CN105943787A - Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method - Google Patents

Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method Download PDF

Info

Publication number
CN105943787A
CN105943787A CN201610420083.9A CN201610420083A CN105943787A CN 105943787 A CN105943787 A CN 105943787A CN 201610420083 A CN201610420083 A CN 201610420083A CN 105943787 A CN105943787 A CN 105943787A
Authority
CN
China
Prior art keywords
group
kidney
deficiency
asthma
rat
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.)
Pending
Application number
CN201610420083.9A
Other languages
Chinese (zh)
Inventor
郑小伟
孔丽娅
余王琴
刘晓谷
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.)
Zhejiang Chinese Medicine University ZCMU
Original Assignee
Zhejiang Chinese Medicine University ZCMU
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 Zhejiang Chinese Medicine University ZCMU filed Critical Zhejiang Chinese Medicine University ZCMU
Priority to CN201610420083.9A priority Critical patent/CN105943787A/en
Publication of CN105943787A publication Critical patent/CN105943787A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/894—Dioscoreaceae (Yam family)
    • A61K36/8945—Dioscorea, e.g. yam, Chinese yam or water yam
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/19—Acanthaceae (Acanthus family)
    • A61K36/195—Strobilanthes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/27—Asclepiadaceae (Milkweed family), e.g. hoya
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • A61K36/315—Isatis, e.g. Dyer's woad
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/40—Cornaceae (Dogwood family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/489—Sophora, e.g. necklacepod or mamani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/79—Schisandraceae (Schisandra family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/83—Thymelaeaceae (Mezereum family), e.g. leatherwood or false ohelo
    • A61K36/835—Aquilaria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/884—Alismataceae (Water-plantain family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/099—Bordetella
    • A—HUMAN NECESSITIES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Medical Informatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Environmental Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)

Abstract

The invention discloses a medicine for treating asthma and kidney qi deficiency, an animal model and an establishing and using method. The medicine for treating asthma and kidney qi deficiency is prepared from 12g of prepared rehmannia roots, 12g of pulp of dogwood fruit, 12g of rhizoma dioscoreae, 12g of rhizoma alismatis, 12g of radix sophorae flavescentis, 12g of Japanese metaplexis pericarp, 12g of tree peony bark, 12g of poria cocos, 5g of schisandra chinensis, 8g of agilawood pieces, 15g of inula flowers, 15g of indigo naturalis and clam powder and 15g of raw radix astragalis. In the early period, a kidney qi deficiency and asthma symptom combined rat model is established, and it is observed that kidney tonifying and asthma relieving soup and glucocorticoid can correct Th1/Th2 unbalance by adjusting expression of transcription factors. Specificity transcription factors, key cell factors and tiny RNA of a T cell subset serve as indexes, an internal mechanism of kidney qi deficiency asthma attacking and Treg/Th balance is proved, and a multi-target-point multi-layered network type regulatory mechanism possibly existing in traditional Chinese medicine for treating asthma is disclosed.

