CN103233059A - A method for preventing and eliminating Candida albicans pollution in vitro cell culture of elite ice and snow athletes - Google Patents
A method for preventing and eliminating Candida albicans pollution in vitro cell culture of elite ice and snow athletes Download PDFInfo
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- CN103233059A CN103233059A CN2013101231102A CN201310123110A CN103233059A CN 103233059 A CN103233059 A CN 103233059A CN 2013101231102 A CN2013101231102 A CN 2013101231102A CN 201310123110 A CN201310123110 A CN 201310123110A CN 103233059 A CN103233059 A CN 103233059A
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Abstract
The present invention discloses a method for efficiently preventing and eliminating Candida albicans pollution in vitro cell culture of elite ice and snow athletes. During the process of in vitro cell culture, microorganisms such as bacteria, fungi and viruses, and some protozoa may result in failure of the cell culture. The Candida albicans is one of the most common fungi; but extensive and long-term use of antifungal drugs causes the emergence of resistant strains easily, which brings great difficulties in eliminating Candida albicans pollution. According to the method of the present invention, chlorhexidine-containing DMEM at a ratio of chlorhexidine: DMEM being 1:100,000 is adopts to prevent and eliminate Candida albicans. The method is efficient, simple and universally applicable.
Description
Technical field
The present invention relates to a kind of outstanding winter sports person's cell in vitro cultivation candidiasis pollution method that prevents and eliminate, namely prevent and eliminate the method for outstanding winter sports person's cell in vitro cultivation candidiasis pollution by chlorhexidine (Chlorhexidine), belong to sports science, microbiology and human cell's biology cross discipline and field.
Background technology
Candida albicans (candida Albicans) claims again extensively to be present in nature by the Candida albicans bacterium, also is present in the normal people oral cavity, the upper respiratory tract, and enteron aisle and vagina, generally quantity is few in normal body, does not cause disease.Be the condition pathogenic fungus.Moniliosis is fungi---the disease of cari oris mucosa that candida infection causes.The main pathogenic fungi is Candida albicans.Have this bacterium in 25%～50% the normal people oral cavity, exist with the blastospore type, and not pathogenic, but under the effect of some factor, spore grows up to behind the mycelia and causes a disease.Because microbiotic and immunosuppressor widespread use clinically in recent years, the probability that the reduction of flora imbalance or immunizing power takes place increases, and makes fungi infestation person increasing, and sickness rate also increases corresponding.
Monilial stomatitis is divided into four types again.1. acute pseudomembrane type is called moniliasis white mouth or white mouth again.Can betide the people at any age, but see at most with the newborn infant and to be mainly in the birth back 2～8 days.Good sending out in cheek, tongue, soft palate and lip, on mucous hyperemia, the soft speckle of white as avenging that is dispersed in arranged, after progressively be fused into the velvet shape patch of well-defined white or pearl opal, and can continue to spread expansion, severe patient can involve tonsilla, pharyngeal, gum etc. to be located, and is profusely flakes shape.Patch adheres to very not tight, slightly firmly can wipe, but expose red mucosal erosion face hyporrhea.Infant is had the fidgets, is cryed, dysilthia, and slight heating is arranged sometimes, and systemic reaction is generally lighter.2. the acute atrophy type is more common in the grownup, often causes owing to Broad spectrum antibiotics and corticosteroid hormone prolonged application.Most patients namely has wasting diseases before with antibiotic etc, as behind leukemia, malnutrition, endocrine regulation, the chemotherapy of tumors etc., or suffers from some tetter such as systemic lupus erythematous, psoriatic, pemphigus etc., so be called the microbiotic stomatitis again.The mucous hyperemia erosion, the back nipple is the agglomerate atrophy, and tongue fur thickens on every side, and parageusia or forfeiture are arranged, xerostomia, mucous membrane cusalgia.3. chronic hypertrophic claims the proliferous type monilial stomatitis again.Be found in buccal mucosa, back and palatine.The sick damage in cheek is positioned at the inboard triangular space of bicker, is nodositas or particulate state hyperplasia, or is set white cutin patch closely, similar general leukoplakia; The sick damage of palatine can be by the denture stomatitis development, and mucous membrane is papillary hyperplasia; The sick damage of back is filiform papillae propagation, and look greyish black, is called hairy tongue.Pseudomembrane and the epithelial lining on this type mucous membrane top layer adhere to closely, are difficult for stripping off.Elderly patient is biopsy early, clarifies a diagnosis.4. chronic atrophic type claims denture stomatitis again.Often betide palate, gum mucous membrane that the palate side of maxillary denture contacts, the women is more than the male sex.Reddening and swelling of mucosa is shinny, or is the streak or mottled pseudomembrane of yellow-white.Can concurrent lip and the infringement of bicker.
