CN110066766A - A kind of abductive approach of the cell activity of neutrophil leucocyte - Google Patents
A kind of abductive approach of the cell activity of neutrophil leucocyte Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, more particularly to a kind of abductive approach of the cell activity of neutrophil leucocyte.The present invention provides a kind of abductive approach of the cell activity of neutrophil leucocyte, comprising: Fiber differentiation neutrophil leucocyte under the conditions of existing for the IFN-α.The method of the neutrophil leucocyte increased activity of induction human body provided by the present invention, utilize the activity of INF- α enhancing neutrophil leucocyte, greatly improve the effect of its killing tumor cell, improve the activity of human body neutrophil leucocyte, have found the method for allowing N2 type (low activity) neutrophil leucocyte to be converted into N1 type (high activity) neutrophil leucocyte, to enhance the immune response of body, reliable theoretical and experiment basis is provided for the method optimization of clinical neutrophil leucocyte immunotherapy of tumors, therapeutic effect.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of abductive approach of the cell activity of neutrophil leucocyte.
Background technique
Neutrophil leucocyte (neutrophil) derives from marrow, accounts for the 50-70% of blood middle leukocytes sum, cell dia
It is 10-12 μm.In Rui Shi-Giemsa staining blood film, cytoplasm is in colourless or pale red, and there are many tiny of Dispersed precipitate
(0.2~0.4 μm) pale red or lilac peculiar particle.
Neutrophil leucocyte is the most abundant leucocyte in blood, has leaflet shape or rod-shaped core, contains in endochylema a large amount of
The neither neutral fine grained of basophilic nor acidophilus.These particles are mostly lysosomes, include myeloperoxidase, lysozyme, alkaline phosphorus
The enzyme abundant such as sour enzyme and acid hydrolase, phagocytosis and digestive function with cell have, it is considered to be during inflammation and infection
The first line of defence.
Neutrophil leucocyte plays good effect in host defense, they ooze out from circulation and enter tissue.?
There, neutrophil leucocyte is by a series of mechanisms damage microorganisms, mainly phagocytosis, the release of antibacterial material, [for example,
Tumor necrosis factor (TNF)-α, interleukins (IL) -1, interferon (IFN) etc.], while it is extracellular to form neutrophil leucocyte
Catches (NETs).
Neutrophil leucocyte can modulate tumor by all means biological processes, it is now recognized that neutrophil leucocyte is secreted
Relevant cell factor (such as MMP-9, VREGF, MMP-8 etc.) growth that tumour cell can be influenced, control tumor-microvessel
It generates, adjusts migration and the invasive ability of tumour cell.There are also research think infiltration degree of the neutrophil leucocyte in tumour with
The prognosis of patient is closely related, a variety of swollen in clear-cell carcinoma, melanoma, bronchovesicular cancer, head and neck squamous cell carcinoma etc.
In tumor, the height infiltration of neutrophil leucocyte can influence the prognosis of patient.Neutrophil leucocyte may participate in the biological function tune of tumour
Control, it is closely related with the clinical prognosis of tumor patient.N1 type (high activity) and N2 type are divided into the activity of tumor-killing according to it
(low activity), N1 can occur for neutrophil leucocyte under different microenvironments and N2 type is converted.Therefore, this field wants to improve
The activity of human body neutrophil leucocyte finds and N2 type (low activity) neutrophil leucocyte is allowed to be converted into N1 type (high activity) neutrophil leucocyte
Method, to enhance the immune response of body, for the method optimization of clinical neutrophil leucocyte immunotherapy of tumors, therapeutic effect
Raising provides reliable theoretical and experiment basis.
Summary of the invention
In view of the foregoing deficiencies of prior art, it is living that the purpose of the present invention is to provide a kind of cells of neutrophil leucocyte
The abductive approach of property, for solving the problems of the prior art.
In order to achieve the above objects and other related objects, one aspect of the present invention provides a kind of cell activity of neutrophil leucocyte
Abductive approach, comprising:
Fiber differentiation neutrophil leucocyte under the conditions of existing for the IFN-α.
In some embodiments of the present invention, at least part of neutrophil leucocyte for being induced culture is that N2 type neutrality grain is thin
Born of the same parents.
