CN108753715A - A kind of immunocyte and its application with oncotherapy function - Google Patents

Abstract

Present invention relates particularly to a kind of immunocyte with oncotherapy function and its applications.Immunocyte provided by the invention is a kind of immunocyte being transfused by external source；It is by combining oncolytic adenovirus, breaking tumour immunity microenvironment first, it is made to generate inflammatory environment, to allow immunocyte preferably to play a role in inflammatory environment, with the effect of enhancing treatment tumour that the immunocyte, which inhibits the ability of tumour,；The immunocyte is cytotoxic T cell (CTL), Dendritic Cells, cytokine induced kill cell (CIK) or tumor infiltrating lymphocyte (TIL).The gene therapy that present oncolytic virus mediates can further release tumor suppression, enhance antitumor immune response, patient's autoimmunity is improved to reach, achieve the purpose that treat tumour by combining immunocyte with oncolytic virus.

Description

A kind of immunocyte and its application with oncotherapy function

Technical field

The present invention relates to biotechnologies, and in particular to a kind of immunocyte with oncotherapy function and its answers With.

Background technology

The diseases such as tumour, cancer have become the major disease for threatening human health now, are the main of human death One of reason.According to statistics, it increases 14,100,000,8,200,000 people of cases of cancer in global range in 2015 newly and dies of cancer, it is contemplated that future 20 Year neopathy number of cases will increase by 70%.Due to aging of population, bad life habits, environmental pollution and traditional means cure rate compared with Low factor, China have become pathogenesis of cancer and dead big country.It counts and shows according to national tumour Register, Chinese cancer in 2015 Disease is 429.16 ten thousand, dead 281.42 ten thousand newly-increased.Wherein lung cancer and gastric cancer are in morbidity and dead front two.Due to diagnosing not In time and the therapeutic scheme of shortage individuation, the inefficiency for the treatment of of cancer are up to 75%.

Now for the therapy of tumour and cancer mainly based on radiotherapy, chemotherapy, operation, tcm therapy is also close The emerging important means for the treatment of cancer and tumour, is clinically used in combination with above-mentioned three kinds of therapies to cancer and tumour over year It is treated, and achieves good effect in the treatment of certain cancers and tumour, people is made to have restored health.But mesh All there are some limitations in preceding radiotherapy, chemotherapy, operation and tcm therapy；For most of tumour, the effect of above-mentioned therapy is still It is ideal not to the utmost, therefore, clinically it is badly in need of studying more efficiently therapy and means.

As therapy emerging at present, it can prepare medicine according to patient body difference and control biotherapy Treatment scheme has many advantages, such as that toxic side effect is less compared with present traditional remedies, tumour specific aim and specific stronger, quilt It is considered to treat the new hope of tumour.

Invention content

For treatment means such as current chemotherapy, radiotherapy, operations for the therapeutic domain of tumour and cancer, also by compared with the day of one's doom System, to the therapeutic effects of some tumours also there is also problem ideal not to the utmost, present invention aims at provide a kind of to have tumour Treat immunocyte and its application of function.

As therapy emerging at present, it can prepare medicine according to patient body difference and control biotherapy Treatment scheme has many advantages, such as that toxic side effect is less compared with present traditional remedies, tumour specific aim and specific stronger, quilt It is considered to treat the important hope of tumour, there is no the direction of the malignant tumour of method healing at present especially for some.In recent years, Immune cell therapy in biotherapy has obtained quick development, and good control is obtained in the treatment of some tumours Therapeutic effect shows it with important medical value and extensive market prospects.

To achieve the above object, the present invention is achieved by the following technical programs：

A kind of immunocyte and its application with oncotherapy function, the immunocyte are cytotoxic T cell (CTL), one kind in natural killer cells, cytokine induced kill cell (CIK).

Preferably, the immunocyte is cytotoxic T cell (CTL) or cytokine induced kill cell (CIK)。

It is highly preferred that the immunocyte is cytokine induced kill cell (CIK).

Preferably, the immune activation factor is interleukin 18.

Preferably, the oncolytic adenovirus be it is artificial reconstructed after can selectively infect and crack the disease of tumour cell Poison.

