[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

CN114200120A - Drug screening kit for treating obesity and using method thereof - Google Patents

Drug screening kit for treating obesity and using method thereof Download PDF

Info

Publication number
CN114200120A
CN114200120A CN202111463254.3A CN202111463254A CN114200120A CN 114200120 A CN114200120 A CN 114200120A CN 202111463254 A CN202111463254 A CN 202111463254A CN 114200120 A CN114200120 A CN 114200120A
Authority
CN
China
Prior art keywords
drug
obesity
buffer
caenorhabditis elegans
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202111463254.3A
Other languages
Chinese (zh)
Inventor
李红玉
尤瑾
王宇
邹玥
李洋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lanzhou University
Original Assignee
Lanzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lanzhou University filed Critical Lanzhou University
Priority to CN202111463254.3A priority Critical patent/CN114200120A/en
Publication of CN114200120A publication Critical patent/CN114200120A/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明属于生物医药技术领域，具体涉及一种治疗肥胖症的药物筛选试剂盒及其使用方法。本发明提供的抗肥胖药物筛选试剂盒包括秀丽隐杆线虫、线虫培养基、大肠杆菌OP50、尼罗红染液，该试剂盒能够用于高通量筛选抗肥胖药物。本发明操作简便，检测快速，适合在筛选抗肥胖药物的领域大规模推广。The invention belongs to the technical field of biomedicine, and in particular relates to a drug screening kit for treating obesity and a use method thereof. The anti-obesity drug screening kit provided by the invention includes Caenorhabditis elegans, nematode culture medium, Escherichia coli OP50, and Nile red staining solution, and the kit can be used for high-throughput screening of anti-obesity drugs. The invention has simple operation and rapid detection, and is suitable for large-scale promotion in the field of screening anti-obesity drugs.

