CN104789528B - Applications of the dodecyl decylenic acid BDSF in RAW264.7 macrophages phagocytic capacities are lifted - Google Patents
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Abstract
The present invention relates to applications of the dodecyl decylenic acid BDSF in RAW264.7 macrophages phagocytic capacities are lifted, belong to biologics technical field technical field.The concentration for adding BDSF can lift RAW264.7 macrophages phagocytosis C.albicans ability when being 3~300 μm of ol/L, BDSF lifting RAW264.7 macrophage phagocytosiss C.Albicans action time is 2h, 3h, 4h, 5h, 6h.BDSF has low bio-toxicity, and the lifting to immunocyte RAW264.7 macrophages phagocytic capacities has remarkable result.
Description
Technical field
The present invention relates to a kind of dodecyl decylenic acid BDSF in the ability of lifting RAW264.7 macrophages phagocytosis
Using belonging to biologics technical field.
Background technology
C.abicans Candida albicans (also known as candida albicans) are the important opportunistic fungus of human body.In normal person
In body, candida albicans is a kind of harmless symbiotic effects；In hypoimmunity crowd, candida albicans can cause Candida
Disease, the lighter can cause mucosa infection, and severe one can develop into systemic disease, or even threat to life.Candida albicans is from Yeast Phase
Morphological Transitions to Hyphal form are one of extremely important pathogenic factors.
Dodecyl decylenic acid (BDSF) is small molecule short chain fatty acids, is secreted and produced by Burkholderia cenocepacia
It is raw.BDSF belongs to that DSF families are quite similar with DSF in structure, and molecular structural formula is
Document (Boon C, Deng Y, Wang LH, He Y, Xu JL, Fan Y, Pan SQ, Zhang LH.A novel DSF-like
signal from Burkholderia cenocepacia interferes with Candida albicans
morphological transition[J].ISME J,2008,2(1):27-36.)
RAW264.7 macrophages (mononuclear macrophage) belong to immunocyte, there is multiple functions, are the phagocytosiss of research cell, thin
The immune important object with molecular immunology of born of the same parents.Once body wound activity starts, macrophage just can largely secrete a variety of lifes
Active substances and a variety of enzyme materials, wherein bioactive substance are also known as Macrophage derived biotic factor, including polypeptide
Conversion growth factor, interleukins, TNF, platelet derived growth factor and nitric oxide etc.；Enzyme thing
Matter mainly includes clostridiopetidase A, elastoser, plasminogen activator etc.；A little bioactive substances directly guide body reparation
Whole process.Meanwhile main phagocyte of the macrophage as inflammation phase, it is responsible for removing tissue and cell at body injury
Necrotic Debris and pathogen etc., these materials have important regulating and controlling effect to wound healing process.Therefore, wound is studied
The changing rule of the species and content different time after wound for the cell factor that macrophage discharges in repair process, it would be possible to
Some significant changes related to trauma time or foundation are provided from molecular level and cellular level；And document report macrophage
The change of cell phagocytosis thing also has and the characteristics of time correlation, and macrophage phagocytosis thing is morphologically easy to observe and detect,
These features cause macrophage to have important Forensic Significance during wound age.
The content of the invention
Present invention solves the technical problem that it is：BDSF new application is provided, i.e. BDSF is in lifting RAW264.7 macrophages
Swallow the application in C.albicans ability.
The technical scheme is that dodecyl decylenic acid BDSF is in RAW264.7 macrophages phagocytic capacities are lifted
Application, the concentration for adding BDSF can lift RAW264.7 macrophages phagocytosis C.albicans when being 3~300 μm of ol/L
Ability.
Preferably, the concentration of the BDSF is 100 μm of ol/L.
Preferably, the action time that BDSF lifts RAW264.7 macrophages phagocytosis C.Albicans is 2h, 3h, 4h, 5h,
6h。
Beneficial effect：
BDSF in this research has low bio-toxicity, and immunocyte RAW264.7 macrophages phagocytic capacities are carried
Rising has remarkable result so that small molecule aliphatic acid BDSF has important research meaning to the influence in terms of animal cell immunity function
Justice, while new material is provided for immunological investigation, there is important Research Significance to microorganism and biological immune aspect.
Brief description of the drawings
The present invention is described further below in conjunction with the accompanying drawings.
Fig. 1 .RAW264.7 macrophages swallow one or more C.albicans cells under BDSF effects
Fig. 2 .BDSF swallow the influence of the phagocytic rate of C.albicans cells to RAW264.7 macrophages
Fig. 3 .BDSF swallow the influence of the phagocytic index of C.albicans cells to RAW264.7 macrophages
Fig. 4 Laser Scanning Confocal Microscopes are continuously shot the process that RAW264.7 macrophages swallow multiple C.albicans cells
Fig. 5 .MTT colorimetric determinations BDSF are to RAW264.7 macrophages effect 12,24,36,48h cell viability situation
Embodiment
The content of patent for a better understanding of the present invention, further illustrate the present invention's below by specific example
Technical scheme.But these embodiments are not intended to limit the present invention.
