CN101940796A - Preparation of gene immunization treating tumor used for vein - Google Patents

Abstract

The invention discloses a preparation of gene immunization treating tumor used for veins, which is composed of replication deficient adenovirus loading human telomerase reverse transcriptase C-terminal polypeptide hTERTC27 and an acceptable pharmaceutic adjuvant. The anti-tumor gene preparation rAdv-hTERTC27 of the invention has high transfection efficiency, and is more effective on the increment and the apoptosis regulation of the tumor cell. The anti-tumor gene preparation of the invention can effectively lead the peptides transfection to enter into the dendritic cell DCs, so as to activate the DCs, secrete IFN-gamma, IL-2, increase and further stimulate T lymphopoiesis, promote the T lymphocyte to generate the special killing effect CTL corresponding to the tumor cell, and immunoregulation is carried out on the tumor.

Description

A kind of intravenous formulation of genetic immunization treatment tumor

Technical field

The present invention relates to a kind of gene formulations, particularly a kind of intravenous formulation of genetic immunization treatment tumor.

Background technology

HTERTC27(C-terminal fragment of the human telomerase reverse transcriptase, hTERTC27), it is the human telomerase reverse transcriptase C-terminal polypeptide of a 27kD, can cause the dysfunction of telomere, end of chromosome is merged fast and do not influence the activity of telomerase, cause hTERT ⁺The apoptosis of HeLa cell and aging, on the nude mice model of HeLa cell, produced tangible tumor killing effect (Huang, J., Lin, M. C., Bai YX, Jing D, Wong BC, Han SW, Lin J, Xu B, Huang CF, Kung HF. Ectopic expression of a COOH-terminal fragment of the human telomerase reverse transcriptase leads to telomere dysfunction and reduction of growth and tumorigenicity in HeLa cells. Cancer Research. 62:3226-3232,2002.; .Huang, J. J., Han SW, Bai YX, Ng SSM, Jing DD, Wong BCY, Huang CF, Kung HF, and Lin MC. A human TERT C-terminal polypeptide sensitizes HeLa cells to H ₂O ₂-induced senescence without affecting telomerase enzymatic activity. Biochemical and Biophysical Research Communications. 301:627-632,2003.).In the nude mice that has inoculated people's glioblastoma cell (U87-MG), hTERTC27 also suppresses growth of tumor (Ng SS significantly, Gao Y, Chau DH, Li GH, Lai LH, Huang PT, Huang CF, Huang JJ, Chen YC, Kung HF, Lin MC.A novel glioblastoma cancer gene therapy using AAV-mediated long-term expression of human TERT C-terminal polypeptide. Cancer Gene Therapy. 14:561-72,2007.).

HTERTC27 can be used as anti-hTERT ⁺The effective gene therapy of tumor cell, simultaneously, hTERTC27 can induce melanoma C57BL/6 mice cells in vivo and humoral immunization, suppresses melanomatous growth significantly.This shows that hTERTC27 can be by Gene regulation and the combined effect of immunomodulating double mechanism in hTERT ⁺Tumor cell plays a kind of broad-spectrum antitumous effect.Discovery hTERTC7 such as Huang Junjian professor and Kong Xiangfu academician can cause that the telomere functional disorder does not influence telomerase activation and suppresses growth of tumour cell at present, successfully apply for and obtain United States Patent (USP) (US 7294708).

In US 7294708, what adopt is that the simple plasmid that carries hTERTC27 is to the tumor cell transfection and observe its influence to tumor cell telomere function, and filter out the HeLa cell strain of stable transfection hTERTC27 and EGFP plasmid, become tumor in the nude mice subcutaneous vaccination, observe the inhibitory action of hTERTC27 tumor growth.The great like this performance that limits the hTERTC27 effect; Simultaneously, the subcutaneous plantation of HeLa cell nude mice of stable transfection hTERTC27 has certain difference with becoming tumor naturally.

Summary of the invention

The object of the present invention is to provide a kind of preparation of genetic immunization treatment tumor.

The technical solution used in the present invention is:

A kind of intravenous formulation of genetic immunization treatment tumor is made up of replication-defective adenoviral loading human telomerase reverse transcriptase C-terminal polypeptide gene hTERTC27 and pharmaceutic adjuvant acceptable.

