, immune system harm, metabolic disorders. The appearance of PSE meat during pig production is the most typical case of stress. The strong stress before slaughtering causes insufficient energy metabolism, increased oxygen consumption, large amount of lactic acid generation, reduced pH value and reduced muscle water retention; the high metabolic rate results in a continuous high temperature, low pH, and severe and significant denaturation of the protein, resulting in a "poached meat". Acute stress can also cause severe changes in animal blood phase, changes in immune cell populations, elevation of serum inflammatory substances, and the like.
The animal experiences stress, a panic response phase, an adaptation or resistance phase, if the stimulus is too intense, and eventually enters a failure phase, accompanied by irreversible failure of multiple organs. Measures for reducing stress response comprise culturing anti-stress varieties, reasonably utilizing populations, regulating nutrition, anti-stress drugs, improving living environment, optimizing breeding procedures, animal welfare and preventing diseases. These measures are mostly aimed at the activities of the farms, and after suffering from stress, the animal individuals still have the opportunity to recover from the stress stimulus; whereas for slaughter products, the stress recovery capacity due to body death will be greatly reduced until it is lost. The stress intervention measures adopted by the traditional breeding industry are not suitable for the development and utilization of special biological resources. Leukocyte active extracts are important biological resources, and are obtained by using blood from slaughtered animals in large quantities. The existing technology for eliminating the stress state of blood cells of slaughtered animals cannot meet the production of high-quality leukocyte extracts.
Disclosure of Invention
Aiming at the problem that the existing technology for eliminating the stress state of blood cells of slaughtered animals cannot meet the production requirement of high-quality white blood cell extracts, the invention provides a treatment solution for eliminating the stress state of blood cells of slaughtered animals and a method for obtaining high-efficiency white blood cell extracts, which aims at relieving or eliminating the stress stimulation of slaughtered programs to immune cells of animals, breaking cells and obtaining white blood cell extracts with more excellent biological properties.
The invention provides a treating fluid for eliminating stress state of blood cells of slaughtered animals, which comprises the following components: (1) The plasma-like isotonic buffer system comprises NaCl with the mass percent concentration of 0.8-1%, hydroxyethyl starch with the mass percent concentration of 0.5-1.5% 100 and glucan 40 with the mass percent concentration of 1-3%; (2) The stress removing component comprises 0.05-0.6% of taurine, 0.1-0.5% of sodium ascorbyl phosphate, 1-2% of ectoin, 0.5-2% of proline and 1-5% of sodium butyrate.
The plasma-like isotonic buffer system comprises NaCl with the mass percent concentration of 0.9%, hydroxyethyl starch with the mass percent concentration of 1% 100 and dextran 40 with the mass percent concentration of 2%.
The invention discloses a preferable treating fluid for eliminating stress state of blood cells of slaughtered animals, wherein the stress eliminating component comprises 0.125% of taurine, 0.325% of sodium ascorbyl phosphate, 1.5% of ectoin, 1% of proline and 2% of sodium butyrate.
The invention also provides a preparation method of the treating fluid for eliminating stress state of blood cells of slaughtered animals, which comprises the following steps:
1) Weighing hydroxyethyl starch (HES 100) with the polymerization degree of 100 and Dextran (Dextran 40) with the polymerization degree of 40, dissolving in NaCl solution, adjusting the mass percent concentration of the hydroxyethyl starch 100 to 0.5-1.5%, adjusting the mass percent concentration of the Dextran with the polymerization degree of 40 to 1-3%, and sterilizing and filtering by a 0.22 mu m filter membrane; preparing a plasma-like isotonic buffer system as a solvent of the treatment fluid;
2) Weighing taurine (taurines), completely dissolving the taurine (taurines) by using the solvent, and adjusting the mass percentage concentration to 0.05-0.6%;
3) Weighing Sodium ascorbyl phosphate (Sodium L-ascorbyl-2-phosphate), completely dissolving with the solvent, and adjusting the mass percentage concentration to 0.1-0.5%;
4) Weighing Ectoine (Ectoine), completely dissolving with the solvent, and adjusting the mass percentage concentration to 1-2%;
5) Weighing a proper amount of Proline (Proline), completely dissolving the Proline in the solvent, and adjusting the mass percentage concentration to 0.5-2%;
6) Weighing Sodium butyrate (Sodium butyrate), completely dissolving with the solvent, and adjusting the mass percentage concentration to 1-5%;
7) The solution is sterilized by a filter membrane and filtered to prepare the treating fluid for eliminating the stress state of the blood cells of slaughtered animals.