Embodiment 1
The experiment that MTT colorimetric determinations BDSF influences on the cell viability of RAW264.7 macrophages:
BDSF is added in RAW264.7 cell culture fluids, concentration is respectively 0,3,30,100,200,300 μm of ol/L.
Secondary Culture RAW264.7 macrophages, by cell be inoculated on 5 piece of 96 orifice plate per the μ L of hole 100 (density be 1 ×
104) every group of 6 multiple holes, in 37 DEG C of 5%CO2 incubators after overnight incubation, the nutrient solution for adding the BDSF containing various concentrations continues
Culture 12,24,36,48h, blank control group is not added with BDSF, reaches to add after MTT is incubated 4h after drug treating time and adds DMSO
150 μ L shake 10min, and absorbance is detected at wavelength 570nm using ELIASA.
As Fig. 5 experimental results are shown, growths and effect of vigor and control group of the BDSF to RAW264.7 macrophages are added
Compared to without significant change, can show, growth increments of the BDSF to RAW264.7 macrophages has no adverse effects, bio-toxicity
It is low, show that BDSF application prospect is good.
Embodiment 2
The lower RAW264.7 macrophages phagocytosis C.albicans120min's of Rui Shi Giemsa stainings method analysis BDSF effects
Experiment：
The RAW264.7 macrophages of Secondary Culture are inoculated in 24 orifice plates with 5 × 105 density, added after cell attachment
After entering the BDSF (concentration is respectively 0,3,30,100,200,300 μm of ol/L) of various concentrations and cultivating 24h, more renew nutrient solution；
The yeast state C.albicans being incubated overnight is added into co-incubation in 24 orifice plates, C.albicans and RAW264.7 macrophages
Ratio be 3：1；After the co-incubation 120min of C.albicans and RAW264.7 macrophages, with 1%PBS washing lotions
Washing 3 times, washes away loose cell, after being fixed with Rui Shi Giemsa stains and dyed 20 minutes, with 1%PBS wash liquids 3
It is secondary, then fluorescence microscopy Microscopic observation and calculate RAW264.7 phagocytosiss C.albicans phagocytic rate (at least swallow one it is true
The macrophage percentage of bacterium cell) and phagocytic index (sum of every 100 macrophages phagocytosis fungal cell).As Fig. 2 is real
Test result to show, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytic rate and be respectively
4.0%th, 6.9% RAW264.7 macrophages when, 11.8%, 11.9%, 11.8%, 8.0%, BDSF is 3~300 μm of ol/L
Phagocytic rate is above the 4.0% of control group, and wherein BDSF concentration is that to reach maximum phagocytic rate be 11.9% to 100 μm of ol/L；Such as Fig. 3
Experimental result shows that various concentrations BDSF acts on the phagocytic index difference of phagocytosis C.albicans after RAW264.7 macrophages
For 0.28,0.46,0.61,0.60,0.69,0.36, BDSF when being 3~300 μm of ol/L the phagocytosis of RAW264.7 macrophages refer to
Number is above the 0.28 of control group.Summarize the phagocytic activity for showing that BDSF effects 2h can improve RAW264.7 macrophages.Number
According to the experiment for being taken at three groups of independence, analysis is at least per 200, hole macrophage.
Embodiment 3
The lower RAW264.7 macrophages phagocytosis C.albicans180min's of Rui Shi Giemsa stainings method analysis BDSF effects
Experiment:
(1) the RAW264.7 macrophages of Secondary Culture are inoculated in 24 orifice plates with 5 × 105 density, cell attachment
After adding the BDSF of various concentrations afterwards and cultivating 24h, more renew nutrient solution；The yeast state C.albicans being incubated overnight is added
Co-incubation in 24 orifice plates, the ratio of C.albicans and RAW264.7 macrophages is 3：1；
(2) after the co-incubation 180min of C.albicans and RAW264.7 macrophages, with 1%PBS washing lotions
Washing 3 times, washes away loose cell, after being fixed with Rui Shi Giemsa stains and dyed 20 minutes, with 1%PBS wash liquids 3
It is secondary, then fluorescence microscopy Microscopic observation and calculate RAW264.7 phagocytosiss C.albicans phagocytic rate (at least swallow one it is true
The macrophage percentage of bacterium cell) and phagocytic index (sum of every 100 macrophages phagocytosis fungal cell).As Fig. 2 is real
Test result to show, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytic rate and be respectively
12.0%th, 15.9% RAW264.7 macrophages are thin when, 24.0%, 34.5%, 27.7%, 30.1%, BDSF is 3~300 μm of ol/L
The phagocytic rate of born of the same parents is above the 12.0% of control group, and wherein BDSF concentration is that to reach maximum phagocytic rate be 34.5% to 100 μm of ol/L；
As Fig. 3 experimental results are shown, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytosis and refers to
RAW264.7 macrophages when number respectively 0.39,0.61,1.01,0.80,1.20,0.84, BDSF is 3~300 μm of ol/L
Phagocytic index is above the 0.39 of control group.Summarize the phagocytosis energy for showing that BDSF effects 3h can improve RAW264.7 macrophages
Power.Data acquisition is in the experiment of three groups of independence, and analysis is at least per 200, hole macrophage.
