Construction method of bone destruction model

Abstract

The invention relates to the technical field of bioengineering, in particular to a construction method of a bone destruction model, and a TM standard strain FRR2161 is adopted to carry out tibia injection infection on a 4-5 week old C57BL / 6N mouse to obtain a non-immunosuppressive mouse talaromyces marneffei bone destruction model. The non-immunosuppressive mouse talaromyces marneffei bone destruction model is successfully established for the first time, the TM bone destruction model is established in a mouse body, the dynamic process of fungi in the tibia is simulated, a model basis is provided for studying the bone infection state of TM in the body, meanwhile, a tool basis is provided for deeply excavating a TM bone destruction mechanism, and the development of the TM bone destruction model is facilitated. A new thought is provided for the application of screening TM bone infection medicines.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for constructing a bone destruction model. Background Art

[0002] Talaromyces marneffei (TM) is the only known thermophilic fungus in the kingdom Fungi, phylum Ascomycota, class Eurotiomycetes, orders Eurotiomycetes, family Trichodermaceae, genus Talaromyces that is pathogenic to mammals, including humans. Talaromycosis (TMS) is an invasive fungal disease caused by TM infection, which is mainly prevalent in tropical and subtropical Asia. The disease is confined to the upper or lower respiratory tract, bones, joints, and intestines, and can spread in multiple organ systems, causing localized or disseminated infections.

[0003] Bone destruction is a common clinical manifestation of TMS. In the 1980s, Professor Deng Zhuolin of Guangxi Medical University first reported in China that TM infection caused bone destruction, which often occurred in the spine and long bones. With the progression of the disease or delayed treatment, about 30% of TM patients will have severe pathological fractures, with a mortality rate of 20% and a permanent disability rate of 50% within 1 year. This not only brings great pain to the patients' families, but also creates a serious socioeconomic burden.

[0004] At present, models of TM infection of other organs or disseminated infection all involve immunosuppression. In reports related to clinical cases, cases of non-HIV infection can be seen, but there are no reports of bone destruction caused by TM accompanied by HIV infection. At the same time, TM bone destruction often cannot be treated specifically due to the lack of clear etiology, which delays the diagnosis and treatment process and causes serious consequences. Therefore, it is necessary to establish a non-immunosuppressive TM bone destruction model to understand the bone destruction caused by TM under non-immunosuppressive conditions and explore its pathogenic mechanism.

[0005] Therefore, it is urgent to construct a bone destruction model. Summary of the invention

[0006] In order to develop a bone destruction model to understand the bone destruction caused by TM in a non-immunosuppressive state and explore its pathogenic mechanism, the present invention provides a method for constructing a bone destruction model. The construction method of the present invention can further clarify the molecular mechanism of TM bone destruction, provide a tool basis for in-depth exploration of the TM bone destruction mechanism, and promote the experimental research and preparation and development of anti-TM bone infection drugs and vaccines.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] A method for constructing a bone destruction model comprises: using the TM standard strain FRR2161 to infect 4-5 week old C57BL / 6N mice by intratibial injection to obtain a Talaromyces marneffei bone destruction model in non-immunosuppressed mice.

[0009] Preferably, the TM standard strain FRR2161 is prepared into a TM standard strain FRR2161 suspension and then injected into the tibia for infection.

[0010] Preferably, the concentration of the TM standard strain FRR2161 suspension is 0.8×10 8 CFU / mL-1.2×10 8 CFU / mL.

[0011] Preferably, the concentration of the TM standard strain FRR2161 suspension is 10 8 CFU / mL.

[0012] Preferably, the suspension injection dose of the TM standard strain FRR2161 is 8 μL-12.5 μL.

[0013] Preferably, the suspension injection dose of the TM standard strain FRR2161 is 10 μL.

[0014] Preferably, the preparation process of the TM standard strain FRR2161 suspension is as follows: after activating the TM standard strain FRR2161, the colonies are scraped off and inoculated into a Tween 20-dd aqueous solution, mixed and centrifuged to discard the supernatant, the bacteria are precipitated, washed, added to PBS and shaken to obtain a TM standard strain FRR2161 suspension.

[0015] Preferably, mice are maintained for 28-30 days following infection by intratibial injection.