The preferred filter is a 0.22 μm filter.
The concentration of each component in the processing liquid for eliminating the stress state of the blood cells of slaughtered animals is preferably prepared as follows: in 0.9% NaCl solution, 1% hydroxyethyl starch 100%, 2% dextran 40,0.125% taurine, 0.325% sodium ascorbyl phosphate, 1.5% ectoin, 1% proline, 2% sodium butyrate are contained.
The invention also provides a method for obtaining the high-efficiency white blood cell extract, which comprises the following steps:
1) Fully mixing the obtained white blood cell suspension with any treatment fluid according to the invention in proportion under the aseptic condition;
2) Inducing incubation of immune cells at sub-low temperature;
3) Centrifuging, and washing the cells to remove the treatment fluid to obtain white blood cells after eliminating stress state (namely stress removal treatment);
a preferred method for obtaining a high-potency leukocyte extract is disclosed, wherein the volume ratio of the treatment fluid to the leukocyte suspension in step 1) is 10:1, a step of; the step 2) is carried out at 32 ℃ and 5% CO 2 Inducing and incubating immune cells for 4-6 hours in a saturated humidity environment; step 3) was centrifuged at 3000rpm for 15min with 4℃pre-chilled PBS.
In one embodiment, the rabbit blood cells are de-stressed after slaughter. Slaughtering rabbit blood, separating at 4deg.C to obtain white blood cells, mixing with the processing solution (processing solution: cells=10:1) at a volume ratio of about 5×10 6 ～1×10 7 individual/mL; incubating for 4-6 hours at a sublow temperature of 32 ℃ to obtain the white blood cells after the stress removal treatment; and obtaining leukocyte extract which can be used as cosmetic raw material according to conventional procedure.
The treatment fluid for eliminating the stress state of the blood cells of the slaughtered animals adopts taurine, vitamin C sodium phosphate, ectoin, proline and sodium butyrate as main raw materials, and is accompanied by physical sub-low temperature treatment, so that the influence of the stress stimulus of the slaughtered animals on the white blood cells of the cultured animals is effectively relieved. The stress stimulus at slaughter stage comprises a variety of stress stimulus mechanisms including endoplasmic reticulum stress stimulus, hypothalamic pituitary adrenal axis stress stimulus, active oxygen radical stimulus, and short-term body temperature elevation stimulus. The invention realizes the improvement of key indexes (such as cell activation rate, morphology and apoptosis state) after leukocyte stress by weakening biological effects of the above approaches; further improving the active protein index of the leukocyte extract, especially as key index of cosmetic raw material (such as inflammatory factor, heat stress index, etc.); all components are listed in the catalogue of raw material medicines in pharmacopoeia, so that the biological safety and compatibility are further improved, all components are listed in the quality control of the 'registration and recording data management of new raw materials for cosmetics', and the stability among batches is high and the safety is high. Wherein, the hydroxyethyl starch and the glucan mainly play a role of buffering agents in the system to provide a plasma-like buffer environment. Proline is a natural biological small molecule, one of important amino acids for synthesizing human protein, and has higher biological safety; vitamin C is the most important active oxygen radical scavenger in organisms, and in the present invention, both functions to reduce oxidative stress damage. Taurine is a natural amino acid that is capable of resisting hypothalamic pituitary adrenal axis stress pressure stimuli. Sodium butyrate inhibits cell proliferation by deacetylation inhibition effect, and reduces endoplasmic reticulum stress stimulation injury. The important stress-resistant biological component of the ectoin is used for maintaining the complete biological functions of cells.
In one embodiment, the treatment fluid is used to treat stressed rabbit leukocytes at a sub-low temperature for ameliorating high temperature stress damage.
