CN108042561B - Method for establishing model of influence of selenized oligomeric amino polysaccharide on immunosuppression-deficient mice - Google Patents
Method for establishing model of influence of selenized oligomeric amino polysaccharide on immunosuppression-deficient mice Download PDFInfo
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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Abstract
The invention discloses a method for establishing a model of influence of selenized oligomeric aminopolysaccharide on an immunosuppression-deficient mouse, wherein the immunosuppression-deficient mouse is obtained by intraperitoneal injection of cyclophosphamide, various influences of the selenized oligomeric aminopolysaccharide on the immunosuppression-deficient mouse are discussed, the model is successfully established, and test results show that the selenized oligomeric aminopolysaccharide can remarkably improve the organism immunity level of the immunosuppression-deficient mouse caused by the cyclophosphamide.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a method for establishing an influence model of selenized oligomeric amino polysaccharide on an immunosuppression-deficient mouse.
Background
The immune system consists of immune organs and tissues, immune cells and immune factors. Many diseases occur largely due to the impairment of the immune function of the body, and hypoimmunity is one of the causes. The most direct manifestation of low immunity is that the body is easy to suffer from diseases, the recovery period is long and the body is easy to repeat. Many factors can cause the decline of immunity of the organism, including congenital deficiency, stress, insomnia, emotional overstrain, etc., and people of all ages are likely to suffer from the decline. With the development of the times and the progress of science and technology, people pay more attention to their health, and the wide biological activity of selenium polysaccharide just fits the need.
Chitosan is also called as aminopolysaccharide and has excellent biocompatibility, biodegradability and no toxic or side effect, a weakly acidic aqueous solution of chitosan has high viscosity, amino groups on the surface of chitosan have positive charges, and the aminopolysaccharide can react with biological macromolecules such as phospholipid with negative charges on the surface of cells, so that drugs are easily combined on the surface of cell membranes to promote the absorption of the drugs, and the chitosan is widely applied to the fields of drug carriers and medical auxiliary materials. The necessity of selenium for human body is a common knowledge of researchers, and is one of the essential trace elements of human body, selenium mainly participates in the metabolic pathways such as antioxidation, thyroid hormone synthesis and immune function in vivo, and the lack of selenium in vivo can cause several chronic diseases such as cancer. With the further understanding of the aminopolysaccharide metal complex and the combination of the good biological activity of the organic selenium compound, the research on the aminopolysaccharide selenium is more and more. Oligomerization promotes the water solubility of the aminopolysaccharide, facilitates the absorption and further exerts the biological activity of the aminopolysaccharide greatly. The selenized oligomeric amino polysaccharide serving as organic selenium has low toxicity and high bioavailability, has biological effects of polysaccharide and selenium, such as immunoregulation, oxidation resistance, tumor resistance and the like, and is a research object in a plurality of fields of medicines, food health care and the like at present.
