CN111066700A - Application of salidroside in combination with isoniazid in prolonging survival time of sea branch bacterial plaque horse fish model - Google Patents
Application of salidroside in combination with isoniazid in prolonging survival time of sea branch bacterial plaque horse fish model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to the technical field of zebra fish models, in particular to application of salidroside combined with isoniazid to prolonging survival time of a sea ramrod dental plaque horse fish model. Salidroside is used for treating fertilized eggs of zebra fish, and isoniazid is used for treating zebra fish injected with mycobacterium marinum. The application process is as follows: providing AB wild type zebra fish, mating and fertilizing the zebra fish, placing fertilized eggs in methylene blue solution, incubating at 28-29 ℃, and after 7-10 hours, soaking the fertilized eggs in a mixed solution of salidroside solution and phenylthiourea solution; according to the fertilization time calculation, injecting mycobacterium marinum bacterial liquid into the fertilized zebra fish for 3 days to obtain a mycobacterium marinum bacterial plaque equine fish model; the next day of injection, culturing the sea mycobacterium plaque horse fish model in isoniazid solution, and replacing the liquid medicine every 24h until the sea mycobacterium zebra fish dies. The research proves that salidroside combined with isoniazid can prolong the survival time of infected zebra fish.
Description
Technical Field
The invention relates to the technical field of zebra fish models, in particular to application of salidroside combined with isoniazid to prolonging survival time of a sea ramrod dental plaque horse fish model.
Background
Tuberculosis is an infectious disease caused by mycobacterium tuberculosis, and is still one of the most major infectious diseases threatening human life and health at present. In recent years, the continuous emergence and rapid propagation of multi-drug resistant and widely drug resistant mycobacterium tuberculosis and the occurrence of the common infection of the mycobacterium tuberculosis and human immunodeficiency virus make the prevention and treatment of tuberculosis more difficult, the traditional antitubercular drugs cannot meet the clinical requirements, and the accelerated research and development of novel antitubercular drugs become urgent.
The pathogenic mechanism of tuberculosis, which is extremely complex. After the mycobacterium tuberculosis invades into the body, the mycobacterium tuberculosis interacts with a host, and survives in the host body for a long time by depending on the virulence, autophagy and immune evasion mechanism of the mycobacterium tuberculosis, and is one of the reasons for the difficulty in treating the tuberculosis. It is estimated that about one third of the world's human population has "latent" mycobacterium tuberculosis, and that these people may not exhibit symptoms of tuberculosis for a lifetime, with only 10% eventually progressing to tuberculosis. After the mycobacterium tuberculosis invades the body, the mycobacterium tuberculosis is firstly phagocytized, killed and eliminated by macrophages participating in non-specific immunity, the mycobacterium tuberculosis can not be attacked by latent diseases in some human bodies for a long time, and the macrophages in part of human bodies become mycobacterium tuberculosis survival places which can not be completely killed and generate drug resistance, so the macrophages become hot spots for researching tuberculosis by experts at home and abroad at present.
Zebrafish are 87% homologous to human at the gene level, and have an early development which is very similar to that of human, and become the best model organism for researching related disease genes. In recent decades, the zebrafish-mycobacterium marinum model has been widely used to study alternative animal models for tuberculosis infection. The similarity between human tuberculosis infection and mycobacterium marinum infection is striking, and the zebrafish-mycobacterium marinum model has many advantages over other mycobacterial infection models. (1) They have similarities to human anatomy, molecules and genetics (2) they are easy to reproduce and low cost to maintain. (3) Based on the optical clarity of embryos and larvae, large scale phenotype-based screening can be performed. (4) The model has the advantages of small harm to human bodies, relatively high growth speed and safe and convenient operation, and the zebra fish-mycobacterium marinum model becomes a novel model for researching the pathogenic mechanism of tuberculosis based on the advantages.
The screening of the medicine for treating tuberculosis by using the zebra fish model has important significance, and the inventor does not find the report of the application of salidroside combined with isoniazid in prolonging the survival time of the sea mycobacterium tuberculosis bacterial plaque horse fish model so far.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of salidroside in combination with isoniazid in prolonging the survival time of a sea branch bacterial plaque horse fish model.
Further, the salidroside is used for preparing a mixed solution with phenylthiourea to soak and culture fertilized eggs of the zebra fish, and the isoniazid is used for soaking and culturing the zebra fish injected with the mycobacterium marinum.
Further, the application comprises the following steps:
s1, providing AB wild type zebra fish, mating and fertilizing the zebra fish, placing fertilized eggs in methylene blue solution, incubating at 28-29 ℃, and after 7-10 hours, soaking the fertilized eggs in a mixed solution of salidroside and phenylthiourea;
s2, according to the fertilization time calculation, injecting mycobacterium marinum bacterial liquid into the zebra fish fertilized for 3 days to obtain a mycobacterium marinum bacterial plaque horse fish model;
s3, injecting for the next day, culturing the sea mycobacterium bacterial plaque horse fish model in isoniazid solution, and replacing the liquid medicine every 24 hours until the sea mycobacterium bacterial horse fish dies.
