Construction method of invasive candida infection model

Abstract

The invention provides a construction method of an invasive candida infection model. The construction method comprises the following steps: S1, laboratory mouse selection, S2, drug preparation, S3, pretreatment, S4, bacterial load observation, S5, candida culture treatment, S6, intragastric administration, and S7, monitoring state evaluation. According to the method, the real state of invasive infection caused by translocation and dissemination of candida colonization from the digestive tract of a clinical candida infected patient can be simulated, the method is closer to clinical practice, the variety and abundance of native flora in the digestive tract of an experimental mouse are reduced through early-stage antibiotic pretreatment, then candida colonization in the digestive tract is achieved through intragastric administration, and the clinical practice is facilitated. Under the action of an inducer, colonized candida is induced to invade and spread through the digestive tract, the invasive candida infection mouse model can be successfully constructed, and the model can highly restore risk factors and pathogenic processes of clinical invasive candida infection and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro detection, and in particular to a method for constructing an invasive Candida infection model. Background Art

[0002] Candida albicans is an important conditional pathogenic fungus. As an important part of the human microbiome, Candida albicans is the most common important conditional pathogenic fungus, colonizing in the oral cavity, gastrointestinal tract, upper respiratory tract and reproductive tract mucosa and skin of most human bodies. The mortality rate of invasive candidiasis is as high as 40%-70%, which has become a serious threat to human life and health and cannot be ignored. Public health issues. Therefore, animal models of Candida infection play a very important role in the study of the pathogenesis and treatment strategies of Candida infection.

[0003] At present, the conventional Candida systemic infection model is mainly the tail vein injection method, which specifically includes injecting Candida spores into mice through the tail vein after enrichment culture, and directly undergoing the process of hematogenous dissemination, Candida destruction occurs in multiple organs, and then pathophysiological processes occur. However, the traditional model starts with the fungus directly entering the blood to produce bacteremia, which does not conform to the general dissemination process of invasive Candida, especially it cannot reproduce the early state of invasive infection. Therefore, the above traditional models cannot fully reflect clinical reality and are difficult to meet the basic scientific research needs of invasive candidiasis.

[0004] Current studies have confirmed that Candida colonization in the human digestive tract is an important fungal source for invasive Candida infection and Candida sepsis in patients with high-risk susceptibility factors, but there is no unified model yet. With the increase in the number of tumor radiotherapy and chemotherapy, immunodeficiency patients (such as HIV-infected patients) and the widespread use of antibiotics and immunosuppressants, Candida colonization in the gastrointestinal tract is very likely to increase abnormally in large quantities in the local microenvironment, thereby destroying the barrier and further invading the infection. The present invention is based on the above pathophysiological mechanism, highly restores the pathogenesis of the real clinical disease, and provides a reliable animal model for basic scientific research on invasive candidiasis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing an invasive Candida infection model to solve the problem that traditional models in the prior art cannot fully reflect clinical reality and are difficult to meet the basic scientific research needs of invasive Candida infection.

[0006] To achieve the above object, the present invention provides the following technical solution: The present invention provides a method for constructing an invasive Candida infection model, comprising the following steps:

[0007] S1. Select experimental mice;

[0008] Select 6-8 week old female KM mice, weighing 18-30 g, and place them in an SPF-grade breeding environment with normal food and water intake for 5 days;

[0009] S2, drug preparation;

[0010] Weigh penicillin G sodium, streptomycin sulfate, vancomycin hydrochloride, and amphotericin B dry powders, dissolve the first three drugs in 30 ml of normal saline, dissolve amphotericin B in DMSO until no solid soluble matter is visible, mix the DMSO solution of amphotericin B with the above normal saline, add water to make up to 40 ml, divide into portions and store at 4°C;

[0011] S3, preprocessing;

[0012] In the early stage, the experimental mice were gavaged with antibiotic solution to achieve the purpose of clearing the normal flora of the digestive tract. The experimental mice were weighed and the above-mentioned antibiotic mixture was gavaged. A single gavage of 100-300 μl / mouse was given once every 12 hours for a total of 5 days. During this period, the feeding was reduced and the drinking water was normal.