[0016] Preferably, the breeding environment has a temperature of 18°C-25°C, and a 12 / 12h day and night cycle is adopted during the breeding process.

[0017] The present invention also provides a method for drug screening, which can act the drug to be screened on a bone destruction model to screen out the drug that can improve or alleviate the bone destruction phenomenon.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention successfully established a bone destruction model for the first time. The construction method provided is suitable for non-immunosuppressed mice, which provides ideas for technical personnel in this field to further develop fungal infection models of specific organs.

[0020] 2. The present invention provides a method for constructing a bone destruction model. The TM bone destruction model constructed in non-immunosuppressed animals simulates the dynamic process of fungi inside human bone tissue, provides a model basis for the diagnosis and targeted treatment of TM bone infection, and provides a tool basis for in-depth exploration of the TM bone destruction mechanism.

[0021] 3. The method for constructing a bone destruction model provided by the present invention provides a tool model for studying TM bone destruction and broadens the research ideas. In particular, TM is a temperature-dependent fungus, and its pathogenic mechanism is different from that of viruses or bacteria. The TM bone destruction model provided by the present invention further provides sufficient basis and solid preliminary foundation for the study of the molecular mechanism of TM bone destruction.

[0022] 4. The method for constructing a bone destruction model provided by the present invention has a short modeling cycle and a stable model, which speeds up the progress of subsequent experimental research based on the model and improves the success rate of the experiment, reduces the investment in manpower, material resources and financial resources, and thus provides a tool basis for screening the application of drugs for TM bone infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Timeline diagram for the construction of the Talaromyces marneffei bone destruction model in non-immunosuppressed mice.

[0025] Figure 2 Figure 2 shows the changes in body weight of mice after TM infection.

[0026] Figure 3 The gross images of the tibiae of mice after TM infection; in the figure, A is the gross image of the tibiae of the Sham group; Figure B is the gross image of the tibiae of the TM group; the Sham group is the sham operation group, and the TM group is the group infected with Talaromyces marneffei.

[0027] Figure 4 This is a gross image of the liver of mice after TM infection; in the figure, A is a gross image of the liver of the Sham group; B is a gross image of the liver of the TM group; the Sham group is the sham operation group, and the TM group is the group infected with Talaromyces marneffei.

[0028] Figure 5These are pictures of the Tibialis fungus culture results after TM infection in mice; in the figure, A is the Tibialis fungus culture result of the Sham group at 25°C; B is the Tibialis fungus culture result of the TM group at 25°C; C is the Tibialis fungus culture result of the Sham group at 37°C; D is the Tibialis fungus culture result of the TM group at 37°C; the Sham group is the sham operation group, and the TM group is the group infected with T. marneffei.

[0029] Figure 6 Figure 2 shows the Micro-CT images of mice infected with TM and the statistical graphs of related indicators. In the figure, A is a representative Micro-CT image of the Sham group; B is a representative Micro-CT image of the TM group; C is a statistical graph of the differences in different indicators between the Sham group and the TM group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; (a) is a statistical graph of the difference in bone volume fraction of cancellous bone between the Sham group and the TM group, (b) is a statistical graph of the difference in the number of trabeculae in cancellous bone between the Sham group and the TM group, (c) (a) is the statistical graph showing the difference in trabecular separation of cancellous bone between the Sham group and the TM group, (d) is the statistical graph showing the difference in structural model index of cancellous bone between the Sham group and the TM group, (e) is the statistical graph showing the difference in bone volume fraction of cortical bone between the Sham group and the TM group, (f) is the statistical graph showing the difference in trabecular pattern factor between the Sham group and the TM group, (g) is the statistical graph showing the difference in average total osteotomy circumference of cortical bone between the Sham group and the TM group, and (h) is the statistical graph showing the difference in average total osteotomy area of ​​cortical bone between the Sham group and the TM group; the Sham group was the sham operation group, and the TM group was the group infected with Talaromyces marneffei.

[0030] Figure 7 HE staining images and statistical graphs of trabecular area percentage after TM infection in mice; in the figure, A is a representative HE staining image of the Sham group; B is a representative HE staining image of the TM group; C is a statistical graph of the difference in trabecular area percentage between the Sham group and the TM group, ** indicates P<0.01; the Sham group is the sham operation group, and the TM group is the group infected with Talaromyces marneffei.