Cyclophosphamide (CPA) is not only an alkylating chemotherapeutic drug, but also an immunosuppressant, which can disrupt cellular DNA structure, leading to apoptosis. In the research of immune toxicology, cyclophosphamide is often applied to the establishment of animal immunosuppression models.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, obtains an immunosuppression-deficient mouse by intraperitoneal injection of cyclophosphamide, discusses a method for establishing an effect model of selenized oligomeric glycosaminoglycan on the immunosuppression-deficient mouse, and provides a certain theoretical basis for developing the selenized oligomeric glycosaminoglycan into an immunomodulator.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for establishing a model of influence of selenized oligomeric amino polysaccharide on an immunosuppression-deficient mouse, which comprises the following steps: (a) taking 80 mice, and randomly dividing into 8 groups according to ten mice in each group;
normal control group: irrigating stomach with normal saline on days 1-14, and performing intraperitoneal injection with normal saline on days 11-14;
cyclophosphamide group: gavage with normal saline on days 1-14, and intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide +0.3 selenized oligomeric aminopolysaccharide group: gavage with 0.3mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide +0.6 selenized oligomeric aminopolysaccharide group: gavage with 0.6mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide +0.9 selenized oligomeric aminopolysaccharide group: gavage with 0.9mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide +0.3 sodium selenite group: intragastric administering 0.3mg/kg BW/day sodium selenite solution in terms of selenium content on days 1-14, and performing intraperitoneal injection on days 11-14 with 50mg CPA/kg BW/day;
cyclophosphamide + oligomeric aminopolysaccharide group: gavage with 9.87mg/kg BW/day oligomeric glycosaminoglycan solution on days 1-14, and intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide + oligomeric aminopolysaccharide +0.3 sodium selenite group: gavage with 0.3mg/kg BW/day sodium selenite solution and 9.87mg/kg BW/day oligomeric glycosaminoglycan solution on days 1-14, and performing intraperitoneal injection on days 11-14 with 50mg CPA/kg BW/day;
(b) after fasting for 24h after the last administration, the mice are weighed, then the eyeballs are bled, the cervical vertebrae are dislocated and killed, then the spleen and the thymus are quickly picked up, the viscera are washed by normal saline, water stains are sucked by filter paper, the weight of the viscera is respectively weighed and recorded, and the viscera index is calculated according to a formula.
Preferably, the organ index formula is: organ index (organ weight mg/mouse body weight g) × 10.
Preferably, the establishing method further includes: (c) dissecting the mice, and evaluating the function of the selenized oligomeric glycosaminoglycan in immunosuppression of the overall immune function of the mice by observing indexes such as spleen tissue morphology, spleen lymphocyte proliferation reaction, mouse serum cytokine and the like.
Preferably, the mice are SPF-grade BALB/C mice, male, 6-8 weeks old and 18-22 g in body weight.
Preferably, the selenized oligomeric aminopolysaccharide has a selenium content of 27.3mg/g, wherein the source of selenium is provided by sodium selenite and the oligomeric aminopolysaccharide used has a molecular weight of about 50 kDa.
Therefore, the invention has the following beneficial effects:
(1) the mouse immunosuppression model established by continuously injecting cyclophosphamide into abdominal cavity for 4 days at a dose of 50mg/kg is more ideal, and compared with a blank group, the thymus and spleen index of the mouse in the CPA group and the level of serum Th1/Th2 cytokines (IL-2/INF-gamma, IL-4/IL-10) are obviously reduced (P is less than 0.05); splenic lymphocytes were significantly inhibited (P < 0.05) in response to ConA or LPS induced proliferation; spleen tissue architecture was compromised.
(2) The injection of LSA at 0.3mg/kg and 0.6mg/kg can obviously improve the index of thymus and spleen of an immunosuppressed mouse (P is less than 0.05).
(3) The LSA of 0.3, 0.6 and 0.9mg/kg can obviously enhance the proliferation reaction (P is less than 0.05) induced by ConA or LPS of splenic lymphocytes of immunosuppressed mice.
(4) The damage condition of spleen tissue structures of immunosuppressive mice can be improved by intragastric administration of 0.3mg/kg and 0.6mg/kg LSA.
(5) The early intake of 0.6mg/kg LSA can obviously improve the levels of INF-gamma and IL-4 of the serum of the mouse (P is less than 0.05); the LSA of gavage amount of 0.3, 0.6 and 0.9mg/kg can obviously improve the IL-2 level of the serum of the mouse (P is less than 0.05), wherein the effect of a medium dose group is best; the LSA of 0.6 and 0.9mg/kg can obviously improve the IL-10 level of the serum of the mouse (P is less than 0.05).
Drawings
FIG. 1 is a graph of the effect of selenized oligomeric aminopolysaccharides on spleen index in immunosuppressed mice.
FIG. 2 is a graph of the effect of selenized oligomeric aminopolysaccharides on thymus index in immunosuppressed mice.
FIG. 3 shows spleen histomorphological changes (HE staining, X100) for each group of mice.
FIG. 4 is a graph of the effect of selenized oligomeric aminopolysaccharides on mouse spleen lymphocyte proliferation.