The method for eliminating the stress state treatment fluid of the slaughtered animal blood cells and obtaining the high-efficiency leukocyte extract can keep and improve the cell viability, morphology, proliferation capacity and anti-apoptosis capacity of the leukocytes in a highly stressed state after slaughtering; and the indexes of the key functional proteins of the leukocyte extract, and the like, are improved in stress parameters.
Furthermore, on the premise of assisting the mature liquid treatment process in industry, the stress state treatment of slaughter animal blood cells can be coupled with the subsequent white blood cell disruption treatment process, so that the full-automatic operation is realized in a closed system, and the influence of human intervention on quality control is reduced to the greatest extent; meets the technical requirements of industrial large-scale preparation of the white blood cell extract.
As an alternative embodiment, the treatment fluid is used for the de-stressing treatment of slaughtered rabbit blood leukocytes. Wherein, the rabbit blood leucocyte is the aggregate of blood white tangible cells, including but not limited to lymphocyte, monocyte and trace stem cell. The source of stressed rabbit blood includes, but is not limited to, that obtained by slaughter means, and a live venous blood sample is also suitable for this process. In addition, due to similar properties of the breeding industry, the method is also suitable for the stress treatment of slaughter blood white blood cells of livestock species such as cattle, horses, sheep, pigs and the like.
Drawings
FIG. 1A is a diagram showing the morphology of blood leukocytes prior to the stress relieving treatment procedure in example 4 of the present application;
FIG. 1B shows the morphology of leukocytes in blood after the de-stressing procedure of example 4 of the present application;
FIG. 2A is a schematic diagram showing the proliferation of immune cells before the stress relieving treatment in example 4 of the present application;
FIG. 2B is a schematic diagram showing the proliferation of immune cells after the stress-free treatment in example 4 of the present application;
FIG. 3 is a graph showing the effect of the stress relieving treatment on the immune cell viability in example 5 of the present application;
FIG. 4 is a graph showing the effect of the destressing treatment on the growth curve of immune cells in example 5 of the present application;
FIG. 5 is a schematic diagram of a standard PI-Annexin V assay for apoptosis function assays in example 6 of the present application;
FIG. 6A is a statistical graph showing the effect of the de-stressing treatment on the viable leukocyte rate in example 6 of the present application;
FIG. 6B is a statistical plot of the effect of the de-stressing treatment on the white blood cell death rate in example 6 of the present application;
FIG. 6C is a statistical graph showing the effect of the de-stressing treatment on the early apoptosis rate of leukocytes in example 6 of the application;
FIG. 6D is a statistical graph showing the effect of the destressing treatment on the apoptosis rate in example 6 of the present application;
FIG. 7 is the effect of the de-stressing treatment on leukocyte stressing markers in example 8 of the present application;
FIG. 8 is the effect of the de-stressing treatment on leukocyte inflammatory markers in example 8 of this application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description. It should be understood that the detailed description is presented merely to illustrate the invention, and is not intended to limit the invention.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
The sources of the products used in the experiment and test of the invention are as follows:
EXAMPLE 1 acquisition of slaughtered Rabbit blood leukocytes
1) Referring to the human blood immune cell separation process, the following procedure is completed in an ultra-clean workshop;
2) 200ml of freshly slaughtered rabbit blood is collected, and the sodium citrate anticoagulant is added according to the proportion of 0.38% of the final concentration;
3) Separating plasma for use;
4) Centrifuging the rest cells by using a lymphocyte separation liquid density gradient, and collecting the mononuclear cells of the tunica albuginea layer;
5) Centrifuging at high speed of 3000rpm for 15min by using PBS precooled at 4deg.C; washing the buffy coat cells twice;
6) Cell density was adjusted to 5X 10 8 ～1×10 9 individual/mL;
7) This is rabbit peripheral blood leukocytes, and a leukocyte suspension is sampled for later use.