Description

For treating asthma syndrome of deficiency of kidney-QI medicine, animal model and building using method
Technical field
The invention belongs to medicine technology field, particularly relate to a kind of for treating asthma syndrome of deficiency of kidney-QI medicine, moving Object model and structure using method.
Background technology
Bronchial asthma (Bronchial asthma is called for short asthma) is one of global modal chronic disease, There are about the adult of up to 10% in western countries to suffer from this with the child of 30%.The whole world there are about 3 at present Hundred million asthmatic patients, prevalence with annual about 1% speed increase, have a strong impact on patient daily life and Work, brings heavy burden to family and society, therefore asthma is carried out further investigation and become the most global Important topic.
Bronchial asthma is as a kind of different substantiality disease, and pathogenesis complexity is various.Regulatory T is thin in recent years Born of the same parents (Treg) and the discovery of helper T lymphocyte 17 (Th17), have modified that traditional Thl/Th2 is unbalance to be drawn Playing the pathogenic theory of asthma immunne response, disclosing complex relationship between Treg and Th is to explore asthma machine One of key link of system and Therapeutic Method.In view of the effectiveness of glucocorticoid treatment asthma and safety etc. Problem, the method exploring Chinese and western medicine therapeutic alliance asthma is expected to obtain the treatment means of more potentiation low toxicity.
The t lymphocyte subset group of functional disorder in Bronchial Asthma
Bronchial asthma is as a kind of different substantiality disease, and pathogenesis complexity is various, it is now recognized that exempt from periphery Epidemic disease tolerance mechanism existing defects is relevant, and T lymphocyte plays central role in asthma immunne response.
Traditional theory thinks helper T cell subgroup (Th) functional disorder, i.e. Thl/Th2 is unbalance Th2 type advantage response, is initiating agent and the maintenance factor of the formation of bronchial asthma pathology process.In recent years Find that Th1/Th2 cell Mechanism of Imbalance can not illustrate the generation of asthma, regulatory T cells (Treg) completely Discovery with Th17 have modified above-mentioned idea, it is believed that Treg Yu Th17 may take part in the Development process of asthma. Th17 key excreted factor IL-17 all increases in asthmatic patient lung tissue, sputum, bronchoalveolar lavage fluid and serum Height, and be proportionate with airway hyperreactivity.Allergic asthma infant sputum Treg is less than normal children； CD4+CD25+Treg can suppress increasing of asthmatic model animal airway eosinophilic granulocyte, alleviates airway inflammation. Treg is the body a kind of negative-feedback regu-lation mechanism to T cell activation, and it can affect Thl cell, also can Act on Th2 cell, also there is the mutual relation of complexity with between Thl7 cell, disclose Treg and Th it Between complex relationship be one of the key link exploring Pathogenesy of Asthma, become the focus of research the most in recent years.
Asthma is recognized very early by the traditional Chinese medical science, though without bronchial wheezing in "Nei Jing", but having the record of " stridulating ", this with Asthma is similar.Chinese medicine is thought: " the lung being the dominator of QI, the kidney being the root of QI ", " at lung, its root is at kidney in cough ", Decline just because of insufficiency of lung-QI, weakened defensive QI, or self-resistance, cause asthma.Internal organs are deficient, essence QI-insufficiency, delay is involved in kidney.The most main gas of void then lung, failure of the kidney in receiving QI, breathe to take the photograph to receive and have no right and breathing heavily breath. So, asthma is the complicated disease of vital QI being weakened and pathogen being violent, simulataneous insufficiency and excessive.Yuan Dynasty's ZHU Dan-xi initiates " asthma " name of disease, carries Go out the Therapeutic Principle of famous " not sending out based on righting gas, both sent out to attack pathogen as urgency ", always by conduct later age The guilding principle for the treatment of asthma.According to clinical experience for many years on the basis of seven taste Duqi Wans, kidney tonifying is used to breathe heavily Peaceful soup (Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Radix Sophorae Flavescentis, Pericarpium Metaplexis, Cortex Moutan, Poria, Fructus Schisandrae Chinensis, Sliced, Flos Inulae, DAIHESAN, Radix Astragali), effect a permanent cure with tonifying the lung kidney, hold concurrently to reduce phlegm, circulation of qi promoting, invigorate blood circulation, Coordinate western medicine asthma, obtain good therapeutic effect, decrease patient's use to hormone etc., strengthen heavy breathing The curative effect breathed heavily, embodies the theory of the traditional Chinese medical science " identification of etiology according to differenciation of symptoms and signs, treatment must aim at the pathogenesis of disease ", is the basic institute effectively treating asthma ?.
Summary of the invention
It is an object of the invention to provide a kind of for treating asthma syndrome of deficiency of kidney-QI medicine, animal model and structure Using method, it is intended to solve to lack Chinese and western medicine therapeutic alliance asthmatic medicament at present, and verify that this medicine is in potentiation The method of the treatment means of low toxicity and the problem of using method.
The present invention is achieved in that a kind of medicine for treating asthma syndrome of deficiency of kidney-QI, described for treating heavy breathing Breathe heavily syndrome of deficiency of kidney-QI medicine Chinese medicinal components by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Radix Sophorae Flavescentis, Pericarpium Metaplexis, The each 12g of Cortex Moutan, Poria, Fructus Schisandrae Chinensis 5g, Sliced 8g, Flos Inulae, DAIHESAN, each 15g of Radix Astragali Composition；Western medicine uses becotide spray, often presses 50 μ g, and 200 press/bottle.
Another object of the present invention is to provide a kind of for verifying for the curative effect of medication treating asthma syndrome of deficiency of kidney-QI The construction method of asthma syndrome of deficiency of kidney-QI animal model, the construction method of this asthma syndrome of deficiency of kidney-QI animal model is: Cleaning grade male Wistar rat 540, body weight 200～250g.Animal is randomly divided into Normal group, roars Breathing heavily model control group, deficiency of kidney-QI asthmatic model group, Western medicine group, Chinese drug-treated group, Chinese medicine and western medicine combines group；
Asthma modeling: in addition to Normal group, each group rat uses ovalbumin sensitization method to carry out asthma modeling；
Deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, each group rat is combined syndrome of deficiency of kidney-QI Modeling.
Further, described asthma modeling method is: test the 1st day, with containing ovalbumin 1mg, hydrogen-oxygen Change alumina gel 10mg and inactivation bordetella pertussis vaccine 5 × 108Individual mixed liquor 1ml injects intraperitoneal, makes it Sensitization.Normal group is with normal saline intraperitoneal injection, standby after 2 weeks；15th day starts, by above-mentioned modeling Rat is placed in the bell glass of 65cm × 45cm × 45cm size, with the ovalbumin Ultrasonic atomising taring of 1% 30min, excites asthma, day 1 time, and continuous agitation is until putting to death；Normal group normal saline substitutes mist Change and suck.
