CN108042561B - Method for establishing model of influence of selenized oligomeric amino polysaccharide on immunosuppression-deficient mice - Google Patents

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

Establishment of a model of the effect of a selenized oligoaminopolysaccharide on immunosuppression-deficient mice method

technical field

The invention relates to the field of biomedicine, in particular to a method for establishing a model for the effect of selenized oligoaminopolysaccharide on immunosuppression-deficient mice.

Background technique

The immune system consists of immune organs and tissues, immune cells and immune factors. The occurrence of many diseases originates from the disturbance of the immune function of the body to a large extent, and low immunity is one of the causes. The most direct manifestation of low immunity is that the body is prone to illness, and the recovery period is very long and easy to repeat. Many factors can lead to the decline of the body's immunity, including congenital insufficiency, stress, insomnia, emotional excess, etc., and it may occur in people of all ages. With the development of the times and the advancement of science and technology, people pay more and more attention to their own health, and the wide range of biological activities of selenium polysaccharides just meet this need.

Chitosan, also known as amino polysaccharide, as a cationic polysaccharide, has excellent biocompatibility and biodegradability, and has no toxic side effects. Its weakly acidic aqueous solution has high viscosity, and the amino groups on its surface are positively charged, which can It interacts with biological macromolecules such as negatively charged phospholipids on the cell surface, making it easy for drugs to bind to the surface of the cell membrane to promote their absorption, so it has been widely used in the fields of drug carriers and medical auxiliary materials. The necessity of selenium for the human body has been the consensus of researchers. It is one of the essential trace elements for the human body. Selenium is mainly involved in metabolic pathways such as antioxidant, thyroid hormone synthesis and immune function in the body. The lack of selenium in the body can cause cancer, etc. several chronic diseases. With the further understanding of aminopolysaccharide-metal complexes, combined with the good biological activity of organoselenium compounds, there are more and more studies on aminopolysaccharide selenium. The oligomerization promotes the water-solubility of aminopolysaccharides, which is conducive to promoting absorption, thereby greatly exerting its biological activity. As an organic selenium, selenized oligoaminopolysaccharide has low toxicity and high bioavailability, and has both the biological effects of polysaccharide and selenium, such as immune regulation, antioxidant, anti-tumor, etc. objects of research in many fields.

Cyclophosphamide (CPA) is not only an alkylating chemotherapeutic drug, but also an immunosuppressant, which can damage the DNA structure of cells and lead to apoptosis. In immunotoxicology studies, cyclophosphamide is often used in the establishment of animal immunosuppression models.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies in the prior art, the present invention obtains immunosuppression-deficient mice by intraperitoneal injection of cyclophosphamide, and discusses a method for establishing a model for the effect of selenized oligoaminopolysaccharide on immunosuppression-deficient mice. The development provides a certain theoretical basis for immunomodulators.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for establishing a model of the effect of selenized oligoaminopolysaccharide on immunosuppression-deficient mice. The steps of the method for establishing the method are as follows: (a) 80 mice are taken and randomly divided into 8 groups according to ten mice in each group;

Normal control group: intragastric administration of normal saline on days 1-14, intraperitoneal injection of normal saline on days 11-14;

Cyclophosphamide group: gavage with normal saline on days 1-14, and intraperitoneal injection with 50 mg CPA/kg BW/day on days 11-14;

Cyclophosphamide + 0.3 selenized oligosaccharide group: 0.3 mg/kg BW/day selenized oligosaccharide solution based on selenium content by gavage on days 1-14, and 50 mg CPA on days 11-14 /kg BW/day for intraperitoneal injection;

Cyclophosphamide + 0.6 selenized oligosaccharide group: 0.6 mg/kg BW/day selenized oligosaccharide solution based on selenium content by gavage on days 1-14, and 50 mg CPA on days 11-14 /kg BW/day for intraperitoneal injection;

Cyclophosphamide + 0.9 selenized oligosaccharide group: 0.9 mg/kg BW/day selenized oligosaccharide solution based on selenium content by gavage on days 1-14, and 50 mg CPA on days 11-14 /kg BW/day for intraperitoneal injection;

Cyclophosphamide + 0.3 sodium selenite group: 0.3 mg/kg BW/day sodium selenite solution based on selenium content by gavage on days 1-14, and 50 mg CPA/kg BW/day on days 11-14 day for intraperitoneal injection;

Cyclophosphamide + oligosaccharide group: 9.87mg/kg BW/day of oligosaccharide solution by gavage on days 1-14, intraperitoneal administration of 50mg CPA/kg BW/day on days 11-14 injection;

Cyclophosphamide + oligomeric amino polysaccharide + 0.3 sodium selenite group: 0.3 mg/kg BW/day sodium selenite solution and 9.87 mg/kg BW/day oligomer based on selenium content were used on days 1-14 Glycosamine solution by gavage and intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;

(b) After fasting for 24 hours after the last administration, the mice were weighed, followed by blood collection from the eyeballs, and sacrificed by cervical dislocation. Then, the spleen and thymus were quickly removed, and the organs were washed with normal saline, and then the water was blotted with filter paper, and weighed separately. , record, and calculate the organ index according to the formula.