Example 2 preparation of a treatment fluid for eliminating stress in blood cells of slaughtered animals
1) Weighing a proper amount of hydroxyethyl starch with the polymerization degree of 100, dissolving the hydroxyethyl starch in 0.9% NaCl solution to a final concentration of 1%, dissolving glucan with the polymerization degree of 40 in 0.9% NaCl solution to a final concentration of 2%, and sterilizing and filtering by a 0.22 mu m filter membrane; preparing a plasma-like isotonic buffer system as a solvent of the treatment fluid;
2) Weighing proper amount of taurine (taurines), completely dissolving with the solvent, and adjusting the concentration to 0.05-0.6%, wherein the preferable final working concentration is 0.125%;
3) Weighing a proper amount of Sodium ascorbyl phosphate (Sodium L-ascorbyl-2-phosphate), completely dissolving with the solvent, and adjusting the concentration to 0.1-0.5%, wherein the preferable final working concentration is 0.325%;
4) Weighing a proper amount of ectoine, completely dissolving with the solvent, and adjusting the concentration to 1-2%, wherein the preferable final working concentration is 1.5%;
5) Weighing a proper amount of Proline (Proline), completely dissolving with the solvent, and adjusting the concentration to 0.5-2%, wherein the preferable final working concentration is 1%;
6) Weighing a proper amount of Sodium butyrate (Sodium butyrate), completely dissolving the Sodium butyrate with the solvent, and adjusting the concentration to 1-5%, wherein the preferable final working concentration is 2%;
7) Preparing a treating fluid for eliminating stress state of blood cells of slaughtered animals: in 0.9% NaCl solution, hydroxyethyl starch 100 (1%), dextran 40 (2%), taurine (0.125%), sodium ascorbyl phosphate (0.325%), ectoin (1.5%), proline (1%), sodium butyrate (2%),
the 0.22 mu m filter membrane is sterilized and filtered to prepare the treating fluid for eliminating the stress state of the blood cells of slaughtered animals.
EXAMPLE 3 leukocyte destressing
1) The white blood cell suspension obtained in example 1 was sterilized with the slaughter animal blood cell stress state eliminating treatment liquid (treatment liquid in volume ratio: cell suspension = 10:1) was thoroughly mixed at which point the cell concentration was approximately 5 x 10 6 ～1×10 7 individual/mL;
2) At 32℃with 5% CO 2 Inducing and incubating immune cells for 4-6 hours in a saturated humidity environment;
3) Centrifuging at high speed of 3000rpm for 15min by using PBS precooled at 4deg.C; washing cells to remove the treatment fluid;
4) This was the de-stressed rabbit leukocytes, sampled.
EXAMPLE 4 Effect of destressing on leukocyte morphology
1) Taking the original rabbit leucocyte suspension obtained in the example 1 and the de-stressing rabbit leucocyte obtained in the example 3 respectively under the aseptic condition;
2) Cell density was adjusted to 5X 10 6 ～1×10 7 Inoculating 1000U/mL of IL-2, IL-1a,
Gamma interferon in lymphocyte serum-free medium; sampling, and observing the cell morphology by microscopic examination; the mononuclear cells obtained by separation show typical sphere morphology, uniformly spread over the whole visual field, and rarely aggregate; no obvious morphological changes were seen in the immune cells after the de-stressing treatment, see fig. 1A and 1B.
3) At 37℃with 5% CO 2 Culturing cells in a saturated humidity environment, performing half liquid exchange and passage on the cells according to the growth condition of the cells, and simultaneously supplementing 3 cytokines in the whole quantity to induce the proliferation of the cells of the cultured immune cells;
4) Sampling, and observing the cell morphology by microscopic examination; monocytes exhibit a clonal colony of spheres morphology, uniformly spreading throughout the field of view; the stress-free cells did not show significant morphological changes, see fig. 2A and 2B.
Example 5 Effect of destressing on leukocyte viability
1) Taking the original rabbit leucocyte suspension obtained in the example 1 and the de-stressed rabbit leucocyte obtained in the example 3 respectively under the aseptic condition;
2) Cell density was adjusted to 5X 10 6 ～1×10 7 Inoculating 1000U/mL of IL-2, IL-1a,
Gamma interferon in lymphocyte serum-free medium;
3) Sampling, using trypan blue staining, and performing cell counting to further calculate the cell viability;
4) At 37℃with 5% CO 2 Culturing cells in a saturated humidity environment, performing half liquid exchange and passage on the cells according to the growth condition of the cells, and simultaneously supplementing 3 cytokines in the whole quantity to induce the proliferation of the cells of the cultured immune cells;
5) At each time of transferring the liquid, checking the cell morphology through a microscope, staining by trypan blue, counting cells, further calculating the cell viability, sampling every day, measuring and drawing a growth curve of the resuscitated cells;
6) The stress-removing treatment can obviously improve the cell activity rate, the initial activity rate of untreated white blood cells is about 70 percent,
slightly decline after 24hr and gradually recover to about 90%; the cell viability after the stress-free treatment was about 85% and increased rapidly to 90%, as shown in FIG. 3.