FIG. 5 is a graph showing the effect of selenized oligomeric amino polysaccharides on the level of mouse serum cytokine IL-2.
FIG. 6 is a graph of the effect of selenized oligomeric amino polysaccharides on the level of mouse serum cytokine IFN-. gamma..
FIG. 7 is a graph showing the effect of selenized oligomeric amino polysaccharides on the level of mouse serum cytokine IL-4.
FIG. 8 is a graph showing the effect of selenized oligomeric amino polysaccharides on the level of mouse serum cytokine IL-10.
Detailed Description
The invention is further described with reference to the following detailed description and accompanying drawings.
A method for establishing a model of influence of selenized oligomeric amino polysaccharide on an immunosuppression-deficient mouse, which comprises the following steps:
(a) taking 80 BALB/C mice with SPF (specific pathogen free) grade, male age of 6-8 weeks and weight of 18-22 g, and randomly dividing the mice into 8 groups according to ten groups;
normal control group (NC): irrigating stomach with normal saline on days 1-14, and performing intraperitoneal injection with normal saline on days 11-14;
cyclophosphamide group (CPA): gavage with normal saline on days 1-14, and intraperitoneal injection with 50mg CPA/kgBW/day on days 11-14;
cyclophosphamide +0.3 selenized oligomeric glycosaminoglycan group (CPA + LSA (0.3)): gavage with 0.3mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14; cyclophosphamide +0.6 selenized oligomeric glycosaminoglycan group (CPA + LSA (0.6)): gavage with 0.6mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14; cyclophosphamide +0.9 selenized oligomeric glycosaminoglycan group (CPA + LSA (0.9)): gavage with 0.9mg/kg BW/day selenized oligomeric glycosaminoglycan solution in terms of selenium content on days 1-14, and performing intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14; cyclophosphamide +0.3 sodium selenite group (CPA + Se-Ni (0.3)): intragastric administering 0.3mg/kg BW/day sodium selenite solution in terms of selenium content on days 1-14, and performing intraperitoneal injection on days 11-14 with 50mg CPA/kg BW/day;
cyclophosphamide + oligomeric glycosaminoglycan group (CPA + LA): gavage with 9.87mg/kg BW/day oligomeric glycosaminoglycan solution on days 1-14, and intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;
cyclophosphamide + oligomeric aminopolysaccharide + sodium selenite group 0.3 (CPA + LA + Se-Ni (0.3)): gavage with 0.3mg/kg BW/day sodium selenite solution and 9.87mg/kg BW/day oligomeric glycosaminoglycan solution on days 1-14, and performing intraperitoneal injection on days 11-14 with 50mg CPA/kg BW/day;
wherein the selenium content of the selenized oligomeric aminopolysaccharide is 27.3mg/g, wherein the sodium selenite provides a selenium source, and the molecular weight of the used oligomeric aminopolysaccharide is about 50 kDa;
(b) after 24h (fasting) of the last administration, the mice were weighed, followed by blood sampling from the eyeballs, cervical dislocation and sacrifice, followed by rapid removal of the spleen and thymus, washing of the organs with physiological saline, blotting of the water stain through filter paper, weighing and recording. Organ indices were calculated according to the formula. Organ index (organ weight mg/mouse body weight g) × 10.
Compared with a normal control group, the mice in the cyclophosphamide group have the clinical manifestations of anorexia, mental committee and the like after continuously injecting cyclophosphamide into the abdominal cavity for 4 days. Since body weight and organ index are visual indicators that reflect the overall health status of animals, the body weight, spleen and thymus index of each group of mice were measured, and the results are shown in table 1 and fig. 1-2. As shown by experimental results, the chemotherapeutic drug Cyclophosphamide (CPA) has no significant effect on the body weight of the mice (P is more than 0.05), but can significantly reduce the spleen and thymus index of the mice (P is less than 0.05). Compared with the cyclophosphamide model group, the spleen and thymus indexes (P is less than 0.05) of the mice can be obviously improved by gavage of 0.3 and 0.6mg/kg LSA, and the spleen and thymus indexes of the mice in the LSA group of 0.9mg/kg are increased compared with the model group, but have no obvious difference (P is more than 0.05). In addition, compared with the mice in the model group, the thymus and spleen indexes of the mice in the sodium selenite group, the oligomeric aminopolysaccharide group and the sodium selenite + oligomeric aminopolysaccharide compound addition group have no significant change (P is more than 0.05).