Description

Drug screening kit for treating obesity and using method thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a drug screening kit for treating obesity and a using method thereof.
Background
With the continuous improvement of living standard of people, obesity becomes an important factor seriously threatening public health in the 21 st century, and can cause various diseases, such as metabolic diseases, skin diseases and hypertension, increase the risk of cardiovascular and cerebrovascular diseases, easily cause psychological disorders and cause troubles to daily life of people. At present, the bariatric surgery is the most effective treatment method for obesity, but has many limitations including irreversibility of surgery, perioperative risks including bleeding, anastomotic leakage, infection, internal hernia, insufficient nutrition, postprandial hypoglycemia, death and high financial cost, so that a drug screening kit for treating obesity is urgently needed to screen more effective drugs for treating obesity.
The caenorhabditis elegans culture conditions are simple, and bacteria (E.coli) can be eaten on agar plates or liquid culture media; the generation cycle is short, and is usually about 3 days; short life, usually around 3 weeks; the caenorhabditis elegans experimental population is easy to amplify and can be synchronized, and the defects of high price and time consumption of rat and mouse models can be avoided, so the caenorhabditis elegans is an ideal model for drug high-flux screening due to the advantages of short life cycle and easy realization of high flux.
Therefore, the invention provides a drug screening kit for treating obesity, which can screen drugs for treating obesity at high flux.
Disclosure of Invention
The invention aims to provide a drug screening kit for treating obesity, and drugs for treating obesity can be screened by using the kit.
Another object of the present invention is to provide a method for using the above drug screening kit for treating obesity.
The invention provides a drug screening kit for treating obesity, which is characterized by comprising the following reagents:
(1) caenorhabditis elegans N2;
(2) NGM medium containing 10mg/mL cholesterol;
(3)5mg/ml of Escherichia coli OP50 bacterial liquid;
(4) nile red dye liquor.
The caenorhabditis elegans N2 is a synchronized nematode, and the nematode synchronizing step is as follows:
(1) flushing the caenorhabditis elegans on the flat plate by using M9 buffer solution, sucking the flushing solution into a centrifugal tube, centrifuging at 7000rpm of 5000-;
(2) adding lysis solution, and shaking on vortex instrument for 7min to break body of caenorhabditis elegans N2 to release egg;
(3) centrifuging at 7000rpm of 5000-:
(4) the buffer solution of M9 is washed twice, then 1ml of buffer solution of M9 is added, and the mixture is placed at the constant temperature of 20 ℃ for culturing for 48 hours, so as to obtain the synchronized caenorhabditis elegans N2.
The preparation method of the M9 buffer solution (taking the volume of 400mL as an example) comprises the following steps: weighing NaCl 1.99g and Na2HPO42.38g，KH2PO41.19g, anhydrous MgSO40.048 g. After being completely dissolved in 400mL of distilled water, the mixture was sterilized by moist heat (121 ℃ C., 20min) and stored at room temperature.
The preparation method of the lysis solution comprises the following steps: suck 320. mu.L of 10% NaClO3The solution was mixed with 4.68mL of M9 buffer, and then mixed with 1M NaOH solution in equal volume, ready for use.
The NGM culture medium is 25ml, the Escherichia coli OP50 bacterial liquid is 450 mul, and the Nile red staining solution is 1.8 mul.
The preparation method of the NGM culture medium (taking 500mL as an example) comprises the following steps: 1.52g NaCl, 1.36g peptone, 1.15gK2HPO4，8.52g KH2PO4After completely dissolving in 500mL of distilled water, 8.58g of agarose was added, shaken, and sterilized by moist heat (121 ℃ C., 20 min). After sterilization, when the temperature of the medium was reduced to 75 ℃ 500. mu.L of 1M MgSO was added4Solution of 500. mu.L CaCl at a concentration of 1M2The solution, 500. mu.L of 5mg/mL cholesterol solution, was shaken well and poured into a glass petri dish for coagulation.