It is highly preferred that the oncolytic adenovirus is the adenovirus by genetic modification.

Preferably, the viral infusion be by oncolytic adenovirus direct injection where tumor focus position.

Preferably, the oncolytic adenovirus be by oncolytic adenovirus direct injection where tumor focus position.

Preferably, the oncolytic adenovirus is the virus that can express immune activation gene IL-18.

Preferably, the cell carrier is slow virus.

A kind of application of the immunocyte with oncotherapy function：

S1. molecular cloning is carried out, plasmid, recycling DNA are prepared and digestion, connection are carried out to DNA；

S2. the recombination of adenovirus vector is carried out；

S3. the slow virus carrier of structure expression Luciferase；

S4. virus preparation and titer determination；

S5.CIK cell culture and cellkilling capacity detection；

S6. fluorescence quantitative PCR detection gene expression；

S7. the expression of adenovirus Hexon and the immune enzyme-linked expression for adsorbing detection IL-18 in immunohistochemistry detection tissue.

Compared with prior art, the present invention has the advantages that：

According to another aspect of the present invention, it the present invention provides a kind of immunocyte with oncotherapy function and its answers With can increase infiltration quantity of the immunocyte in tumor tissues, enhance the antitumor action of immunocyte, realize " cell- The therapeutic effect of virus-gene " combination among the strong ones has positive effect for the treatment of liver neoplasm.

Specific implementation mode

The present invention is further elaborated with reference to specific embodiment, the embodiment is served only for explaining this hair It is bright, it is not intended to limit the scope of the present invention.Test method used in following embodiments is routine unless otherwise specified Method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.

Embodiment 1

In the present embodiment, the anti-swollen of combined immunization cell therapy is treated by internal and external test research " virus-gene " Tumor acts on, and the antitumous effect of related mechanism in combination；And pass through single therapy group (AdCN215-GFP, AdCN215- IL18, CIK) and combination therapy group (AdCN215-GFP and CIK, AdCN215-IL18 and CIK) compared, check it is thin with CIK Born of the same parents as carrier, be carried through interleukin 18 carried out the oncolytic adenovirus of genetic modification for the therapeutic effect of liver neoplasm and Effect.It is specific as follows：

A kind of immunocyte with oncotherapy function

The measurement of the preparation and titer of virus

5 type oncolytic adenovirus AdCN215-GFP of double regulation and control；5 type adenovirus Ad-EGFP1 of not replicated is that voluntarily structure preserves. Slow virus skeleton plasmid pCDH-EF1 α are purchased from addgene, slow virus packaging plasmid pMD2.G, pMDLg/pRRE and pRSV-Rev It is purchased from Addgene；The PGL3-basic plasmid origins of Luciferase genes are expressed in Promega companies.Express mcherry bases The plasmid Nanog-2A-mCherry of cause is purchased from Addgene.

Experiment cell strain used and its source, condition of culture property

HEK293 (human embryonic kidney cell line of adenovirus E 1 area conversion), HEK293T (adenovirus E 1 area and large T antigen conversion Human embryonic kidney cell line), PLC/PRF/5, SMMC7721 and HuH-7 (Bel7402) DMEM+10%FBS, adherent growth

Test method

1. molecular cloning

The preparation of 1.1 plasmids

1.2 Plasmid DNA are largely extracted:(using Omega kits)

1.3 recycle DNA from Ago-Gel

1.4 digestions with connect

1.4.1 Plasmid DNA digestion

It is separately added into 10 × digestion Buffer of 4 μ l, the DNA of 2ug is added, 0.5 μ l restriction enzymes is first added inward Enzyme is then added aseptic double-distilled water and complements to 40 μ l volumes, and mixing is put in digestion 1-4h in the water-bath that temperature is 37 DEG C.

1.4.2 the connection of DNA fragmentation and carrier

10 × connection Buffer of 2 μ l is first added, adds two DNA fragmentations, 0.5 μ l ligases is first added inward, with It is 20 μ l that aseptic double-distilled water to system is added afterwards, and mixing, and 4h is connected at 16 DEG C.

