CN107192656A - A kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer - Google Patents
A kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer Download PDFInfo
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- 230000000242 pagocytic effect Effects 0.000 title claims abstract description 39
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 206010057249 Phagocytosis Diseases 0.000 claims abstract description 30
- 230000008782 phagocytosis Effects 0.000 claims abstract description 30
- 239000012530 fluid Substances 0.000 claims abstract description 28
- 210000004027 cell Anatomy 0.000 claims description 51
- 239000004005 microsphere Substances 0.000 claims description 38
- 238000001914 filtration Methods 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 239000004744 fabric Substances 0.000 claims description 10
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 9
- 241000167880 Hirundinidae Species 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 6
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- 238000010586 diagram Methods 0.000 claims description 5
- 238000000205 computational method Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 150000007942 carboxylates Chemical class 0.000 claims description 2
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- 235000003140 Panax quinquefolius Nutrition 0.000 abstract 1
- 235000008434 ginseng Nutrition 0.000 abstract 1
- 230000009747 swallowing Effects 0.000 abstract 1
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- 238000004519 manufacturing process Methods 0.000 description 3
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
-
- G01N15/01—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Abstract
The invention discloses a kind of stichopus japonicus Coelomocytes phagocytic rate detection method based on flow cytometer, stichopus japonicus Coelomocytes are extracted including live body, fluorescence phagocytosis microballoon is incubated altogether with coelomocyte of Apostichopus japonicus, the phagocytic activity of coelomocyte of Apostichopus japonicus is finally determined using flow cytometer.Coelomocyte directly with being incubated altogether under microballoon in vitro lower temperature, is reduced the influence of temperature by the present invention；Coelomic fluid is extracted in short thrust ginseng anal orifice, it is ensured that the fresh and alive state of stichopus japonicus, make data that there is coherent, validity；It is resuspended using the coelomic fluid after sterilizing instead of traditional PBS, reduces influence of the chemical reagent to coelomocyte.Instant invention overcomes phagocytic rate relatively low deficiency when using fluorescent material mark rate relatively low in existing utilization flow cytomery phagocyte active technologies and swallowing microballoon using internal injection, method is quick, accurate, simple and easy to apply.
Description
Technical field
The present invention relates to field of marine biotechnology, more specifically to a kind of stichopus japonicus body based on flow cytometer
Chamber cell phagocytic rate detection method.
Background technology
The stichopus japonicus aquaculture of China, secondary industry growth momentum are swift and violent, are provided for marine economy market extremely wide
Prospect.To the end of the year 2015, China's stichopus japonicus yield has reached 20.5 ten thousand tons.But compared with the fast development of stichopus japonicus industry, stichopus japonicus
Study on Ecophysiological Characteristics of Narrow relatively lags behind, and causes its cultivation, processing, transportation technology not science, and market management in addition does not have with running
Carried out under the guidance of science, occur in that such as rottenization skin phenomenon takes place frequently, the problems such as seed natural resources shortage, caused huge
Economic loss, as one of restriction industry health, main bottleneck of sustainable development.
Stichopus japonicus lacks acquired immune system as other invertebrates, and its immune system is nospecific immunity
System.Stichopus japonicus, which has, compares broad deuterocoel, and the coelomic fluid in body cavity, lymph is functionally similar to, wherein there is participation immune
The cell of reaction.By coelomocyte and a variety of body cavity immune factors it is common and directly act on invasion cause of disease constitute its it is immune should
Answer.Stichopus japonicus body immune system is mainly made up of cellular immunity and humoral immunity.Cellular immunity relies primarily on the body largely existed
Chamber cell is completed；Humoral immunity is then to act on completing by the immune factor being secreted by coelomocyte.Both mutually collaborations,
Internal foreign matter is identified jointly, is become innocuous substance or directly excludes foreign matter in vitro, repaiied so as to complete wound
Multiple effect.There are some researches show the phagocytosis of echinoderm coelomocyte is one of most important immunologic function of body, and it swallows
The size of ability directly affects the height of immunological effect.
There is the method that many measure cell swallows at present, such as traditional microscope inspection counting method and flow cytometer fluorescence are examined
Survey method etc..Wherein, Microscopical Method For Detection passes through microscopical observation analysis, it is adaptable to morphological observation and individual cells the phagocytosis work(of cell
Can qualitative research, but due to the shortcomings of there is subjectivity strong and poor repeatability in the technology, its apply to a certain extent by
Limitation.Low cytometric analysis (Flow-cytometry, FCM) is using phagocytic function of the different fluorescence labelings to cell
Studied, the characteristics of high, reproducible with accuracy, and easy to operate quick.Before this, people are often with Escherichia coli etc.