The nile red dye solution is prepared from acetone, and the final concentration is 0.5 mg/mL.
Preferably, the rotation speed during centrifugation is 6000 rpm.
The invention also provides a use method of the drug screening kit for treating obesity, which comprises the following steps:
(1) screening the medicine for treating obesity by adopting a medicine screening kit for treating obesity, and setting a model control group and a medicine effect evaluation group;
(2) a pharmacodynamic evaluation group, adding 25ml of NGM culture medium containing the drug to be detected into a 90mm culture dish under aseptic condition, and waiting for solidification; uniformly coating an Escherichia coli OP50 bacterial liquid and a Nile red staining solution on an NGM culture medium, wherein the volume ratio of the Escherichia coli OP50 bacterial liquid to the Nile red staining solution is 250:1, then adding 400 pieces of synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture in a constant temperature culture at 20 ℃ for 48h, and setting three parallels;
(3) in the model control group, 25ml of NGM culture medium containing sterile water or solvent which is equal to the drug solution to be detected is added into a 90mm culture dish under the aseptic condition, and the NGM culture medium is solidified for later use; uniformly coating an Escherichia coli OP50 bacterial liquid and a Nile red staining solution on an NGM culture medium, wherein the volume ratio of the Escherichia coli OP50 bacterial liquid to the Nile red staining solution is 250:1, then adding 400 pieces of synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture in a constant temperature culture at 20 ℃ for 48 hours, establishing an obesity model as a model control group, and setting three parallels;
(4) post-treatment of caenorhabditis elegans N2: washing caenorhabditis elegans N2 in the model control group and the drug effect evaluation group respectively from a culture medium by using M9 buffer solution, collecting the caenorhabditis elegans N2 in a centrifuge tube, washing by using M9 buffer solution, removing supernatant, repeating for 3 times, adding 150 mu L M9 buffer solution again, decoloring for 2 hours at 20 ℃, sucking 150 mu LM9 buffer solution for washing after decoloring is completed, removing supernatant, repeating for 2 times, sucking 20 mu L of caenorhabditis elegans on a glass slide, sucking 20 mu L of 20mM NaN3Fixing;
(5) observing the dyeing result under a fluorescence microscope, taking a picture and recording, processing and analyzing experimental data by adopting statistical software, and if the fluorescence intensity of the efficacy evaluation group is lower than that of the model control group, proving that the drug to be detected has the activity of treating obesity; if the fluorescence intensity of the efficacy evaluation group is not significantly different from that of the model control group, the drug to be tested is proved to have no activity of treating obesity; thus, the activity of the test drug for treating obesity is evaluated.
Preferably, the Escherichia coli OP50 bacterial liquid and the Nile red staining liquid are respectively 450 mu L and 1.8 mu L.
Preferably, the number of caenorhabditis elegans N2 nematodes added to the NGM medium is 500.
The invention has the beneficial effects that: the caenorhabditis elegans model-based drug screening kit for treating obesity provided by the invention is simple in use method, high in accuracy and high in screening speed, and can be widely used for screening drugs for treating obesity.
Specific examples are provided below to realize the screening kit for obesity treating drugs according to the present invention, but are not limited to these examples.
Drawings
FIG. 1 evaluation of the efficacy of L-carnitine for the treatment of obesity Using a drug screening kit for the treatment of obesity
Detailed Description
Experimental biology:
caenorhabditis elegans N2, Caenorhabditis elegans N2 is a wild-type strain purchased from The Caenorhabditis Genetics Center (CGC).
Coli OP50, uracil auxotroph, as standard food for culture of c.
EXAMPLE 1 Assembly of the kit
A screening kit for drugs for treating obesity comprises the following materials:
(1) caenorhabditis elegans N2;
