CN110402853A - The detection model and its construction method of immunocyte killing tumour ability and application - Google Patents
The detection model and its construction method of immunocyte killing tumour ability and application Download PDFInfo
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Classifications
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- 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
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of construction methods of the detection model of immunocyte killing tumour ability, it include: after (1) tears egg membrane open to 24-96 hours zebrafish embryos of after fertilization, it will be in the tumor cell injection to zebra fish body of the first living cells dye marker, then it is cultivated in the culture water containing melanin inhibitor, filters out the zebra fish of the visible first living cells dye marker in tail portion；(2) after to the zebra fish anesthesia being collected into, it is divided into immunocyte injection group and control group, the immunocyte of the second living cells dye marker is injected, to control group injection carrier solution to immunocyte injection group in a manner of microinjection, is subsequently placed in the culture water containing melanin inhibitor and is continued to cultivate.The period of the construction method is short, and time saving, laborsaving, modeling cost is low, and the detection model of building accurately can objectively evaluate the effect of tumour immunotherapy.The present invention also provides based on detection model and its application made from this method.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of immunocyte kills detection model and its building of tumour ability
Methods and applications.
Background technique
Malignant tumour has become the arch-criminal for threatening human life, and disease incidence is at ascendant trend.Traditional therapeutic scheme, such as
Operation, radiation and chemotherapy etc., although can improve the prognosis of patient to a certain extent, needle is to patients with advanced cancer
Income is little, and the huge toxic side effect of chemicotherapy further limits its extensive use.Therefore new tumour is found to control
Treatment method is always the popular direction of people's research.Immunization therapy in recent years achieved in therapeutic field of tumor significantly into
Step, it is a big hope for the patient that operation can not be resistant to, chemicotherapy is insensitive and not tolerated.
Immunization therapy can effectively extend the life cycle of patient, improve the quality of life of patient.Especially monoclonal antibody
Para-immunity checkpoint inhibitor, such as programmed death receptor (PD-1) antibody, Cytotoxic T lymphocyte-associated antigen (CTLA-4)
Antibody obtains good curative effect in non-small cell lung cancer, the medium multinomial clinical test of melanoma cells.For another example tumour mistake
It is also concerned after property cellular immunotherapy as a part of immunization therapy, it, which refers to, utilizes the immune thin of itself or donor
Born of the same parents make it have tumour-specific killing ability after induction, screening or gene modification in vitro, are then expanded to one again
Patient's body is fed back after fixed number amount, to achieve the purpose that killing tumor cell, adjusting and enhancing body's immunity.It wraps among these
Natural killer cells (NK), cytokine induced kill cell (CIK), tumor infiltrating lymphocyte (TIL), cell are included
Toxic T lymphocyte (CTL), Combined with Dendritic cytokine induced kill cell (DC-CIK), Chimeric antigen receptor T
A variety of therapeutic schemes such as cell (CAR-T).
Future, immunization therapy will become the important method of oncotherapy.And the drug of immunization therapy how is selected, and
How to screen specific good, immunocyte subgroup that killing ability is strong etc. is particularly important.Therefore, it is necessary to provide one kind
The living animal detection model of immunotherapy can be detected, the research of immunotherapy is objectively completed.It yet there are no this respect
Report.
Summary of the invention
In view of this, the present invention provides a kind of detection moulds of simple, quick, economic immunocyte killing tumour ability
Type and its construction method and application.Using zebra fish as model animal, vascular system and the mankind of zebra fish connect the detection model
Closely, embryo it is transparent, can be in individual cell level, objectively evaluate the immunization therapies preparation such as immunocyte to the killing energy of tumour
Power, the model kill ability for vivo detection immunocyte and provide extremely convenient method and believable platform, have filled up this
The blank of one side.The construction schedule of the detection model is short, and uniformity, reproducible, disturbing factor is few, is conducive to whereby model
To study the effect of immunotherapy.