[0031] Figure 8 Figure 2 shows the TRAP staining images of mice infected with TM and the statistical graph of the percentage of TRAP staining positive areas. In the figure, A is a representative image of TRAP staining in the Sham group; B is a representative image of TRAP staining in the TM group; C is a representative image of TRAP staining in the Sham group and the TM group. + Statistical chart of area percentage differences, * indicates P < 0.05; Sham group is the sham operation group, and TM group is the group infected with Talaromyces marneffei. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Example 1: A method for constructing a bone destruction model.

[0034] S1, Construction of TM infected mice:

[0035] ① Animal husbandry: After obtaining animal ethics review, mice were kept in an animal biosafety laboratory. The humidity in the animal room was constant, and a 12 / 12h day and night cycle was adopted during the feeding process. Sterilized mouse feed and water were fed.

[0036] ② Preparation of TM standard strain FRR2161 suspension: Take out the TM standard strain FRR2161 stored in a -80℃ low-temperature refrigerator, inoculate it into PDA medium by plate streaking method, observe the growth of the strain 7 days after inoculation, and take the plate with good growth to prepare the suspension. Use an inoculation loop to scrape the colony and put it into 0.001% Tween 20-dd aqueous solution, then vortex and oscillate for 5 minutes, centrifuge at 12000r / min for 5 minutes, discard the supernatant, wash the precipitate twice with sterile PBS solution, add 1mL PBS and shake to mix, count with a hemocytometer and dilute to 10 8 CFU / mL.

[0037] ③TM infected mice: the concentration of 10 8 CFU / mL suspension of TM standard strain FRR2161 was injected into the tibia to achieve TM infection in mice. Mice were raised for 28 days after infection, and mice were killed on the 1st, 3rd, 5th, 7th, 14th and 28th day to obtain blood, liver, tibia and other tissue specimens.

[0038] S2. Monitoring of physiological parameters of mice and detection of TM bone destruction

[0039] ① Observe vital signs and record body weight: Observe whether the vital weight of mice is stable on 1, 3, 5, 7, 14, and 28 days after infection, weigh and record.

[0040] ② Animal dissection and specimen processing: After intraperitoneal injection of sodium pentobarbital, the mice were anesthetized, the eyeballs were removed to obtain blood, and the blood was placed in an EDTA anticoagulant tube, and then euthanized by cervical dislocation. The mice were dissected aseptically, the abdominal cavity was opened to observe the liver lesions and samples were taken for photography, the skin and muscles of the lower limbs were opened, the tibia was taken out and photographed.

[0041] ③ Tibia fungus culture: Take part of the tibia and cut it into pieces under a sterile environment. Inoculate the tibia fragments into PDA medium, culture at 25℃ for 7 days, observe and take pictures. If colonies grow, select gray-green colonies with rich spore production to collect spores, inoculate them into SDA medium, culture at 37℃ for 7 days, observe and take pictures.

[0042] ④ Part of the tibial tissue was fixed in paraformaldehyde, scanned and analyzed by Micro-CT (Bruker, Skyscan1275), and three-dimensionally reconstructed using NRecon software. CTAN software was used to analyze and compare the reconstructed bone volume percentage (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), structural model index (SMI) and connection density (Conn-Dens) and other related parameters.

[0043] ⑤ Part of the tibia was fixed in paraformaldehyde, dehydrated, decalcified, waxed and embedded, and 5 μm serial sections were sliced ​​and stained with HE and TRAP. The pathological changes were observed under an optical microscope and pictures were obtained.

[0044] Example 2: Based on Example 1, the method for constructing the model is further described. Figure 1 shown.

[0045] First, 5-week-old C57BL / 6N male mice were selected. Then, on day 0, the mice were allowed to adapt to the environment for 7 days, and ear-marking forceps were used to punch holes and mark them for subsequent experiments.

[0046] On the 8th day, the TM standard strain was used to administer drugs to the mice, and the physiological indices of the mice were regularly monitored, including vital signs and body weight of the mice.

[0047] When the bacteria from tibia tissue were cultured successfully and met the biphasic growth characteristics, it indicated that TM-infected mice were successfully established, and then TM-infected mice were continuously bred.