(2) NGM medium containing 10mg/mL cholesterol;
(3)5mg/ml of Escherichia coli OP50 bacterial liquid;
(4) nile red dye liquor.
The culture method of the Escherichia coli OP50 comprises the following steps: pouring the LB solid culture medium sterilized by high temperature and moist heat into a glass culture dish with the diameter of 90mm, cooling and solidifying the LB solid culture medium, dipping a small amount of bacterial liquid by using an inoculating ring, separating bacterial colonies by using a plate marking method, then putting the bacterial colonies into a constant temperature incubator at 37 ℃ for culturing overnight to obtain single bacterial colonies of Escherichia coli OP50, and storing the single bacterial colonies at 4 ℃ for later use.
The preparation method of the LB solid medium (taking 200mL as an example) comprises the following steps: analytically pure NaCl and peptone each 2.00g and yeast powder 1.00g are weighed respectively, and after being completely dissolved in 200mL of distilled water, agar 3.42g is added. Sterilizing with damp heat (121 deg.C, 20min) for use.
The nile red dye solution is prepared from acetone, and the final concentration is 0.5 mg/mL.
The caenorhabditis elegans N2 is a synchronized nematode, and the nematode synchronizing step is as follows:
(1) flushing down the caenorhabditis elegans on the plate by using M9 buffer solution, sucking the flushing solution into a centrifuge tube, centrifuging at 7000rpm of 5000-;
(2) adding lysis solution, and shaking on vortex instrument for 7min to break the body of caenorhabditis elegans and release ovum;
(3) centrifuging at 7000rpm of 5000-:
(4) the buffer solution of M9 was washed twice, 1ml of M9 buffer solution was added, and the mixture was incubated at 20 ℃ for 48 hours to obtain the synchronized C.elegans N2.
Preferably, the rotation speed during centrifugation is 6000 rpm.
Example 2 kit Effect verification
The L-carnitine is an amino acid analog for promoting fat to be converted into energy and is widely applied to the treatment of obesity, so that the effect of the kit is verified by using the L-carnitine as a medicine to be detected.
1 setting drug efficacy evaluation group
Under aseptic condition, adding 25ml of NGM culture medium containing 25 mul of L-carnitine liquid medicine into a 90mm culture dish, and waiting for solidification; mixing 450 mu l of escherichia coli OP50 bacterial liquid and 1.8 mu l of Nile red staining solution to ensure that the final concentration of Nile red is 0.002mg/ml, uniformly coating the mixed solution on an NGM culture medium, adding 500 synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture at the constant temperature of 20 ℃ for culturing for 48 hours, and setting three parallel strains;
2 setting model control group
Under aseptic condition, adding 25ml of NGM culture medium containing aseptic water or solvent with the same quantity of L-carnitine solution into a 90mm culture dish, and solidifying for later use; mixing 450 mu l of escherichia coli OP50 bacterial liquid and 1.8 mu l of Nile red staining solution to ensure that the final concentration of Nile red is 0.002mg/ml, uniformly coating the mixed solution on an NGM culture medium, adding 500 synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture at the constant temperature of 20 ℃ for culturing for 48 hours, and setting three parallel strains;
3 post-treatment of C.elegans N2
Separating with M9 buffer solutionWashing caenorhabditis elegans N2 in the model control group and the drug effect evaluation group from the culture medium, collecting the washed caenorhabditis elegans N2 in a centrifuge tube, washing the centrifuge tube with M9 buffer solution, removing the supernatant, repeating the washing for 3 times, adding 150 mu l M9 buffer solution again, decoloring the mixture at 20 ℃ for 2 hours, and removing the supernatant; additional 150 μ L M9 buffer wash was added, the supernatant removed and repeated 2 times, then 20 μ L of nematodes pipetted onto the slide and 20 μ L of 20mM NaN added3Fixing for observing dyeing results;
4 evaluation of Effect