TABLE 1 Effect of selenized oligomeric aminoglycans on body weight, thymus index, and spleen index in mice
Note: multiple comparisons were performed using the LSD assay. The table indicates significant differences between groups with different lower case letters (P < 0.05) and the table indicates insignificant differences between groups with the same lower case letters (P > 0.05).
2 morphological Observation of spleen tissue
Washing fresh spleen tissue with normal saline for multiple times, washing to remove residual blood, fixing with 10mL of 4% paraformaldehyde in a clean EP tube at normal temperature for 24h, dehydrating with ethanol, clearing xylene, embedding paraffin, cutting tissue paraffin blocks into continuous 6 μm tissue sections with a microtome, randomly selecting 2 tissue blocks, performing hematoxylin-eosin (HE) staining according to a conventional process, and observing histopathological changes under a microscope.
The maintenance of the intact tissue structure of an organ is the material basis for its function. The spleen parenchyma is composed of three parts, white marrow, red marrow and marginal zone. The effect of selenized oligomeric glycosaminoglycan on mouse spleen tissue morphology is shown in fig. 3, which shows that the normal control group (fig. 3A): the spleen structure is clear, the boundaries of white marrow and red marrow are obvious, and the white marrow area is large; cyclophosphamide group (fig. 3B): spleen structures are fuzzy, boundaries of white marrow and red marrow are not clear, and the area of the white marrow is obviously reduced; LSA processing group (3C-3E): compared with the cyclophosphamide group, spleen structures of the intragastric and high-dose LSA mice are clearer, boundaries of red marrow and white marrow are obvious, and the area of the white marrow is increased. Results suggest that early LSA uptake protects mouse spleen tissue.
3 spleen lymphocyte proliferation response assay
Spleen lymphocyte suspension is prepared firstly, and the steps are as follows:
① obtaining materials, killing mice after 24h (fasting) of last administration by dislocation of cervical vertebrae, soaking in 75% ethanol for 3min for disinfection, transferring into a super clean bench, placing on sterile kraft paper, cutting a small opening in the middle of the abdomen with the left side facing upwards, exposing spleen, lifting spleen with forceps, separating connective tissue below with ophthalmic scissors, taking out spleen, placing in a centrifuge tube filled with 3mL Hank's solution, storing on ice, and performing subsequent treatment as soon as possible;
② grinding, placing the culture dish on ice, placing 10mL Hank's liquid, placing a 200-mesh screen on the culture dish, grinding the spleen on the screen by using a piston rubber head of a disposable injector, flexibly fixing the screen by the left hand in the grinding process, and slightly and quickly grinding the spleen which is not attached to the culture dish but below the Hank's liquid until the spleen is completely whitened;
③ lysis erythrocyte, transferring spleen suspension in the culture dish to 15mL centrifuge tube, centrifuging for 5min at 300g, reversing and discarding supernatant, adding 2mL erythrocyte lysate (sterilized by filtration with 0.22 μm filter membrane) into each tube, centrifuging for 5min at normal temperature at 300g, reversing and discarding supernatant;
④ washing, suspending Hank's solution to 12mL, centrifuging at 300g for 5min, discarding supernatant, and repeating the steps for 2 times;
⑤ the spleen lymphocytes obtained are resuspended in a proper amount of PRMI-1640 complete culture medium (containing 10% FBS, 100U/mL streptomycin, 50. mu.M β -mercaptoethanol), counted by a cell counting plate, and the cell concentration is adjusted to 3X 106 cells/mL for subsequent index determination.
