Cryptosporidium parvum infection immunosuppression mouse model and construction method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cryptosporidium parvum infection immunosuppression mouse model and a construction method thereof. Aiming at the problem that the development of cryptosporidiosis drugs and vaccines is seriously limited due to the fact that no model for successfully infecting mice with cryptosporidium parvum exists at present, the invention provides the construction method of the cryptosporidium parvum infection immunosuppression mouse model, which comprises the following steps: firstly, carrying out gavage treatment on the mice for 4-7 days by adopting cyclophosphamide; constructing to obtain an immunosuppressive mouse; feeding PBS (Phosphate Buffer Solution) containing cryptosporidium parvum oocysts through the mouth, and infecting for 7-21 days; and when oocysts are continuously detected from excrement of the mouse and the content of the oocysts is higher than the infection amount, constructing the cryptosporidium parvum infection immunosuppression mouse model. The cryptosporidium parvum infection immunosuppression mouse model is successfully constructed, can be used for subsequent drug and vaccine development tests for cryptosporidium parvum diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Cryptosporidium parvum-infected immunosuppressed mouse model and a method for constructing the same. Background Art

[0002] Cryptosporidium is a genus of single-celled parasites and a member of the phylum Apicomplexa, including 25 well-known genera such as Plasmodium and Toxoplasma. It can infect a variety of animals including humans. The disease caused by Cryptosporidium is called cryptosporidiosis. Cryptosporidium mainly infects the small intestinal epithelial cells of humans and mammals, often causing self-limiting diarrhea in normal immune organisms, but it is likely to cause severe watery diarrhea in immunodeficient individuals such as AIDS patients or young individuals, and can even lead to the death of patients in severe cases. There is currently no effective drug or vaccine for the treatment of cryptosporidiosis. Nitazoxanide is the only drug approved for cryptosporidiosis, but its efficacy is limited in individuals with normal immune function and ineffective in immunocompromised individuals, especially HIV / AIDS patients. For a long time, the screening of drugs and vaccines for Cryptosporidium has always been a research hotspot, and it is very crucial to select a suitable animal model. Therefore, establishing a stable, reliable and safe Cryptosporidium animal infection model is helpful for the research of Cryptosporidium.

[0003] Cryptosporidium parvum is the most important zoonotic species, which can infect humans and a variety of animals and is the most studied Cryptosporidium species at present. The infectivity of Cryptosporidium parvum varies in different hosts. Calves are the best parasitic hosts, and a large number of oocysts can be excreted after successful infection. However, using calves as an animal model is expensive, has strict requirements for the test site, and has low practical value. Mice are common animals in animal models, which are cheap, easy to infect and obtain. However, mice with normal immune function are not susceptible hosts for Cryptosporidium parvum, and mice with immune deficiency or immunosuppression need to be selected as hosts.

[0004] At present, no stable and safe Cryptosporidium mouse infection model has been successfully constructed, and there is an urgent need for development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that there is currently no model for successful infection of mice with Cryptosporidium parvum, which severely restricts the development of drugs and vaccines for cryptosporidiosis.

[0006] The technical solution of the present invention to solve the above technical problem is to provide a method for constructing a Cryptosporidium parvum-infected immunosuppressed mouse model. The method includes the following steps:

[0007] a. Construct immunosuppressed mice

[0008] The mice were intragastrically administered with cyclophosphamide for 4 - 7 days to construct immunosuppressed mice;

[0009] b. Infect the immunosuppressed mice with Cryptosporidium parvum

[0010] The immunosuppressed mice obtained in step a were orally administered with PBS containing Cryptosporidium parvum oocysts and infected for 7 - 21 days;

[0011] c. When oocysts were continuously detected in the feces of the mice and the oocyst content was higher than the infection amount, an immunosuppressed mouse model infected with Cryptosporidium parvum was constructed.

[0012] Among them, in the method for constructing the above - mentioned immunosuppressed mouse model infected with Cryptosporidium parvum, the cyclophosphamide described in step a was purchased from Shanghai Yuanye Bio - technology Co., Ltd.

[0013] Among them, in the method for constructing the above - mentioned immunosuppressed mouse model infected with Cryptosporidium parvum, the concentration of the cyclophosphamide described in step a was 97%.

[0014] Among them, in the method for constructing the above - mentioned immunosuppressed mouse model infected with Cryptosporidium parvum, the dosage of the cyclophosphamide described in step a was 50 mg / kg / d per mouse.