Some bacteriums detect the phagocytic activity of phagocyte, but because bacterium does not have fluorescence in itself, it is necessary to use isosulfocyanic acid fluorescence
Fluorescence such as plain (FITC) is marked, but mark rate is relatively low, so the detection for phagocytic rate is inaccurate.Lijin's longevity etc. utilizes
The method of internal injection phagocytosis microballoon detects the change of phagocytic activity, it is found that the phagocytic rate for utilizing the method to detect is generally relatively low,
And stichopus japonicus belongs to open cycle system in itself, in vivo in phagocytosis experiment, body fluid is the main composition of stichopus japonicus, agglutination phenomenon compared with
To be universal, and agglutination phenomenon is one of index of humoral immunity, it is taken as that being substantially carried out body fluid in phagocytosis experiment in vivo and exempting from
Epidemic disease is reacted, and is more not suitable for the method with internal injection, so needing in original utilization flow cytomery phagocyte activity
On the basis of explore a set of more accurate, reliable, easy method.
The content of the invention
It is an object of the invention to overcome the shortcomings of it in existing utilization flow cytomery phagocyte active technologies
Place, sets up external phagocytosis detection model, the phagocytic rate of stichopus japonicus is generally improved, while eliminating unopsonized fluorescent microsphere to analysis result
Influence, can to coelomocyte swallow fluorescent microsphere number carry out quantitative analysis, detection quick, accurate, simple and easy to apply.
To reach above-mentioned purpose, the invention provides a kind of coelomocyte of Apostichopus japonicus phagocytic rate detection based on flow cytometer
Method, comprises the following steps：
S1, prepare coelomocyte suspension：The coelomic fluid at stichopus japonicus anal orifice nearly 1/3 is taken, by 3~5 stichopus japonicus coelomic fluids etc.
Volume mixture, after 300 mesh filter-cloth filterings, takes 2000~3000r/min of filtrate to centrifuge after 5~10min and removes after supernatant, use
Sterilized stichopus japonicus coelomic fluid is resuspended, and it is 10 to prepare cell concentration6~107Cells/mL coelomocyte suspension；
S2, fluorescent microsphere mark cell：Take the fluorescence of 0.30~2.00 μm of diameter to swallow microballoon, be added to step S1 preparations
Coelomocyte suspension in, at 15 DEG C~25 DEG C at a slow speed dark reaction shake 30~90 minutes after, 300 mesh filtering test sample to be checked is made
Product；The fluorescence phagocytosis microballoon and coelomocyte suspension volume ratio prepared by step S1 are 1:250~2000；
S3, utilize flow cytomery coelomocyte phagocytic activity：
By sample with 300 mesh filter-cloth filterings before S31, loading；
S32, take in step S1 not plus fluorescent microsphere coelomocyte suspension as negative control, carry out after loading, loading
Cell colony is drawn a circle to approve according to FSC/SSC scatter diagrams, then by adjusting FITC fluorescence channel voltages, makes resuspension coelomocyte colony peak
Position be located at fluorescence voltage transverse axis 102Left side, as negative control group, and collects coelomocyte；
S33, in the case where ensuring that each fluorescence voltage parameter of flow cytometer set by negative control group and threshold value are constant, take
The sample of addition fluorescent microsphere prepared by step S2, is collected to sample, is had according to the cell of phagocytosis fluorescent microsphere
Fluorescence signal, the cell and unopsonized fluorescence that phagocytosis fluorescent microsphere is distinguished in the one-parameter histograms of fluorescence channel number FITC is micro-
The cell of ball, the cell gating of unopsonized fluorescent microsphere is P2, and the cell gating of phagocytosis fluorescent microsphere is P3；
Phagocytic rate computational methods are as follows：Cell colony (P3)/TCS (P2 of cell phagocytic rate=phagocytosis fluorescent microsphere
+ P3) × 100%.
Flow cytometer used of the invention is the production of BD companies, model FACSVerseTM.
Under preferred embodiment, sterilized stichopus japonicus coelomic fluid described in step S1 is takes fresh and alive stichopus japonicus coelomic fluid, and HTHP is
121 DEG C, 103Under kPa, sterilize 30min, 0~4 DEG C of preservation；This stichopus japonicus coelomic fluid is the coelomic fluid at stichopus japonicus anal orifice nearly 1/3.
Under preferred embodiment, the fluorescence phagocytosis microballoon of 0.30~2.00 μm of diameter described in step S2 is Fluoresbrite YG
Carboxylate Microspheres fluorescence swallows microballoon.