Preferably, the organ index formula is: organ index=(organ weight mg/mice weight g)×10.

Preferably, the method for establishing further comprises: (c) dissecting the mice, and evaluating the effect of selenized oligosaccharides on the overall immune-suppressed mice by observing spleen tissue morphology, spleen lymphocyte proliferation response, and mouse serum cytokines and other indicators. role in immune function.

Preferably, the mice are SPF grade BALB/C mice, male, 6-8 weeks old, weighing 18-22 g.

Preferably, the selenium content of the selenized oligomeric aminopolysaccharide is 27.3 mg/g, wherein the selenium source is provided by sodium selenite, and the molecular weight of the used oligomeric aminopolysaccharide is about 50 kDa.

Therefore, the present invention has the following beneficial effects:

(1) The mouse immunosuppression model established by continuous intraperitoneal injection of cyclophosphamide at a dose of 50 mg/kg for 4 days is ideal. Compared with the blank group, the thymus, spleen index, serum Th1/Th2 cytokine (IL- 2/INF-γ, IL-4/IL-10) levels were significantly decreased (P < 0.05); spleen lymphocytes were significantly inhibited by ConA or LPS-induced proliferation (P < 0.05); spleen tissue structure was damaged. Happening.

(2) LSA at 0.3 and 0.6 mg/kg could significantly increase the thymus and spleen index of immunosuppressed mice (P<0.05).

(3) LSA at 0.3, 0.6 and 0.9 mg/kg by gavage could significantly enhance the proliferation of spleen lymphocytes induced by ConA or LPS in immunosuppressed mice (P<0.05).

(4) LSA at 0.3 and 0.6 mg/kg could improve the damage of spleen tissue structure in immunosuppressed mice.

(5) LSA intake of 0.6 mg/kg in the early stage can significantly increase the levels of serum INF-γ and IL-4 in mice (P < 0.05). 2 levels (P<0.05), among which the middle-dose group had the best effect; intragastric administration of 0.6 and 0.9 mg/kg LSA could significantly increase the level of serum IL-10 in mice (P<0.05).

Description of drawings

Figure 1 shows the effect of selenized oligosaccharide on the spleen index of immunosuppressed mice.

Figure 2 is the effect of selenized oligosaccharide on the thymus index of immunosuppressed mice.

Figure 3 shows the morphological changes of the spleen of mice in each group (HE staining, ×100).

Figure 4 is the effect of selenized oligosaccharide on the proliferation of mouse spleen lymphocytes.

Figure 5 is the effect of selenized oligosaccharide on the content of serum cytokine IL-2 in mice.

Figure 6 is the effect of selenized oligosaccharide on the content of serum cytokine IFN-γ in mice.

Figure 7 shows the effect of selenized oligosaccharide on the content of serum cytokine IL-4 in mice.

Figure 8 is the effect of selenized oligosaccharide on the content of serum cytokine IL-10 in mice.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

A method for establishing a model for the effect of selenized oligoaminopolysaccharide on immunosuppression-deficient mice, wherein the steps of the method for establishing the method are as follows:

(a) 80 SPF grade, male, 6-8 weeks old BALB/C mice weighing 18-22 g were randomly divided into 8 groups according to ten mice in each group;

Normal control group (NC): gavage with normal saline on days 1-14, and intraperitoneal injection with normal saline on days 11-14;

Cyclophosphamide group (CPA): gavage with normal saline on days 1-14, and intraperitoneal injection with 50 mg CPA/kgBW/day on days 11-14;