Chlorhexidine (chloehexidine, former name: Tubulicid, chlorhexidine) is that the fifties in last century is by the antiseptic-germicide of Britain Imperial Chemical Industries exploitation.Widespread use in medicine, medical treatment product and personal hygiene article (as collutory, toothpaste etc.).Chlorhexidine is strong basicity, and physiological pH is the dication state.It is water insoluble, but its pair gluconate and diacetate water soluble.It is extremely bitter that the aqueous solution is colourless, nothing is smelt, distinguished the flavor of.During topical application, the protein of its chloro-phenyl-and skin mucosa produces lasting anti-microbial effect with the phospholipid moiety that is covalently bonded in the bacteria cell wall protein component.High density is germicidal action.Enter endochylema and damage its endochylema film, form irreversible throw out with ATP in the cell and nucleic acid.The mycocidal effect of pressing down is arranged, part virus is also had killing action.MIC comparison G-to G+ is lower, and is bigger with the cell walls avidity of G+.Prolonging exposure duration will increase killing action to most of bacteriums.
The cell in peripheral blood source only could be grown at solid or semisolid surface, and poor growth, and incubation time is long.Compare with microorganism culturing, harsher for the requirement of nutrition, except amino acid, VITAMIN, salt, glucose or semi-lactosi, also need serum.Adaptability for environment is poorer, and is extremely responsive to environment, comprises that pH, dissolved oxygen, temperature, shearing force etc. have very high requirement, generally needs strict monitoring.Cell also needs to prevent pollution problem in culturing process, microorganism such as bacterium, fungi, virus and some protozoons all can cause the pollution of animal cell culture.Candida albicans is modal a kind of fungi, mainly invades in skin, mucous membrane, also can cause internal organ or systemic infection.Because the carrying out of the chemicotherapy of the application of Broad spectrum antibiotics, tumour, invasive treatment, organ transplantation, the extensive application of immunosuppressor causes normal flora imbalance in recent years, and the infection of Candida albicans had tangible increase more in the past.At present, because the extensive and life-time service of a large amount of antifungal drugs causes the appearance of Resistant strain easily, bring very big difficulty for the treatment of monilial infection.Since Tubulicid generally use and to have extremely strong broad-spectrum antibacterial, germicidal action and toxicity low, to the anti-microbial effect of Gram-positive, negative bacterium, bacterial spore, fungi and virus etc., even still effective when having serum, blood etc. to exist.
Summary of the invention
The purpose of this invention is to provide a kind of method that suppresses and eliminate the pollution of elite's cell in vitro cultivation Candida albicans.
For achieving the above object, the present invention takes following technical scheme:
(1) cultivation of bacterial strain: on husky Bao Shi substratum, after waiting to grow bacterium colony, picking list colony inoculation forms chlamydospore on the corn nutrient agar, and then is identified in conjunction with sugar-fermenting or other biochemical test with bacterial classification inoculation in elder generation.
(2) inoculation bacterium liquid is in cell: with 2 * 10
7The inoculation of/ml Candida albicans bacterium liquid is good with 30 bottles of upgrowth situations, the cell 0.1ml/ bottle of density about 60%.