[0013] S4, observe bacterial load;

[0014] Collect mouse feces before and after the cleaning cycle, weigh them, add 1 ml PBS buffer, grind them thoroughly and let them settle for 10 minutes, then apply 10 μl of supernatant on TSA and YPD culture media, culture them at 37°C for 48 hours, count the bacterial load, and the treatment is considered qualified if the bacterial load decreases significantly;

[0015] S5, Candida cultivation and treatment;

[0016] Candida albicans SC5314 was revived on YPD medium, and a single colony was reselected in YPD liquid medium, placed in a 37°C shaker, shaken at 200 rpm overnight, and the fungi were collected by centrifugation, washed twice with PBS buffer, placed in 1640 medium containing 10% special grade fetal bovine serum, and placed in a 37°C shaker again, shaken at 200 rpm for 2 hours, and the fungi were collected by centrifugation, resuspended in an appropriate amount of physiological saline, and gavaged into mice, 300 μl / mouse;

[0017] S6, intragastric administration;

[0018] After oral administration of Candida albicans, appropriate amount of cyclophosphamide powder was weighed at an appropriate time, dissolved in sterile saline, and administered to the experimental mice once by intraperitoneal injection at a dosage of 60 mg / kg. After cyclophosphamide administration, dexamethasone was injected subcutaneously into the neck of the experimental mice every other day at a dosage of 1 mg / kg.

[0019] S7. Monitoring status assessment;

[0020] Drug administration was stopped after the mice showed mental and behavioral abnormalities, and they were allowed to eat and drink normally. The heart rate, respiration, body temperature, hair, and mental and behavioral states of the mice were continuously monitored until the mice died. The mice were dissected and pathological staining was performed to evaluate the degree of Candida invasion of the mice's organs and the degree of organ damage.

[0021] The present invention has at least the following beneficial effects:

[0022] The present invention provides a method for constructing an invasive Candida infection model. The method can simulate the actual state of invasive infection caused by the translocation and dissemination of Candida colonization in the digestive tract of patients with clinical Candida infection, and is closer to clinical practice. The technology uses preliminary antibiotic pretreatment to reduce the types and abundance of native flora in the digestive tract of experimental mice, and then achieves Candida digestive tract colonization by gavage, and induces the colonized Candida to invade and disseminate through the digestive tract under the action of an inducer. This technical point can successfully construct an invasive Candida infection mouse model. The model can highly restore the risk factors and pathogenic process of clinical invasive Candida infection, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Provides a graph of intestinal bacterial load of mice after treatment for the present invention;

[0024] Figure 2 Provide a Candida albicans load variation graph for the present invention;

[0025] Figure 3 Provide a respiratory monitoring diagram of experimental mice for the present invention;

[0026] Figure 4 Provide a heart rate monitoring diagram of experimental mice for the present invention;

[0027] Figure 5 Provide a temperature monitoring diagram of experimental mice for the present invention;

[0028] Figure 6 Provide a monitoring diagram of experimental mouse hair for the present invention;

[0029] Figure 7 Provides a behavior monitoring diagram of experimental mice for the present invention;

[0030] Figure 8 Provide fungal load graphs for the present invention;

[0031] Fig. 9 Provide digestive tract fungal invasion map for the present invention;

[0032] Fig.10 Provided for the present invention are images of the presence of Candida hyphae or fatty degeneration in the liver and inflammatory cell infiltration in the portal area;

[0033] Fig.11Provide the present invention with a picture of kidneys showing glomerular reduction and surrounding inflammatory cell infiltration;

[0034] Fig.12 The invention provides an active map of the proliferation of lymphatic germinal centers in the spleen. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by technicians in the technical field to which the invention belongs. The terms used in this specification and in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more related listed items.

[0036] Example

[0037] A method for constructing an invasive Candida infection model may include the following steps:

[0038] S1. Select experimental mice;

[0039] Select 6-8 week old female KM mice, weighing 18-30 g, and place them in an SPF-grade breeding environment with normal food and water intake for 5 days;

[0040] S2, drug preparation;

[0041] Weigh penicillin G sodium, streptomycin sulfate, vancomycin hydrochloride, and amphotericin B dry powders, dissolve the first three drugs in 30 ml of normal saline, dissolve amphotericin B in DMSO until no solid soluble matter is visible, mix the DMSO solution of amphotericin B with the above normal saline, add water to make up to 40 ml, divide into portions and store at 4°C;

[0042] S3, preprocessing;

[0043] In the early stage, the experimental mice were gavaged with antibiotic solution to achieve the purpose of clearing the normal flora of the digestive tract. The experimental mice were weighed and the above-mentioned antibiotic mixture was gavaged. A single gavage of 100-300 μl / mouse was given once every 12 hours for a total of 5 days. During this period, the feeding was reduced and the drinking water was normal.