In some embodiments of the present invention, the culture medium in MEM culture medium, 1640 culture medium of RPMI one
Kind or a variety of combinations.
In some embodiments of the present invention, the concentration of the IFN-α in culture medium is >=10IU/mL, preferably >=50IU/
ML, more preferably >=102IU/mL。
In some embodiments of the present invention, the neutrophil leucocyte is human neutrophil.
In some embodiments of the present invention, the neutrophil leucocyte isolates and purifies acquisition by peripheral blood.
In some embodiments of the present invention, the cell activity is tumor cytotoxicity activity.
Another aspect of the present invention provides purposes of the IFN-α in neutrophil leucocyte Fiber differentiation.
In some embodiments of the present invention, the purposes is particularly for enhancing neutrophil leucocyte killing tumor cell
Effect.
In some embodiments of the present invention, the purposes is specially to convert N2 type neutrality grain for N1 type neutrophil leucocyte
Cell.
Detailed description of the invention
Fig. 1 be shown as the present invention split it is red after WBC sub-population streaming divide group's schematic diagram.
Fig. 2 is shown as the present invention, and neutrophil leucocyte streaming divides group's schematic diagram after purification.
Fig. 3 is shown as neutrophil leucocyte of the present invention to the fragmentation effect figure schematic diagram of tumour cell.
Fig. 4 is shown as the effect of IFN-α enhancing N2 type neutrophil leucocyte direct killing tumour cell in the embodiment of the present invention
Schematic diagram.
Fig. 5 is shown as in the embodiment of the present invention neutrophil leucocyte to the killing functions of immunocytes schematic diagram of Daudi cell.
Specific embodiment
Inventor passes through numerous studies, is lured under the conditions of having been surprisingly found that neutrophil leucocyte existing for the IFN-α
It leads, can be N1 type cell by N2 type cell transformation, to provide a kind of induction of the cell activity of new neutrophil leucocyte
Method completes the present invention on this basis.
First aspect present invention provides a kind of abductive approach of the cell activity of neutrophil leucocyte, comprising:
Fiber differentiation neutrophil leucocyte under the conditions of existing for the IFN-α.
In abductive approach provided by the present invention, at least part of neutrophil leucocyte for being induced culture is N2 type neutrality grain
Cell.In the present invention, the cell activity is often referred to the tumor cytotoxicity activity of neutrophil leucocyte, and neutrophil leucocyte is according to it
N1 type and N2 type can be divided into the effect of tumor-killing, are induced in the neutrophil leucocyte of culture to may include N1 type neutrality grain
Cell and/or N2 type neutrophil leucocyte.The N1 type neutrophil leucocyte usually to tumour killing activity with higher, for example,
In real-time n cell analytical technology (Realtime Cellular Analysis, RTCA) measurement neutrophil leucocyte to adherent
In the experiment of the cytotoxicity of tumour cell, if the tumour cell of larger proportion is killed (for example, 70% or more can be killed
Tumour cell), then it has been generally acknowledged that the N1 type neutrophil leucocyte containing larger proportion on the whole；The N2 type neutrophil leucocyte is logical
Often there is lower killing activity to tumour, for example, in real-time n cell analytical technology (Realtime Cellular
Analysis, RTCA) measurement neutrophil leucocyte is in the experiment of the cytotoxicity of attached tumor cells, if small percentage is swollen
Oncocyte is killed (for example, can kill 20% tumour cell below), then it has been generally acknowledged that on the whole containing larger proportion
N2 type neutrophil leucocyte.
In abductive approach provided by the present invention, the culture medium can be the various neutrophil leucocytes that are suitable in this field and train
Feeding culture medium, for example, it may be 1640 culture medium of Minimum Essential Medium (MEM) culture medium or RPMI etc.
One of or it is a variety of.