1.5 adenovirus vectors recombinate

The slow virus carrier structure of 1.6 expression Luciferase

1.7 viruses prepare and titer determination

1.7.1 AdIL18, AdCN215-IL18, AdCN215-GFP adenovirus virus are packed, purifying and titre identification

1) restriction enzyme Pac I is used to linearize virus particle.

2.5 μ l Pac of adenoviral plasmid, I 0.5 μ l

L Buffer 2.5μl ddH₂O 19.5μl

25 μ l of total system

37 DEG C of water-bath 2h, digestion products are recycled by ethanol precipitation.

2) packaging virus in HEK293 cells.

Will recycling post-fragment transfect into HEK293 cells, transfection use Effectene transfection reagent boxes, by specification into Row operation.

Eve every hole in 6 orifice plates is transfected to spread into 1 × 10⁶HEK293 cells.Next day by I enzymes of Pac of about 1 μ g it is linearized after Plasmid add in people EC buffer to 150 μ l of total volume, add 8 μ l of Enhancer buffer, vibrated on vortex oscillation instrument 3s mixings, stand 5min at room temperature.25 μ l of transfection reagent Effectene are added in said mixture, shake on vortex oscillation instrument 15s mixings are swung, are incubated 10min at room temperature.Supernatant is abandoned in the suction of HEK293 cells, and the PBS being added after 2ml warm bath washed once, and is added Culture medium after the fresh warm bath of 1ml, separately plus 0.5ml cultures are based in above-mentioned plasmid-transfection reagent mixtures, add after mixing well Into cell, containing 5%CO₂, culture medium (fresh cultures of the 2ml containing 2%FBS) is replaced after culture 6-18h at 37 DEG C.Observation Cell growth status, virus plaque about occurred at 9-14 days.

4) amplification Yu purifying of adenovirus and titer determination

Viral AdCN215-IL18, AdCN215-GFP, AdIL8 are expanded, purifying and titer determination.Viral is big Amount amplification is with purifying by HEK293 cells with 3 × 10⁶The density of/dish is inoculated in 30-40 10cm dish, and (10ml contains 2% The DMEM culture mediums of FBS), when cell growth is to degrees of fusion 70-80%, it is added into each dish proper amount of packaged Virus (about 10⁷-10⁸PFU/ml)10μl.Observe cell, infection 2-3 days after when cell major part portion's lesion, 1200rpm from The heart abandons supernatant and collects cell.Cell precipitation is that 1.10g/ml CsCl solution is resuspended with 2ml density.By multigelation 5 times, break Chopping fine born of the same parents.5000rpm centrifuges 10min and collects broken supernatant, and supernatant is further removed with 0.45 μm of membrane filtration Cell fragment.Spread the 1.40g/ml CsCl into 2.0ml successively respectively in the special 15ml centrifuge tubes of Beckman ultracentrifuges Solution, top layer spread the filtered viral supernatants into about 5ml.22800rpm centrifuges 2.5h at 4 DEG C.In 1.30- at that time 1.40g/ml joins interface and collects viral item, is carefully transferred to virus band with Pasteur pipe and boils processed bag filter In.Bag filter is being positioned over elution buffer (50g sucrose, the Tris-HCl liquid that 10ml 1M pH are 8.0,2ml 1M MgCl₂Solution is settled to 1000ml) in, the dialysed overnight in 4 DEG C of refrigerators replaces a dialyzate per 6h.Collect disease afterwards for 24 hours Poison is placed in -80 DEG C of refrigerators after packing and preserves for use.

The CsCl solution of different densities is prepared, and in 4 DEG C of preservations.