The dyeing result is observed under a fluorescence microscope, the photographing record is carried out, statistical software is adopted to process and analyze experimental data, the result is shown in figure 1, compared with a model control group, the L-carnitine of the drug group to be detected obviously reduces the fat accumulation in the caenorhabditis elegans N2 strain, the L-carnitine is shown to have the effect of treating obesity, and the detection result of the kit is proved to be accurate and effective.
The preparation method of the L-carnitine liquid medicine comprises the following steps: weighing 16.12mg of L-carnitine, adding 1mL of distilled water, dissolving into 100mM of mother solution, keeping out of the sun, and storing at 2-8 ℃ for later use.
The preparation method of the drug-containing culture medium comprises the following steps: accurately sucking the solution of the drug to be tested, adding the solution into sterilized NGM (Next Generation broadcasting) which is cooled to about 75 ℃ according to a certain dilution ratio under the aseptic condition, fully shaking up, pouring the solution into a sterilized glass culture dish, uniformly coating 450 mu L of Escherichia coli OP50 bacterial solution after the culture medium is cooled and solidified, placing the solution at the constant temperature of 37 ℃ for culturing for 12 hours, and storing the solution at 15 ℃ for later use.
EXAMPLE 3 use of the kit
(1) Screening the medicine for treating obesity by adopting a medicine screening kit for treating obesity, and setting a model control group and a medicine effect evaluation group;
(2) setting a drug effect evaluation group: under the aseptic condition, 25ml of NGM culture medium of the liquid medicine to be detected is added into a 90mm culture dish, and the liquid medicine to be detected is solidified for later use; mixing 450 mu l of Escherichia coli OP50 bacterial liquid and 1.8 mu l of Nile red staining solution to ensure that the final concentration of Nile red is 0.002mg/ml, uniformly coating the mixed solution on an NGM culture medium, adding 400 and 600 synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture at the constant temperature of 20 ℃ for culturing for 48 hours, and setting three parallels;
(3) setting a model comparison group: under aseptic condition, adding 25ml of NGM culture medium containing sterile water or solvent with the same amount as the liquid medicine to be detected into a 90mm culture dish, and solidifying for later use; mixing 450 mu l of Escherichia coli OP50 bacterial liquid and 1.8 mu l of Nile red staining solution to ensure that the final concentration of Nile red is 0.002mg/ml, uniformly coating the mixed solution on an NGM culture medium, adding 400 and 600 synchronized caenorhabditis elegans N2 into the NGM culture medium, placing the mixture at the constant temperature of 20 ℃ for culturing for 48 hours, establishing an obesity model as a model control group, and setting three parallels;
(4) post-treatment of caenorhabditis elegans N2: washing caenorhabditis elegans N2 in the model control group and the drug effect evaluation group from the culture medium by using M9 buffer solution, collecting the washed caenorhabditis elegans N2 in a centrifuge tube, washing the washed caenorhabditis elegans N2 in M9 buffer solution, removing supernatant, repeating the washing for 3 times, adding 150 mu l M9 buffer solution again, decoloring the mixture at 20 ℃ for 2 hours, and removing the supernatant; additional 150 μ L M9 buffer wash was added, the supernatant removed and repeated 2 times, then 20 μ L of nematodes pipetted onto the slide and 20 μ L of 20mM NaN added3Fixing;
(5) observing the dyeing result under a fluorescence microscope, taking a picture and recording, processing and analyzing experimental data by adopting statistical software, and if the fluorescence intensity of the efficacy evaluation group is lower than that of the model control group, proving that the drug to be detected has the activity of treating obesity; if the active oxygen level of the efficacy evaluation group has no significant difference with the model control group, the drug to be tested is proved to have no activity of treating obesity; thus, the activity of the test drug for treating obesity is evaluated.
Preferably, the number of caenorhabditis elegans N2 nematodes added to the NGM medium is 500.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (6)