Under preferred embodiment, step S2 using shaking table, shake 30~90 minutes at a slow speed by dark reaction.
Step S1 extracts coelomic fluid using asepsis injector at stichopus japonicus belly nearly 1/3, by 3~5 stichopus japonicus coelomic fluids etc.
Volume mixture is mixed in equal volume, with 300 mesh filter-cloth filterings in 10mL Eppendorf (EP) pipe, takes the mixture of appropriate volume
Chamber liquid is in 1.5mL EP pipes, and 2000~3000r/min centrifuges 5~10min.
Step S1 takes 3~5 stichopus japonicus coelomic fluids to ensure the fresh and alive state of stichopus japonicus after extracting as far as possible, can be achieved continuous
Detect phagocytic activity of the same stichopus japonicus in different sampling stages point, it is ensured that the coherent, validity of experiment gathered data.
Live body of the present invention extracts stichopus japonicus Coelomocytes, fluorescence phagocytosis microballoon is incubated altogether with coelomocyte of Apostichopus japonicus, finally
The phagocytic activity of coelomocyte of Apostichopus japonicus is determined using flow cytometer.
The technological innovation of the present invention is：
1st, coelomocyte is directly incubated by the present invention altogether at 15 DEG C~25 DEG C in vitro with microballoon, more traditional incubation bar
Part, employs relatively low incubation temperature, reduces influence of the temperature to coelomocyte of Apostichopus japonicus, can obtain closer to nature
The phagocytic activity of lower coelomocyte of Apostichopus japonicus.
2nd, present invention asepsis injector ensures to take out away from coelomic fluid is extracted at stichopus japonicus anal orifice (afterbody) nearly 1/3, as far as possible
The fresh and alive state of rear stichopus japonicus is put forward, can be achieved continuously to detect phagocytic activity of the same stichopus japonicus in different sampling stages point, it is ensured that
Test the coherent of gathered data, validity.
3rd, the present invention is resuspended using the coelomic fluid after autoclave sterilization instead of traditional PBS, as far as possible
Influence of the reduction chemical reagent to coelomocyte, the acid or alkali environment of stichopus japonicus coelomic fluid under access expansion state can be maintained, it is ensured that
The activated state of coelomocyte.
4th, instant invention overcomes fluorescent material mark is used in existing utilization flow cytomery phagocyte active technologies
The universal relatively low deficiency of phagocytic rate, is incubated in vitro to coelomocyte of Apostichopus japonicus when note rate is relatively low and swallows microballoon using internal injection
Educate, set up external phagocytosis detection model, improve the phagocytic rate of stichopus japonicus, improve the accuracy of experimental result, at the same to phagocytosis with
Unopsonized cell circle door processing, eliminates influence of the unopsonized fluorescent microsphere to analysis result, can swallow fluorescence to coelomocyte
The number of microballoon carries out quantitative analysis, easy to operate quick, and accuracy is high, reproducible.
Brief description of the drawings
Fig. 1 is the two-dimentional scatter diagram that coelomocyte swallows result.
Fig. 2 is that coelomocyte swallows result histogram, and wherein P2 is the cell of unopsonized fluorescent microsphere, and P3 is phagocytosis fluorescence
The cell of microballoon.
Fig. 3 is that ultraviolet is irradiated after 30min, stands the phagocytic rate testing result of different time (0~4h).
Embodiment
Following non-limiting examples can make one of ordinary skill in the art be more fully understood the present invention, but not with
Any mode limits the present invention.
Embodiment 1
Internal phagocytic rate detection：The much the same stichopus japonicus of size volume is taken, using asepsis injector from stichopus japonicus afterbody to internal
30 μ L fluorescent microsphere stoste is injected, is incubated in vivo after 30min, 60min, 120min respectively, using asepsis injector in stichopus japonicus
Coelomic fluid is extracted at anal orifice (afterbody) nearly 1/3, many stichopus japonicus coelomic fluids are mixed in equal volume, 300 mesh filter-cloth filterings are made and treated
Detect sample.