Further, described deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, respectively organize rat Compound syndrome of deficiency of kidney-QI modeling, concrete grammar: 1. experiment starts on the 1st day, and morning every day puts in the same time Quiet room stimulates in terror, each 10min, i.e. broadcasts the tape of mew, kowtows with percussopunctator simultaneously Hitting 20 times/min of rat, simulation cat grabs and takes the sight attacking rat, vibrates mouse cage simultaneously；2. afternoon every day will Animal swimming with a load attached to the body: be wound around the electric fuse that weight is this rat body weight 10% in rat root of the tail portion, put into the depth of water 50cm, the tank went swimming of water temperature 20 DEG C, exhaust with power and do not have upstream face 10s for degree rat nose.Day 1 Secondary, totally 14 days, i.e. excite the previous day.
Another object of the present invention is to provide a kind of construction method utilizing asthma syndrome of deficiency of kidney-QI animal model to set up Animal model.
Another object of the present invention is to provide a kind of construction method utilizing asthma syndrome of deficiency of kidney-QI animal model to set up The using method of animal model, described using method includes:
It is administered and processes: Western medicine group, Chinese drug-treated group and Chinese medicine and western medicine are combined group rat and within the 15th day, started to be administered in experiment； Day dosage is: Western medicine group is to beclometasone 50 μ g/ only；Chinese drug-treated group is to Chinese medicine 2.0g/kg；Chinese medicine and western medicine group is given Corresponding Chinese medicine 2.0g/kg, beclometasone 50 μ g/；Normal group, asthmatic model group, asthmatic model group of suffering from a deficiency of the kidney To normal saline gavage；Each group rat intervenes process in 15 days respectively, weighs, and eyeball takes blood；Deficiency of kidney-QI is roared Breathe heavily model group to carry out four diagnostic methods quantifying index and carry out deficiency of vital energy degree judge；
Symptom, sign are observed: observe each group of rat appetite, fur, body weight, activity, outstanding tail resistance, Swimming time, double lung show；
Virus monitory: detection thyroxine (T4), hydrocortisone (Cor), testosterone (T) level.
Pathological observation: take lung tissue segment row hematoxylin--Yihong (HE) dyes observation.
Bronchoalveolar lavage fluid (BALF) detection leukocyte differential count and counting: and use EL ISA method Detection BALF centrifuged supernatant in cytokine IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, The content of IFN-γ, TGF-β etc..
Lung tissue detects: take out the 1/3 of lung tissue, uses reverse-transcription polymerase chain reaction (RT-PCR) Factor mRNA such as T-bet, GATA-3, ROR γ t, Foxp3, IL-6, TGF-β in algoscopy detection lung tissue Expression；With immunoblotting (Western blot) detection lung tissue T-bet, GATA-3, ROR γ t, Foxp3, IL-6, the expression of TGF-β albumen.Take out the 1/3 of other lung tissue, for miRNA phase Close detection；
Spleen tissue detection: mononuclearcell in separating spleen, fixing, contaminate FITC-CD4, then rupture of membranes, Add specific antibody IL-4 of Th1, Th2, Th17 and Treg cell, IFN-γ, IL-17A and Foxp3 respectively. Machine testing resuspended, upper after hatching.
MicroRNA microarrayed genes detects: rat is put to death, and lungs are promptly released and are placed in cryopreservation tube, directly Connect and put into freezing in liquid nitrogen, in the short time, complete whole operation.With MirVana microRNAisolation kit Collect total serum IgE to specifications.Take appropriate RNA solution, quantitative determine RNA through ultraviolet spectrophotometer Sample OD260 and OD280 value, calculate RNA concentration.1% sepharose electrophoresis observes total serum IgE, takes 2-5 μ g Filter with Microcon centrifugal filter micro-centrifugal filtration post, obtain fragment less than 300 nucleotide Tiny RNA.3 ' ends of tiny RNA plus poly (A) tail, are reconnected one by application Poly (A) polymerase Oligonucleotide labelling, for follow-up fluorescent labeling.Complete μ ParafloTM microRNA microarray base Because of express experiment and analyze: by micro circulation pump hybridization instrument on μ ParafloTM micro-fluid chip overnight, Using the SSPE buffer Han ammonium formate to hybridize, rinsing dries, and gathers hybridization image with laser scanner, Hybridization image is digitally converted by Array--Pro software, data process and analyzes.Calculate two groups to detect The ratio of signal and the p value of t inspection, be defined as signal there were significant differences property, analysis difference table with p < 0.01 The site reached.Rat micro-array chip includes all 454 microRNA reported at present, comprises data base In all of ripe microRNA and the complementary strand of Partial mature microRNA；Detection probe on chip is equal Carrying out 9 repetitions, experiment carries out 2 times.
Real-time fluorescence quantitative PCR detects: receive to specifications with MirVana microRNAisolation kit Collection total serum IgE, calculates RNA concentration.Special reverse transcriptase primer obtains from Taqman MicroRNAAssays, And be that cDNA carries out reality with Taqman MicroRNAReverseTranscription Kit by RNA reverse transcription Time fluorescence quantitative PCR detection, reaction terminate after by ABI Prism7900 software analysis result.PCR experiment Carry out 3 times, with comparing threshold cycle methods analyst data, obtain Ct value, needed for i.e. fluorescence reaches threshold value PCR cycle number, and analyze gene relative expression quantity.
Bioinformatics Prediction: application 3 bioinformatics target bases of miRanda, TargetScan and PicTar Because of forecasting software, the target gene of microRNA select in microRNA chip results is made prediction；
Statistical procedures: according to character, distribution and the design feature of data, applies SPSS13.0 statistical software Data are analyzed.
Further, the data analysing method of microRNA chip:
Use GenePix Pro 6.0 to read chip scanning image, and extract the signal value of probe；
Identical probe takes intermediate value and merges, and is retained in all samples all > whole chips are entered by=the probe of 30.0 Row Median Normal, screens differential expression probe；
Fold change and P-value is used to screen the differential expression miRNA of two groups of sample rooms；
Use the differential expression miRNAs of Fold change two sample rooms of screening；
Finally, differential expression miRNAs clustered and draw dendrogram.
Fold change and P-value is used to screen two groups of samples in the differential expression miRNA of two groups of sample rooms Between have repetition；Use two sample rooms in the differential expression miRNAs of Fold change two sample rooms of screening Do not repeat.
Another object of the present invention is to provide a kind of Th1, Th2, Th17, Treg assay method, described Th1, Th2, Th17, Treg assay method is:
Flow cytometer is used to measure the content of Th1, Th2, Th17, Treg in spleen tissue, to each medication Group Th1/Th2 and Th17/Treg average statistical analysis.