Cyclophosphamide + 0.3 selenized oligosaccharide group (CPA+LSA(0.3)): 0.3 mg/kgBW/day selenized oligosaccharide solution based on selenium content was administered by gavage on the 1st to 14th day. Use 50mg CPA/kg BW/day for intraperitoneal injection on days 11-14; cyclophosphamide + 0.6 selenized oligoaminoglycan group (CPA + LSA(0.6)): use 0.6 mg based on selenium content on days 1-14 /kgBW/day selenized oligosaccharide solution by gavage, intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14; )): 0.9 mg/kg BW/day selenized oligosaccharide solution by gavage on days 1-14 and 50 mg CPA/kg BW/day by intraperitoneal injection on days 11-14; cyclophosphine Amide + 0.3 sodium selenite group (CPA+Se-Ni(0.3)): 0.3 mg/kg BW/day sodium selenite solution based on selenium content was administered by gavage on days 1-14, and on days 11-14 daily intraperitoneal injection of 50 mg CPA/kg BW/day;

Cyclophosphamide+oligosaccharide group (CPA+LA): 9.87mg/kg BW/day oligosaccharide solution was administered by gavage on days 1-14, and 50mg CPA/kg on days 11-14 BW/day for intraperitoneal injection;

Cyclophosphamide + Oligoaminopolysaccharide + 0.3 Sodium Selenite Group (CPA+LA+Se-Ni(0.3)): 0.3mg/kg BW/day Sodium Selenite based on selenium content on days 1-14 solution and 9.87mg/kg BW/day oligosaccharide solution by gavage, intraperitoneal injection with 50mg CPA/kg BW/day on days 11-14;

The selenium content of the selenized oligoaminopolysaccharide is 27.3mg/g, wherein the selenium source is provided by sodium selenite, and the molecular weight of the oligoaminopolysaccharide used is about 50kDa;

(b) 24 hours after the last administration (fasting), the mice were weighed, then blood was collected from the eyeballs, and then sacrificed by cervical dislocation, and then the spleen and thymus were quickly removed, and the organs were washed with normal saline and then blotted with filter paper. Weigh and record. Calculate the organ index according to the formula. Organ index=(organ weight mg/mice body weight g)×10.

Compared with the normal control group, the mice in the cyclophosphamide group developed clinical symptoms such as anorexia and mental fatigue after continuous intraperitoneal injection of cyclophosphamide for 4 days. Because body weight and organ index are intuitive indicators to reflect the overall health of animals, the body weight, spleen and thymus index of mice in each group were measured. The results are shown in Table 1 and Figures 1-2. The experimental results showed that the chemotherapeutic drug cyclophosphamide (CPA) had no significant effect on the body weight of mice (P>0.05), but could significantly reduce the spleen and thymus indices (P<0.05). Compared with cyclophosphamide model group, gavage of 0.3 and 0.6 mg/kg LSA could significantly increase the spleen and thymus indices of mice (P<0.05). There was an upward trend, but there was no significant difference (P>0.05). In addition, compared with the mice in the model group, there was no significant change in the thymus and spleen indices of the mice in the sodium selenite group, the oligosaccharide group and the sodium selenite + oligosaccharide compound addition group (P>0.05). ).

Table 1 Effects of selenized oligosaccharide on body weight, thymus index and spleen index of mice

NOTE: Multiple comparisons were performed with the LSD test. Different lowercase letters in the table represent significant differences between groups (P<0.05), and those marked with the same lowercase letters represent insignificant differences between groups (P>0.05).

2 Histomorphological observation of spleen

Fresh spleen tissue was washed with physiological saline for several times to remove residual blood, fixed with 10 mL of 4% paraformaldehyde at room temperature for 24 hours in a clean EP tube, followed by ethanol dehydration, transparent in xylene, embedded in paraffin, and sliced into paraffin blocks with a microtome. Cut into continuous 6 μm tissue sections, and randomly select 2 pieces of each tissue block for hematoxylin-eosin staining (HE staining) according to the routine procedure, and observe the histopathological changes under the microscope.

Organs maintain a complete tissue structure is the material basis for their functions. The spleen parenchyma consists of three parts: white pulp, red pulp and marginal zone. The effect of selenized oligoaminopolysaccharide on the morphology of the spleen of mice is shown in Figure 3. It can be seen from the figure that the normal control group (Figure 3A): the structure of the spleen is clear, the boundaries between white pulp and red pulp are clear, and the area of white pulp is large; Phosphamide group (Fig. 3B): the structure of the spleen was blurred, the boundary between white pulp and red pulp was unclear, and the area of white pulp was significantly reduced; LSA treatment group (3C-3E): compared with the cyclophosphamide group, the middle and high doses were administered orally The spleen structure of LSA mice was clearer, the boundary between red pulp and white pulp was more obvious, and the area of white pulp was enlarged. The results suggest that the early intake of LSA has a protective effect on the spleen tissue of mice.