7) Typical logarithmic cell growth characteristics exhibited by both the de-stressed and control groups; the control group without stress treatment showed a slightly longer resting period at the initial stage of the culture, followed by a rapid growth in the logarithmic growth phase; the de-stressing treatment can significantly shorten the resting period of the cells, about 12-24 hours earlier. At the end of the culture, both groups of cells showed similar cell densities, see fig. 4. EXAMPLE 6 determination of leukocyte apoptosis function by destressing
1) Taking the original rabbit leucocyte suspension obtained in the example 1 and the de-stressed rabbit leucocyte obtained in the example 3 respectively under the aseptic condition, and blowing off to form single cell suspension;
2) 5min at 1500rpm, centrifuging to collect cell supernatant, and washing the cells twice with pre-chilled physiological saline at 4deg.C;
3) Cells were suspended using 400. Mu.l Annexin V binding solution and cell concentration was adjusted to about 10 6 cells/ml；
Adding 5 mu l Annexin V-FITC staining solution into the cell suspension, and reacting for 15min at 2-8 ℃ in a dark place;
4) 5-10 μl PI-RNase solution is reacted at 2-8deg.C for 5min in dark;
5) Flow cytometry detection, measuring apoptosis levels. As shown in FIG. 5, the PI-Annexin V method is a classical method for measuring apoptosis, and living cells, dead cells, early apoptotic cells and late apoptotic cells can be distinguished by measuring the double parameters of membrane permeability and apoptosis-specific protein expression of cells during apoptosis.
6) From FIGS. 6A-D, it can be seen that the process of separation and purification of white blood cells affects the cell viability, and stress reaction also aggravates the apoptosis phenomenon; the cell activity rate is totally reduced in the initial stage, and the apoptosis aggravates to reach the peak in 12-24 hours; and stabilizing step by step; the de-stressing treatment effectively increases the rate of initial viable cells, which eventually becomes slightly higher than untreated cells as the culture time increases, see FIGS. 6A,6B; the protection effect of the stress-relieving treatment on the cells is to inhibit the aggravation of the apoptosis, and compared with an untreated control group, the early apoptosis rate and the apoptosis rate of the cells are inhibited to a certain extent after the stress-relieving treatment, and the apoptosis rate of the untreated cells is higher than that of the stress-relieving treated cells at any time point; these above all demonstrate that the de-stressing treatment has an inhibitory protective effect on apoptosis, see fig. 6C, fig. 6D.
EXAMPLE 7 leukocyte lysate extraction
1) Collecting the cells in the example 3, and fully lysing the erythrocytes by using an erythrocyte lysate;
2) The rest white part is resuspended by normal saline and placed in a 5ml freezing tube;
3) Freezing the freezing tube containing the cells in liquid nitrogen for 2 minutes, and then quickly taking out of a 37 ℃ water bath for resuscitation for 1 minute;
4) Repeating the steps for 2 times;
5) Combining cell suspensions after repeated freezing and thawing, and centrifuging at a high speed of 3000rpm for 15min;
6) Collecting supernatant after cell lysis to obtain leukocyte extract, and storing for later use.