Preferably, replication-defective adenoviral is Ad5.

Antioncogene preparation rAdv-hTERTC27 of the present invention, the transfection efficiency height is regulated more effective to the increment and the apoptosis of tumor cell.Simultaneously, Antioncogene preparation of the present invention, can effectively the transfection of purpose peptide section be entered dendritic cell DCs, DCs is activated, secretion of gamma-IFN, IL-2, increase and the further T of stimulation lymphopoiesis, and impel the T lymphocyte that corresponding tumor cell is produced specific lethal effect CTL, tumor is carried out immunomodulating.

Description of drawings

Fig. 1 is that the seed culture of viruses of hTERTC27 is identified electrophoretogram;

Fig. 2 is RT-PCR, the Western blot electrophoretogram behind the rAdv-hTERTC27 viral infection target cell;

Fig. 3 is the influence figure of rAdv-hTERTC27 to hepatocarcinoma (Hepa 1-6), glioma (U87) cell proliferation and apoptosis;

Fig. 4 is the figure that influences of rAdv-hTERTC27 pair cell telomerase activation;

Fig. 5 is a rAdv-hTERTC27 transfection derived from bone marrow DCs design sketch;

Fig. 6 is the influence of rAdv-hTERTC27 transfection to the DCs secrete cytokines;

Fig. 7 is that rAdv-hTERTC27-DCs promotes the lymphopoietic effect of T;

Fig. 8 is the effect that rAdv-hTERTC27-DCs promotes T lymphocyte killing tumor cells target cell;

Fig. 9 is to the figure that influences of rat liver cancer gross tumor volume and life cycle behind the injection rAdv-hTERTC27.

The specific embodiment

A kind of intravenous formulation of genetic immunization treatment tumor is made up of replication-defective adenoviral loading human telomerase reverse transcriptase C-terminal polypeptide gene hTERTC27 and pharmaceutic adjuvant acceptable.Pharmaceutic adjuvant acceptable is that ability waveguide technology personnel are known, as normal saline etc.

Preferably, replication-defective adenoviral is Ad5.

Replication-defective adenoviral Ad5 is by skeleton plasmid pBHGlox_E1, two plasmid co-transfection 293 cells of 3Cre and shuttle plasmid pDC316-hTERTC27-EGFP, and homologous recombination produces recombinant adenovirus.

Below in conjunction with embodiment, further specify the present invention.

RAdv-hTERTC27(recombinant adenovirus-hTERTC27-EGFP) preparation and evaluation:

1) with 293 cell inoculations in six orifice plates, every hole 5 * 10 ⁵Individual cell, culture medium are DMEM+10% Hyclone hyclone, put 37 ℃ and contain 5% CO ₂Incubator in overnight incubation;

2) change liquid, continue to be cultured to cell with the fresh DMEM culture medium that contains 10% hyclone and grow to 80～90% of floor space; Get 24 μ g skeleton plasmids and 6 μ g shuttle plasmids, mix homogeneously, wherein skeleton plasmid is selected pBHGlox_E1 for use, 3Cre, shuttle plasmid is selected pDC316-hTERTC27-EGFP for use;

3) plasmid of the mix homogeneously DMEM culture fluid with 1800 μ l is diluted, room temperature is placed 5min; The Lipofectamine2000 liposome of getting 60 μ l dilutes with the DMEM culture fluid of 1800 μ l, and room temperature is placed 5min;

The mixing plasmid that 4) will dilute mixes with the liposome of dilution, and the room temperature lucifuge is placed 30imn; The mixture that the room temperature lucifuge is placed equably with cultured mixing with cells, carry out transfection with reference to Lipofectamine2000 liposome description;

5) after the transfection second day, with the passage that covers with in 25cm ²In the Tissue Culture Flask, continue to cultivate with the DMEM culture medium that contains 5% hyclone, observe every day, treat cell cover with bottle at the bottom of the time, repeated transmission is gone into 75 cm ²In the Tissue Culture Flask,, be botryoidalis, and begin to occur obvious plaque, show that cell goes out poison when cell becomes the big circle that becomes, treat the most of pathological changes of cell and come off from the bottom after collect out poison cell;