Early stage of the present invention is set up deficiency of kidney-QI asthma disease and is combined rat model, and observes that kidney tonifying breathes heavily the associating of peaceful soup It is unbalance that glucocorticoid can correct Th1/Th2 by the expression of regulation transcription factor, and the present invention is in early stage work On the basis of work, with idiosyncratic transcription factor, the key of T cell subgroup (Th1, Th2, Th17, Treg) Cytokine and Microrna are index, it was demonstrated that the inherent mechanism that deficiency of kidney-QI asthma balances with Treg/Th, Disclose the network type regulatory mechanism that compound of Chinese medicine asthma Mutiple Targets that may be present is multi-level.
The present invention is by screening, prediction deficiency of kidney-QI asthmatic model group and asthmatic model matched group pulmonary differentiation table Reach miRs and the target factor and mutual relation thereof, disclose in the part science that on molecular level, syndrome of deficiency of kidney-QI is essential Contain.Demonstrate the complex relationship between the macrophenotypic of syndrome and microbiological molecule.
Have not yet to see and in asthma analysis, Th1, Th2, Th17, Treg are carried out multi-angle and comprehensively examine Consider.Therefore the present invention from Treg/Th Analysis on Unbalance Pathogenesy of Asthma.
On model: successfully animal model in asthma to asthma because of and study of pathogenesis in have highly important Meaning.The deficiency of kidney-QI asthma disease combination model close to clinical disease is established under instruction of Chinese Medicine theory, than The most simple asthmatic model more meets theory of Chinese medical science, is also more conducive to exploration side's card phase on deficiency of kidney-QI asthmatic model To kidney tonifying breathe heavily peaceful soup treatment asthma mechanism；
On heuristic approach: the reticent regulation and control of microRNA wide participation said target mrna, and it is that Mutiple Targets is many Hierarchical network formula regulates and controls, and often presents space-time formula in growth promoter and lysis and expresses.It is suitable for Syndrome in TCM The research of type.The scientific meaning of syndrome of deficiency of kidney-QI essence is made some useful explorations by present invention application miR technology, Diagnosis and treatment for multiple disease provide new thinking.
Accompanying drawing explanation
Fig. 1 is the construction method flow chart of the asthma syndrome of deficiency of kidney-QI animal model that the embodiment of the present invention provides.
Fig. 2 is the lung tissue of rats HE coloration result normal group figure that the embodiment of the present invention provides.
Fig. 3 is the lung tissue of rats HE coloration result asthmatic model group figure that the embodiment of the present invention provides.
Fig. 4 is the lung tissue of rats HE coloration result deficiency of kidney-QI asthmatic model group figure that the embodiment of the present invention provides.
Fig. 5 is the lung tissue of rats HE coloration result Chinese medicine and western medicine group figure that the embodiment of the present invention provides.
Fig. 6 is the lung tissue of rats HE coloration result Western medicine group figure that the embodiment of the present invention provides.
Fig. 7 is the lung tissue of rats HE coloration result Chinese drug-treated group figure that the embodiment of the present invention provides.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, The present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to Explain the present invention, be not intended to limit the present invention.
Below in conjunction with the accompanying drawings and the application principle of the present invention is further described by specific embodiment.
A kind of medicine for treating asthma syndrome of deficiency of kidney-QI, the described medicine for treating asthma syndrome of deficiency of kidney-QI Chinese medicinal components by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Radix Sophorae Flavescentis, Pericarpium Metaplexis, Cortex Moutan, each 12g of Poria, Fructus Schisandrae Chinensis 5g, Sliced 8g, Flos Inulae, DAIHESAN, each 15g of Radix Astragali form；Western medicine uses beclometasone Aerosol, often presses 50 μ g, and 200 press/bottle.
Another object of the present invention is to provide a kind of for verifying for the curative effect of medication treating asthma syndrome of deficiency of kidney-QI The construction method of asthma syndrome of deficiency of kidney-QI animal model, as it is shown in figure 1, this asthma syndrome of deficiency of kidney-QI animal model Construction method be:
S101: cleaning grade male Wistar rat 540, body weight 200～250g, animal is just randomly divided into Often matched group, asthmatic model matched group, deficiency of kidney-QI asthmatic model group, Western medicine group, Chinese drug-treated group, Chinese medicine and western medicine joins Charge-coupled；
S102: asthma modeling: in addition to Normal group, each group rat uses ovalbumin sensitization method to roar Breathe heavily modeling；
S103: deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, each group rat is combined kidney Syndrome of deficiency of QI modeling.
Further, described asthma modeling method is: test the 1st day, with containing ovalbumin 1mg, hydrogen-oxygen Change alumina gel 10mg and inactivation bordetella pertussis vaccine 5 × 108Individual mixed liquor 1ml injects intraperitoneal, makes it Sensitization.Normal group is with normal saline intraperitoneal injection, standby after 2 weeks；15th day starts, by above-mentioned modeling Rat is placed in the bell glass of 65cm × 45cm × 45cm size, with the ovalbumin Ultrasonic atomising taring of 1% 30min, excites asthma, day 1 time, and continuous agitation is until putting to death；Normal group normal saline substitutes mist Change and suck.
Further, described deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, respectively organize rat Compound syndrome of deficiency of kidney-QI modeling, concrete grammar: 1. experiment starts on the 1st day, and morning every day puts in the same time Quiet room stimulates in terror, each 10min, i.e. broadcasts the tape of mew, kowtows with percussopunctator simultaneously Hitting 20 times/min of rat, simulation cat grabs and takes the sight attacking rat, vibrates mouse cage simultaneously；2. afternoon every day will Animal swimming with a load attached to the body: be wound around the electric fuse that weight is this rat body weight 10% in rat root of the tail portion, put into the depth of water 50cm, the tank went swimming of water temperature 20 DEG C, exhaust with power and do not have upstream face 10s for degree rat nose.Day 1 Secondary, totally 14 days, i.e. excite the previous day.
Another object of the present invention is to provide a kind of construction method utilizing asthma syndrome of deficiency of kidney-QI animal model to set up Animal model.
Another object of the present invention is to provide a kind of construction method utilizing asthma syndrome of deficiency of kidney-QI animal model to set up The using method of animal model, described using method includes:
It is administered and processes: Western medicine group, Chinese drug-treated group and Chinese medicine and western medicine are combined group rat and within the 15th day, started to be administered in experiment； Day dosage is: Western medicine group is to beclometasone 50 μ g/ only；Chinese drug-treated group is to Chinese medicine 2.0g/kg；Chinese medicine and western medicine group is given Corresponding Chinese medicine 2.0g/kg, beclometasone 50 μ g/；Normal group, asthmatic model group, asthmatic model group of suffering from a deficiency of the kidney To normal saline gavage；Each group rat intervenes process in 15 days respectively, weighs, and eyeball takes blood；Deficiency of kidney-QI is roared Breathe heavily model group to carry out four diagnostic methods quantifying index and carry out deficiency of vital energy degree judge；
Symptom, sign are observed: observe each group of rat appetite, fur, body weight, activity, outstanding tail resistance, Swimming time, double lung show；
Indexs measure: index and the deficiency of vital energy observed by four diagnostic methods quantifying judge: play the video recording of spacious field, note with computer Record each animal level across the liftoff number of times of standing of lattice number and forelimb.Measure by YLS-13A type large and small Mus grip Instrument detection grip.It is the most secondary that deficiency of vital energy degree=each animal moves horizontally measured value/normal group mean × each animal of 0.3+ Number/normal group mean × 0.2+ each animal grip measured value/normal group mean × 0.5.CM syndrome differentiation criterion: with normal group ratio Relatively, it is arrogant more than 1.25, is the deficiency of vital energy less than 0.75.
Virus monitory: detection thyroxine (T4), hydrocortisone (Cor), testosterone (T) level.
Pathological observation: take lung tissue segment row hematoxylin--Yihong (HE) dyes observation.
Bronchoalveolar lavage fluid (BALF) detection leukocyte differential count and counting: and use EL ISA method Detection BALF centrifuged supernatant in cytokine IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, The content of IFN-γ, TGF-β etc..
Lung tissue detects: take out the 1/3 of lung tissue, uses reverse-transcription polymerase chain reaction (RT-PCR) Factor mRNA such as T-bet, GATA-3, ROR γ t, Foxp3, IL-6, TGF-β in algoscopy detection lung tissue Expression；With immunoblotting (Western blot) detection lung tissue T-bet, GATA-3, ROR γ t, Foxp3, IL-6, the expression of TGF-β albumen.Take out the 1/3 of other lung tissue, for miRNA phase Close detection；
Spleen tissue detection: mononuclearcell in separating spleen, fixing, contaminate FITC-CD4, then rupture of membranes, point Do not add specific antibody IL-4 of Th1, Th2, Th17 and Treg cell, IFN-γ, IL-17A and Foxp3. Machine testing resuspended, upper after hatching.
MicroRNA microarrayed genes detects: rat is put to death, and lungs are promptly released and are placed in cryopreservation tube, directly Connect and put into freezing in liquid nitrogen, in the short time, complete whole operation.With MirVana microRNAisolation kit Collect total serum IgE to specifications.Take appropriate RNA solution, quantitative determine RNA through ultraviolet spectrophotometer Sample OD260 and OD280 value, calculate RNA concentration.1% sepharose electrophoresis observes total serum IgE, takes 2-5 μ g Filter with Microcon centrifugal filter micro-centrifugal filtration post, obtain fragment less than 300 nucleotide Tiny RNA.3 ' ends of tiny RNA plus poly (A) tail, are reconnected one by application Poly (A) polymerase Oligonucleotide labelling, for follow-up fluorescent labeling.Complete μ ParafloTM microRNA microarray base Because of express experiment and analyze: by micro circulation pump hybridization instrument on μ ParafloTM micro-fluid chip overnight, Using the SSPE buffer Han ammonium formate to hybridize, rinsing dries, and gathers hybridization image with laser scanner, Hybridization image is digitally converted by Array--Pro software, data process and analyzes.Calculate two groups to detect The ratio of signal and the p value of t inspection, be defined as signal there were significant differences property, analysis difference table with p < 0.01 The site reached.Rat micro-array chip includes all 454 microRNA reported at present, comprises data base In all of ripe microRNA and the complementary strand of Partial mature microRNA；Detection probe on chip is equal Carrying out 9 repetitions, experiment carries out 2 times.
Real-time fluorescence quantitative PCR detects: receive to specifications with MirVana microRNAisolation kit Collection total serum IgE, calculates RNA concentration.Special reverse transcriptase primer obtains from Taqman MicroRNAAssays, And be that cDNA carries out reality with Taqman MicroRNAReverseTranscription Kit by RNA reverse transcription Time fluorescence quantitative PCR detection, reaction terminate after by ABI Prism7900 software analysis result.PCR experiment Carry out 3 times, with comparing threshold cycle methods analyst data, obtain Ct value, needed for i.e. fluorescence reaches threshold value PCR cycle number, and analyze gene relative expression quantity.
Bioinformatics Prediction: application 3 bioinformatics target bases of miRanda, TargetScan and PicTar Because of forecasting software, the target gene of microRNA select in microRNA chip results is made prediction；
Statistical procedures: according to character, distribution and the design feature of data, applies SPSS13.0 statistical software Data are analyzed.
The data analysing method of microRNA chip:
Use GenePix Pro 6.0 to read chip scanning image, and extract the signal value of probe；
Identical probe takes intermediate value and merges, and is retained in all samples all > whole chips are entered by=the probe of 30.0 Row Median Normal, screens differential expression probe；
Fold change and P-value is used to screen the differential expression miRNA of two groups of sample rooms (having repetition)；
Fold change is used to screen the differential expression miRNAs of two sample rooms (not having to repeat)；
Finally, differential expression miRNAs clustered and draw dendrogram.
Early stage of the present invention is set up deficiency of kidney-QI asthma disease and is combined rat model, and observes that kidney tonifying breathes heavily the associating of peaceful soup It is unbalance that glucocorticoid can correct Th1/Th2 by the expression of regulation transcription factor, and the present invention is in early stage work On the basis of work, with idiosyncratic transcription factor, the key of T cell subgroup (Th1, Th2, Th17, Treg) Cytokine and Microrna are index, it was demonstrated that the inherent mechanism that deficiency of kidney-QI asthma balances with Treg/Th, Disclose the network type regulatory mechanism that compound of Chinese medicine asthma Mutiple Targets that may be present is multi-level.
The present invention is by screening, prediction deficiency of kidney-QI asthmatic model group and asthmatic model matched group pulmonary differentiation table Reach miRs and the target factor and mutual relation thereof, disclose in the part science that on molecular level, syndrome of deficiency of kidney-QI is essential Contain.Demonstrate the complex relationship between the macrophenotypic of syndrome and microbiological molecule.
The present invention is in theory: discovered in recent years Th1/Th2 cell Mechanism of Imbalance can not illustrate asthma completely Occurring, after finding Treg and Th17, prompting Treg/Th17 cell is unbalance may play weight wherein Want role, but have not yet to see, in asthma is studied, Th1, Th2, Th17, Treg are carried out multi-angle Consider every possible angle.Therefore the present invention has inquired into Pathogenesy of Asthma from Treg/Th is unbalance；