Example 8 Effect of destressing treatment on slaughter Rabbit blood leukocyte stress indicators
1) Another about 500 ten thousand cells were washed 2 times with PBS;
2) Adding 1ml of RNA extracting solution Trizol, standing on ice for 5min, and blowing and mixing;
3) Adding 200ul of chloroform, and standing at room temperature for 5min;
4) Collecting the upper transparent RNA solution at 4℃and 12000rpm for 20min;
5) Adding isopropanol with the same volume, and standing at room temperature for 10min;
6) Centrifuging at 4deg.C at 12000rpm for 20min, collecting precipitate, and washing with 75% ethanol once;
7) 4 ℃,12000rpm,20min; after drying, the total RNA was dissolved in 30ul DEPC water;
8) Reverse transcription into cDNA using an RNA reverse transcription kit;
9) Using the rt-pcr kit of HSP60, HSP70, HSP90 to detect the level of transcription of the stress marker, using GAPDH transcription level as an internal reference; fold changes in transcript levels of the pre/post-stress genes were compared.
10 Heat stress protein family (HSP family) is a recognized marker of cellular stress; the change of HSP expression quantity is not only against heat stress, but also against multiple stress signals such as pressure stress, endoplasmic reticulum stress, ROS stress and the like. In this example, the white blood cells obtained by direct isolation were in a highly stressed state (untreated-0 hr);
after the stress is removed (treatment fluid is-32 ℃ C. To 4 hr), the expression of the stress markers is greatly returned; directly placing the leukocyte sample under 4 deg.C, wherein the expression of stress marker is not changed (4 deg. -4 hr); and the white blood cells are directly treated at a sub-low temperature without using a treatment liquid, so that the white blood cells can be returned to a stress state (32-4 hr) with a certain degree of help. The results are shown in FIG. 7. Example 9 destressing to improve the quality of leukocyte extracts
1) Reverse transcription was performed as in example 8 to obtain leukocyte cDNA;
2) The transcription level of oncorene was detected using the rt-pcr kit for IL-1 beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha, using GAPDH transcription level as an internal reference; comparison before stress removal
Fold change in transcript levels of each gene;
3) Inflammatory proteins (including pro-inflammatory factors and inflammation inhibitors) are important bioeffective proteins in leukocyte extracts; in the field of skin regeneration medicine, excessive inflammatory response is directly related to skin photodamage, ROS stress damage, regeneration and scar repair, desiccation damage, and skin aging. Therefore, when used as a cosmetic raw material, the inflammatory index is actually the most important performance index. In this example, the white blood cells obtained by direct isolation were in a highly stressed state (untreated for-0 hr), and the inflammatory protein markers were all in a highly stressed state; after the stress is removed (treatment fluid is-32 ℃ C. To 4 hr), the expression of the inflammatory markers is greatly returned; directly placing the leukocyte sample under 4 deg.C condition, wherein the expression of inflammatory marker is not changed (4 deg. -4 hr); the treatment of leukocytes directly at sub-low temperatures without the use of treatment fluids will help to some extent the inflammatory protein response index to be reversed (32-4 hr). In the case of the inflammatory inhibitor protein IL-10, the expression level was substantially stable, and the results are shown in FIG. 8. Such results are in line with the expectations of leukocyte active ingredient optimisation.
Example 10
A treatment solution for eliminating stress in blood cells of slaughtered animals was prepared as in example 2, and the following concentrations of the treatment solutions were prepared:
treatment fluid one: the 0.8 percent NaCl solution contains 100 parts of hydroxyethyl starch (1.5 percent), 40 parts of glucan (1 percent), 0.05 percent of taurine (0.5 percent), 0.5 percent of sodium ascorbyl phosphate (2 percent) of ectoin (0.5 percent), 0.5 percent of proline (5 percent) of sodium butyrate (5 percent) and a 0.22 mu m filter membrane for sterilization and filtration to prepare a treatment solution for eliminating the stress state of blood cells of slaughtered animals;
and (2) treating fluid II: the 1.0% NaCl solution contains 100% hydroxyethyl starch (0.5%), 40% dextran (3%), 0.6% taurine, 0.1% sodium ascorbyl phosphate, 1% ectoin (2%), 1% sodium butyrate and 0.22 μm filter membrane for sterilization and filtration to obtain the treatment fluid for eliminating blood cell stress state of slaughtered animals.