[0015] Among them, in the method for constructing the above - mentioned immunosuppressed mouse model infected with Cryptosporidium parvum, the concentration of Cryptosporidium parvum oocysts in the PBS described in step b was 5×10 4 -5×10 5 oocysts in every 200 μL of the solution.

[0016] The present invention also provides an immunosuppressed mouse model infected with Cryptosporidium parvum constructed by the above method.

[0017] The present invention also provides a use of the above - mentioned immunosuppressed mouse model infected with Cryptosporidium parvum for the development of drugs and vaccines against cryptosporidiosis.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention provides an immunosuppressed mouse model infected with Cryptosporidium parvum and a method for constructing the same. First, the mice were immunosuppressed with cyclophosphamide to obtain immunosuppressed mice, and then infected with an appropriate concentration of Cryptosporidium parvum. It was found that the infected mice could stably excrete Cryptosporidium oocysts. Therefore, an immunosuppressed mouse model infected with Cryptosporidium parvum was successfully constructed, which can be used for subsequent drug and vaccine development tests for cryptosporidiosis and has good application prospects. Brief Description of the Drawings

[0020] Figure 1The figure shows the changes in body weight of each group of mice after being infected with Cryptosporidium parvum;

[0021] Figure 2 The figure shows the excretion pattern of Cryptosporidium oocysts in infected mice;

[0022] Figure 3 The figure shows the survival curve of infected mice;

[0023] Figure 4 The figure shows epithelial cells; Figure A is the image of the ileal epithelial cell site of mice infected with Cryptosporidium in the CXT + _1×10 4 Cp + group, Figure B is the image of the damage of intestinal villi of ileal epithelial cells of mice in the CXT + _1×10 4 Cp + group, and Figure C is the image of the NC group.

[0024] Figure 5 The figure shows the change levels of IL-10 and IL-17 in mice on the 4th and 7th days of immunosuppression. Specific implementation mode

[0025] The present invention provides a method for constructing a model of immunosuppressed mice infected with Cryptosporidium parvum, comprising the following steps:

[0026] a. Construct immunosuppressed mice

[0027] The mice are gavaged with cyclophosphamide for 4 - 7 days to construct immunosuppressed mice;

[0028] b. Infect the immunosuppressed mice with Cryptosporidium parvum

[0029] The immunosuppressed mice in step a are orally gavaged with PBS containing Cryptosporidium oocysts for 7 - 21 days;

[0030] c. When oocysts are continuously detected in the feces of the mice and the oocyst content is higher than the infection amount, a model of immunosuppressed mice infected with Cryptosporidium parvum is constructed.

[0031] The present invention first obtains immunosuppressed mice by gavaging the mice with cyclophosphamide. Cyclophosphamide (CTX) is a cell-active precursor substance, which is mainly used in clinical practice for diseases such as tumors and leukemia. Cyclophosphamide also has the function of immunosuppression, by blocking the growth and development of lymphoblasts and blocking the differentiation of T and B lymphocytes to inhibit cellular and humoral immunity. Currently, there is no precedent for using cyclophosphamide to immunize mice to obtain immunosuppressed mice.

[0032] The present invention for the first time clearly defines the process of obtaining immunosuppressed mice by immunosuppressing mice with cyclophosphamide and determines the dosage of cyclophosphamide. At the same time, starting from the first day of immunosuppression, the physiological indicators of the mice are detected, such as body weight, appetite, mental state, etc. On the 4th and 7th days of immunosuppression, blood is collected by orbital blood collection, and the levels of IL-10 and IL-17 in the mice are detected by ELISA method. The present invention for the first time verifies whether immune mice are obtained by detecting the levels of IL-10 and IL-17 in the blood of mice. It is found in the experiment that after 7 days of cyclophosphamide immunosuppression, the hair of the mice in the immunosuppression group is relatively messy, not smooth, the appetite decreases, the movement is slow, and the body weight growth rate decreases. On the 4th and 7th days of immunosuppression, both IL-10 and IL-17 cytokines show a downward trend. Thus, by using the method of the present invention to treat mice with cyclophosphamide, immunosuppressed mice are obtained.