6) Tissue Culture Flask that will go out poison is placed in-70 ℃ of refrigerators and the 37 ℃ of water-baths multigelation earlier three times, and virus is fully discharged from sick cell, with the centrifugal 5min of freeze thawing liquid 3000rpm, collects the supernatant that contains virus, wins for seed culture of viruses (P1);

7) use the P1 rotaring redyeing 293 cell, receive poison after cultivating, pcr amplification, electrophoresis identify errorless after, obtain second filial generation seed culture of viruses (P2);

8) use the P2 rotaring redyeing 293 cell, receive poison after cultivating, with the viral supernatant of results with the DNase enzymic digestion after, with the membrane filtration of 0.45 μ m, then the ion column purification, be further purified virus after molecular sieve afterwards;

9) virus of purification is stored in adenovirus and preserves in the liquid, after desalination is handled with 0.22 μ m disposable filter filtration sterilization, thereby obtain aseptic purified virus recombinant adenovirus human telomerase polypeptide gene rAdv-hTERTC27.

Electrophoresis is identified figure as shown in Figure 1.

RT-PCR, Western blot detect the expression of recombinant adenovirus in the Hepa1-6 cell strain:

1) growth conditions is good, 70%～80% Hepa1-6 cell dissociation counting that merges, 5 * 10 ⁵Cells/ goes down to posterity in the hole and is inoculated in 6 well culture plates, presses MOI=20 transfection rAdv-hTERTC27, rAdv-EGFP, with containing RPMI 1640 complete mediums of 10%FCS in 37 ℃, 5%CO ₂Hatch 48h in the incubator, be used for RT-PCR and Western blot and detect;

2) getting the total RNA reverse transcription of 1 μ g becomes cDNA, and reaction is 42 ℃ of 70min, 70 ℃ of 15min, 4 ℃ of preservations; 10 μ l cDNA mix 56 ℃ of 30min with 0.4 μ l protease k, boiling water bath 10min, and quenching on ice is with the viral capsid that breaks; Carry out polymerase chain reaction PCR with Premix Taq, the primer of hTERTC27 is: hTERTC27-F (5'-ATGACAGTGGTGAACTTCC-3') (SEQ ID NO:1), hTERTC27-R (5'-TCAGTCCAGGATGGTCTTG-3') (SEQ ID NO:2) (PCR product 716bp); PCR response procedures: 94 ℃ of 5min, 30 circulation (94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 1min), 72 ℃ of 10min, sample is to the 1.0 % agaroses that contain SYBR Safe DNA dyestuff (0.1 μ g/ml) on the PCR product, and the voltage with 100V in TBE runs 30min, with gel imaging instrument observed result;

3) get 100 ℃ of degeneration 5min of 15 μ g total proteins, add equal-volume 2 * SDS protein electrophoresis sample-loading buffer, sample on the mixing, and add protein molecular weight and dye Marker electrophoresis (200V, to bromophenol blue arrive the separation gel bottom or enter electrophoresis tank) together in advance; Electricity changes film, 70V, and as seen 1h dyes Marker in advance and also goes on the pvdf membrane; TBST washes film, 5min * 3 time; The sealing of confining liquid room temperature, 2h(has sealed and has not washed film); Add one anti-(1:1000) and hatch 4 ℃ of shaken overnight; TBST washes film, 5min * 6 time; Two anti-(1:1000) that add the HRP labelling, incubated at room 1h; TBST washes film, 5min * 6 time; Darkroom exposure: now join luminescent solution, it is blended that the A liquid of ECL test kit and B liquid are pressed 1:1, drops on the film, exposes in the darkroom.

The foundation of mice original position liver cancer model

Laboratory animal: 24 of C57BL/6 mices, male and female half and half, body weight 18-21g; Tumor strain: adopt rat liver cancer cell Hepa1-6, provide by the Shanghai cell bank.

Method: the Hepa1-6 cell preparation density of the phase growth of taking the logarithm is 5 * 10 ⁷The cell suspending liquid of individual/ml adopts microsyringe to inject cell 0.1ml(5 * 10 down in every Mouse Liver peplos ⁶Individual), sub-cage rearing is under the SPF environment, and the free diet of all nude mices is observed body weight change and continued to feed to producing hepatocarcinoma, and is standby.