3 Spleen lymphocyte proliferation assay

First prepare the spleen lymphocyte suspension, the steps are as follows:

① Take materials. 24 hours after the last administration (fasting), the mice were sacrificed by cervical dislocation, immersed in 75% ethanol for 3 min for disinfection, and then moved into a clean table and placed on sterile kraft paper. With the left abdomen facing upward, a small opening was cut in the middle of the abdomen to expose the spleen, lift the spleen with forceps, separate the underlying connective tissue with ophthalmic scissors, take out the spleen, put it in a centrifuge tube containing 3 mL of Hank's solution, and store on ice follow up as soon as possible;

② Grinding. Place the petri dish on ice, put 10 mL of Hank's solution, and place a 200-mesh sieve on the petri dish. Grind the spleen on the sieve with the plunger tip of a disposable syringe. During the grinding process, the spleen is flexibly fixed with the left hand, so that the spleen is both There is no petri dish under the Hank's liquid surface, and grind gently and quickly until the spleen is completely white;

③ lysis of red blood cells. Transfer the spleen suspension in the petri dish to a 15mL centrifuge tube, centrifuge at 300g for 5min, invert and discard the supernatant; add 2mL erythrocyte lysate to each tube (sterilized by filtration with a 0.22μm filter), centrifuge at 300g for 5min at room temperature, invert and discard supernatant;

④ Wash. The Hank's solution was resuspended to 12mL, centrifuged at 300g for 5min, and the supernatant was discarded, and this was repeated twice;

⑤ The obtained spleen lymphocytes were resuspended with an appropriate amount of PRMI-1640 complete medium (containing 10% FBS, 100 U/mL penicillin, 50 μM β-mercaptoethanol), counted on a cell counter, and adjusted to a cell concentration of 3×106 cells/ mL was used for subsequent index determination.

Spleen lymphocyte proliferation response assay:

Spleen lymphocytes (3×106 cells/mL) were inoculated into 96-well plates, 100 μL per well, and then 100 μL of PRMI-1640 blank medium or 100 μL of PRMI-1640 complete medium containing ConA or LPS (the final mixture of ConA and LPS) was added. The concentrations were 5 μg/mL and 10 μg/mL, respectively), placed in a 37°C 5% CO2 cell incubator for 72 hours, and 50 μL of MTT was added to each well (sterilized by 0.22 filter, the final concentration of MTT was 0.5 mg/mL) , continue to culture for 4h, centrifuge at 4000r/min for 10min, discard the liquid in each well, add 150 μL of DMSO to each well to dissolve the generated formazan, measure the OD value of each well at a wavelength of 490nm with a microplate reader, and use 630nm as the calibration wavelength. Stimulation index (SI) was used as an index to judge the degree of lymphocyte transformation and proliferation. The calculation formula of SI was as follows.

SI=(OD value of stimulated well-blank well)/(OD value of stimulated well-blank well)

The effect of LSA on the proliferation of spleen lymphocytes in immunosuppressed mice was investigated, and the results are shown in Figure 4. As can be seen from the figure, compared with the normal control group, the splenic lymphocytes in the cyclophosphamide group were significantly inhibited by ConA or LPS-induced proliferation (all P<0.05), while LSA could reverse the inhibitory effect caused by CPA to a certain extent. The specific performance is as follows: compared with the cyclophosphamide group, LSA in each dose group can significantly improve the proliferation response of spleen lymphocytes to ConA-induced by immunosuppressed mice (P<0.05); The proliferative response of spleen lymphocytes to LPS-induced in immunosuppressed mice was increased (P<0.05). In addition, compared with mice in the cyclophosphamide group, the spleen lymphocytes of the mice fed with sodium selenite, oligosaccharide or sodium selenite + oligosaccharide had no significant proliferative response to ConA or LPS induction. Sexual changes (P>0.05).

4. Detection of serum cytokines in mice

24 hours after the last administration (fasting), blood was collected from the eyeballs of the mice, packed in clean and dry EP tubes, left standing at room temperature for 2 hours, centrifuged at 2000 rpm/min at 4°C for 10 minutes, and the serum was collected and stored in a -80°C refrigerator for future use. The content of cytokines (IL-2/IFN-γ, IL-4/IL-10) in serum was determined by ELISA kit, and the specific operation was carried out according to the instructions of the kit.