In abductive approach provided by the present invention, the presence of IFN-α can effectively turn the neutrophil leucocyte of low activity
Turn to the neutrophil leucocyte of high activity, the concentration of the IFN-α in culture medium can for >=10IU/mL, >=50IU/mL, >=
102IU/mL、≥103IU/mL、≥104IU/mL、≥105IU/mL、≥106IU/mL or >=107IU/mL.The embodiment of the present invention
In be unequivocally established, 105The IFN-α of IU/mL or so concentration significantly can convert height for the neutrophil leucocyte of low activity
Active neutrophil leucocyte enhances the effect of neutrophil leucocyte killing tumor cell, when IFN-α concentration is further up,
The Induction Transformation effect of neutrophil leucocyte can be further strengthened.The condition of culture of suitable neutrophil leucocyte is for ability
It should be known for field technique personnel, for example, can be in 37 DEG C, 5%CO2Condition of culture under Fiber differentiation neutrality grain it is thin
Born of the same parents.
In abductive approach provided by the present invention, Fiber differentiation neutrality grain is thin under the conditions of can be existing for the tumour cell
Born of the same parents, neutrophil leucocyte and tumour cell co-culture, can be in order to detecting the anti-tumor activity of neutrophil leucocyte.For example, described
Tumour cell can be attached tumor cells, and the attached tumor cells density can be 5~8 × 104Cells/ml cell, in
The ratio of property granulocyte and tumour cell can be 3:1~10:1.
In abductive approach provided by the present invention, neutrophil leucocyte is obtained, is incubated under the conditions of with existing for the IFN-α
The method of culture should be known to those skilled in the art, for example, the neutrophil leucocyte can be people's neutrality
Granulocyte, for another example the neutrophil leucocyte can be the neutrophil leucocyte for isolating and purifying and obtaining from human peripheral, then example
Such as, the acquisition methods of the neutrophil leucocyte may include: by Blood Sedimentation, centrifugation, to obtain neutrophil leucocyte.
Second aspect of the present invention provides purposes of the IFN-α in neutrophil leucocyte Fiber differentiation, and the purposes specifically can be with
It is the effect for enhancing neutrophil leucocyte killing tumor cell, more specifically can be and convert N2 type for N1 type neutrophil leucocyte
Neutrophil leucocyte.
There is the process of an aging in neutrophil leucocyte, cell viability gradually decreases, and dead cell gradually increases in vitro,
Function gradually decreases, but up to the present, there is not yet N1 and N2 type neutrophil leucocyte is shifted to new management mechanisms in human tissue sample
Report.And the method for the neutrophil leucocyte increased activity of induction human body provided by the present invention, enhance neutral grain using INF- α
The activity of cell, greatly improve the effect of its killing tumor cell (can determine the ratio of N1 cell by cell killing vigor
Example increases), the activity of human body neutrophil leucocyte is improved, has found and N2 type (low activity) neutrophil leucocyte is allowed to be converted into N1 type (height
Activity) neutrophil leucocyte method, to enhance the immune response of body, for the side of clinical neutrophil leucocyte immunotherapy of tumors
Method optimization, therapeutic effect provide reliable theoretical and experiment basis.
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
Neutrophil leucocyte isolation and purification and CNAA (cytotoxic neutrophil antitumor activity) are living
Property method for measuring, step are specific as follows:
1) it takes 200 μ l new bloods in centrifuge tube, splits red rear outer with flow cytometer (model: BD C6 Plus) detection
Leukocyte component and ratio in all blood.Testing result is as shown in Figure 1, be lymphocyte, ratio 32.4%, in R2 in R1
For monocyte, ratio 4.28%, it is neutrophil leucocyte, ratio 52.0% that R3 interior.
2) residual blood is put into centrifuge tube, the 30g/L glucan for adding same volume mixes, and stands 45min, takes
Clean centrifuge tube configuration layering liquid.The Percoll liquid that 80%, 70%, 55%, 45% is sequentially added in centrifuge tube (is purchased from GE
Healthcare company, product article No. are 17-0891-09).
3) supernatant is sucked out after erythrocyte sedimentation, is slowly added on layering liquid.And 30min is centrifuged with 500xg.
4) granulocyte is located between layer second from the bottom and third layer layering liquid after being centrifuged, and sops up supernatant, uses liquid relief
Rifle draws neutrophil leucocyte, collects in centrifuge tube, and take 200 μ l flow cytometry analysis.Analyze result as shown in Fig. 2,
It is neutrophil leucocyte in neutrophil, ratio reaches 95.5%.