Further, in S1, the methylene blue solution is prepared by dissolving methylene blue in embryo culture water, and the concentration is 0.5 mg/L.
Further, the mixed solution is prepared by dissolving phenylthiourea and salidroside in embryo culture water.
Further, the final concentration of salidroside in the mixed solution was 1.5-1.8mM/ml, and the final concentration of phenylthiourea was 0.295. mu. mol/L.
Further, in S2, 10 μ l of mycobacterium marinum solution with OD of 1 was selected.
Furthermore, the mycobacterium marinum liquid is also added with 3 mul of phenol red.
Further, in S3, the concentration of the isoniazid solution is 0.12-0.13 mM/ml.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers for the first time that salidroside combined with isoniazid can prolong the survival time of infected zebra fish and provides a basis for further screening antituberculosis drugs. Through verification, salidroside also has the capability of enabling macrophages to migrate to wounds in an acute injury state, and is favorable for deep research on the technical fields of normal development, homeostasis, tissue repair and pathogen immunoreaction of the macrophages.
Drawings
FIG. 1 is a graph showing the comparison of the survival times of the zebra fish models of example 1 and comparative examples 1 to 5.
FIG. 2 is a comparison of fluorescence of Mycobacterium marinum in vivo for comparative example 2 and comparative example 3.
FIG. 3 is a comparison of macrophage numbers at the tail fin off-tips of salidroside treated and untreated zebra fish of example 2.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
Application of salidroside in combination with isoniazid in prolonging survival time of sea branch bacterial plaque horse fish model
The method comprises the following steps:
s1, preparing AB wild type zebra fish, fertilizing the zebra fish on the second day, pulling out a plate to collect sperms and eggs, putting the fertilized eggs into methylene blue solution with the concentration of 0.295 mu mol/L, incubating at 28.5 ℃, wherein the methylene blue solution is prepared by dissolving methylene blue in embryo culture water with the concentration of 0.5 mg/L; after 8 hours, transferring the fertilized eggs into a mixed solution of salidroside and phenylthiourea for soaking, wherein the mixed solution is prepared by dissolving the phenylthiourea and the salidroside in embryo culture water, and the final concentration of the salidroside in the mixed solution is 1.5-1.8mM/ml and the final concentration of the phenylthiourea is 0.295 mu mol/L;
s2, according to the fertilization time, injecting 10 mul of mycobacterium marinum bacterial liquid with OD being 1 into the zebra fish fertilized for 3 days, and adding 3 mul of phenol red into the bacterial liquid to obtain a sea branch bacterial plaque horse fish model;
s3, the next day of injection, culturing the zebra fish injected with the mycobacterium marinum in an isoniazid solution with the concentration of 0.125mM/ml, and replacing the liquid medicine every 24 hours until the death statistical growth curve of the zebra fish injected with the mycobacterium marinum.
Comparative example
Comparative examples 1-5 the parameter settings are shown in table 1, with the rest of the procedure being as in example 1.
TABLE 1 comparative examples 1-5 parameter settings
The results are shown in FIG. 1, where a corresponds to the statistical result of example 1, and b-f correspond to the statistical results of comparative examples 1 to 5 in order, and the results show that comparative example 1 has a difference in significance (P <0.0001) compared to comparative example 5, comparative example 1 has a difference in significance (P <0.0001) compared to comparative example 4, comparative example 1 has a difference in significance (P <0.0001) compared to comparative example 3, comparative example 1 has a difference in significance (P <0.0001) compared to example 1, comparative example 3 has a difference in significance (P <0.0001) compared to comparative example 4, example 1 has a difference in significance (P <0.0001) compared to comparative example 3, and example 1 has a difference in significance (P <0.0001) compared to comparative example 5.
And (3) culturing the zebra fish of the comparative example 3 in the isoniazid exposure solution for the same day, taking a picture under a high content microscope on the second day, detecting the fluorescence of mycobacterium marinum in the zebra fish body, continuously taking four days, and finally processing the fluorescence of the picture by Image J software. The zebra fish of comparative example 2 was photographed at the same time, and the results are shown in fig. 2.
The results show that compared with the comparative example 2, after continuously treating the mycobacterium marinum infected zebra fish for three days and four days, the bacterial fluorescence is significantly reduced by difference (P <0.05 and P <0.01) compared with the control group, which indicates that the 0.125mM/ml concentration has obvious bacteriostasis, but the zebra fish of the comparative example 2 has longer survival time than the zebra fish of the comparative example 3, and the salidroside has no bactericidal effect in vitro, but the salidroside (1.6mM/ml) knot and isoniazid (0.125mM/ml) are combined to prolong the survival time of the mycobacterium marinum infected zebra fish.