[0048] The continuous feeding time is 1 day, 3 days, 5 days, 7 days, 14 days and 28 days.

[0049] The continuous feeding time arrangement adopted in the present embodiment is as follows:

[0050] The mice were maintained for 28 days after infection and were killed on days 1, 3, 5, 7, 14 and 28 to obtain blood, liver, tibia and other tissue specimens.

[0051] The monitoring of various physiological indicators of mice may adopt the method of this embodiment, or may adopt other monitoring methods such as in vivo monitoring of mice according to actual needs.

[0052] Secondly, TM could be detected in the tibia of TM-infected mice at every time point, indicating that TM could successfully colonize in the tibia and TM successfully infected the tibia.

[0053] Finally, detect the state of TM bone destruction: monitor the physiological indicators of mice and detect the state of TM bone destruction. The specific main process is:

[0054] ① Weight monitoring: monitor the weight changes of mice on the 1st, 3rd, 5th, 7th, 14th and 28th days after infection to evaluate the growth status.

[0055] ② Observe and record the gross condition of the tibia and liver: dissect the mice at the specified time points, remove the organs and rinse them with pre-cooled sterile saline, absorb the water and take pictures. The gross morphology can reflect the pathological changes of various organs caused by TM infection.

[0056] ③ Tibia fungal culture: The results of fungal culture can reflect whether the mouse tibia is infected with fungi and the colony morphology of the infected fungi to determine whether it is TM infection.

[0057] ④Micro-CT: Micro-CT can reflect the changes in the cancellous bone mass, the number, thickness and structural characteristics of trabeculae in the tibia of mice, as well as the changes in the cortical bone mass, bone area and circumference, and trabecular structure. It is a key indicator of bone destruction.

[0058] ⑤HE and TRAP staining: HE staining can more intuitively see the situation inside the tibial tissue, while TRAP staining reflects the area occupied by osteoclasts (the only bone-absorbing cells, which are closely related to bone destruction), which can verify whether bone destruction has occurred and its intensity at another level.

[0059] Example 3: Step S1 constructs the process of TM infection in mice.

[0060] Step S1, construct TM to infect mice.

[0061] After obtaining animal ethics review, mice were kept in an animal biosafety laboratory. The humidity of the animal room was constant, and a 12 / 12h day and night cycle was adopted during the feeding process. They were fed sterilized mouse feed and water. The mice used in the experiment were C57BL / 6N mice, and they were 5-week-old male mice.

[0062] On days 0-7, the mice were allowed to adapt to the environment and marked with ear tags. Then, starting from day 7, the mice were weighed regularly and the data were recorded for subsequent experiments.

[0063] On the 8th day, take out the TM standard strain suspension stored at 4°C. The concentration of the suspension can be determined by direct counting with a hemocytometer. The concentration is 10 8 CFU / mL, the TM standard strain (FRR2161) used in this experiment was donated by Professor Cao Cunwei, Department of Dermatology, First Affiliated Hospital of Guangxi Medical University. 10 μL of suspension was injected into the tibia to achieve TM infection in mice. TM-infected mice were maintained and their vital signs were monitored.

[0064] On day 1 after TM infection, mice were weighed and their vital signs were monitored.

[0065] On day 3 after TM infection, mice were weighed and their vital signs were monitored.

[0066] On day 5 after TM infection, mice were weighed and their vital signs were monitored.

[0067] On day 7 after TM infection, mice were weighed and their vital signs were monitored.

[0068] On the 14th day after TM infection, it was found that the body weight of TM-infected mice was significantly lower than that of the sham operation group (Sham group), and the body weight gain of mice slowed down.

[0069] On the 28th day after TM infection, it was found that the body weight of TM-infected mice was significantly lower than that of the Sham group, and the weight gain of mice slowed down. Figure 2 shown.

[0070] This example only describes the condition of mice in the first 21 days. According to the experimental needs, the mice were kept and their vital signs were monitored.

[0071] Example 4: Introducing the process of detecting TM bone destruction in step S2.

[0072] After completing step S1 to construct TM-infected mice, continue to feed the mice and monitor their vital signs.