RAdv-hTERTC27 is to the influence of hepatocarcinoma (Hepa 1-6) cell proliferation and apoptosis:

1) with the Hep1-6 cell of exponential phase, with 6000 cell inoculations in every hole in 96 orifice plates; Be divided into 3 groups, rAdv-hTERTC27, rAdv-EGFP24 and PBS group, 4 every group multiple holes; RAdv-hTERTC27 and rAdv-EGFP infect rAdv-hTERTC27 and rAdv-EGFP according to MOI=30 respectively after 24 hours, and the PBS group adds and the isopyknic PBS of rAdv-hTERTC27; Again place 37 ℃, 5%CO ₂Cultivated 48 hours in the incubator;

2) CCK-8 clolrimetric assays(Dojindo Molecular Technologies is adopted in the increment of cell, Inc., and Gaithersbury, MD USA) measures; Take out 96 orifice plates, add 10 μ l CCK-8 solution in each hole, continue to cultivate 4 hours, the room temperature vibration was determined at the light absorption value (reference wavelength 650nm) at 450nm place after 5 minutes by the chemiluminescence microplate reader;

3) observe the apoptotic difference of different disposal group; With step 1, respectively organize morphocytology with observation by light microscope after 24 hours and change; Adding Hochest 33258 then in each porocyte respectively makes its final concentration be respectively 10 μ g/ml and 100 μ g/ml with PI, on fluorescence microscope, observe after keeping in Dark Place 10 minutes for 4 ℃, Hochest dyeing excites with ultraviolet light 340nm wavelength, and fluorescence microscope is observed down.

Apoptotic cell dyeing is strong blue-fluorescence, and normal cell only is faint fluorescence, and dead cell then is not colored, and can detect apoptosis thus; Select for use under fluorescence microscope greater than the exciting light of 536nm and observe the PI coloration result, the dyeing fluorescence that takes on a red color is dead cell, by counting apoptosis and dead cell quantity difference between the different disposal group relatively on the same group.Its result as shown in Figure 3.

The influence of rAdv-hTERTC27 pair cell telomerase activation:

Telomerase activation adopts PCR ELISA test kit, and (Roche, Mannheim Germany) measure.

1) the take the logarithm Hep1-6 cell of trophophase is used 0.25 trypsinization when cell grows to about 90% when merging, and 0.01M pH7.3 PBS washes cell 3 times, makes single cell suspension in the 15mL centrifuge tube; With cell dilution to concentration is 2 * 10 ⁵Cells/ml, with every hole 500 μ l cell suspension with cell inoculation in 24 orifice plates; Shake gently, make cell even, place 37 ℃, 5%CO ₂Cultivated 48 hours in the incubator;

2) each porocyte is divided into 3 groups, establishes 4 multiple holes for every group, rAdv-hTERTC27 and rAdv-EGFP are respectively according to the MOI=30 target cell infection, and the 3rd group adds the PBS with volume with rAdv-hTERTC27, place 37 ℃, 5%CO again ₂Cultivated 24 hours in the incubator;

3) telomerase activation detects: each EP pipe adds 200 μ l cell pyrolysis liquids, hatched on ice 30 minutes, during shake the EP pipe and make the abundant cracking of cell, 4 ℃ of 16000 * g are centrifugal 20 minutes; Get supernatant, be transferred to a new EP pipe; Other gets a histone sample through 100 ℃ of water-baths degeneration in 10 minutes, as negative control group, measure each histone concentration with the BCA method, calculate applied sample amount and go up sample by each sample 2 μ g protein refractometer, the concrete operations step is seen Telomerase PCR ELISA Kit description.

Adopt argentation and ELISA method that telomerase activation is carried out labelling respectively, each organizes the telomerase activation result as shown in Figure 4.