[0033] After that, for immunosuppressed mice, the present invention determines the method of infecting immunosuppressed mice with parasites. Immunosuppressed mice are treated with Cryptosporidium parvum, and the daily oocyst excretion of KM mice is detected by fecal purification and counting method. In this experiment, after 3 days of infection, the mice in the drug-treated and parasite-infected group can continuously excrete oocysts, and the harvested oocysts are higher than the infection amount, indicating that Cryptosporidium parvum successfully multiplies in the immunosuppressed mice. Compared with the non-immunosuppressed mice, the body weight of the immunosuppressed mice of the present invention decreased by an average of 10% after being infected with Cryptosporidium parvum. It can be seen that the animal model of immunosuppressed mice infected with Cryptosporidium parvum of the present invention is successfully constructed, which plays a guiding role in further studying the pathogenic mechanism of Cryptosporidium parvum and screening drugs and vaccines.

[0034] The following will further explain the specific implementation manners of the present invention through examples, but it does not mean that the protection scope of the present invention is limited to the scope described in the examples.

[0035] The Cryptosporidium parvum used in the examples is preserved in the Animal Parasitology Laboratory of the College of Animal Science and Veterinary Medicine, Southwest Minzu University.

[0036] The mice used are Kunming (KM) mice, clean grade, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0037] The cyclophosphamide used is purchased from Shanghai Yuanye Bio-Technology Co., Ltd., batch number F13IS206786.

[0038] The remaining reagents and raw materials are all ordinary commercially available products.

[0039] Example: Construction of a mouse model infected with Cryptosporidium parvum after immunosuppression with cyclophosphamide

[0040] Thirty-six Kunming mice, 4 weeks old and weighing about 25 g, were randomly divided into 6 groups of 6 mice each. Among them, the CXT + _Cp - group was immunosuppressed and not challenged with parasites; the CXT - _5×10 4 Cp + group was not immunosuppressed and challenged with parasites (5×10 4 ); the CXT + _5×10 3 Cp + group was immunosuppressed and challenged with parasites at a low concentration (5×10 3 ); the CXT + _1×10 4 Cp + group was immunosuppressed and challenged with parasites at a medium concentration (1×10 4 ); the CXT + _5×10 4 Cp + group was immunosuppressed and challenged with parasites at a high concentration (5×10 4 ); the NC group was not immunosuppressed and not challenged with parasites.

[0041] One week before infection, the mice in the CXT + _Cp - group, the CXT + _5×10 3 Cp + group, and the CXT+_1×10 4 Cp + group were gavaged with CXT at a dose of 50 mg / kg / d for 7 consecutive days; then, the mice in the CXT + _5×10 4 Cp + group, the CXT - _5×10 4 Cp + group, the CXT + _5×10 3 Cp + group, and the CXT+_1×10 4 Cp + group were orally administered 200 μL of PBS containing different doses of Cryptosporidium parvum oocysts, and the CXT + _5×10 4 Cp + group and the NC group were administered an equal dose of PBS. + _Cp - (1) Changes in the body weight of mice during model construction

[0042] (1) Changes in the body weight of mice during model construction

[0043] Starting from the day of intragastric administration of CXT, the body weight of each mouse was measured daily until 20 days after infection with the parasite. The average body weight of each group was calculated, and the changes in body weight of mice in each group were analyzed. Two weeks after infection with Cryptosporidium parvum, all mice showed poor spirit, messy hair, decreased skin elasticity, reduced appetite and activity. In the later stage of experimental infection, the color of mouse feces changed from brownish-yellow to light yellow. The body weight of the experimental groups increased more slowly and fluctuated more greatly compared with the control group, as Figure 1 shown. At the end of the experiment, the body weights of all experimental groups were lower than those of the control group. And throughout the experiment, the CXT + _5×10 3 Cp + group, the CXT + _1×10 4 Cp + group and the CXT + _5×10 4 Cp + group all showed significant differences in body weight compared with the control group. Combining the survival curve and the results of body weight changes, it shows that in this model, the use of CXT and infection with Cryptosporidium will not have a significant impact on the survival of mice, but may reduce the overall body weight index by affecting the absorption and digestion of the intestine.

[0044] The result data was first sorted using Excel, and then one-way ANOVA and multiple comparisons were performed using Graph Prism 10.1.2 software. The results were expressed as "mean ± standard deviation", and P < 0.05 indicated significant differences.