Spleen lymphocyte proliferation response assay:
splenic lymphocytes (3X 106/mL) were plated into 96-well plates at 100. mu.L per well, 100. mu.L of either PRMI-1640 blank medium or PRMI-1640 complete medium containing ConA or LPS (final concentrations of ConA and LPS were 5. mu.g/mL and 10. mu.g/mL, respectively), incubated at 37 ℃ for 72 hours in a 5% CO2 cell incubator, 50. mu.L of MTT (sterile filtered through a 0.22 filter, final concentration of MTT was 0.5mg/mL) per well, incubated for 4 hours, centrifuged at 4000r/min for 10min, the wells were discarded, 150. mu.L of DMSO was added per well to dissolve formazan, OD was measured at 490nm using a microplate reader for each well, and 630nm was used as the calibration wavelength. The Stimulation Index (SI) is used as an index to judge the conversion and proliferation degree of the lymphocytes, and the calculation formula of the SI is as follows.
SI ═ (stimulated OD value-blank well)/(stimulated OD value-blank well)
The effect of LSA on the ability of immunosuppressed mouse spleen lymphocytes to proliferate was examined and the results are shown in fig. 4. As can be seen from the figure, compared with the normal control group, the spleen lymphocytes of the mice in the cyclophosphamide group are obviously inhibited from the proliferation reaction induced by ConA or LPS (mean P is less than 0.05), while LSA can reverse the inhibition effect caused by CPA to a certain extent, which is specifically shown as follows: compared with the cyclophosphamide group, LSA of each dose group can obviously improve the proliferation response of splenic lymphocytes of the immunosuppressed mice to ConA induction (P is less than 0.05); LSA dose of 0.6 and 0.9mg/kg BW can obviously improve the proliferation response of splenic lymphocytes of immunosuppressed mice to LPS induction (P is less than 0.05). In addition, spleen lymphocytes of the gavage sodium selenite, oligomeric aminopolysaccharide or sodium selenite + oligomeric aminopolysaccharide mice had no significant change in the proliferation response induced by ConA or LPS (P > 0.05) compared to the cyclophosphamide group mice.
4 mouse serum cytokine detection
After 24h (fasting) of the last administration, blood is taken from the eyeballs of the mice, the blood is subpackaged in clean and dry EP tubes, the blood is stood for 2h at room temperature, the centrifugation is carried out for 10min at 2000rpm/min at 4 ℃, and serum is taken and stored in a refrigerator at 80 ℃ below zero for standby. The content of cytokines (IL-2/IFN-gamma, IL-4/IL-10) in serum is determined by an ELISA kit, and the specific operation is carried out according to the instruction of the kit.
The effect of selenized oligomeric aminoglycans on the levels of mouse serum IL-2, IFN- γ, IL-4, and IL-10 was examined and the results are shown in FIGS. 5-8. As can be seen from the figure, compared with the normal control group, the serum IL-2, IFN-gamma, IL-4 and IL-10 contents of the mice in the cyclophosphamide group are all obviously reduced (P is less than 0.05). Compared with the cyclophosphamide group, the IL-2 content of the serum of the low, medium and high LSA mice is obviously improved (P is less than 0.05); the lavage of 0.3 and 0.9mg/kg LSA has certain improvement effect on the reduction of IFN-gamma and IL-4 caused by cyclophosphamide, but has no statistical difference (P is more than 0.05), and the content of IFN-gamma and IL-4 in the serum of 0.6mg/kg LSA mice is obviously improved (P is less than 0.05); LSA of 0.6 and 0.9mg/kg can obviously improve the IL-10 content (P is less than 0.05) of the mouse serum. In addition, compared with the mice in the cyclophosphamide group, the contents of IL-2, IFN-gamma, IL-4 and IL-10 in the serum of the mice in the sodium selenite group, the oligomeric glycosaminoglycan group and the sodium selenite + oligomeric glycosaminoglycan composite addition group have no significant change (P is more than 0.05).