5) measurement (using Cellbiolabs companies adenovirus virus detection kit) of adenovirus titre

1.7.2 slow virus poison is packed, purifying and titre identification

1) packaging virus in HEK293 cells (calcium phosphate transfection method packs slow virus)

A) 12h before virus packaging, HEK293T cells are with 1 × 10⁷The density of a/bottle is laid on 75cm²Tissue Culture Flask in；

B) 2h before virus coating, it is newer to change cell culture fluid；

C) 30 μ g pCDH-EF1 α-luciferase-2A-mCherry, 20 μ g PGK-GFP；5μg pMDLg/pRRE； 3μg pRSV-Rev；12 μ gpMD2.G are mixed in proportion, and 2 × HEPES of 1ml is added to be uniformly mixed；

D) 1ml 0.5M CaCl are added dropwise in mixed solution₂Solution blows and beats mixing with pipettor repeatedly；

E) mixed liquor after mixing is in being placed at room temperature for 20min；

F) 75cm is added in mixed solution dropwise with liquid-transfering gun²Tissue Culture Flask in, be uniformly mixed；

G) after transfecting 5-8h, original cell culture fluid is discarded, the culture solution containing DMEM that the new warm bath of 15ml is added (contains 2% FBS)；

H) after transfecting 48h, culture supernatant is collected, and the culture solution containing DMEM for continuously adding the new warm bath of 15ml (contains 2% FBS)；

I) after transfecting 72h, culture supernatant is collected, is merged with the 48h supernatants collected, with being protected after 0.45 μm of aperture membrane filtration - 80 DEG C of refrigerators are stored in, follow-up test is used for.

2) purifying (using kits) of slow virus

3) slow virus titer determination

A) HEK293T cells are pressed 2 × 10 by 12h before infecting⁵It is incubated in 24 well culture plates per hole；

B) 2h replaces culture medium before infecting, and uses the training liquid of the DMEM containing 5%FBS instead and continues to cultivate；

When c) infecting, 1 a concentration of 16mg/mLPolybrene of μ l are added per hole；

D) virus (0.1 μ l, 1 μ l, 10 μ l) that purifying obtains is separately added into cell.Utilize streaming thin after cultivating 72h Born of the same parents' instrument detects HEK293 cell fluorescence positive ratios；

E) the viral effective titre in virus stock solution used is calculated by formula.

T=(P × N)/(D × V)

In formula：T indicates the i.e. viral titre of the infectious unit for including in every milliliter, unit TU/ml；

P indicates the ratio of mCherry positive cells；

N indicates the quantity of cell when infection；

D indicates diluted multiple；

V indicates the virus liquid volume being added.

1.8 CIK cell cultures

1.8.1 PBMC cells detach

Peripheral blood mononuclear cells detaches, by the way of glucan density gradient separation.

The specific steps are：

1. average mark after the 50ml peripheral bloods being collected 20ml syringe collectings is filled to two 50ml sterile tubes (A, B Pipe) in, it is separately added into 6%HES (hydroxyethyl starch) 5ml.

2. standing 30min after mixing, red blood cell and HES are combined and are fallen to centrifugation bottom of the tube.

3. the blood plasma that leucocyte is contained on A, B pipe top is moved to new centrifuge tube (C pipes).By A and B pipes centrifugation (400 × g, After 10min), take a small amount of blood plasma on red blood cell top that C pipes are also added.

4. centrifuge tube C is centrifuged 10min with 400 × g, supernatant moves to new centrifuge tube (D pipes)；The cell of C pipes lower part is used It is detached in PBMC.

5. D pipes are put into 56 DEG C of water-baths and inactivate complement component 3 0min.

6.D pipes centrifuge (800 × g, 20min) and remove flocky precipitate, and supernatant is moved to new centrifuge tube (E pipes).

Supernatant (about 25ml) in 7.E pipes can add use as the autologous plasma of patient in cell culture.

8. 15ml PBS are added in above-mentioned C pipes, overturn 5-6 times and mix well repeatedly.

9. taking 2 new 50ml sterile tubes, paving plus 15ml human lymphocyte separating liquids will be given birth to disposable sterilized pipette Blood 15ml after reason brine dilution is slowly added to separating liquid upper layer.750 × g centrifuges 20min at 23 DEG C.

10. drawing middle layer white ribbon to 50ml sterile centrifugation tubes, the physiological saline of drawn 4 times of volumes of liquid is added 10min is centrifuged after mixing.

11. abandoning supernatant, cell precipitation is collected, 10ml RPMI1640 culture mediums are added.5min is centrifuged under 1200rpm.And It is primary to repeat above step.Cell is resuspended 10ml training liquid.