On model: successfully animal model in asthma to asthma because of and study of pathogenesis in have highly important Meaning.The deficiency of kidney-QI asthma disease combination model close to clinical disease is established under instruction of Chinese Medicine theory, than The most simple asthmatic model more meets theory of Chinese medical science, is also more conducive to exploration side's card phase on deficiency of kidney-QI asthmatic model To kidney tonifying breathe heavily peaceful soup treatment asthma mechanism；
On heuristic approach: the reticent regulation and control of microRNA wide participation said target mrna, and it is that Mutiple Targets is many Level
Network type regulates and controls, and often presents space-time formula in growth promoter and lysis and expresses.It is suitable for Syndrome in TCM The invention of type.The scientific meaning of syndrome of deficiency of kidney-QI essence is made some useful explorations by present invention application miR technology, Diagnosis and treatment for multiple disease provide new thinking.
The present invention provides a kind of Th1, Th2, Th17, Treg assay method, described Th1, Th2, Th17, Treg assay method is:
Flow cytometer is used to measure the content of Th1, Th2, Th17, Treg in spleen tissue, to each medication Group Th1/Th2 and Th17/Treg average statistical analysis.
Below in conjunction with concrete analysis, the present invention is further described.
One, the result of micRNA is screened
1. the miRNA screening results between asthmatic model group, asthma due to kidney-deficiency group, normal group
1.1 asthma group compare with normal group, and Fold chang is more than 3 times or less than 0.33 times, and P-value
MicRNA less than 0.05 has 13, see table 1,3 rises, 10 downwards, wherein rno-miR-370-3p Downward reaches more than 7 times.
Table 1 asthma group compares with normal group
Name Fold change P-value
rno-miR-450a-5p 3.672907474 0.030700678
rno-miR-181c-5p 3.180354232 0.008559343
rno-let-7a-1-3p/rno-let-7c-2-3p 3.263503027 0.018867658
rno-miR-291a-5p 0.253366323 0.001651507
rno-miR-652-5p 0.276631295 0.002304921
rno-miR-370-3p 0.129882271 0.029640336
rno-miR-292-5p 0.302037645 0.007247725
rno-miR-465-3p 0.278758669 0.008775467
rno-miR-667-3p 0.276021935 0.045812777
rno-miR-331-5p 0.276273909 0.006955694
rno-miR-487b-5p 0.319087445 0.039504933
rno-miR-2985 0.223001768 0.000262881
rno-miR-3573-3p 0.292860106 0.037137704
1.2 asthma due to kidney-deficiency groups compare with normal group, and Fold chang is more than 3 times or less than 0.33 times, and P-value is less than The micRNA of 0.05 has 2, see table 2,2 rises, and wherein rno-miR-450a-5p is similar to asthma group, All raise.
Table 2 asthma due to kidney-deficiency group compares with normal group
Name Fold change P-value
rno-miR-27b-3p 2.195809383 0.013101224
rno-miR-450a-5p 3.339141265 0.012433015
3. asthma due to kidney-deficiency group compares with asthma group, and the Fold chang micRNA more than 3 times or less than 0.33 times has 8, See table 3,6 rises, 2 downwards, wherein rno-miR-488-5p raises more than 13 times, rno-miR-1-3p Downward reaches 28 times.
Table 3 asthma due to kidney-deficiency group compares with asthma group
Name Fold change P-value
rno-miR-3596d 3.372381099 0.329809835
rno-miR-653-3p 6.099956147 0.343530206
rno-miR-370-3p 3.732235481 0.403736101
rno-miR-3557-5p 4.210096767 0.307129158
rno-miR-344a-5p 3.646240471 0.292299088
rno-miR-488-5p 13.2511768 0.298227266
rno-miR-133a-3p 0.35730468 0.346101851
rno-miR-1-3p 0.035765059 0.367075199
Owing to sample content is less, although asthma due to kidney-deficiency group and asthma group P value are all higher than 0.05, but biological differences is obvious, Can be further analyzed.
2. miRNA screening results between Western medicine group, Chinese drug-treated group, Chinese medicine and western medicine group:
2.1 Chinese drug-treated group compare with Western medicine group, and Fold chang is more than 3 times or less than 0.33 times, and P-value is less than Having 23 rises in the micRNA of 0.05,19 downwards (omiting), wherein rno-miR-487b-3p is up to 300 Many times, rno-miR-376b-3p is up to 60 times, and also 7 miRNA differential expressions are up to more than 10 times.
2.2 Chinese medicine and western medicine groups compare with Western medicine group, and Fold chang is more than 3 times or less than 0.3 times, and P-value is little MicRNA in 0.05 has 3, is shown in Table 4, and wherein rno-miR-487b-3p raises and reaches 42 times.
Table 4 Chinese medicine and western medicine group compares with Western medicine group
Name Fold change P-value
rno-miR-487b-3p 41.99498626 0.000343931
rno-miR-450a-5p 0.19895338 0.000388542
rno-miR-181c-5p 0.186421241 0.001652361
2.3 Chinese medicine and western medicine compare with Chinese medicine, and Fold chang is more than 3 times or less than 0.33 times, and P-value is less than 0.05 MicRNA have 20 (omiting), 6 rises, 12 downwards, wherein rno-miR-376b-3p lower reach 40 Times, rno-miR-138-2-3p lowers and reaches 10 times.
3. from the foregoing, it will be observed that on this basis, can be further:
3.1 enlarged sample content, the difference that screening asthma due to kidney-deficiency group is expressed with asthma group micRAN；
3.2 checkings: Chinese medicine and the Western medicine difference that micRNA expresses in effect with asthma and asthma due to kidney-deficiency model.
3.3 functions understanding the strong miRNA site of differential expression.
Two, Th1, Th2, Th17, Treg are measured
Flow cytometer is used to measure the content of Th1, Th2, Th17, Treg in spleen tissue, from six groups The column of Th1/Th2 and Th17/Treg average is found out, trend is ideal, but sample content is less, each medication Statistical significance is there is not yet between group.Next step can continue enlarged sample content, repeats above-mentioned experiment.Packet Th1/Th2 (mean ± standard deviation) Th17/Treg (mean ± standard deviation)
Three, the ELISA testing result of each correlation factor of bronchoalveolar lavage fluid in model:
Each group each index level of rat compares
Note: * compares P < 0.05, △ and compares P < 0.05 with deficiency of kidney-QI asthmatic model group with normal group.
Note: * compares P < 0.05, △ and compares P < 0.05 with deficiency of kidney-QI asthmatic model group with normal group.
Each group lung tissue of rats HE coloration result:
Fig. 2 is the lung tissue of rats HE coloration result normal group figure that the embodiment of the present invention provides；
Fig. 3 is the lung tissue of rats HE coloration result asthmatic model group figure that the embodiment of the present invention provides；
Fig. 4 is the lung tissue of rats HE coloration result deficiency of kidney-QI asthmatic model group figure that the embodiment of the present invention provides；
Fig. 5 is the lung tissue of rats HE coloration result Chinese medicine and western medicine group figure that the embodiment of the present invention provides；
Fig. 6 is the lung tissue of rats HE coloration result Western medicine group figure that the embodiment of the present invention provides；
Fig. 7 is the lung tissue of rats HE coloration result Chinese drug-treated group figure that the embodiment of the present invention provides.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this Any amendment, equivalent and the improvement etc. made within bright spirit and principle, should be included in the present invention Protection domain within.