In a first aspect, the present invention provides a kind of construction method of the detection model of immunocyte killing tumour ability, packet
Include following steps:
(1) 24-96 hours zebrafish embryos of after fertilization are taken, after tearing egg membrane open to it, are anaesthetized using anesthetic, then with aobvious
Zebra fish to the internal of zebra fish, is then set the tumor cell injection of the first living cells dye marker by the mode of microinjection
Cultivate 3-6h in the culture water containing melanin inhibitor, fluorescence microscopy under the microscope, take pictures, collect tail portion visible described the
The zebra fish of the transfer stove of one living cells dye marker transports metastasis model to get to zebra fish tumour blood；
(2) immunocyte of the second living cells dye marker is provided, is resuspended in carrier fluid, obtains immunocyte
Suspension；
Take the zebra fish being collected into step (1), after being anaesthetized, be divided into immunocyte injection group and control group, with aobvious
The mode of microinjection injects the immunocyte suspension to immunocyte injection group, injects isometric carrier fluid to control group,
It is subsequently placed in the culture water containing melanin inhibitor and continues to cultivate 10-15h, obtain the detection of immunocyte killing tumour ability
Model.
Wherein, the carrier fluid is serum free medium, phosphate buffer or water.Immunocyte injection group and control group
The substance injected contains carrier fluid of the same race, and only control group is free of immunocyte.
Optionally, it is described continue culture after, further includes: seen under fluorescent instrument (such as confocal microscope)
Examine, record and the immunocyte injection group and control group in the first living cells dye marker transfer stove number,
To evaluate immunocyte killing tumour ability.
In the present invention, the first living cells coloring agent is different from the fluorescence emission of the second living cells coloring agent.
It can be distinguished under exciting light irradiation.For example, the first living cells coloring agent is the Calcein-AM of green light, institute
Stating the second living cells coloring agent is the Dil (cell membrane red fluorescence probe) to glow.
Wherein, the tumour cell of the first living cells dye marker is that zebra fish is injected into a manner of cell suspension
In vivo, the preparation method is as follows: being diluted to dimethyl sulfoxide (DMSO) solution containing the first living cells coloring agent with PBS buffer solution
(8-12 μM is in the system of the first living cells coloring agent upon mixing to the first living cells dyeing agent solution that concentration is 8-12 μM
Concentration), it is then added into the culture medium containing primary tumor cell, culture is incubated for 30-60 minutes at 37 DEG C, under room temperature
Cell is washed 2-4 times with PBS buffer solution, after being digested with pancreatin, sucks pancreatin, cell is resuspended in serum free medium, obtain
To the tumor cell suspension of the first living cells dye marker.Optionally, in the tumor cell suspension tumour cell concentration
It is 105-107A/mL.Such as 106A/mL.
Wherein, in step (2), the immunocyte suspension the preparation method is as follows: using PBS buffer solution come compound concentration
Agent solution is dyed for 1-3 μM of the second living cells, second living cells dyeing agent solution is added to the culture of immunocyte
In base suspension, culture is incubated for 30-60 minute at 37 DEG C, and centrifugation discards supernatant liquid, washed under room temperature with PBS buffer solution obtained by
Then cell deposition is resuspended with carrier fluid (such as serum free medium), obtains tumor cell suspension.Optionally, described immune
The concentration of immunocyte is 10 in cell suspension5-107A/mL, such as 5 × 106A/mL.
Optionally, the tumour cell be selected from melanoma cells, cervical cancer cell, breast cancer cell, pancreatic cancer cell,
One of liver cancer cells, lung carcinoma cell, stomach cancer cell and colon cancer cell are a variety of.Clinical patient test immunotherapy or
When drug, the primary tumor cell of each patient should be taken to cultivate.
Optionally, the immunocyte be selected from tumor infiltrating lymphocyte (TIL), natural killer cells (NK), cell because
Killing cell (CIK), the cytotoxic T lymphocyte (CTL), Combined with Dendritic cytokine-induced killer cell of son induction
One of cell (DC-CIK), Chimeric antigen receptor T cell (CAR-T) and T cell receptor mosaic type T cell (TCR-T) or
It is a variety of.When clinical patient tests immunotherapy or drug, the primary immune cells that can use each patient are cultivated, a to realize
Body diagnosis and treatment.
Wherein, the zebrafish embryo is wild type or transgenic zebrafish embryo.Optionally, the wild type is AB product
System, the transgenic zebrafish are Tg (fli1:EGFP) strain.The present invention chooses zebra fish and makees modeling animal, cheap,
Modeling period is short, it is often more important that, it is approached compared to mammals, the vascular system of zebra fish and the mankind such as mouse, rabbit, dogs, embryo
Tire is transparent, can in individual cell level, accurate objectively evaluate the killings of the immunization therapies preparation to tumour cell such as immunocyte
Ability.
In addition, the purpose for carrying out tearing egg membrane open to zebrafish embryo before modeling is to reinforce zebrafish embryo to anesthetic, black
The absorption of the drugs such as plain inhibitor promotes the form of zebra fish to stretch, convenient for the observation of subsequent experimental and quantitative.