1.8.2 CIK cell culture

1. cell after separation is resuspended with RPM1640 culture mediums of the 10ml containing 10%FBS, cell concentration is adjusted to 2.0 × 10⁶/ ml is laid on 75cm²In Flask.In 5%CO₂, 37 DEG C of incubator culture 2h.

2. jiggling Flask, non-attached cell is collected, cell density is adjusted to 1.0 × 10⁶INF- γ are added in/ml To 1000U/ml, in 5%CO₂, 37 DEG C of incubator cultures for 24 hours.

After 3.24h, CD3 monoclonal antibodies OKT-3 (50ng/ml), IL-1 α (100U/ml), IL-2 (300U/ are added into cell ml).In 5%CO₂, cultivate in 37 DEG C of incubators.

4. it is isometric per 2-3 days that fresh medium is added according to cell growth status, IL-2 (300U/ml) is added every time. The cell of culture 10-14 days is used for follow-up test.

1.8.3 the expression detection of the surface marker of CIK cell

1. collecting cell

Culture is collected to 14 days CIK cells in centrifuge tube, with rotating speed 1000rpm centrifugation 3min, supernatant is abandoned and collects and sink Cell is resuspended to density 1.0 × 10 with the PBS containing 1%BSA in shallow lake cell⁶/ml。

2. fluorescent marker

The cell that PBS is resuspended is separately added into the antibody or Isotype control of 10 μ l fluorescent markers, gently with the 100 μ l packing of every pipe Bomb tube bottom, mixes well cell, and 30min is incubated in 4 DEG C of refrigerators.After incubation, washed 2 times with PBS, 1200rpm rotating speeds Centrifuge 3min.Finally cell is suspended in 300 PBS of the μ l containing 1%BSA, carries out flow cytomery.

1.9 cellkilling capacities detect

1.9.1 CCK8 detects killing ability of the adenovirus to liver cancer cells

(1) in 96 porocyte culture plates, by 1.5 × 10⁵Normal cell or liver cancer cells are laid on 96 holes by the density of/ml In plate, per 100 μ l of hole；

(2) after 12h, original culture medium is abandoned in suction, and 100 adenovirus of the μ l containing specified MOI, every group of 8 repetitions are added per hole；

(3) 10 μ l CCK8 were added into cell and detect liquid the time point specified after virus is added, and 37 DEG C are continued to cultivate 2h Afterwards each hole absorbance value under 490nm is detected with microplate reader.

(4) inhibiting rate of the adenovirus to cell growth is calculated by formula：

Cell survival rate=(experimental group OD values-blank control group OD values)/(negative control group OD values-blank control group OD Value).

1.9.2 the killing of Luciferase luminous intensities method detection CIK cell and CIK joint oncolytic virus to target cell Ability

1.9.2.1. it utilizes pCDH-EF1 α-Luciferase-2A-mCherry slow virus to build and stablizes expression The cell line of Luciferase

1) cell line for infecting slow virus will be needed with 1.5 × 10⁵/ ml density is laid in 6 porocyte culture plates, per hole 2ml liquid；

2) after cell adherent growth 12h, the α-Luciferase-2A-mCherry slow virus of pCDH-EF1 containing 5MOI is replaced, The fresh culture of 8 μ g/ml Polybrene；

3) after 6h is added in virus, original culture medium is abandoned in suction, and fresh complete medium is added；

4) after virus infection 72h, trypsin digestion and cell is collected cell progress airflow classification and (is carried out for mCherry Sorting).

1.9.2.2. Luciferase luminous intensities are verified and cell number is linear

1) cell line of expression Luciferase will be stablized respectively from 1 × 10³-2×10⁴By 1 × 10³Density gradient is laid on In 96 orifice plates；

2) after the adherent raw 6h of cell, supernatant is abandoned in suction, and 50 μ l Promega companies luciferase assay detection kits are added In PLB 1 × cracking buffer, place 5min at room temperature；

3) 2000rp m centrifugations 5min removes cell fragment at 4 DEG C；

4) it draws in supernatant 10 μ l to 96 orifice plate of white background, 50 μ l of luminous substrate is added；