Example 2
Method of producing a composite material
1) And (3) respectively putting Tg (mfap4 EGFP) zebra fish (transgenic zebra fish with green fluorescent protein labeled macrophages) in a sexual maturity period into a mating tank with a baffle inserted in the middle for one male and one female, standing overnight, automatically controlling light and darkness for 14/10h, pulling out the baffle after the second day of light illumination, and immediately mating and spawning the zebra fish. Collecting roe with mesh screen, washing with pure water, adding roe into methylene blue solution (0.5 mg/L for inhibiting fungi), and replacing with phenylthiourea solution (0.295. mu. mol/L for inhibiting melanin of zebrafish) after 8 hr. And separately picking 80 eggs and placing the eggs into a mixed solution of a phenylthiourea solution and a salidroside solution (the final concentration of salidroside is 1.5-1.8mM/ml, and the final concentration of phenylthiourea is 0.295 mu mol/L), marking as a drug group when 3dfp is hatched, and marking as a control group when the salidroside solution is not mixed in the phenylthiourea solution.
2) 3dfp (day post fertilization) zebrafish were anesthetized with tricaine (0.612 nmol/L) and the tail fin was cut off under a microscope with a sterile syringe needle.
3) The phenylthiourea solution (concentration 0.295. mu. mol/L) was changed, and the two groups of zebrafish were placed in the petri dishes (d 10mm) of the drug group and the control group, respectively, and incubated for 2 hours at 28.5 ℃.
4) Two groups of zebrafish were removed from the incubator and each dish was fixed in 10ml of 4% by volume formaldehyde solution for 1 hour (the dish was placed in a 4 ℃ freezer and the dish was wrapped in aluminum foil paper to protect it from light. )
5) Zebrafish were placed into an EP tube containing 1ml of pbs-t (addition of tween20 at a volume fraction of 0.5%), and the zebrafish was washed 3 times for 5 minutes each, again on a shaker.
6) After washing, each fish was individually placed in a 48-well plate, and then macrophages expressing green fluorescence at the tail fin end (within 300 μm) of each zebra fish were counted under a high content fluorescence microscope green fluorescence channel (WL 470nm) and a 10-fold objective lens and photographed.
Experimental results and conclusions:
the results are shown in fig. 3, the absolute value of macrophages at the tail fin cut end of salidroside-treated mfap4 EGFP zebra fish is statistically different from that of the blank control group (P <0.0001), which indicates that salidroside has the ability of migrating macrophages to wounds in an acute injury state, and macrophages are used as main immune effector cells to prolong the survival time of zebra fish infected with mycobacterium marinum by enhancing the immune response capability of the body.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.
Claims (9)
1. Application of salidroside combined with isoniazid in prolonging survival time of Paris polyphylla bacterial plaque horse fish model is provided.
2. The use of salidroside in combination with isoniazid for prolonging the survival time of a mycobacterium marinum dental plaque horse fish model as claimed in claim 1, wherein the salidroside is used for preparing a mixed solution with phenylthiourea for soaking and culturing fertilized eggs of the zebra fish, and the isoniazid is used for soaking and culturing the mycobacterium marinum injected zebra fish.
3. The use of salidroside in combination with isoniazid for prolonging the survival time of a sea Branch rod plaque horse fish model as claimed in claim 1, comprising the steps of:
s1, providing AB wild type zebra fish, mating and fertilizing the zebra fish, placing fertilized eggs in methylene blue solution, incubating at 28-29 ℃, and after 7-10 hours, soaking the fertilized eggs in a mixed solution of rhodiola rosea solution and phenylthiourea;
s2, according to the fertilization time calculation, injecting mycobacterium marinum bacterial liquid into the zebra fish fertilized for 3 days to obtain a mycobacterium marinum bacterial plaque horse fish model;
s3, injecting for the next day, culturing the sea mycobacterium bacterial plaque horse fish model in isoniazid solution, and replacing the liquid medicine every 24 hours until the sea mycobacterium bacterial horse fish dies.
4. The use of salidroside in combination with isoniazid for prolonging the survival time of a sea Branch rod bacterial plaque horse fish model as claimed in claim 3, wherein in S1, the methylene blue solution is prepared by dissolving methylene blue in embryo culture water at a concentration of 0.5 mg/L.
5. The use of salidroside in combination with isoniazid for prolonging the survival time of a sea Branch rod bacterial plaque horse fish model as claimed in claim 3, wherein the mixed solution is prepared by dissolving phenylthiourea and salidroside in the embryo culture water.
6. The use of salidroside in combination with isoniazid for prolonging the survival time of a sea Branch rod bacterial plaque horse fish model according to claim 5, wherein the final concentration of salidroside in the mixed solution is 1.5-1.8mM/ml and the final concentration of phenylthiourea is 0.295. mu. mol/L.
7. The use according to claim 3, wherein in S2, 10. mu.l of M.marinum strain with OD-1 is selected.
8. The use of claim 7, wherein the Mycobacterium marinum solution further comprises 3 μ l phenol red.
9. The use according to claim 3, wherein the concentration of isoniazid solution in S3 is 0.12-0.13 mM/ml.
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