1.一种治疗肥胖症的药物筛选试剂盒，其特征在于，所述试剂盒包括如下材料：1. a drug screening test kit for the treatment of obesity, is characterized in that, described test kit comprises following material: (1)秀丽隐杆线虫N2；(1) Caenorhabditis elegans N2; (2)含有10mg/mL胆固醇的NGM培养基；(2) NGM medium containing 10 mg/mL cholesterol; (3)5mg/ml的大肠杆菌OP50菌液；(3) Escherichia coli OP50 bacterial liquid of 5mg/ml; (4)尼罗红染液。(4) Nile red staining solution. 2.根据权利要求1所述的治疗肥胖症的药物筛选试剂盒，其特征在于，所述的试剂盒所用的秀丽隐杆线虫N2为同步化后的线虫，线虫同步化的步骤为：2. the drug screening test kit for the treatment of obesity according to claim 1, is characterized in that, the used Caenorhabditis elegans N2 of described test kit is the nematode after synchronization, and the step of nematode synchronization is: (1)用M9缓冲液将平板上的秀丽隐杆线虫N2冲下来，将冲洗液吸入离心管，5000-7000rpm离心7分钟，去除上清；(1) Wash down the C. elegans N2 on the plate with M9 buffer, suck the washing liquid into a centrifuge tube, centrifuge at 5000-7000 rpm for 7 minutes, and remove the supernatant; (2)加入裂解液，在涡旋仪上震荡7min，使秀丽隐杆线虫N2的虫体破裂释放卵；(2) adding lysate and shaking on a vortexer for 7min to rupture the worm body of Caenorhabditis elegans N2 to release eggs; (3)5000-7000rpm离心7min，去除上清，收集沉淀获得大量的秀丽隐杆线虫卵：(3) Centrifuge at 5000-7000rpm for 7min, remove the supernatant, and collect the precipitate to obtain a large amount of C. elegans eggs: (4)M9缓冲液冲洗两次，再加入1ml M9缓冲液，置于20℃培养箱中孵化48小时，获得同步化后的秀丽隐杆线虫N2。(4) Rinse twice with M9 buffer, add 1 ml of M9 buffer, and incubate in a 20° C. incubator for 48 hours to obtain synchronized Caenorhabditis elegans N2. 3.根据权利要求1所述的一种治疗肥胖症的药物筛选试剂盒，其特征在于，所述的尼罗红染液由丙酮配制为0.5mg/mL。3 . The drug screening kit for treating obesity according to claim 1 , wherein the Nile Red staining solution is prepared to be 0.5 mg/mL by acetone. 4 . 4.一种如权利要求1任意一项所述的治疗肥胖症的药物筛选试剂盒的使用方法，其特征在于，所述使用方法如下：4. the use method of the drug screening kit for the treatment of obesity as described in any one of claim 1, is characterized in that, described use method is as follows: (1)采用治疗肥胖症的药物筛选试剂盒筛选治疗肥胖症的药物，设定模型对照组和药效评价组；(1) Use a drug screening kit for the treatment of obesity to screen drugs for the treatment of obesity, and set up a model control group and a drug efficacy evaluation group; (2)药效评价组，将大肠杆菌OP50菌液和尼罗红染液均匀涂布在含有待测药物的NGM培养基上，所述大肠杆菌OP50菌液和尼罗红染液的体积比为250:1，然后将400—600条秀丽隐杆线虫N2加入NGM培养基并置于20℃恒温培养48h；(2) drug efficacy evaluation group, the Escherichia coli OP50 bacterial solution and Nile red staining solution were evenly coated on the NGM medium containing the drug to be tested, and the volume ratio of the Escherichia coli OP50 bacterial solution and the Nile red staining solution was 250:1, then 400-600 Caenorhabditis elegans N2 were added to NGM medium and incubated at 20°C for 48h; (3)模型对照组，将大肠杆菌OP50菌液和尼罗红染液均匀涂布在含有与待测药物溶液等量的无菌水或溶媒的NGM培养基上，所述大肠杆菌OP50菌液和尼罗红染液的体积比为250:1，然后将400—600条秀丽隐杆线虫N2加入NGM培养基并置于20℃恒温培养48h，建立成为肥胖模型作为模型对照组；(3) model control group, the Escherichia coli OP50 bacterial solution and Nile red staining solution were evenly coated on the NGM medium containing sterile water or solvent equal to the drug solution to be tested, the Escherichia coli OP50 bacterial solution The volume ratio of Nile red staining solution was 250:1, then 400-600 C. elegans N2 were added to NGM medium and placed at 20°C for constant temperature cultivation for 48h to establish an obesity model as a model control group; (4)秀丽隐杆线虫N2的后处理：用M9缓冲液分别将模型对照组和药效评价组中的秀丽隐杆线虫N2从培养基上洗下,收集到离心管中，用M9缓冲液清洗，去除上清，重复3次，再次加入150μlM9缓冲液,20℃脱色2h，去除上清；再加入150μL