Claims (5)
1. A method for establishing a model of influence of selenized oligomeric amino polysaccharide on an immunosuppression-deficient mouse is characterized by comprising the following steps:
taking 80 mice, and randomly dividing into 8 groups according to ten mice in each group;
normal control group: irrigating stomach with normal saline on days 1-14, and performing intraperitoneal injection with normal saline on days 11-14;
cyclophosphamide group: gavage with normal saline on days 1-14, and performing intraperitoneal injection on cyclophosphamide aqueous solution 50mg/kg body weight on days 11-14;
cyclophosphamide +0.3 selenized oligomeric aminopolysaccharide group: intragastric administering 0.3mg per kg body weight of selenized oligomeric glycosaminoglycan solution in terms of selenium content per day on days 1-14, and administering 50mg per kg body weight of cyclophosphamide aqueous solution per day on days 11-14 for intraperitoneal injection;
cyclophosphamide +0.6 selenized oligomeric aminopolysaccharide group: intragastric administering 0.6mg per kg body weight of selenized oligomeric glycosaminoglycan solution in terms of selenium content per day on days 1-14, and administering 50mg per kg body weight of cyclophosphamide aqueous solution per day on days 11-14 for intraperitoneal injection;
cyclophosphamide +0.9 selenized oligomeric aminopolysaccharide group: intragastric administering selenized oligomeric glycosaminoglycan solution 0.9mg per kg body weight per day on days 1-14 using a selenium content meter, and intraperitoneal administering cyclophosphamide aqueous solution 50mg per kg body weight per day on days 11-14;
cyclophosphamide +0.3 sodium selenite group: intragastric administering 0.3mg sodium selenite solution per kg body weight per day on days 1-14 with selenium content meter, and administering 50mg cyclophosphamide aqueous solution per kg body weight per day on days 11-14 for intraperitoneal injection;
cyclophosphamide + oligomeric aminopolysaccharide group: administering 9.87mg of the oligomeric glycosaminoglycan solution per kg of body weight per day for intragastric administration on days 1-14, and administering 50mg of the cyclophosphamide aqueous solution per kg of body weight per day for intraperitoneal injection on days 11-14;
cyclophosphamide + oligomeric aminopolysaccharide +0.3 sodium selenite group: intragastric administering 0.3mg sodium selenite solution and 9.87mg oligomeric glycosaminoglycan solution per kg body weight per day on days 1-14 using a selenium content meter, and administering 50mg cyclophosphamide aqueous solution per kg body weight per day on days 11-14 for intraperitoneal injection;
(b) after fasting for 24h after the last administration, the mice are weighed, then the eyeballs are bled, the cervical vertebrae are dislocated and killed, then the spleen and the thymus are quickly picked up, the viscera are washed by normal saline, water stains are sucked by filter paper, and the viscera indexes are respectively weighed and recorded and calculated according to a formula.
2. The method for establishing the model of the influence of the selenized oligomeric glycosaminoglycan on the immunosuppression deficient mice as claimed in claim 1, wherein the organ index formula is as follows: organ index = (organ weight mg/mouse body weight g) × 10.
3. The method for modeling the effect of selenized oligomeric glycosaminoglycan on immunosuppression-deficient mice of claim 1, further comprising:
(c) dissecting the mice, and evaluating the function of the selenized oligomeric glycosaminoglycan in immunosuppression of the overall immune function of the mice by observing spleen tissue morphology, spleen lymphocyte proliferation reaction and mouse serum cytokine indexes.
4. The method for establishing the model of the influence of the selenized oligomeric aminopolysaccharide on the immunosuppression deficient mice according to claim 1 or 3, wherein the mice are SPF-grade BALB/C mice, male mice, 6-8 weeks old and 18-22 g in weight.
5. The method of claim 1, wherein the selenium content of the selenized oligomeric glycosaminoglycan is 27.3mg/g, wherein the selenium source is provided by sodium selenite, and the molecular weight of the used oligomeric glycosaminoglycan is 50 kDa.
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