Embodiment 4
The lower RAW264.7 macrophages phagocytosis C.albicans240min's of Rui Shi Giemsa stainings method analysis BDSF effects
Experiment:
(1) the RAW264.7 macrophages of Secondary Culture are inoculated in 24 orifice plates with 5 × 105 density, cell attachment
After adding the BDSF of various concentrations afterwards and cultivating 24h, more renew nutrient solution；The yeast state C.albicans being incubated overnight is added
Co-incubation in 24 orifice plates, the ratio of C.albicans and RAW264.7 macrophages is 3：1；
(2) after the co-incubation 240min of C.albicans and RAW264.7 macrophages, with 1%PBS washing lotions
Washing 3 times, washes away loose cell, after being fixed with Rui Shi Giemsa stains and dyed 20 minutes, with 1%PBS wash liquids 3
It is secondary, then fluorescence microscopy Microscopic observation and calculate RAW264.7 phagocytosiss C.albicans phagocytic rate (at least swallow one it is true
The macrophage percentage of bacterium cell) and phagocytic index (sum of every 100 macrophages phagocytosis fungal cell).As Fig. 2 is real
Test result to show, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytic rate and be respectively
12.2%th, 30.5% RAW264.7 macrophages are thin when, 32.9%, 55.3%, 37.5%, 34.0%, BDSF is 3~300 μm of ol/L
The phagocytic rate of born of the same parents is above the 12.2% of control group, and wherein BDSF concentration is that to reach maximum phagocytic rate be 55.3% to 100 μm of ol/L；
As Fig. 3 experimental results are shown, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytosis and refers to
RAW264.7 macrophages gulps down when number respectively 0.35,0.63,1.9,1.73,1.86,1.23, BDSF is 3~300 μm of ol/L
Bite index is above control group 0.35.Summarize the phagocytosis energy for showing that BDSF effects 4h can improve RAW264.7 macrophages
Power.Data acquisition is in the experiment of three groups of independence, and analysis is at least per 200, hole macrophage.
Embodiment 5
The lower RAW264.7 macrophages phagocytosis C.albicans300min's of Rui Shi Giemsa stainings method analysis BDSF effects
Experiment
(1) the RAW264.7 macrophages of Secondary Culture are inoculated in 24 orifice plates with 5 × 105 density, cell attachment
After adding the BDSF of various concentrations afterwards and cultivating 24h, more renew nutrient solution；The yeast state C.albicans being incubated overnight is added
Co-incubation in 24 orifice plates, the ratio of C.albicans and RAW264.7 macrophages is 3：1；
(2) after the co-incubation 300min of C.albicans and RAW264.7 macrophages, with 1%PBS washing lotions
Washing 3 times, washes away loose cell, after being fixed with Rui Shi Giemsa stains and dyed 20 minutes, with 1%PBS wash liquids 3
It is secondary, then fluorescence microscopy Microscopic observation and calculate RAW264.7 phagocytosiss C.albicans phagocytic rate (at least swallow one it is true
The macrophage percentage of bacterium cell) and phagocytic index (sum of every 100 macrophages phagocytosis fungal cell).As Fig. 2 is real
Test result to show, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytic rate and be respectively
13.1%th, 33.5% RAW264.7 macrophages are thin when, 33.8%, 54.1%, 41.5%, 35.1%, BDSF is 3~300 μm of ol/L
The phagocytic rate of born of the same parents is above the 13.1% of control group, and wherein BDSF concentration is that to reach maximum phagocytic rate be 54.1% to 100 μm of ol/L；
As Fig. 3 experimental results are shown, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytosis and refers to
RAW264.7 macrophages when number respectively 0.38,0.67,1.16,1.90,1.78,1.27, BDSF is 3~300 μm of ol/L
Phagocytic index is above the 0.38 of control group.Summarize the phagocytosis energy for showing that BDSF effects 5h can improve RAW264.7 macrophages
Power.Data acquisition is in the experiment of three groups of independence, and analysis is at least per 200, hole macrophage
Embodiment 6
The lower RAW264.7 macrophages phagocytosis C.albicans360min's of Rui Shi Giemsa stainings method analysis BDSF effects
Experiment
(1) the RAW264.7 macrophages of Secondary Culture are inoculated in 24 orifice plates with 5 × 105 density, cell attachment