M9缓冲液清洗，去除上清，重复2次，然后吸取20μL线虫于载玻片上，并加入20μL 20mM NaN3固定；(4) Post-treatment of Caenorhabditis elegans N2: The Caenorhabditis elegans N2 in the model control group and the drug efficacy evaluation group were washed from the culture medium with M9 buffer, collected in a centrifuge tube, and washed with M9 buffer. Wash, remove the supernatant, repeat 3 times, add 150 μl M9 buffer again, decolorize at 20°C for 2 h, remove the supernatant; then add 150 μL M9 buffer to wash, remove the supernatant, repeat twice, and then pipette 20 μL of nematodes onto the glass slide, And add 20μL 20mM NaN 3 to fix; (5)在荧光显微镜下观察染色结果并拍照记录，采用统计软件对实验数据进行处理、分析，如果药效评价组的荧光强度低于模型对照组，则证明待测药物具有治疗肥胖的活性；如果药效评价组的荧光强度与模型对照组无显著性差异，则证明待测药物不具有治疗肥胖的活性；以此评价待测药物的治疗肥胖的活性。(5) Observing the staining results under a fluorescence microscope and taking pictures and recording, using statistical software to process and analyze the experimental data, if the fluorescence intensity of the efficacy evaluation group is lower than that of the model control group, it proves that the drug to be tested has the activity of treating obesity; If there is no significant difference between the fluorescence intensity of the drug efficacy evaluation group and the model control group, it proves that the drug to be tested does not have the activity of treating obesity; thereby evaluating the activity of the drug to be tested for treating obesity. 5.根据权利要求4所述的一种治疗肥胖症的药物筛选试剂盒的使用方法，其特征在于，所述的秀丽隐杆线虫N2为同步化后的线虫，线虫同步化的步骤为：5. the using method of a kind of drug screening test kit for the treatment of obesity according to claim 4, is characterized in that, described Caenorhabditis elegans N2 is the nematode after synchronization, and the step of nematode synchronization is: (1)用M9缓冲液将平板上的秀丽隐杆线虫冲下来，将冲洗液吸入离心管，5000-7000rpm离心7分钟，去除上清；(1) Wash the C. elegans on the plate with M9 buffer, suck the washing liquid into a centrifuge tube, centrifuge at 5000-7000 rpm for 7 minutes, and remove the supernatant; (2)加入裂解液，在涡旋仪上震荡7min，使秀丽隐杆线虫的虫体破裂释放卵；(2) adding lysate and shaking on a vortexer for 7min to rupture the worm body of C. elegans to release eggs; (3)5000-7000rpm离心7min，去除上清，收集沉淀获得大量的秀丽隐杆线虫卵：(3) Centrifuge at 5000-7000rpm for 7min, remove the supernatant, and collect the precipitate to obtain a large amount of C. elegans eggs: (4)M9缓冲液冲洗两次，再加入1ml M9缓冲液，置于20℃培养箱中孵化48小时，获得同步化后的秀丽隐杆线虫。(4) Rinse twice with M9 buffer, add 1 ml of M9 buffer, and incubate in a 20° C. incubator for 48 hours to obtain synchronized Caenorhabditis elegans. 6.根据权利要求4所述的一种治疗肥胖症的药物筛选试剂盒的使用方法，其特征在于，所述的尼罗红染液在涂布前加入大肠杆菌OP50菌液，使尼罗红染液终浓度为0.002mg/mL。6. the using method of a kind of drug screening test kit for the treatment of obesity according to claim 4, is characterized in that, described Nile red staining solution adds Escherichia coli OP50 bacterial liquid before coating, makes Nile red The final concentration of the dye solution was 0.002 mg/mL.
CN202111463254.3A 2021-12-02 2021-12-02 Drug screening kit for treating obesity and using method thereof Withdrawn CN114200120A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111463254.3A CN114200120A (en) 2021-12-02 2021-12-02 Drug screening kit for treating obesity and using method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111463254.3A CN114200120A (en) 2021-12-02 2021-12-02 Drug screening kit for treating obesity and using method thereof