Derived from bone marrow dendritic cell (DCs) and rAdv-hTERTC27 infect the DCs transfection efficiency to be observed:

1) separation of derived from bone marrow dendritic cell: get the 5-6 C57BL/6 mice in age in week, disconnected cervical vertebra is put to death, aseptic femur and the tibia peeled off, cut to red bone marrow, draw RPMI1640 with 30 mL syringes bone marrow is dashed to culture dish, 100 eye mesh screens filter the back centrifugal 6min of 458 * g and collect medullary cell, and cell precipitation adds the 0.83%Tris-NH4Cl 3mL splitting erythrocyte of pre-temperature, add centrifugal 6 min of 40 mL RPMI 1640:458 * g behind the cracking 5min, abandon supernatant;

2) trypan blue exclusion method living cell counting number (measuring cell viability) greater than 95%, cell precipitation adds RPMI1640 complete medium (containing 10% hyclone), be seeded to 6 well culture plates, add 10ng/mL rmGM-CSF, 5 ng/mL IL-4, every hole adds complete culture medium to 4 mL; To the 3rd day, change the complete culture solution that contains the same concentrations cytokine; To the 5th day, half amount was changed liquid and is supplied cytokine; To the 6th day, all suspension cells were collected in piping and druming gently, were the DCs in the mouse bone marrow cells source of amplification in vitro; Use the growth and the metamorphosis of phase contrast microscope observation of cell every day.

3) with 200 ng/mL LPS cultivate altogether support 24 h after, add rAdv-hTERTC27 respectively or rAdv-EGFP is hatched 24 h according to MOI=200, under fluorescence microscope, observe transfection efficiency.

Express green fluorescence and be rAdv-hTERTC27 or the rAdv-EGFP that effectively is transfected into DCs.The result as shown in Figure 5.

RAdv-hTERTC27 stimulates DCs secrete cytokines Il-2, INF-γ to increase:

1, collect rAdv-hTERTC27, rAdv-EGFP or PBS infect 24 hours respectively organize the DC culture supernatant;

2, the content of INF-γ and IL-2 in the ELISA method detection DC culture supernatant:

1)?NF-γ-ELISA（R&D?Systems，USA）：

1. use the standard substance of 1 * Calibrator Diluent RD5P tonsure dilution preparation 5000pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312 pg/mL, 156 pg/mL and 78 pg/mL, contrast as 0pg/mL(zero) with 1 * Calibrator Diluent RD5P;

2. use 1 * Calibrator Diluent RD5P with 50 times of detected sample dilutions;

3. in ELISA Plate, add Assay Diluent RD1-55, every hole 100--L;

4. add 100--L standard substance, 100--L zero contrast and 100--L testing sample respectively in corresponding hole, respectively establish 2 multiple holes, paste the film capping, place on the horizontal rail oscillator, 2h is hatched in room temperature, 500rpm vibration;

5. sop up liquid, with the about 500--L/ of Wash Buffer hole washing 4 times, seal dry cleaning liquid on filter paper;

6. add 6Ckine Conjugate 200--L/ hole, paste the film capping, put on the horizontal rail oscillator, 2h is hatched in room temperature, 500rpm vibration;

7. repeating step is 6. 1 time;

8. add Substrate Solution 200--L/ hole, lucifuge, incubated at room 20min;

9. add Stop Solution 50--L/ hole;

10. measuring optical density (OD) value at 450nm wavelength place in 30min with microplate reader, is reference wavelength with 570nm;

The drawing standard curve, the concentration of calculation sample.

2)?IL-2-ELISA（R&D?Systems，USA）：

The concrete operations step is the same, and the result as shown in Figure 6.

The DCs of transfection rAdv-hTERTC27 is to the observation of T cell increment and cytotoxic T cell (CTL) effect:

(1) the lymphocytic cultivation of T:

Disconnected neck is put to death mice, soaks 1～2 minute in the ethanol of immersion 75%.In super-clean bench, carefully cut off the abdominal part crust of mice, fix, cut off the abdominal cavity of mice again, take out mouse spleen with tweezers with pin; In the 35mm culture dish, put into 4-5mL EZ-Sep--Mouse 1X lymphocyte separation medium (shaking up lymphocyte before the use separates); With the fixing nylon wire of tweezers, grind mouse spleen gently with syringe piston then, make the dispersive unicellular nylon wire that sees through enter in the lymphocyte separation medium; There is the separating medium of spleen cell to transfer to immediately in the centrifuge tube outstanding, covers 1640 culture medium that go up about 200 μ l again before centrifugal.