In the present invention, the mode of the microinjection includes cuvierian duct injection, pericardial injection or yolk sac injection.It is excellent
Selection of land, the mode of the microinjection are cuvierian duct injection or yolk sac injection.It is more suitable for zebrafish embryo in this way, makes to infuse
The medicament penetrated more smoothly reaches blood vessel.
Wherein, in step (1) and step (2), the cultivation temperature of zebra fish is 34 DEG C.Optionally, step (1) and step
(2) in, the dissolved oxygen concentration of the culture water is 6-8mg/L, water temperature is 34 DEG C, pH 7.2-7.6, total hardness 200-
250mg/L.Temperature, dissolved oxygen of the culture water etc. are more suitable for embryo rather than adult fish.
In the present invention, the anesthetic is the substance that zebra fish can not be made lethal.Optionally, the anesthetic is tricaine
(tricain)。
Optionally, in step (1), every zebra fish injection has 100-300 tumour cell.
Optionally, in the culture water, the concentration of melanin inhibitor is 0.1-0.3mmol/L.
Optionally, the melanin inhibitor is phenylthiourea (1-phenyl 2-thiourea, PTU).
In step (1) of the invention, the effect that melanin inhibitor is added is: inhibiting the pigment deposition of zebra fish, prevents
Trunk blackening, in case influence the later period observes the first living cells coloring agent and the second living cells coloring agent under fluorescent instrument
Fluorescence.
In the present invention, the processing through step (1), under fluorescence microscope, tail portion is it is observed that first living cells contaminates
The zebra fish of the fluorescence of toner can be described as " zebra fish of the visible transfer stove in tail portion ", and can be described as " zebra fish tumour blood operating shifting
Model ", it is subsequent to be used for selection tumour immunotherapy and screen in tumour immunity drug.And tail portion transfer stove is deposited
Illustrating that tumour cell successfully passed blood vessel and be transferred in zebra fish body.
Wherein, in the immunocyte injection group of step (2), injection has 1.0 × 10 in every zebra fish3-1.6×103It is a to exempt from
Epidemic disease cell.
The construction method of the detection model for the immunocyte killing tumour ability that first aspect present invention provides is simple, with spot
Horse fish makees modeling animal, and cheap, modeling period is short, and the vascular system of zebra fish and the mankind are close, and embryo is transparent,
It can be observed in individual cell level.The construction method can realize the quick, effective, time saving, laborsaving of Animal Model.The inspection of building
The uniformity, reproducible for surveying model, provides completely new Study of Support for the research of immunotherapy, accurately can objectively evaluate
The immunization therapies such as immunocyte preparation, and can be in physiology, pathologically to for clinically tumour to the killing ability of tumour cell
Treatment provide certain reference frame.
Second aspect, the present invention provides a kind of detection models of immunocyte killing tumour ability.The detection model can
Using constructed by construction method as described in relation to the first aspect.
The third aspect, the present invention provides the detection models of immunocyte killing tumour ability as described in relation to the first aspect
Construction method, or as described in second aspect immunocyte killing tumour ability detection model selection tumour immunotherapy in
Application.Specifically, can be used for evaluating different immunocytes to the killing ability of tumour cell, select specific a kind of or more
Immunocyte is planted to carry out immunotherapy of tumors.
Fourth aspect, the present invention provides the detection models of immunocyte killing tumour ability as described in relation to the first aspect
Construction method, or as described in second aspect immunocyte killing tumour ability detection model screening tumour immunity drug in
Application.Specifically, different immunotherapy preparations (such as tumour immunity medicine can be given to the experimental group of injecting immune cell
Object), it selects specific tumour immunity drug that can promote the killing tumour ability of above-mentioned immunocyte, is exempted from preferably carrying out tumour
Epidemic disease treatment.
Advantages of the present invention will be illustrated partially in the following description, and a part is apparent according to specification
, or can implementation through the embodiment of the present invention and know.
Detailed description of the invention
Fig. 1 is the construction method process for the detection model that immunocyte provided in an embodiment of the present invention kills tumour ability
Figure.
Fig. 2 is the control group (A column) and tumour cell and the experimental group of immunocyte co-injection (B column) for injecting tumour cell
Fluorescence imaging figure of the zebra fish blood fortune metastasis model after 0 hour (I), 12 hours (II).