Publications (1)

Publication Number Publication Date
CN114200120A true CN114200120A (en) 2022-03-18

Family

ID=80650259

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111463254.3A Withdrawn CN114200120A (en) 2021-12-02 2021-12-02 Drug screening kit for treating obesity and using method thereof

Country Status (1)

Country Link
CN (1) CN114200120A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115067283A (en) * 2022-07-06 2022-09-20 完美（广东）日用品有限公司 Construction method and application of a Caenorhabditis elegans obesity model

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008140178A1 (en) * 2007-05-09 2008-11-20 Ginseng Science Inc. Composition comprising an extract of processed ginseng for preventing and treating obesity and the use thereof
CN104928208A (en) * 2015-04-30 2015-09-23 江苏紫石微康生物科技有限公司 Lactobacillus plantarum Lp90, and screening method and application thereof
CN105560269A (en) * 2016-03-07 2016-05-11 甘肃东方天润玫瑰科技发展有限公司 Application of rose polysaccharide in preparing medicine for treating obesity
US20160143963A1 (en) * 2013-07-18 2016-05-26 Biopolis, S.L. Novel strain of bifidobacterium animalis subsp. lactis cect 8145 and use thereof for the treatment and/or prevention of excess weight and obesity and associated diseases
CN112229803A (en) * 2020-09-11 2021-01-15 江苏大学 Application of carbendazim in fat accumulation and construction of biological model thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008140178A1 (en) * 2007-05-09 2008-11-20 Ginseng Science Inc. Composition comprising an extract of processed ginseng for preventing and treating obesity and the use thereof
US20160143963A1 (en) * 2013-07-18 2016-05-26 Biopolis, S.L. Novel strain of bifidobacterium animalis subsp. lactis cect 8145 and use thereof for the treatment and/or prevention of excess weight and obesity and associated diseases
CN104928208A (en) * 2015-04-30 2015-09-23 江苏紫石微康生物科技有限公司 Lactobacillus plantarum Lp90, and screening method and application thereof
CN105560269A (en) * 2016-03-07 2016-05-11 甘肃东方天润玫瑰科技发展有限公司 Application of rose polysaccharide in preparing medicine for treating obesity
CN112229803A (en) * 2020-09-11 2021-01-15 江苏大学 Application of carbendazim in fat accumulation and construction of biological model thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115067283A (en) * 2022-07-06 2022-09-20 完美（广东）日用品有限公司 Construction method and application of a Caenorhabditis elegans obesity model

Similar Documents

Publication Publication Date Title
Sacchetto et al. Modeling cardiovascular diseases with hiPSC-derived cardiomyocytes in 2D and 3D cultures
CN109633140B (en) Method for evaluating neurodevelopment toxicity of perfluorinated compounds by using zebra fish
Esposito et al. Enterovirus D68 infection
Ramírez et al. Mosquito excreta: A sample type with many potential applications for the investigation of Ross River virus and West Nile virus ecology
Božanić Urbančič et al. The importance of early genetic diagnostics of hearing loss in children
JP2016523550A (en) Methods for predicting responses to chemical or biological substances
Aguilar-Díaz et al. In vitro induction of Entamoeba histolytica cyst-like structures from trophozoites
Badenhorst et al. No evidence of mosquito involvement in the transmission of equine hepacivirus (flaviviridae) in an epidemiological survey of Austrian horses
CN113583973B (en) High-lytic Klebsiella pneumoniae RDP-KP-20007 and application thereof
CN114200120A (en) Drug screening kit for treating obesity and using method thereof
Schuh et al. Natural reservoir Rousettus aegyptiacus bat host model of orthonairovirus infection identifies potential zoonotic spillover mechanisms
Becatti et al. Blood leukocyte ROS production reflects seminal fluid oxidative stress and spermatozoa dysfunction in idiopathic infertile men
Hu et al. Rapid nucleic acid extraction for aquatic animal DNA virus determination using Chelex 100 resin via conventional PCR and digital droplet PCR detection
Golender et al. Identification and genetic characterization of viral pathogens in ruminant gestation abnormalities, Israel, 2015–2019
Rogers et al. Impaired geotaxis as a novel phenotype of Nora virus infection of Drosophila melanogaster
De Jong et al. Ivacaftor or lumacaftor/ivacaftor treatment does not alter the core CF airway epithelial gene response to rhinovirus
Guo et al. Survival and replication of dengue-2 virus in diapausing eggs of Aedes albopictus (Diptera: Culicidae)
Haley Amplification techniques for the detection of misfolded prion proteins in experimental and clinical samples
Stancu et al. Unusual Canine Distemper Virus Infection in Captive Raccoons (Procyon lotor)
CN114200119A (en) A kind of drug screening kit for treating type 2 diabetes and using method thereof
CN117757725A (en) Construction method and application of type 2diabetes zebra fish embryo model
Takemura et al. SARS-CoV-2 infection in beaver farm, Mongolia, 2021
Chen et al. Epidemiological and genetic characteristics of rabies virus transmitted through organ transplantation
Grassi et al. Recreating chronic respiratory infections in vitro using physiologically relevant models
Kang et al. Accurate diagnosis of COVID-19 from self-collectable biospecimens using synthetic apolipoprotein H peptide-coated nanoparticle assay

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20220318

WW01 Invention patent application withdrawn after publication