(2) DCs is to lymphocytic proliferation experiment:

RAdv-hTERTC27-DC, rAdv-EGFP-DC, untreated DC and T lymphocyte after recombinant adenovirus infects 24h are cultivated altogether; In irritation cell (S): the ratio of effector lymphocyte (T)=1:5,1:10,1:20,1:40 adds 1 * 10 in 96 orifice plates add ⁴Each 100 μ l and 5 * 10 of 3 kinds of DCs in/hole ⁴μ l/ hole, 1 * 10 ⁵μ l/ hole, 2 * 10 ⁵μ l/ hole, 4 * 10 ⁵μ l/ hole lymphocyte 100 μ l set up contrast simultaneously, establish 3 multiple holes for every group; Cell culture incubator is incubated 72h, adds CCK-8 reagent 20 μ l/ holes, continue to hatch 3 h after, measure absorbance (reference wavelength 650nm) with microplate reader at the 450nm wavelength.The result as shown in Figure 7.

(3) the T lymphocyte that stimulates of rAdv-hTERTC27-DC is to the lethal effect of tumor cell:

Collect the Hepa1-6 cell of In vitro culture, by every hole 1 * 10 ⁴The density inoculating cell of individual cell is in 96 orifice plates, as target cell.With respectively with rAdv-hTERTC27-DC, rAdv-EGFP-DC, untreated DC and T lymphocyte co-cultivation 24h, collect T lymphocyte action effect cell.Imitating target be 5:1,20:1,40:1 than (E:T), establishes the control wells of having only target cell and having only the effector lymphocyte in addition, cultivates altogether behind the 48h to detect with the CCK-8 method and respectively organizes absorbance (reference wavelength 650nm) under wavelength 450 nm.Be calculated as follows the killing activity of CTL

Kill rate=[1-(is imitated wad cutter value-effect hole value)/target cell hole value] * 100%

The result as shown in Figure 8.

Laboratory observation rAdv-hTERTC27 is to the inhibitory action of tumor growth in the body:

1) animal is divided into 3 groups, every group of 8 mices: group 1: rAdv-hTERTC27(5 * 10 ⁷Pfu); Group 2:rAdv-EGFP(5 * 10 ⁷Pfu); Group 3: with the isopyknic PBS of rAdv-hTERTC27;

2) with the microsyringe Glisson's capsule down injection Hepa1-6 rat liver cancer cell set up mice liver in situ tumor model, Glisson's capsule inoculation back the 7th day down injects rAdv-hTERTC27, rAdv-EGFP or PBS according to grouping tail vein;

3) each treated animal treatment back is observed and the measurement of tumor size life cycle: observe mice animation and life cycle;

4) mice is promptly got liver organization before dying: anesthetized animal, cut off rat abdominal cavity, and cut liver organization, observe the tumor growth situation of respectively organizing, and measure tumor size, gross tumor volume=a by vertical and horizontal direction ²* b * π/6(a is the minor axis of tumor, and b is the major diameter of tumor).

The result as shown in Figure 9.

The result:

Fig. 1 is that the seed culture of viruses of hTERTC27 is identified electrophoretogram, by as seen bright band being arranged below slightly at the 750bp band, similar with the position and the brightness of positive control, and two sample standard deviations produce this band, can determine that from figure hTERTC27 has successfully packed in the adenovirus vector.

Fig. 2 is RT-PCR and the Western Blot electrophoretogram behind the rAdv-hTERTC27 viral infection target cell, as seen from the figure, successfully detected dna fragmentation and the protein fragments of hTERTC27 in the rat liver cancer cell, hence one can see that, and recombinant adenovirus successfully carries hTERT in the target cell.

Fig. 3 is the influence figure of rAdv-hTERTC27 to hepatocarcinoma (Hepa 1-6) cell proliferation and apoptosis.In order to detect the inhibitory action of hTERTC27 to growth of tumour cell, rAdv-hTERTC27, rAdv-EGFP or PBS target cell infection hatched 48 hours after, cell dyes with PI and Hochest respectively.Inducing apoptosis of tumour cell or death obviously are better than rAdv-EGFP and PBS group (Fig.3A) behind the rAdv-hTERTC27 infection cell as seen from the figure.Can see that by the CCK-8 detection method rAdv-hTERTC27 compares the obvious increment that suppresses tumor cell (Fig. 3 B) than rAdv-EGFP or PBS.