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Outer without illustrating in the embodiment of the present invention, agents useful for same and consumptive material are commercial goods.
Embodiment 1
Fig. 1 is the construction method process for the detection model that immunocyte provided in an embodiment of the present invention kills tumour ability
Figure, in conjunction with Fig. 1, a kind of construction method of the detection model of immunocyte killing tumour ability provided in this embodiment, including such as
Lower step:
(1) building zebra fish tumour blood transports metastasis model:
A, the tumour cell of fluorescent marker is prepared
All kinds of primary tumor cells are suitable for this model, show here using melanoma cells as specific tumour cell
Example is introduced.It is with the PBS buffer solution (phosphate buffer) of pH=7.4 that the DMSO of the Calcein-AM of coloring agent containing living cells is molten
The Calcein-AM solution that concentration is 10 μM is made in liquid dilution, and the Calcein-AM solution of 1mL is added to containing primary black
In the 10cm culture dish of plain oncocyte.The melanoma cells are incubated at 37 DEG C 30 minutes, then with room temperature PBS buffer solution come
Wash cell twice, after the digestion of 0.25% pancreatin, exhaust pancreatin, adds the RPMI-1640 fresh cultured base weight of serum-free
Outstanding, obtaining the density marked by Calcein-AM is 106The melanoma cells suspension of/mL.
B, zebrafish embryo preparation and tumor cell injection
The embryo for taking after fertilization 48h hours wild type AB strain zebra fish, tears egg membrane open to it, then uses 0.04mg/mL
Anesthetic tricaine (tricain) anesthesia, then with microinjector by the above-mentioned melanoma that Calcein-AM is marked
Cell infusion about injects 200 tumour cells into the cuvierian duct of zebra fish to every zebra fish every time.After injection,
Zebra fish is placed in the culture water of the PTU of melanin inhibitor containing 0.2mmol/L (1-phenyl 2-thiourea, phenylthiourea)
In, and it is placed on 34 DEG C of constant incubator culture.After cultivating 4h, in confocal microscope (Nikon Corporation, Eclipse
C1 it takes pictures under).The zebra fish of the visible obvious transfer stove (i.e. the green fluorescence of Calcein-AM) in tail portion is collected, after
Continuous culture.Collection, which obtains zebra fish, can be described as " zebra fish tumour blood transports metastasis model ", can be used to study sending out for tumour.
(2) preparation of detection model
C, the immunocyte of fluorescent marker is prepared
All kinds of immunocytes are suitable for the preparation of required detection model, are with tumor infiltrating lymphocyte (TILs) here
Example is introduced.Use room temperature PBS buffer solution compound concentration spare for the solution 10mL of 2 μM of cell membrane fluorescence probe (Dil).It will
The solution of 10mL, Dil are added 1 × 106In the RPMI-1640 culture medium suspension of a TILs, in 37 DEG C of incubation 30min to dye,
Then it is centrifuged with the speed of 1500rpm, discards supernatant liquid, cleaned cell precipitation 2 times with room temperature PBS buffer solution, later with no blood
Clear fresh RPMI-1640 culture medium is resuspended, and obtaining density is 5 × 106The TILs immunocyte suspension of a/mL.
D, immunocyte is injected
It takes the zebra fish being collected into above-mentioned (c) to be anaesthetized, is classified as control group and immunocyte injection group is (i.e. real
Test group).Control group injection carrier liquid (isometric serum-free RPMI-1640 culture medium).Experimental group is used after zebra fish is anaesthetized
The immunocyte suspension for having contaminated Dil is injected into the position of cuvierian duct by microinjector, is placed in the culture water at 34 DEG C containing PTU
Constant incubator in cultivate.After cultivating 12h, observes, photograph to record under confocal microscope, by recording and comparing
Marked in experimental group and control group the tumour cell of Calcein-AM green fluorescence number (i.e. transfer stove number) evaluate
Killing ability of the immunocyte to tumour.
Fig. 2 is the fluorescence imaging result of the zebra fish of the embodiment of the present invention 1.In Fig. 2, (I) and (II) small figure is respectively spot
Horse fish blood is same after transporting metastasis model tail portion transfer stove (relative to injection 0 hour) and injecting tumor infiltrating immunocyte 12 hours
Tail zebra fish tail portion transfer stove situation of change, A are control group (only tumour cell and carrier solution), and B column are the bases in A column
Variation diagram (experimental group of tumour cell and immunocyte co-injection) after having added TILs immunocyte on plinth.In Fig. 2, blue is empty
Frame is following figure position in upper figure.The position of red triangular instruction transfer stove.