[0044] S4, observe bacterial load;

[0045] Collect mouse feces before and after the cleaning cycle, weigh them, add 1 ml PBS buffer, grind them thoroughly and let them settle for 10 minutes, then apply 10 μl of supernatant on TSA and YPD culture media, culture them at 37°C for 48 hours, count the bacterial load, and the treatment is considered qualified if the bacterial load decreases significantly;

[0046] S5, Candida cultivation and treatment;

[0047] Candida albicans SC5314 was revived on YPD medium, and a single colony was reselected in YPD liquid medium, placed in a 37°C shaker, shaken at 200 rpm overnight, and the fungi were collected by centrifugation, washed twice with PBS buffer, placed in 1640 medium containing 10% special grade fetal bovine serum, and placed in a 37°C shaker again, shaken at 200 rpm for 2 hours, and the fungi were collected by centrifugation, resuspended in an appropriate amount of physiological saline, and gavaged into mice, 300 μl / mouse;

[0048] S6, intragastric administration;

[0049] After oral administration of Candida albicans, appropriate amount of cyclophosphamide powder was weighed at an appropriate time, dissolved in sterile saline, and administered to the experimental mice once by intraperitoneal injection at a dosage of 60 mg / kg. After cyclophosphamide administration, dexamethasone was injected subcutaneously into the neck of the experimental mice every other day at a dosage of 1 mg / kg.

[0050] S7. Monitoring status assessment;

[0051] Drug administration was stopped after the mice showed mental and behavioral abnormalities, and they were allowed to eat and drink normally. The heart rate, respiration, body temperature, hair, and mental and behavioral states of the mice were continuously monitored until the mice died. The mice were dissected and pathological staining was performed to evaluate the degree of Candida invasion of the mice's organs and the degree of organ damage.

[0052] Among them, experimental mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., certificate number: SCXK(Lu)20220006;

[0053] Penicillin G sodium (purity ≥99%), streptomycin sulfate (purity ≥99%), and cyclophosphamide (≥99.9%) were purchased from Maclean;

[0054] Vancomycin hydrochloride (purity ≥99%) was purchased from Aladdin;

[0055] Amphotericin B (purity ≥99%) and dimethyl sulfoxide (DMSO) (cell culture grade) were purchased from SIGMA, USA;

[0056] Normal saline was purchased from Shandong Chenxin Pharmaceutical;

[0057] TSA medium, Yeast extract peptone dextrose agar medium (YPD medium) (containing chloramphenicol), and YPD liquid medium were purchased from Qingdao Haibo Company;

[0058] 1640 culture medium, special grade fetal bovine serum and phosphate buffered saline (PBS buffer) were purchased from Prosai Company.

[0059] SPF wild-type KM mice, female, 6 weeks old, weighing 18-23 g / mouse, were taken, 10 mice per group, and adaptively raised for 5 days. On the 6th day, antibiotic solution (prepared according to the technical steps) was given by gavage, 200 μl / time per mouse, twice a day, for 5 consecutive days. The amount of antibiotics prepared is shown in Table 1.

[0060] Table 1 Antibiotic configuration quality

[0061]

[0062] During the treatment period, mice were allowed to drink water freely and eat less. Before and after the treatment, the feces of the mice were collected and placed in a sterile centrifuge tube, and 1 ml of PBS buffer was added to fully grind. After standing for 10 minutes, 10 μl of the supernatant was spread on trypticase soy agar medium (TSA medium) and yeast extract peptone glucose agar medium (YPD medium) (containing chloramphenicol), respectively, and cultured at 37°C for 48 hours. The bacterial load was counted. After the treatment, the intestinal bacterial load of the mice decreased significantly and diarrhea occurred (see Figure 1 );

[0063] Candida albicans SC5314 was revived on yeast peptone glucose agar medium (YPD medium) (containing chloramphenicol), and a single colony was resuspended in YPD liquid medium, placed in a 37°C shaker, shaken at 200 rpm overnight, and the fungi were collected by centrifugation, rinsed twice with PBS buffer, placed in 1640 medium containing 10% special grade fetal bovine serum, and placed in a 37°C shaker again, shaken at 200 rpm for 2 hours. The fungi were collected by centrifugation, resuspended in an appropriate amount of physiological saline, and gavaged into mice treated by the process of Example 1, 300 μl / mouse, and the mice were fed and drank normally after gavage. From the first day after gavage, the feces of the mice were continuously collected, 1 ml of PBS buffer was added, and 10 μl of the supernatant was applied to yeast peptone glucose agar medium (YPD medium) (containing chloramphenicol) after grinding, and cultured at 37°C for 24 hours. The load of Candida albicans in mice was observed. The load of Candida albicans in mice gradually stabilized from the 5th day, and the mice were in good condition (see Figure 2 );