Claims (8)

1. the medicine being used for treating asthma syndrome of deficiency of kidney-QI, it is characterised in that described for treating asthma kidney The Chinese medicinal components of the medicine of syndrome of deficiency of QI is by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Radix Sophorae Flavescentis, Pericarpium Metaplexis, male The each 12g of Cortex Moutan, Poria, Fructus Schisandrae Chinensis 5g, Sliced 8g, Flos Inulae, DAIHESAN, Radix Astragali each 15g group Become；Western medicine uses becotide spray, often presses 50 μ g, and 200 press/bottle.
2. the structure being used for verifying the asthma syndrome of deficiency of kidney-QI animal model of curative effect of medication described in claim 1 Method, it is characterised in that the construction method of this asthma syndrome of deficiency of kidney-QI animal model is:
Cleaning grade male Wistar rat 540, body weight 200～250g；Animal is randomly divided into Normal group, Asthmatic model matched group, deficiency of kidney-QI asthmatic model group, Western medicine group, Chinese drug-treated group, Chinese medicine and western medicine combines group；
Asthma modeling: in addition to Normal group, each group rat uses ovalbumin sensitization method to carry out asthma modeling；
Deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, each group rat is combined syndrome of deficiency of kidney-QI Modeling.
3. the construction method of asthma syndrome of deficiency of kidney-QI animal model as claimed in claim 2, it is characterised in that Described asthma modeling method is: test the 1st day, with containing ovalbumin 1mg, gel aluminum hydroxide 10mg And inactivation bordetella pertussis vaccine 5 × 108Individual mixed liquor 1ml injects intraperitoneal, makes its sensitization；
Normal group is with normal saline intraperitoneal injection, standby after 2 weeks；15th day starts, by big for above-mentioned modeling Mus is placed in the bell glass of 65cm × 45cm × 45cm size, with the ovalbumin Ultrasonic atomising taring of 1% 30min, excites asthma, day 1 time, and continuous agitation is until putting to death；
Normal group normal saline substitutes Neulized inhalation.
4. the construction method of asthma syndrome of deficiency of kidney-QI animal model as claimed in claim 2, it is characterised in that Described deficiency of kidney-QI modeling: in addition to Normal group, asthmatic model matched group, each group rat is combined syndrome of deficiency of kidney-QI Modeling, concrete grammar:
Testing the 1st day and start, morning every day puts a quiet room in the same time to stimulate, every time in terror 10min, i.e. broadcasts the tape of mew, and simultaneously with percussopunctator 20 times/min of knocking rat, simulation cat grabs and takes Attack the sight of rat, vibrate mouse cage simultaneously；
Afternoon every day is by animal swimming with a load attached to the body: be wound around the guarantor that weight is this rat body weight 10% in rat root of the tail portion Danger silk, puts into depth of water 50cm, the tank went swimming of water temperature 20 DEG C, exhausts with power and submerges for degree rat nose Water surface 10s, every day 1 time, totally 14 days.
5. the animal model that the construction method as described in claim 2～4 any one claim is set up.
6. the using method of an animal model as claimed in claim 5, it is characterised in that described use Method includes:
It is administered and processes: Western medicine group, Chinese drug-treated group and Chinese medicine and western medicine are combined group rat and within the 15th day, started to be administered in experiment； Day dosage is: Western medicine group is to beclometasone 50 μ g/ only；Chinese drug-treated group is to Chinese medicine 2.0g/kg；Chinese medicine and western medicine group is to phase Answer Chinese medicine 2.0g/kg, beclometasone 50 μ g/；
Normal group, asthmatic model group, asthmatic model group of suffering from a deficiency of the kidney are to normal saline gavage；Each group rat is divided Within 15 days, Gan Yu not process, weigh, eyeball takes blood；Deficiency of kidney-QI asthmatic model group carries out four diagnostic methods quantifying index Carry out deficiency of vital energy degree judge；
Symptom, sign are observed: observe each group of rat appetite, fur, body weight, activity, outstanding tail resistance, Swimming time, double lung show；
Virus monitory: detection thyroxine, hydrocortisone, testosterone levels；
Pathological observation: take lung tissue segment row hematoxylin--eosin stains is observed；
Bronchoalveolar lavage fluid detection leukocyte differential count and counting: and use ELISA method to detect BALF Cytokine IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TGF-β in centrifuged supernatant Content；
Lung tissue detects: take out the 1/3 of lung tissue, uses reverse-transcription polymerase chain reaction algoscopy detection lung T-bet, GATA-3, ROR γ t, Foxp3, IL-6, the expression of TGF-β factor mRNA in tissue； With immunoblotting detection lung tissue T-bet, GATA-3, ROR γ t, Foxp3, IL-6, TGF-β albumen Expression, takes out the 1/3 of other lung tissue, for miRNA coherent detection；
Spleen tissue detection: mononuclearcell in separating spleen, fixing, contaminate FITC-CD4, then rupture of membranes, point Do not add specific antibody IL-4 of Th1, Th2, Th17 and Treg cell, IFN-γ, IL-17A and Foxp3, Machine testing resuspended, upper after hatching；
MicroRNA microarrayed genes detects: rat is put to death, and lungs are promptly released and are placed in cryopreservation tube, directly Connect and put into freezing in liquid nitrogen, in the short time, complete whole operation；With MirVana microRNA isolation kit Collect total serum IgE to specifications；Take appropriate RNA solution, quantitative determine RNA through ultraviolet spectrophotometer Sample OD260 and OD280 value, calculate RNA concentration, and 1% sepharose electrophoresis is observed total serum IgE, taken 2-5 μ g Filter with Microcon centrifugal filter micro-centrifugal filtration post, obtain fragment less than 300 nucleotide Tiny RNA；3 ' ends of tiny RNA plus poly (A) tail, are reconnected an oligomerization by application Poly polymerase Nucleotide marker, for follow-up fluorescent labeling, completes μ ParafloTM microRNA microarrayed genes；
Express experiment and analyze: by micro circulation pump hybridization instrument on μ ParafloTM micro-fluid chip overnight, Using the SSPE buffer Han ammonium formate to hybridize, rinsing dries, and gathers hybridization image with laser scanner, Hybridization image is digitally converted by Array--Pro software, data process and analyzes, and calculates two groups and detects The ratio of signal and the p value of t inspection, be defined as signal there were significant differences property, analysis difference table with p < 0.01 The site reached, rat micro-array chip includes 454 microRNA, comprises maturation in data base MicroRNA and the complementary strand of Partial mature microRNA；Detection probe on chip all carries out 9 repetitions, Experiment carries out 2 times；
Real-time fluorescence quantitative PCR detects: receive to specifications with MirVana microRNA isolation kit Collection total serum IgE, calculates RNA concentration；Special reverse transcriptase primer obtains from Taqman MicroRNA Assays, And be that cDNA carries out reality with Taqman MicroRNA ReverseTranscription Kit by RNA reverse transcription Time fluorescence quantitative PCR detection, reaction terminate after by ABI Prism7900 software analysis result, PCR experiment Carry out 3 times, with comparing threshold cycle methods analyst data, obtain Ct value, needed for i.e. fluorescence reaches threshold value PCR cycle number, and analyze gene relative expression quantity；
Bioinformatics Prediction: application 3 bioinformatics target bases of miRanda, TargetScan and PicTar Because of forecasting software, the target gene of microRNA select in microRNA chip results is made prediction；
Statistical procedures: according to character, distribution and the design feature of data, applies SPSS13.0 statistical software Data are analyzed.
7. the using method of animal model as claimed in claim 6, it is characterised in that microRNA core The data analysing method of sheet:
Use GenePix Pro 6.0 to read chip scanning image, and extract the signal value of probe；
Identical probe takes intermediate value and merges, and is retained in all samples all > whole chips are entered by=the probe of 30.0 Row Median Normal, screens differential expression probe；
Fold change and P-value is used to screen the differential expression miRNA of two groups of sample rooms；
Use the differential expression miRNAs of Fold change two sample rooms of screening；
Finally, differential expression miRNAs clustered and draw dendrogram.
8. the using method of animal model as claimed in claim 7, it is characterised in that use Fold change Screen two groups of sample rooms in the differential expression miRNA of two groups of sample rooms with P-value and have repetition；Use Fold In the differential expression miRNAs of change two sample rooms of screening, two sample rooms do not repeat.
CN201610420083.9A 2016-06-15 2016-06-15 Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method Pending CN105943787A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610420083.9A CN105943787A (en) 2016-06-15 2016-06-15 Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610420083.9A CN105943787A (en) 2016-06-15 2016-06-15 Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method