5) PBS is added in centrifuge tube, it is soft to mix, 10min is centrifuged with 300xg.It is cultivated when during this period, by bed board
Hela cell inspects 6 holes by random samples from E-Plate detection plate, removes and pancreatin is added after culture solution digests and count, and obtains every hole and is averaged cancer
Cell number.
6) centrifugation terminates, and removes supernatant, and 1ml MEM culture medium (Corning MEM, article No. is added with 1ml liquid-transfering gun
10-010-CVR), blow it is even, take 20 μ l count, obtain neutrophil leucocyte concentration.
7) 50 μ l neutrophil leucocyte suspension (concentration 0.5x10 are added according to required ratio on E-Plate6~
1.8x106Cell/ml) (neutrophil leucocyte: tumour cell=3:1~10:1), each ratio does three holes.To control group
Each hole adds 50 μ l culture solutions.
8) the E-Plate plate for adding neutrophil leucocyte is put on monitor station to (monitor station is placed in 37 DEG C, 5%CO2 incubator
In), continue RTCA (model: ACEA Bioscience.Inc xCELLigence RTCA) reading, carries out dynamic in real time thin
Born of the same parents are proliferated detection to obtain cell Proliferation curve.
9) 10h, 12h, 14h, 16h, cell reading (three holes of same ratio are averaged) for 24 hours are recorded respectively, with
Reading of the control group in the same time compares, and the neutrophil leucocyte of you can get it the blood sample kills cancer activity.According to anti-
Cancer activity can get high activity (N1 type) with the neutrophil leucocyte of low activity (N2 type) two different subgroups as shown in figure 3, in figure,
After different condition handles tumour cell, using the survival condition of RTCA method detection tumour cell, survivorship curve is drawn.It is red bent
Line: normally culture group；Green curve: N2 type neutrophil leucocyte processing group；Blue curve: N1 type neutrophil leucocyte processing group.
10) in the tumour cell of E-Plate equally be added and preceding step same ratio N2 type neutrophil leucocyte and
The IFN-α (10 of various concentration2~103、104~105、106~107IU/ml after) being incubated for 2 hours, confirmed using RTCA technology
IFN-α can induce N2 type and be converted into N1 type neutrophil leucocyte, improves the effect of its killing tumor cell, is illustrated in figure 4 difference
The survivorship curve drawn after condition processing tumour cell using the survival condition of RTCA method detection tumour cell, curve is by upper
Successively under are as follows:
Normal culture group (the 1st article of red line)；
3:1 experimental group (N2 type neutrophil leucocyte: tumour cell): the untreated subgroup of IFN-α (the 2nd bar of light green line) and its 3
The processing subgroup (the 3rd article, 4,5 curves) of a difference IFN-α concentration；
10:1 experimental group (N2 type neutrophil leucocyte: tumour cell): untreated subgroup (the 6th bar of dark blue line) is different with 3
The processing subgroup (the 7th, 8,9 bar of line) of IFN-α concentration.
In addition, adding 50 μ l neutrophil leucocyte suspensions according to required ratio on E-Plate, and various concentration is added
IFN-α(102~103、104~105、106~107IU/ml), each ratio does three holes, record respectively 10h, 12h, 14h,
16h, cell for 24 hours read (three holes of same ratio are averaged), it can be found that in the survival condition and addition of tumour cell
Property granulocyte is related with the concentration of IFN-α.
Embodiment 2
The method for detecting neutrophil leucocyte killing lympha tumour cell (suspension cell), the specific steps are as follows:
1) the CFSE solution for configuring 50nM, first takes 1 pipe CFSE, and the DMSO dissolution of 36 μ l is added thereto, obtains 5mM's
CFSE stoste；It takes the 5mM CFSE of 1 μ l to add the PBS of 1ml, is made into 5 μM of CFSE solution；5 μM of CFSE solution for taking 10 μ l, matches
At the CFSE solution of 1ml 50nM.
2) Daudi cell is taken, 850rpm5min is centrifuged, goes supernatant that fresh culture is added and is resuspended.