The A column of (I) small figure can be seen that from Fig. 2, can be slender injecting melanoma cells 0h to zebrafish embryo
Sending out for melanoma cells (green fluorescence of corresponding Calcein-AM) is observed in born of the same parents' level.And (II) small figure in Fig. 2
A, the comparison of the column of B two can be seen that, can observe that immunocyte inhibits in experimental group after injecting immune cell 12h on individual cell level
The sending out of tumour cell.In addition, being carried out at different pharmaceutical by comparing different immunocytes, or to same immunocyte
Reason, this model can preferably it is optimum inhibit tumour cell method, thus guidance clinically to tumour immunotherapy
Specific choice.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of construction method of the detection model of immunocyte killing tumour ability, which comprises the steps of:
(1) 24-96 hours zebrafish embryos of after fertilization are taken, after tearing egg membrane open to it, are anaesthetized using anesthetic, then with micro- note
The mode penetrated by the tumor cell injection of the first living cells dye marker to zebra fish it is internal in, then zebra fish is placed in
3-6h is cultivated in culture water containing melanin inhibitor, in fluorescence microscopy microscopic observation, takes pictures, collects tail portion visible described first
The zebra fish of the transfer stove of living cells dye marker；
(2) immunocyte of the second living cells dye marker is provided, is resuspended in carrier fluid, it is outstanding to obtain immunocyte
Liquid；
Take the zebra fish being collected into step (1), after being anaesthetized, be divided into immunocyte injection group and control group, with micro- note
The mode penetrated injects the immunocyte suspension to immunocyte injection group, injects isometric carrier fluid to control group, then
It is placed in the culture water containing melanin inhibitor and continues to cultivate 10-15h；Wherein, the carrier fluid is serum free medium, phosphoric acid
Salt buffer or water.
2. the construction method of detection model as described in claim 1, which is characterized in that in step (2), continue to cultivate described
Later, further includes: observed under fluorescent instrument, the first living cells in record and the immunocyte injection group and control group
The transfer stove of dye marker, to evaluate immunocyte killing tumour ability.
3. the construction method of detection model as described in claim 1, which is characterized in that described in step (1) and step (2)
It cultivates in water, the concentration of melanin inhibitor is 0.1-0.3mmol/L.
4. the construction method of detection model as described in claim 1, which is characterized in that in step (1), every zebra fish injection
There is 100-300 tumour cell.
5. the construction method of detection model as described in claim 1, which is characterized in that in step (2), the immunocyte note
It penetrates in group, injection has 1.0 × 10 in every zebra fish3-1.6×103A immunocyte.
6. the construction method of detection model as described in claim 1, which is characterized in that the first living cells dye marker
Tumour cell be to be injected into a manner of cell suspension in zebra fish body, the preparation method is as follows: first will be contained with PBS buffer solution
The dimethyl sulphoxide solution of living cells coloring agent is diluted to the first living cells that concentration is 8-12 μM and dyes agent solution, then by it
It is added in the culture medium containing primary tumor cell, culture is incubated for 30-60 minutes at 37 DEG C, uses phosphate buffer under room temperature
Washing cell 2-4 times, after being digested with pancreatin, sucks pancreatin, cell is resuspended in serum free medium, and it is outstanding to obtain tumour cell
Liquid.
7. the construction method of detection model as described in claim 1, which is characterized in that the tumour cell is selected from melanoma
Cell, cervical cancer cell, breast cancer cell, pancreatic cancer cell, liver cancer cells, lung carcinoma cell, stomach cancer cell and colon cancer cell
One of or it is a variety of；The immunocyte is selected from tumor infiltrating lymphocyte, natural killer cells, cytokine induction and kills
It is thin to hurt cell, cytotoxic T lymphocyte, Combined with Dendritic cytokine induced kill cell, Chimeric antigen receptor T
One of born of the same parents and T cell receptor mosaic type T cell are a variety of.
8. the detection model of the killing tumour ability of immunocyte made from construction method as claimed in any one of claims 1 to 6.
9. construction method as claimed in any one of claims 1 to 6 or immunocyte as claimed in claim 8 kill tumour energy
Application of the detection model of power in selection tumour immunotherapy.
10. construction method as claimed in any one of claims 1 to 6 or immunocyte as claimed in claim 8 kill tumour energy
Application of the detection model of power in screening tumour immunity drug.
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