5) plate is detected to luminescent lumen intensity value in microplate reader

6) linear analysis is carried out by cell number and luminescent lumen intensity value；

1.9.2.3. the killing ability of CIK cell and CIK joint oncolytic virus to target cell is detected

1) target cell for stablizing expression Luciferase is pressed 1 × 10⁴It is laid in 96 orifice plates；

2) effector cell is pressed into effect target than 5:CIK cell is added in target cell 1 ratio；

3) viral group or viral combined CIK groups, by virus MOI numbers by viral adding hole, single CIK groups or negative control group The PBS of respective volume is added；

4) for 24 hours, 48h, 72h, 96h different time points collect target cell carry out Luciferase luminous intensity detection.

5) tumor control rate=1- (experimental group lumen value-blank control group lumen value)/(negative control group lumen value-sky White control group lumen value) × %.

2.0 fluorescence quantitative PCR detection gene expressions

2.1 immunohistochemistry detect the expression of adenovirus Hexon in tissue

1, dewaxing and aquation

1) paraffin section, which is placed in dimethylbenzene, impregnates 10min；

2) in soaked in absolute ethyl alcohol 5min；

3) 5min is impregnated in 95% ethyl alcohol；

4) 5min is impregnated in 70% ethyl alcohol；

2.1.1 antigen retrieval

Antigen thermal high hot repair is multiple, and EDTA (pH8.0) buffer solution is added in boiling water.Slide is placed in metallochromy frame On, after Pressure method 10min, heat source is removed, is placed in cold water.

2.1.2 immunohistochemical staining

1) PBS impregnates 2~3 times, each 5min；

2) serum block is added dropwise, is stored at room temperature 20 minutes, gets rid of surplus liquid；

3) I anti-50 μ l after diluting are added dropwise, 4 DEG C stand overnight；

4) 4 DEG C overnight after in 37 DEG C of rewarming 45min；

5) PBS impregnates 2~3 times, each 5min；

6) II anti-50 μ l after diluting are added dropwise, are stored at room temperature 1h；

7) PBS impregnates 2~3 times, each 5min

8) 5~10min of DAB colour developings, grasps dye levels under the microscope；

9) 10min is rinsed under tap water；

10) haematoxylin redyeing 2min, hydrochloride alcohol differentiation；

11) tap water rinses 10~15min；

12) dehydration, transparent, mounting, microscopy.

2.1.3 the expression (being detected using ELISA Set kits) of enzyme-linked absorption detection IL-18 is immunized

By by oncolytic virus AdCN215-IL18 in tumor cell lines such as BEL7404, HuH-7, SMMC7721 and normal Virus reproductivity in liver cell AKN-1 compares and analyzes, and test result shows that oncolytic virus AdCN215-IL18 exists It can be replicated in the tumor cell lines such as BEL7404, HuH-7, SMMC7721, virus amplification is about 120 times, and AdCN215- IL18 is substantially difficult to replicate in normal liver cell, and replication capacity is poor, and the duplication of this display oncolytic virus has tumour special It is anisotropic；And BEL7404 and HuH-7 tumor cell lines after ELSIA detection display infection oncolytic virus AdCN215-IL18 and The IL18 expressions of normal liver cell AKN-1, as a result the IL18 expressions in tumor cells showed system be also substantially higher in Expression in normal liver cell AKN-1 shows that foreign gene IL18 expression has tumour-specific；By comparing oncolytic Viral AdCN215-IL18 is also obviously better than it to just to the killing effect in vitro of tumour cell BEL7404, HuH-7, Hep3B The killing effect in vitro of normal liver cell AKN-1, killing capacity variance obviously have biological significance, this shows AdCN215-IL18 There is target killing ability for tumour.

Whether have to CIK cell killing ability in the lethal effect and discussion IL-12 of detection CIK cell pairing effect cell There is the test result of facilitation to show, simple IL-18 is to the no inhibiting effect of the growth of tumour cell, in effect target 20:1 In the case of, IL-18 promotes the extracorporeal anti-tumor function of CIK cell, 36.55% is promoted to by 14.5% ± 2.45% ± 1.56%, P=0.000488,40:In the case of 1 antitumor action be promoted to 73.36% by 50.25% ± 1.35% ± 1.05%, p=0.002355 prompt IL-18 that can be obviously improved inhibiting effect of the CIK cell to liver cancer cells.