[0045] (2) Oocyst excretion in mice infected with Cryptosporidium parvum in the model

[0046] Fresh feces of mice in each group were collected every other day, weighed and counted. The mouse cages were changed daily after disinfection to improve the accuracy of fecal counting. The experiment was terminated on the 20th day after infection. The method for oocyst counting was as follows: Take 2 g of feces and place it in a dispensing box, add an appropriate amount of saturated saline for dilution. The mixed suspension was filtered through an 80-mesh sieve into a 15-ml centrifuge tube, and then saturated sucrose solution was added until it was full. After centrifugation at 3000 r / min for 10 minutes, 2 ml of the upper liquid was taken, and then 8 ml of pure water was added. The liquid was centrifuged at 3000 r / min for 10 minutes, and the upper clear liquid was removed. Pure water was added to 1 ml, and after mixing, 10 μL of the liquid was taken onto a hemocytometer and counted under a ×40 magnification field of view to calculate OPG. The regular changes in oocyst excretion were observed.

[0047] Fresh mouse feces were collected every two days after intragastric infection with Cryptosporidium parvum oocysts. After filtration and centrifugation of the feces through saturated sucrose solution, they were counted using a hemocytometer. The results were as Figure 2 shown. The results showed that oocysts were detected in the feces of all infected groups on the 3rd day. The CXT+ _5×10 3 Cp + group, CXT + _1×10 4 Cp + group and CXT + _5×10 4 Cp + The oocysts of the group showed a gradually increasing trend and reached the highest value on the 7th day, CXT + _5×10 3 Cp + The group was 4.2×10 5 , CXT + _1×10 4 Cp + was 4.8×10 5 , CXT + _5×10 4 Cp + The group was 6.7×10 5 . It can be seen that among the three parasite challenge doses we selected, as the parasite challenge dose increased, the number of oocysts we could obtain also increased, showing a positive correlation trend between the two. Considering only the number of Cryptosporidium finally obtained, the high-dose parasite challenge group was better and could obtain the largest number of oocysts, but too high a dose might cause the death of mice. Therefore, it was not that the higher the parasite challenge dose, the better.

[0048] In the above results, the number of oocysts gradually decreased from the 7th day to the 13th day, and the ovulation amount increased on the 14th day, then decreased and tended to be stable. This indicates that Cryptosporidium has a rapid reproduction stage in the mouse intestine from 3 to 7 days, reaches the peak on the 7th day, and discharges the most oocysts. If time cost needs to be saved, the parasite challenge can be carried out for 3 - 7 days. However, with the increase of the parasite challenge time, more oocysts can be obtained. Therefore, the parasite challenge time can also be extended to 7 - 14 days.

[0049] And CXT - _5×10 4 Cp + The number of oocysts discharged by the group fluctuated little and showed an overall downward trend from the oocysts discharge to the end of the experiment. The number of oocysts discharged was lower than that of each immunosuppressive group, and there was no peak period of oocyst discharge.

[0050] We also examined the survival of the experimental mice. The survival curve is as Figure 3 shown. No death occurred in the CXT + _Cp - group, and the survival rate was 100%; CXT - _5×10 4 Cp +One mouse died in each of the groups on the second and seventh days of immunosuppression, and the survival rate was 66.7%; CXT + _5×10 3 Cp + Mice in the group died on the sixth and seventh days of immunosuppression and on the second day after infection, and the survival rate was 50%; CXT + _1×10 4 One mouse in the Cp+ group died on the fifth day after infection, and the survival rate was 83.3%; CXT + _5×10 4 Cp + One mouse died in each of the groups on the seventh day of immunosuppression and on the fourth day after infection, and the survival rate was 66.7%; no death occurred in the NC group, and the survival rate was 100%. Therefore, when selecting the infection dose, we gave priority to the CXT + _1×10 4 Cp+ group, that is, the medium-concentration infection group.

[0051] Based on the above experimental results, mice infected with different doses of Cryptosporidium parvum under immunosuppression could excrete oocysts, and an animal model infected with Cryptosporidium parvum could be successfully established. After comparing a series of indicators, we finally selected CXT + _1×10 4 Cp + infection as the best infection dose.

[0052] (3) Autopsy of model mice and observation of pathological changes in intestinal mucosa tissue

[0053] The mice were sacrificed on the 20th day after infection. The intestinal tissues of the mice were taken and fixed in paraformaldehyde for 24 hours, then dehydrated through a gradient of 70%, 80%, 90%, and 100% ethanol, cleared with xylene, and embedded in paraffin to make paraffin sections. The sections were dewaxed and hydrated, stained with HE, and sealed with neutral gum. After the gum solidified, they were observed under a microscope.

[0054] Pathological sections were made from the ileum tissues of mice on the 20th day after infection and observed by HE staining. The results showed that Cryptosporidium parvum was attached in the ileum tissues of the infected group, and some epithelial cells fell off (as Figure 4 shown), while Cryptosporidium parvum was not observed in the ileum tissues of the control group, and there were no obvious pathological changes.