Publications (1)

Publication Number Publication Date
CN105943787A true CN105943787A (en) 2016-09-21

Family

ID=56905551

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610420083.9A Pending CN105943787A (en) 2016-06-15 2016-06-15 Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method

Country Status (1)

Country Link
CN (1) CN105943787A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113491251A (en) * 2021-07-02 2021-10-12 山西中医药大学 Kidney-yang deficiency type Parkinson disease rat model and preparation method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101396486A (en) * 2007-09-26 2009-04-01 北京亚东生物制药有限公司 Traditional Chinese medicine composition for treating cough and asthma and preparation and quality control method thereof
CN103877383A (en) * 2014-03-26 2014-06-25 陈运平 Traditional Chinese medicine preparation for treating bronchial asthma with syndrome of intermin-gled phlegm and qi
CN104436172A (en) * 2014-09-30 2015-03-25 中国医学科学院医学实验动物研究所 Method for preparing novel animal model of asthma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101396486A (en) * 2007-09-26 2009-04-01 北京亚东生物制药有限公司 Traditional Chinese medicine composition for treating cough and asthma and preparation and quality control method thereof
CN103877383A (en) * 2014-03-26 2014-06-25 陈运平 Traditional Chinese medicine preparation for treating bronchial asthma with syndrome of intermin-gled phlegm and qi
CN104436172A (en) * 2014-09-30 2015-03-25 中国医学科学院医学实验动物研究所 Method for preparing novel animal model of asthma

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
卞涛等: "支气管哮喘大鼠转录因子T-bet/GATA-3失衡表达与气道炎症的关系", 《中华结核和呼吸杂志》 *
郑小伟等: "肾气虚哮喘模型及中西药联合干预的实验研究", 《浙江中医杂志》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113491251A (en) * 2021-07-02 2021-10-12 山西中医药大学 Kidney-yang deficiency type Parkinson disease rat model and preparation method thereof

Similar Documents

Publication Publication Date Title
Sadeghi Dousari et al. COVID-19 (Coronavirus disease 2019): a new coronavirus disease
Chen et al. Epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children
Choi et al. Genetics and epigenetics in allergic rhinitis
Walters et al. Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area
Piralla et al. Frequency of respiratory viruses among patients admitted to 26 intensive care units in seven consecutive winter-spring seasons (2009–2016) in Northern Italy
CN102732520B (en) The preparation method of the serum miRNAs that a kind of active tuberculosis is sick special
El Moussi et al. Virological surveillance of influenza viruses during the 2008–09, 2009–10 and 2010–11 seasons in Tunisia
Ali et al. Clinical features, diagnosis and treatment of COVID-19
CN101096704A (en) A set of fluorescent quantitative PCR primer and probe for detecting human boca virus
Liu et al. Comparison of clinical features and outcomes of medically attended COVID-19 and influenza patients in a defined population in the 2020 respiratory virus season
Olveira et al. Development and validation of a SYBR green real time PCR protocol for detection and quantification of nervous necrosis virus (NNV) using different standards
Qiao et al. Herbal small RNAs in patients with COVID-19 linked to reduced DEG expression
Soffritti et al. Modulation of micrornome by human cytomegalovirus and human herpesvirus 6 infection in human dermal fibroblasts: Possible significance in the induction of fibrosis in systemic sclerosis
CN102007208B (en) Human rhinovirus HRV-C and detection method thereof and test kit
Eyileten et al. Alterations in circulating MicroRNAs and the relation of MicroRNAs to maximal oxygen consumption and intima–media thickness in ultra-marathon runners
Assou et al. The transcriptome landscape of the in vitro human airway epithelium response to SARS-CoV-2
CN105943787A (en) Medicine for treating asthma and kidney qi deficiency, animal model and establishing and using method
CN104017806A (en) MicroRNA (Ribose Nucleic Acid) and application thereof in preparation of active tuberculosis detection reagent
Chen et al. Identification and expression profiling of circulating MicroRNAs in serum of Cysticercus pisiformis-infected rabbits
Young Sort by
Yang et al. MicroRNA expression profile in peripheral blood lymphocytes of sheep vaccinated with nigeria 75/1 peste des petits ruminants virus
CN107641652A (en) Reference gene and its screening technique applied to one group of real-time quantitative PCR relative quantification of hepatocellular carcinoma
CN101942524B (en) Combined nucleic acid real-time fluorescent detection method for influenza A H1N1 virus and influenza A virus and kit
Pereira et al. Functional capacity and respiratory muscle strenght of candidates to hepatic transplant
Li et al. Mpox virus Clade IIb infected Cynomolgus macaques via mimic natural infection routes closely resembled human mpox infection

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Zheng Xiaowei

Inventor after: Kong Liya

Inventor after: Yu Wangqin

Inventor after: Liu Xiaogu

Inventor after: Deng Jincha

Inventor before: Zheng Xiaowei

Inventor before: Kong Liya

Inventor before: Yu Wangqin

Inventor before: Liu Xiaogu

CB03 Change of inventor or designer information
RJ01 Rejection of invention patent application after publication

Application publication date: 20160921

RJ01 Rejection of invention patent application after publication