The test result of extracorporeal anti-tumor function for enhancing CIK cell in detection expression IL-18 show, oncolytic virus and Whether the united antitumor action of CIK cell and the oncolytic virus combination therapy for expressing IL-18 can further be promoted anti-swollen Tumor ability；In effect target than 10:1, in the case that viral multiplicity of infection is 10MOI, for liver cancer cell lines HuH-7,48h when Between point CIK cell combine the more single CIK groups of AdCN215-GFP groups, tumor cell survival reduce, significant difference, CIK cell combine The more single CIK groups tumor cell survival of AdCN215-IL18 groups reduces, and difference is extremely notable；Time point CIK of 72h combines AdCN215-IL18 groups are stronger compared with the tumor inhibition effect of joint AdCN215-GFP groups, significant difference.

Transplanted tumor model treatment results are shown in vivo, single therapy group (AdCN215-GFP, AdCN215-IL18, CIK) phase There is certain tumor inhibitory effect, and significant difference for PBS groups, but significant difference is had no between single therapy group. CIK cell combines AdCN215-GFP groups has significant difference compared with single therapy group AdCN215-GFP and CIK.CIK cell is combined There is AdCN215-IL18 groups strongest antitumor action, more all single therapy groups to have pole significant difference, combine compared with CIK AdCN215-GFP group antitumous effects are more notable.Find there are 3 in CIK cell joint AdCN215-IL18 groups in therapeutic process Mouse tumor completely disappears.

Finally, double targeting oncolytic virus AdCN215-IL18 of CIK cell Combined expression IL-18 genes have collaboration anti- Function of tumor finds that the tumor lysis effect of oncolytic virus and the expression of tumor by local high concentration IL-18 increase CIK cell and exist Infiltration quantity in tumor tissues, enhances the antitumor action of CIK cell, realizes " cell-virus-gene " combination among the strong ones Therapeutic effect, for liver neoplasm treatment have positive effect.(test result is as shown in table 1)

Table 1

Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art answer Work as understanding, technical scheme of the present invention can be modified or replaced equivalently, without departing from the reality of technical solution of the present invention Matter and range.

Claims (9)

1. a kind of immunocyte and its application with oncotherapy function, which is characterized in that the immunocyte is cell One kind in cytotoxic T cell, cytokine induced kill cell or natural killer cells.

2. the immunocyte according to claim 1 with oncotherapy function, which is characterized in that the immunocyte For cytotoxic T cell or cytokine induced kill cell.

3. the immunocyte according to claim 1 with oncotherapy function, which is characterized in that the immune activation The factor is interleukin 18.

4. the immunocyte according to claim 1 with oncotherapy function, which is characterized in that the oncolytic adenopathy Poison be it is artificial reconstructed after can selectively infect and crack the virus of tumour cell.

5. the immunocyte according to claim 1 with oncotherapy function, which is characterized in that the virus infusion Mode be by oncolytic adenovirus direct injection where tumor focus position.

6. the immunocyte according to claim 4 with oncotherapy function, which is characterized in that the oncolytic adenopathy Poison is the mosaic type adenoviral by genetic modification.

7. the immunocyte according to claim 5 with oncotherapy function, which is characterized in that the oncolytic adenopathy Poison is the virus that can express immune activation gene IL-18.

8. the immunocyte according to claim 7 with oncotherapy function, which is characterized in that the carrier is slow Virus.

9. a kind of application of the immunocyte with oncotherapy function：

S1. molecular cloning is carried out, plasmid, recycling DNA are prepared and digestion, connection are carried out to DNA；

S2. the recombination of adenovirus vector is carried out；

S3. the slow virus carrier of structure expression Luciferase；

S4. virus preparation and titer determination；

S5.CIK cell culture and cellkilling capacity detection；

S6. fluorescence quantitative PCR detection gene expression；

S7. the expression of adenovirus Hexon and the immune enzyme-linked expression for adsorbing detection IL-18 in immunohistochemistry detection tissue.