After adding the BDSF of various concentrations afterwards and cultivating 24h, more renew nutrient solution；The yeast state C.albicans being incubated overnight is added
Co-incubation in 24 orifice plates, the ratio of C.albicans and RAW264.7 macrophages is 3：1；
(2) after the co-incubation 360min of C.albicans and RAW264.7 macrophages, with 1%PBS washing lotions
Washing 3 times, washes away loose cell, after being fixed with Rui Shi Giemsa stains and dyed 20 minutes, with 1%PBS wash liquids 3
It is secondary, then fluorescence microscopy Microscopic observation and calculate RAW264.7 phagocytosiss C.albicans phagocytic rate (at least swallow one it is true
The macrophage percentage of bacterium cell) and phagocytic index (sum of every 100 macrophages phagocytosis fungal cell).As Fig. 2 is real
Test result to show, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytic rate and be respectively
12.3%th, 33.4% RAW264.7 macrophages are thin when, 34.1%, 47.7%, 40.0%, 36.3%, BDSF is 3~300 μm of ol/L
The phagocytic rate of born of the same parents is above the 12.3% of control group, and wherein BDSF concentration is that to reach maximum phagocytic rate be 47.7% to 100 μm of ol/L；
As Fig. 3 experimental results are shown, various concentrations BDSF, which is acted on after RAW264.7 macrophages, to be swallowed C.albicans phagocytosis and refers to
RAW264.7 macrophages when number respectively 0.36,0.65,0.99,1.98,1.85,1.40, BDSF is 3~300 μm of ol/L
Phagocytic index is above the 0.36 of control group.Summarize the phagocytosis energy for showing that BDSF effects 6h can improve RAW264.7 macrophages
Power.Data acquisition is in the experiment of three groups of independence, and analysis is at least per 200, hole macrophage
Embodiment 7
Laser Scanning Confocal Microscope is continuously shot in the case where 100 μm of ol/LBDSF are acted on, the phagocytosis of RAW264.7 macrophages
C.albicans experiment.
The RAW264.7 macrophages of step i. Secondary Cultures are inoculated in culture dish with 5 × 105 density, cell attachment
100 μm of ol/L BDSF is added afterwards and cultivates 24h；
The yeast state C.albicans being incubated overnight is added co-incubation in the culture in step i by step ii.,
The ratio of C.albicans and RAW264.7 macrophages is 3：1；
Step iii. is continuously shot shape of the function every cell in a 10s culture dish of shooting using Laser Scanning Confocal Microscope
State, it is continuously shot five hours, observation wherein RAW264.7 macrophages and C.albicans operative condition.
As shown in figure 1, in the case where 100 μm of ol/LBDSF are acted on, RAW264.7 macrophages swallow one and multiple
C.albicans phenomenon.The vigor enhancing of cell, phagocytic activity get a promotion.
As shown in figure 4, RAW264.7 macrophages in figure constantly swallow multiple C.albicans out of A-L this periods
Phenomenon, it can be seen that BDSF is remarkably improved the phagocytic activity of RAW264.7 macrophages.
The concrete technical scheme being not limited to described in above-described embodiment of the present invention, all technologies formed using equivalent substitution
Scheme is the protection domain of application claims.
Claims (3)
1. dodecyl decylenic acid BDSF is preparing the application in lifting RAW264.7 macrophages phagocytic capacity medicines, its feature
It is：The concentration for adding BDSF can lift RAW264.7 macrophages phagocytosis C.albicans energy when being 3~300 μm of ol/L
Power.
2. dodecyl decylenic acid BDSF according to claim 1 is preparing lifting RAW264.7 macrophages phagocytic capacities
Applied in medicine, it is characterised in that：The concentration of the BDSF is 100 μm of ol/L.
3. dodecyl decylenic acid BDSF according to claim 1 is preparing lifting RAW264.7 macrophages phagocytic capacities
Applied in medicine, it is characterised in that：BDSF lifting RAW264.7 macrophage phagocytosiss C.Albicans action time is 2h,
3h, 4h, 5h, 6h.
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| CN103382220A (en) * | 2012-05-03 | 2013-11-06 | 北京大学 | Cell factor FAM19A4 with anti-infection and antineoplastic activity and application thereof |
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