[0064] SPF grade KM mice, 6 weeks old, female, were taken and adapted for 5 days. After the mice were treated according to the schemes of Example 1 and Example 2, an appropriate amount of cyclophosphamide (≥99.9%) was dissolved in sterile physiological saline and injected into the mice once (60 mg / kg). After the single injection, dexamethasone (1 mg / kg) was injected subcutaneously into the neck of the experimental mice one day after the single injection. The mental and behavioral states of the mice were observed. After the mental and behavioral states of the mice were abnormal, the respiration of the mice was monitored (see Figure 3 ), heart rate (see Figure 4 ), body temperature (see Figure 5 ), hair (see Figure 6), mental behavior (cf. Figure 7 After the mice died, they were dissected and their esophagus, stomach, intestines, liver, kidneys, spleen, heart, brain and other organs were taken for histopathological examination and bacterial load evaluation. The results showed that the fungal load of each organ of the mice, except for the digestive tract, was greater than or equal to 2 organs (see Figure 8 ), histopathology showed fungal invasion of the mouse digestive tract (see Fig. 9 ), Candida hyphae or fatty degeneration and inflammatory cell infiltration in the portal area of ​​the liver (see Fig.10 ), the kidneys showed glomerular reduction and peripheral inflammatory cell infiltration (see Fig.11 ), the spleen showed active proliferation of lymphoid germinal centers (see Fig.12 ), which indicates that the model has consistent pathophysiological manifestations with sepsis caused by Candida albicans in clinical practice.

[0065] This model can highly restore the risk factors and pathogenic process of clinical invasive Candida infection and has good application prospects.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may also be combined, the steps may be implemented in any order, and there are many other changes in different aspects of the present invention as above, which are not provided in detail for the sake of simplicity. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an invasive Candida infection model, characterized in that: The following steps are involved: S1. Select experimental mice; Select 6-8 week old female KM mice, weighing 18-30 g, and place them in an SPF-grade breeding environment with normal food and water intake for 5 days; S2, drug preparation; Weigh penicillin G sodium, streptomycin sulfate, vancomycin hydrochloride, and amphotericin B dry powders, dissolve the first three drugs in 30 ml of normal saline, dissolve amphotericin B in DMSO until no solid soluble matter is visible, mix the DMSO solution of amphotericin B with the above normal saline, add water to make up to 40 ml, divide into portions and store at 4°C; S3, pre-processing; In the early stage, the experimental mice were gavaged with antibiotic solution to achieve the purpose of clearing the normal flora of the digestive tract. The experimental mice were weighed and the above-mentioned antibiotic mixture was gavaged. A single gavage of 100-300 μl / mouse was given once every 12 hours for a total of 5 days. During this period, the feeding was reduced and the drinking water was normal. S4, observe bacterial load; Collect mouse feces before and after the cleaning cycle, weigh them, add 1 ml PBS buffer, grind them thoroughly and let them settle for 10 minutes, then apply 10 μl of supernatant on TSA and YPD culture media, culture them at 37°C for 48 hours, count the bacterial load, and the treatment is considered qualified if the bacterial load decreases significantly; S5, Candida cultivation and treatment; Candida albicans SC5314 was revived on YPD medium, and a single colony was reselected in YPD liquid medium, placed in a 37°C shaker, shaken at 200 rpm overnight, and the fungi were collected by centrifugation, washed twice with PBS buffer, placed in 1640 medium containing 10% special grade fetal bovine serum, and placed in a 37°C shaker again, shaken at 200 rpm for 2 hours, and the fungi were collected by centrifugation, resuspended in an appropriate amount of physiological saline, and gavaged into mice, 300 μl / mouse; S6, intragastric administration; After oral administration of Candida albicans, appropriate amount of cyclophosphamide powder was weighed at an appropriate time, dissolved in sterile saline, and administered to the experimental mice once by intraperitoneal injection at a dosage of 60 mg / kg. After cyclophosphamide administration, dexamethasone was injected subcutaneously into the neck of the experimental mice every other day at a dosage of 1 mg / kg. S7. Monitoring status assessment; Drug administration was stopped after the mice showed mental and behavioral abnormalities, and they were allowed to eat and drink normally. The heart rate, respiration, body temperature, hair, and mental and behavioral states of the mice were continuously monitored until the mice died. The mice were dissected and pathological staining was performed to evaluate the degree of Candida invasion of the mice's organs and the degree of organ damage.

2. The method for constructing an invasive Candida infection model according to claim 1, characterized in that: The mass ratio of penicillin G sodium, streptomycin sulfate, vancomycin hydrochloride and amphotericin B dry powder in the S1 is 10:10:5:

1.

3. The method for constructing an invasive Candida infection model according to claim 1, characterized in that: The purity of penicillin G sodium is ≥99%, the purity of streptomycin sulfate is ≥99%, the purity of cyclophosphamide is ≥99.9%, the purity of vancomycin hydrochloride is ≥99%, and the purity of amphotericin B is ≥99%.

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