[0073] On the 28th day, the experiment found that after dissection, compared with the Sham group, the upper end of the tibia of the mice in the TM group became brittle and easy to break, the upper joint surface became soft, hyperplasia and abscesses appeared, the bone marrow cavity turned white, and the liver tissue color changed, and white spots were visible. Figure 3-Figure 4As shown. After culturing in PDA medium at 25°C for one week, no colony growth was observed in the Sham group, while the TM group cultured obvious yellow fluffy colonies with red in the middle and white hyphae. They grew densely and vigorously, and spread in concentric circles toward the edge of the plate, producing soluble wine-red pigments that diffused into the culture medium. After inoculation into SDA medium and culturing at 37°C for one week, white yeast-like colonies appeared, which were moist, raised, and waxy. After continued cultivation, the colonies showed gyrus-like appearance. It meets the characteristics of biphasic growth, so the bacteria were confirmed to be TM. Figure 5 Next, we tested the bone destruction.

[0074] The test found that in the Micro-CT results, compared with the Sham group, the TM group had a decrease in tibia cancellous bone mass, broken and thinned trabeculae, disappeared connections, decreased number, and a scattered structure. The cortical bone mass was lost, the bone area and circumference decreased, and the continuity of the trabecular structure changed. Figure 6 shown.

[0075] In order to more intuitively see the internal situation of the tibia tissue, we performed HE staining and found that the bone tissue in the TM group was destroyed, the structure was disordered, and the trabecular bone / tissue area was significantly smaller than that in the Sham group (P<0.001). Figure 7 shown.

[0076] In order to observe the number and area of ​​osteoclasts in the tibia, we performed tartrate-resistant acid phosphatase (TRAP) staining and found that compared with the Sham group, the activity of osteoclasts in the TM group was enhanced, the number increased, and the bone absorption capacity was improved. Figure 8 shown.

[0077] Example 5: Based on any one of Examples 1, 2, 3 or 4, further supplementary explanation is given on model construction.

[0078] The infection model was established by intratibial injection (1×10 8 CFU), can directly infect the mouse tibia in situ, and TM can spread through the blood system to the liver, etc., retaining the characteristics of TM disseminated infection.

[0079] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0080] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for constructing a bone destruction model, characterized in that: The construction method comprises: using the TM standard strain FRR2161 to infect C57BL / 6N mice aged 4-5 weeks by intra-tibia injection, so as to obtain a T. marneffei bone destruction model in non-immunosuppressed mice.

2. The method for constructing a bone destruction model according to claim 1, characterized in that: The TM standard strain FRR2161 was prepared into a TM standard strain FRR2161 suspension and then injected into the tibia for infection.

3. The method for constructing a bone destruction model according to claim 1, characterized in that: The concentration of the TM standard strain FRR2161 suspension was 0.8×10 8 CFU / mL-1.2×10 8 CFU / mL.

4. The method for constructing a bone destruction model according to claim 1, characterized in that: The concentration of the TM standard strain FRR2161 suspension was 10 8 CFU / mL.

5. The method for constructing a bone destruction model according to claim 1, characterized in that: The suspension injection dosage of the TM standard strain FRR2161 is 8 μL-12.5 μL.

6. The method for constructing a bone destruction model according to claim 1, characterized in that: The injection dose of the suspension of the TM standard strain FRR2161 is 10 μL.

7. The method for constructing a bone destruction model according to claim 1, characterized in that: The preparation process of the TM standard strain FRR2161 suspension is as follows: after activating the TM standard strain FRR2161, the colonies are scraped off and inoculated into a Tween 20-dd aqueous solution, mixed and centrifuged, the supernatant is discarded, the bacteria are precipitated, PBS is added after washing, and then oscillated and mixed to obtain the TM standard strain FRR2161 suspension.

8. The method for constructing a bone destruction model according to claim 1, characterized in that: Mice were maintained for 28-30 days after intra-tibia infection.

9. The method for constructing a bone destruction model according to claim 1, characterized in that: The breeding environment has a temperature of 18°C-25°C, and a 12 / 12h day and night cycle is adopted during the breeding process.

10. A method for drug screening, characterized in that: include: A bone destruction model is constructed using the bone destruction model construction method according to any one of claims 1 to 9, and the drug to be screened is applied to the bone destruction model to screen out the drug that can improve or alleviate the bone destruction phenomenon.

Images