(3) grouping experiment: 30 bottles of cells are divided be divided into three groups at random, 10 bottles every group be respectively control group, contain 3% sodium bicarbonate DMEM, (the two chlorobenzene biguanides: DMEM) two chlorobenzene biguanides DMEM that contain 1: 100000
Advantage of the present invention and benefit: Candida albicans is the normal microflora that resides on normal human's digestive tube, respiratory tract and the female reproduction mucous membrane.Generally not pathogenic, only when long-term granulocytopenia, blood system malignant tumour, prolonged application glucocorticosteroid, bone marrow transplantation or solid organ transplantation and acquired immune deficiency syndrome (AIDS) (AIDS) etc. caused immunity of organisms and weaken, the invasion of a large amount of Candida albicanss just may be fallen ill.There is data to show that (biological re-sponse modifer, BRM) the control deep fungal infection is feasible method to the applying biological reaction control agent.Traditional B RM such as bacille Calmette-Guerin vaccine (BCG), IL-2, transfer factor etc. all exist the different toxic side effect of degree and price than shortcomings such as costlinesses, make it to be subjected to certain restriction in clinical application.Because the extensive and life-time service of a large amount of antifungal drugs causes the appearance of Resistant strain easily, brings very big difficulty for the treatment of monilial infection.Since Tubulicid generally use and to have extremely strong broad-spectrum antibacterial, germicidal action and toxicity low, anti-microbial effect to Gram-positive, negative bacterium, bacterial spore, fungi and virus etc., even when having serum, blood etc. to exist, still effectively have the broad-spectrum antimicrobial antiinflammation, contain a certain amount of pair of chlorobenzene biguanides and also can prevent and eliminate cell in vitro and cultivate oidiomycetic pollution.
Specific embodiments:
Used key instrument and reagent source among the embodiment:
(1) main instruments and equipment
DL-CJ-2N high-performance aseptic experiment platform: east, Chinese Harbin connection electronic technology development corporation, Ltd.
37.5 ℃ 5%CO2 incubator: German Heraeus company
TDL-40B horizontal centrifuge: the smart grand experimental instrument and equipment of Chinese Shanghai company limited
AR1530/C electronic balance: U.S. OHAUS company
IX-71 inverted phase contrast microscope: Japanese Olympus company
Electric-heated thermostatic water bath, blood counting chamber, liquid nitrogen container: BeiJing, China 61 factories
Pall ultrapure water instrument: German Pall company
Electronic pipettor: German Eppendorff company
Ultralow Temperature Freezer: company of China's Haier
Culture plate: U.S. Corning company
Grads PCR instrument: U.S. Applied Biosystems
The ultrapure water instrument Millipore U.S.
Gel imaging system: U.S. Alpha Innotech
Ultraviolet spectrophotometer: U.S. Ultrospec1100pro GE
Ultraviolet spectrophotometer: U.S. ND-1000Nanodrop
Desk type high speed refrigerated centrifuge: German Eppendorf
(2) main agents and compound method
(1) 0.2M L-Glutamine: take by weighing 1.46g L-Glutamine and be dissolved in the 50ml tri-distilled water, place 37 ℃ of water-bath heating for dissolving, 0.22 μ m membrane filtration degerming ,-20 ℃ of preservations are standby.
(2) 3%NaHCO
3: take by weighing 3g NaHCO
3Be dissolved in the 100ml tri-distilled water, 0.22 μ m membrane filtration degerming, 4 ℃ of preservations are standby.
(3) DMEM substratum: with DMEM dry powder and the 2.2gNaHCO of a packing
3Be dissolved in an amount of tri-distilled water, be settled to 1000ml, transfer about pH7.0,0.22 μ m filtering with microporous membrane degerming, the packing of 500ml/ bottle, 4 ℃ of preservations are standby.
(4) the full substratum of DMEM: RPMI1640 basic medium+10%FBS+1% is two to be resisted+1%L-Glutamine, regulates pH7.0,0.22 μ m filtering with microporous membrane degerming, and 4 ℃ of preservations are standby.