The effects of selenized oligoaminopolysaccharide on the content of serum IL-2, IFN-γ, IL-4 and IL-10 in mice were investigated, and the results are shown in Figures 5-8. As can be seen from the figure, compared with the normal control group, the serum levels of IL-2, IFN-γ, IL-4 and IL-10 of the mice in the cyclophosphamide group were significantly decreased (P<0.05). Compared with the cyclophosphamide group, the levels of serum IL-2 in the low, medium and high LSA mice were significantly increased (P<0.05). 4 reduction had a certain improvement effect, but there was no statistical difference (P>0.05), the serum IFN-γ and IL-4 contents of 0.6mg/kg LSA mice were significantly increased (P<0.05); 0.6, 0.9mg/kg LSA could significantly increase the serum IL-10 content in mice (P<0.05). In addition, compared with the mice in the cyclophosphamide group, the serum IL-2, IFN-γ, IL-4 and There was no significant change in IL-10 content (P>0.05).

Claims (5)

1. the establishment method of a selenized oligoamino polysaccharide to the immunosuppression deficiency mouse influence model, it is characterised in that the described establishment method steps are as follows: 80 mice were randomly divided into 8 groups according to ten mice in each group; Normal control group: intragastric administration of normal saline on days 1-14, intraperitoneal injection of normal saline on days 11-14; Cyclophosphamide group: gavage with normal saline on days 1-14, and intraperitoneal injection of 50 mg cyclophosphamide aqueous solution per kilogram of body weight every day on days 11-14; Cyclophosphamide + 0.3 selenized oligosaccharide group: 0.3 mg selenized oligosaccharide solution per kg body weight per day on the basis of selenium content by gavage on days 1-14, and per kilogram per day on days 11-14 Intraperitoneal injection of 50 mg of cyclophosphamide in water by body weight; Cyclophosphamide + 0.6 selenized oligosaccharide group: 0.6 mg selenized oligosaccharide solution per kilogram of body weight per day based on selenium content was administered by gavage on days 1-14, and per kilogram per day on days 11-14 Intraperitoneal injection of 50 mg of cyclophosphamide in water by body weight; Cyclophosphamide + 0.9 selenized oligosaccharide group: 0.9 mg selenized oligosaccharide solution per kg body weight per day based on selenium content by gavage on days 1-14, and per kilogram per day on days 11-14 Intraperitoneal injection of 50 mg of cyclophosphamide in water by body weight; Cyclophosphamide + 0.3 sodium selenite group: 0.3 mg sodium selenite solution per kg body weight per day based on selenium content was administered by gavage on days 1-14, and 50 mg per kg body weight per day on days 11-14 Cyclophosphamide aqueous solution for intraperitoneal injection; Cyclophosphamide + oligosaccharide group: 9.87 mg oligosaccharide solution per kg body weight per day on days 1-14, and 50 mg cyclophosphamide aqueous solution per kg body weight per day on days 11-14. intraperitoneal injection; Cyclophosphamide + oligomeric amino polysaccharide + 0.3 sodium selenite group: 0.3 mg sodium selenite solution and 9.87 mg oligo amino polysaccharide solution were administered by gavage per kg body weight per day based on selenium content on days 1-14 , 50 mg of cyclophosphamide in water per kilogram of body weight per day for intraperitoneal injection on days 11-14; (b) After fasting for 24 hours after the last administration, the mice were weighed, followed by blood collection from the eyeballs, and sacrificed by cervical dislocation. Then, the spleen and thymus were quickly removed. Weight, record, and calculate the organ index according to the formula. 2. a kind of selenized oligoaminopolysaccharide according to claim 1 to the establishment method of immunosuppression deficiency mouse influence model, it is characterized in that, described organ index formula is: organ index=(organ weight mg/mouse body weight g) × 10. 3. a kind of selenized oligoaminopolysaccharide according to claim 1 to the establishment method of immunosuppression deficiency mouse influence model, it is characterized in that, also comprises: (c) The mice were dissected, and the effect of selenized oligoaminoglycan on the overall immune function of immunosuppressed mice was evaluated by observing spleen tissue morphology, spleen lymphocyte proliferation response and mouse serum cytokine indexes. 4. a kind of Selenized Oligoaminopolysaccharide according to claim 1 and 3 described a kind of establishment method of immunosuppression deficiency mouse influence model, it is characterized in that, described mouse is SPF grade BALB/C mouse, male , 6-8 weeks old, weighing 18-22 g. 5. the establishment method of a kind of selenized oligoaminopolysaccharide according to claim 1 on the influence model of immunosuppression deficiency mice, it is characterized in that, the selenium content of selenized oligoaminopolysaccharide is 27.3 mg/g, wherein by Sodium selenite provides the source of selenium, and the molecular weight of the oligomeric aminopolysaccharide used is 50 kDa.

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