Similar experimental results were obtained by the methods of the above treatment fluid one and two reference examples 3 to 9.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (7)
1. A treatment fluid for eliminating stress states of blood cells of slaughtered animals, the treatment fluid comprising: (1) The plasma-like isotonic buffer system comprises NaCl with the mass percent concentration of 0.8-1%, hydroxyethyl starch with the mass percent concentration of 0.5-1.5% 100 and glucan 40 with the mass percent concentration of 1-3%; (2) The stress removing component comprises 0.05-0.6% of taurine, 0.1-0.5% of sodium ascorbyl phosphate, 1-2% of ectoin, 0.5-2% of proline and 1-5% of sodium butyrate.
2. The processing liquid for eliminating stress state of blood cells of slaughtered animals according to claim 1, wherein the plasma-like isotonic buffer system comprises 0.9% of NaCl, 100% of hydroxyethyl starch with 1% of mass percent and 40% of glucan with 2% of mass percent.
3. The processing liquid for eliminating stress state of blood cells of slaughtered animals according to claim 1 or 2, wherein the stress eliminating component comprises 0.125% taurine, 0.325% sodium ascorbyl phosphate, 1.5% ectoin, 1% proline and 2% sodium butyrate.
4. The preparation method of the treating fluid for eliminating stress state of blood cells of slaughtered animals is characterized by comprising the following steps:
1) Weighing hydroxyethyl starch with the polymerization degree of 100 and dextran with the polymerization degree of 40, dissolving the hydroxyethyl starch 100 and the dextran with the polymerization degree of 40 in NaCl solution, adjusting the mass percentage concentration of the hydroxyethyl starch 100 to 0.5-1.5%, adjusting the mass percentage concentration of the dextran with the polymerization degree of 40 to 1-3%, and performing filter membrane sterilization and filtration to prepare a plasma-like isotonic buffer system as a solvent of a treatment fluid;
2) Weighing taurine, completely dissolving the taurine by using the solvent, and adjusting the mass percentage concentration to 0.05-0.6%;
3) Weighing sodium ascorbyl phosphate, completely dissolving with the solvent, and adjusting the mass percentage concentration to 0.1-0.5%;
4) Weighing the ectoin, completely dissolving the ectoin by using the solvent, and adjusting the mass percentage concentration to 1-2%;
5) Weighing a proper amount of proline, completely dissolving the proline by using the solvent, and adjusting the mass percentage concentration to 0.5-2%;
6) Weighing sodium butyrate, completely dissolving the sodium butyrate by using the solvent, and adjusting the mass percentage concentration to 1-5%;
7) The solution is sterilized by a filter membrane and filtered to prepare the treating fluid for eliminating the stress state of the blood cells of slaughtered animals.
5. The method for preparing the treatment fluid for eliminating the stress state of the blood cells of the slaughtered animals according to claim 4, wherein the filter membrane is a 0.22 μm filter membrane, and the prepared treatment fluid for eliminating the stress state of the blood cells of the slaughtered animals has the following concentration of each component: in NaCl solution with the mass percent concentration of 0.9%, hydroxyethyl starch with the mass percent concentration of 1% is contained in 100, dextran with the mass percent concentration of 2% is contained in 40, taurine with the mass percent concentration of 0.125%, sodium vitamin C phosphate with the mass percent concentration of 0.325%, ectoin with the mass percent concentration of 1.5%, proline with the mass percent concentration of 1% and sodium butyrate with the mass percent concentration of 2%.
6. A method for obtaining a high potency leukocyte extract comprising the steps of:
1) Thoroughly mixing the obtained leukocyte suspension with the treating liquid for eliminating stress state of blood cells of slaughtered animals in any one of examples 1 to 3 in proportion under the aseptic condition;
2) Inducing incubation of immune cells at sub-low temperature;
3) Centrifuging, and washing the cells to remove the treatment liquid to obtain the white blood cells after the stress treatment.
7. A method for obtaining a high-efficiency white blood cell extract, which is characterized in that the volume ratio of the processing liquid for eliminating the stress state of the blood cells of slaughtered animals to the white blood cell suspension in the step 1) is 10:1, a step of; the step 2) is carried out at 32 ℃ and 5% CO 2 Inducing and incubating immune cells for 4-6 hours in a saturated humidity environment; step 3) was centrifuged at 3000rpm for 15min with 4℃pre-chilled PBS.
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