Utilize flow cytomery coelomocyte phagocytic activity：Flow cytometer used is the production of BD companies, model
For FACSVerseTM, by sample with 300 mesh filter-cloth filterings before loading, (1) take not plus fluorescent microsphere resuspension coelomic fluid as the moon
Property control, carry out loading, cell colony drawn a circle to approve according to FSC/SSC scatter diagrams after loading, then by adjusting FITC fluorescence channels electricity
Pressure, makes the position at resuspension coelomocyte colony peak be located at fluorescence voltage transverse axis 102Left side, and collect coelomocyte；(2) in streaming
In the case that each fluorescence voltage parameter of cell instrument keeps constant with threshold value, detected sample is removed, (Fig. 1) is collected to sample,
The fluorescence signal being had according to the cell of phagocytosis fluorescent microsphere, distinguishes in the one-parameter histograms of fluorescence channel number FITC and gulps down
The cell of fluorescent microsphere and the cell of unopsonized fluorescent microsphere are bitten, the cell gating of unopsonized fluorescent microsphere is P2, swallows fluorescence
The cell gating of microballoon is P3 (Fig. 2), and phagocytic rate computational methods are as follows：The cell colony of cell phagocytic rate=phagocytosis fluorescent microsphere
(P3)/TCS (P2+P3) × 100%.Detection and result of calculation such as table 2.
Table 1
From table 1, fluorescent microsphere content is more needed for phagocytosis in vivo, and incubation time is longer, but generally speaking, phagocytic rate
It is all relatively low, it is unfavorable for experiment and carries out.
Embodiment 2
Take the much the same stichopus japonicus of size volume, setting normal group (Control) and experimental group, (ultraviolet (UV) A is quiet after irradiating
Put 1,2,3,4h).Each testing site ensures：The coelomic fluid of 3 stichopus japonicus is mixed as once testing in equal volume, replication three
It is secondary.
Prepare coelomocyte suspension：Body cavity is extracted at stichopus japonicus stichopus japonicus anal orifice (afterbody) nearly 1/3 using asepsis injector
Liquid, many stichopus japonicus coelomic fluids are mixed in equal volume, 300 mesh filter-cloth filterings.The mixocoel liquid of appropriate volume is taken in 1.5mL EP
Guan Zhong, 2000~3000r/min remove supernatant after centrifuging 5~10min, utilize HTHP (121 DEG C, 103kPa, 30min)
Sterilizing, 0~4 DEG C preservation, the stichopus japonicus coelomic fluid without coelomocyte be resuspended, prepare cell concentration be 106~
107Cells/mL coelomocyte suspension.
Fluorescent microsphere marks cell：Take 750 μ L coelomocytes suspensions to add 0.5 μ L fluorescence phagocytosis microballoon stoste to mix, 15
DEG C~25 DEG C of shaking table dark reactions are incubated 30min, detected sample is made in 300 mesh filter-cloth filterings.
Utilize flow cytomery coelomocyte phagocytic activity：Flow cytometer used is the production of BD companies, model
For FACSVerseTM, by sample with 300 mesh filter-cloth filterings before loading, (1) take not plus fluorescent microsphere resuspension coelomic fluid as the moon
Property control, carry out loading, cell colony drawn a circle to approve according to FSC/SSC scatter diagrams after loading, then by adjusting FITC fluorescence channels electricity
Pressure, makes the position at resuspension coelomocyte colony peak be located at fluorescence voltage transverse axis 102Left side, and collect coelomocyte；(2) in streaming
In the case that each fluorescence voltage parameter of cell instrument and threshold value keep constant, detected sample is removed, sample is collected, according to gulping down
The fluorescence signal that the cell of fluorescent microsphere has is bitten, phagocytosis fluorescence is distinguished in the one-parameter histograms of fluorescence channel number FITC
The cell of the cell of microballoon and unopsonized fluorescent microsphere, the cell gating of unopsonized fluorescent microsphere is P2, phagocytosis fluorescent microsphere
Cell gating is P3, and phagocytic rate computational methods are as follows：Cell colony (P3)/cell of cell phagocytic rate=phagocytosis fluorescent microsphere is total
Number (P2+P3) × 100%.Detection and result of calculation such as table 2, statistical result such as Fig. 3.