3) 1 × 106/ml of cell concentration is aligned, Daudi cell is added to the CFSE solution of 1ml 50nM, is kept away at room temperature
Light incubated cell 20 minutes.
4) it is dyed by being added 5 times of the original dyeing volume of the cell culture medium containing 10%FBS to be quenched.
5) it is centrifuged 850rpm 5min, is resuspended in the cell culture medium of 200 μ l preheating, is protected from light incubated cell 10 minutes.
6) experimental group is set:
A) neutrophil leucocyte control group: is not added
B) sample group 1: neutrophil leucocyte: Daudi=3:1
C) sample group 2: neutrophil leucocyte: Daudi=10:1
7) by 48 orifice plate of Daudi plating cells, 100 μ l Daudi cells are added in every hole
8) it is separately added into neutrophil leucocyte by experimental group, co-cultured for 24 hours.
9) after for 24 hours, cell is collected, CD19 dyeing is added.
10) 4 DEG C of incubation 20min are protected from light, flow cytomery is used.
In embodiment 2 neutrophil leucocyte to tumour cell there are lethal effect as shown in figure 5, in figure A be control group, dye
CFSE, be unstained CD19, C, E, G be respectively neutrophil leucocyte: Daudi=0,3,10 co-culture later for 24 hours situation, B, D,
F, H is respectively situation in CFSE+ in A, C, E, G figure.As the result is shown: neutrophil leucocyte: the CD19 positive in Daudi=0 is
87.9%, Daudi cell survival rate are that 87.9%, Daudi understands some natural death, neutrophil leucocyte: in Daudi=3
The CD19 positive be 50.6%, neutrophil leucocyte: the CD19 positive in Daudi=10 is 20.9%, the addition of neutrophil leucocyte
Declining the survival rate of Daudi cell, i.e. to tumour cell, there are lethal effects.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. a kind of abductive approach of the cell activity of neutrophil leucocyte, comprising:
Fiber differentiation neutrophil leucocyte under the conditions of existing for the IFN-α.
2. abductive approach as described in claim 1, which is characterized in that it is at least part of be induced culture neutrophil leucocyte be
N2 type neutrophil leucocyte.
3. abductive approach as described in claim 1, which is characterized in that the culture medium is selected from MEM culture medium, RPMI 1640
One of culture medium or a variety of combinations.
4. abductive approach as described in claim 1, which is characterized in that the concentration of the IFN-α in culture medium is >=10IU/mL,
It is preferred that >=50IU/mL, more preferably >=102IU/mL。
5. abductive approach as described in claim 1, which is characterized in that the neutrophil leucocyte is human neutrophil.
6. abductive approach as described in claim 1, which is characterized in that the neutrophil leucocyte is isolated and purified by peripheral blood to be obtained
?.
7. abductive approach as described in claim 1, which is characterized in that the cell activity is tumor cytotoxicity activity, excellent
It is selected as increasing the ratio of N1 cell.
Purposes of the 8.IFN- α in neutrophil leucocyte Fiber differentiation.
9. purposes as claimed in claim 8, which is characterized in that the purposes is swollen particularly for enhancing neutrophil leucocyte killing
The effect of oncocyte.
10. purposes as claimed in claim 8, which is characterized in that the purposes is specially to convert N1 type neutrophil leucocyte to
N2 type neutrophil leucocyte, the ratio of N1 cell preferably in increase neutrophil leucocyte.
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| TW201122479A (en) * | 2009-12-30 | 2011-07-01 | Univ Taipei Medical | Kit and method for the capture of tumor cells |
| US20180250336A1 (en) * | 2015-08-31 | 2018-09-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods of enhancing anti-tumor response using hybrid neutrophils |
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2019
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| TW201122479A (en) * | 2009-12-30 | 2011-07-01 | Univ Taipei Medical | Kit and method for the capture of tumor cells |
| US20180250336A1 (en) * | 2015-08-31 | 2018-09-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods of enhancing anti-tumor response using hybrid neutrophils |
| CN111356475A (en) * | 2017-09-18 | 2020-06-30 | 波士顿大学董事会 | Methods for treating NETosis and neutrophil activation |
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