(5) contain the full substratum of sodium bicarbonate DMEM: DMEM basic medium+10%FBS+1% is two to be resisted+1%L-Glutamine+4ml 3%NaHCO
3, regulate pH7.0,0.22 μ m filtering with microporous membrane degerming, 4 ℃ of preservations are standby.
(6) containing two full substratum of chlorobenzene biguanides DMEM: DMEM basic medium+10%FBS+1% is two to be resisted+the two chlorobenzene biguanides of 1%L-Glutamine+150 μ l, regulate pH7.0,0.22 μ m filtering with microporous membrane degerming, 4 ℃ of preservations are standby.
(7) the husky Bao Shi liquid nutrient medium (CMO147) of husky Bao Shi substratum: OXIOD Britain
(8) Candida albicans is available from Chinese industrial microbial strains preservation center
The cultivation of embodiment 1 Candida albicans
With bacterial classification inoculation on husky Bao Shi substratum, 37 ℃ of cultivations, 1-2d grows bacterium colony, picking list bacterium colony is inoculated in respectively in corn nutrient agar and the corn tween 80 substratum, covers aseptic cover plate then.Cultivate in 37 ℃ of incubators, carry out microscopy respectively at 24,48,72h.
Result: 48h observes, and all grows bacterium colony in the substratum, the bacterium colony circle, and white or little cream color that is, flash of light, the edge is smooth, surperficial corrugationless.
Embodiment 2 inoculation bacterium liquid are in cell and packet transaction
With 2 * 10
7The inoculation of/ml Candida albicans bacterium liquid is good with 30 bottles of upgrowth situations, the cell 0.1ml/ bottle of density about 60%, 30 bottles of cells branches are divided into three groups at random, every group 10 bottles be respectively control group, contain 3% sodium bicarbonate DMEM, (two chlorobenzene biguanides: DMEM) two chlorobenzene biguanides DMEM place 37.5 ℃, 5%CO to contain 1: 100000
2Cultivate in the incubator, observe it every day.It is carried out DNA extraction, PCR product augmentation detection cultivating 7
Conclusion: through all tests, contain two full substratum of chlorobenzene biguanides DMEM and can control and eliminate outstanding winter sports person's cell in vitro effectively and cultivate Candida albicans and pollute, therefore contain a certain amount of pair of chlorobenzene biguanides and also can prevent and eliminate cell in vitro and cultivate oidiomycetic pollution.
Claims (1)
1. one kind is efficiently prevented and eliminates winter sports person's cell in vitro and cultivate the method that Candida albicans pollutes, it is characterized in that, (two chlorobenzene biguanides: DMEM) (eliminated, and detects two chlorobenzene biguanides through PCR and can prevent and eliminate cell in vitro and cultivate oidiomycetic pollution by the cell of 3 * 107/ml) outstanding winter sports person's vitro culture of polluting to Candida albicans for two chlorobenzene biguanides DMEM (containing 3% sodium bicarbonate) with 1: 100000.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104560760A (en) * | 2013-10-11 | 2015-04-29 | 晏健强 | Pathogenic bacterium sorting and picking method in blood examination |
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| CN1324638A (en) * | 2000-05-20 | 2001-12-05 | 李儒镠 | External-use medicine for treating vaginitis |
| CN102988335A (en) * | 2012-11-29 | 2013-03-27 | 王便草 | Antifungal composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1324638A (en) * | 2000-05-20 | 2001-12-05 | 李儒镠 | External-use medicine for treating vaginitis |
| CN102988335A (en) * | 2012-11-29 | 2013-03-27 | 王便草 | Antifungal composition |
Non-Patent Citations (2)
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| 崔春梅等人: "1‰洗必泰液治疗顽固性霉菌性阴道炎20 例", 《中外医疗》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104560760A (en) * | 2013-10-11 | 2015-04-29 | 晏健强 | Pathogenic bacterium sorting and picking method in blood examination |
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Application publication date: 20130807 |