Table 2
| Control | 0 | 1 | 2 | 3 | 4 | |
| P2 | 9971 | 9687 | 9328 | 9668 | 9911 | 9732 |
| P3 | 602 | 1088 | 1527 | 906 | 865 | 829 |
| Phagocytic rate=P3/ (P2+P3) × 100% | 5.69% | 10.10% | 14.06% | 8.56% | 8.03% | 7.85% |
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (4)
1. a kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer, it is characterised in that comprise the following steps：
S1, prepare coelomocyte suspension：The coelomic fluid at stichopus japonicus anal orifice nearly 1/3 is taken, 3~5 stichopus japonicus coelomic fluids are isometric
Mixing, after 300 mesh filter-cloth filterings, takes 2000~3000r/min of filtrate to centrifuge after 5~10min and removes after supernatant, with having gone out
The stichopus japonicus coelomic fluid of bacterium is resuspended, and it is 10 to prepare cell concentration6~107Cells/mL coelomocyte suspension；
S2, fluorescent microsphere mark cell：Take the fluorescence of 0.30~2.00 μm of diameter to swallow microballoon, be added to the body of step S1 preparations
In chamber cell suspension, after dark reaction is shaken 30~90 minutes at a slow speed at 15 DEG C~25 DEG C, detected sample is made in the filtering of 300 mesh；
The fluorescence phagocytosis microballoon and coelomocyte suspension volume ratio prepared by step S1 are 1:250~2000；
S3, utilize flow cytomery coelomocyte phagocytic activity：
By sample with 300 mesh filter-cloth filterings before S31, loading；
S32, take in step S1 not plus fluorescent microsphere coelomocyte suspension as negative control, carry out basis after loading, loading
FSC/SSC scatter diagrams draw a circle to approve cell colony, then by adjusting FITC fluorescence channel voltages, make the position at resuspension coelomocyte colony peak
Setting in fluorescence voltage transverse axis 102Left side, as negative control group, and collects coelomocyte；
S33, in the case where ensuring that each fluorescence voltage parameter of flow cytometer set by negative control group and threshold value are constant, take step
The sample of addition fluorescent microsphere prepared by S2, is collected to sample, the fluorescence being had according to the cell of phagocytosis fluorescent microsphere
Signal, distinguishes the cell and unopsonized fluorescent microsphere of phagocytosis fluorescent microsphere in the one-parameter histograms of fluorescence channel number FITC
Cell, the cell gating of unopsonized fluorescent microsphere is P2, and the cell gating of phagocytosis fluorescent microsphere is P3；
Phagocytic rate computational methods are as follows：The cell colony (P3) of cell phagocytic rate=phagocytosis fluorescent microsphere/TCS (P2+P3)
× 100%.
2. a kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer, its feature according to claim 1
It is, sterilized stichopus japonicus coelomic fluid described in step S1 is takes fresh and alive stichopus japonicus coelomic fluid, and HTHP is 121 DEG C, 103Under kPa,
Sterilize 30min, 0~4 DEG C of preservation.
3. a kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer, its feature according to claim 1
It is, the fluorescence phagocytosis microballoon of 0.30~2.00 μm of diameter described in step S2 is Fluoresbrite YG Carboxylate
Microspheres fluorescence swallows microballoon.
4. a kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer, its feature according to claim 1
It is, step S2 using shaking table, shake 30~90 minutes at a slow speed by dark reaction.
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| CN107873587A (en) * | 2017-11-09 | 2018-04-06 | 大连工业大学 | A kind of method for delaying stichopus japonicus body wall self-dissolving |
| CN110029143A (en) * | 2019-04-28 | 2019-07-19 | 宁波大学 | A kind of screening technique of resistance Portunus trituberculatus Miers |
| CN110714048A (en) * | 2019-11-15 | 2020-01-21 | 集美大学 | Screening method of plant-derived immunity-enhanced feed additive for eels |
| CN110849851A (en) * | 2019-11-22 | 2020-02-28 | 辽宁省检验检测认证中心 | Novel method for rapidly evaluating immunity enhancement of medicine/health-care product based on flow cytometry fluorescent microsphere dual-standard method |
| CN111019813A (en) * | 2019-12-25 | 2020-04-17 | 深圳赛动生物自动化有限公司 | Intelligent cell constant volume system and constant volume method thereof |
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| CN107873587A (en) * | 2017-11-09 | 2018-04-06 | 大连工业大学 | A kind of method for delaying stichopus japonicus body wall self-dissolving |
| CN107873587B (en) * | 2017-11-09 | 2021-09-24 | 大连工业大学 | Method for delaying autolysis of stichopus japonicus body wall |
| CN110029143A (en) * | 2019-04-28 | 2019-07-19 | 宁波大学 | A kind of screening technique of resistance Portunus trituberculatus Miers |
| CN110714048A (en) * | 2019-11-15 | 2020-01-21 | 集美大学 | Screening method of plant-derived immunity-enhanced feed additive for eels |
| CN110849851A (en) * | 2019-11-22 | 2020-02-28 | 辽宁省检验检测认证中心 | Novel method for rapidly evaluating immunity enhancement of medicine/health-care product based on flow cytometry fluorescent microsphere dual-standard method |
| CN111019813A (en) * | 2019-12-25 | 2020-04-17 | 深圳赛动生物自动化有限公司 | Intelligent cell constant volume system and constant volume method thereof |
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