[0055] (4) Determination of IL-10 and IL-17 contents by enzyme-linked immunosorbent assay

[0056] Blood samples of mice on the 4th and 7th days of immunosuppression were collected by orbital blood collection, stored at -20°C, and then the serum levels of interleukin 10 and interleukin 17 were detected by an enzyme-linked immunosorbent assay kit. All samples were measured in triplicate.

[0057] The results are as follows Figure 5 As shown, CXT + is CXT + _Cp - group, CXT + _5×10 3 Cp + group, CXT + _1×10 4 Cp + group, CXT + _5×10 4 Cp + group, CXT - The group is CXT - _5×10 4 Cp + groups and the NC group, Figure 5 Shown are the changing levels of IL-10 and IL-17 in immunosuppressed mice at 4 dpi and 7 dpi. At 4 days and 7 days after immunosuppression, both IL-10 and IL-17 cytokines showed a downward trend. Thus, by using the method of the present invention, mice were treated with cyclophosphamide to obtain immunosuppressed mice.

[0058] The result data was first sorted using Excel and then subjected to two-way ANOVA and multiple comparisons using Graph Prism 10.1.2 software. The results are expressed as "mean ± standard deviation", and P < 0.05 indicates significant differences.

[0059] During immunosuppression, it was found that the levels of IL-10 and IL-17 in mice after cyclophosphamide immunosuppression were lower compared to the control group. At 4 days after immunosuppression, compared to the control group, the IL-10 level in the immunosuppression group decreased by approximately 22%, and the IL-17 level decreased by approximately 23%. At 7 days after immunosuppression, the IL-10 level in the immunosuppression group decreased by approximately 60% compared to the control group, and the IL-17 level decreased by 360%.

[0060] As can be seen from the above examples, using KM mice as an animal model, immunosuppression was carried out by intragastric administration of cyclophosphamide to reduce the resistance of mice, and then different doses of C. parvum oocysts were inoculated to successfully construct a murine Cryptosporidium parvum infection model. In each infection model, C. parvum oocysts were successfully detected, but the excretion amounts were different. Inoculation with 1×0 4 C. parvum oocysts excreted a large number of oocysts during the experiment without death; while inoculation with 5×10 4 C. parvum oocysts, the clinical symptoms of the mice were the most significant. Although the number of oocysts excreted was the largest, the mortality rate was higher than that of the group inoculated with 1×10 4 C. parvum group. Therefore, we believe that inoculation with 1×104 It is most suitable for infecting with 4 C. parvum oocysts.

[0061] In summary, in this experiment, immunosuppressed mice were used by cyclophosphamide to preliminarily establish an animal model of C. parvum infection in KM mice, and the optimal infection dose was further screened out, which plays a guiding role in further studying the pathogenic mechanism of C. parvum, as well as the screening of drugs and vaccines.

Claims

1. A method for constructing a mouse model infected with Cryptosporidium parvum immunosuppressed, characterized in that, Comprising the following steps: a. Construct immunosuppressed mice The mice were gavaged with cyclophosphamide for 4 - 7 days to construct immunosuppressed mice; b. Infect the immunosuppressed mice with Cryptosporidium parvum The immunosuppressed mice obtained in step a were orally gavaged with PBS containing Cryptosporidium parvum oocysts for 7 - 21 days; c. When oocysts were continuously detected in the feces of the mice and the oocyst content was higher than the infection dose, an immunosuppressed mouse model infected with Cryptosporidium parvum was constructed.

2. The method for constructing a murine model of immunosuppressed mice infected with Cryptosporidium parvum according to claim 1, characterized in that: The concentration of cyclophosphamide described in step a is 97%.

3. The method for constructing a mouse model of immunosuppressed mice infected with Cryptosporidium parvum according to claim 1, wherein: The dosage of cyclophosphamide described in step a is 50 mg / kg / d per mouse.

4. The method for constructing a murine model of immunosuppressed mice infected with Cryptosporidium parvum according to claim 1, wherein: The concentration of Cryptosporidium parvum oocysts in the PBS described in step b is 5×10 4 -5×10 5 per 200 μL of solution.

5. An immunosuppressed mouse model infected with Cryptosporidium parvum constructed by the method according to any one of claims 1 - 4.

6. Use of the immunosuppressed mouse model infected with Cryptosporidium parvum according to claim 5, characterized in that: For the development of drugs and vaccines for cryptosporidiosis.

Images