Fig. 4 is the figure that influences of rAdv-hTERTC27 pair cell telomerase activation.In order to prove whether hTERTC27 plays the promotion apoptotic effect by influencing telomerase activation, with the recombinant adenovirus target cell infection, collecting cell extracts albumen after 48 hours, adopt TRAP-ELISA to detect telomerase activation, as shown in the figure, at rAdv-hTERTC27, rAdv-EGFP and three groups of Telomerase Activity of PBS all is male, does not have significant difference between the three.ELISA method and argentation have all confirmed same result.

Fig. 5 is a rAdv-hTERTC27 transfection derived from bone marrow DCs design sketch.Wherein A is depicted as from mouse bone marrow cells and separates the DCs that obtains, and is cultured to the 8th day, and the DCs maturation can be carried out next step experiment.B was depicted as viral infection DCs after 24 hours, and green fluorescence is depicted as the virion that is transfected into DCs.When MOI=200, transfection efficiency can reach 80%.

Fig. 6 is the influence of rAdv-hTERTC27 Ad-6Ckine/IFN γ transfection to the DC secrete cytokines; RAdv-hTERTC27, rAdv-EGFP or PBS infected after DCs24 hour, IFN-γ, IL-2 concentration in the culture supernatant are measured with the ELISA test kit, as shown in Figure 7, rAdv-hTERTC27 infects IFN-γ (328.6 ± 11.26 pg/ml) in the DCs group supernatant, IL-2 (8.18 ± 0.45 pg/ml) is apparently higher than rAdv-EGFP and PBS group, and IFN-γ and IL-2 concentration are respectively IFN- γ (234.50 ± 10.56 pg/ml, P＜0.05 ), IL-2 (3.06 ± 0.42 pg/ml, P＜0.05), IFN- γ(251.63 ± 13.0 pg/ml, P＜0.05), IL-2 (2.65 ± 0.38 pg/ml, P＜0.05).This part experimental result has proved that rAdv-hTERTC27 can promote DC emiocytosis cytokine to increase.This also is one of important mechanisms of rAdv-hTERTC27 immunization therapy.

Fig. 7 is that rAdv-hTERT27-DCs promotes the T lymphopoiesis; Stimulation index (SI) is used for calculating T lymphocyte increment degree behind the DC cytositimulation, and rAdv-hTERTC27, rAdv-EGFP, PBS stimulate T lymphocyte increment level to be respectively 0.91 ± 0.09,0.43 ± 0.01,0.45 ± 0.02(S/T=1:5) after infecting the DC cell respectively as shown in Figure 8; 0.56 ± 0.02,0.37 ± 0.01,0.37 ± 0.02(S/T=1:10), 0.46 ± 0.01,0.37 ± 0.01,0.37 ± 0.02(S/T=1:20); 0.41 ± 0.01,0.35 ± 0.00,0.39 ± 0.012(S/T=1:40), behind the visible rAdv-hTERTC27 infection of result DC cell, when S/T 〉=1:10, the value-added ability of DC cytositimulation T lymphocyte obviously is better than rAdv-EGFP and PBS group (P＜0.05), and difference does not have statistical significance (P〉0.05) between rAdv-EGFP and the PBS.

Fig. 8 is the effect that rAdv-hTERTC27-DCs promotes T lymphocyte killing tumor cells target cell; E/T ratio is respectively 5:1,20:1,40:1 as shown in the figure, and the result shows that rAdv-hTERTC27-DCs activated T lymphocyte is respectively (16.16 ± 2.75) %, (44.44 ± 3.11) % and (65.21 ± 2.98) % when E:T=5:1, E:T=20:1 and the E:T=40:1 to the kill rate of Hepa1-6 cell; RAd-EGFP-DCs and PBS-DCs activated T lymphocyte are respectively (17.79 ± 2.95) %, (33.65 ± 3.16) %, (40.54 ± 3.18) % and (16.99 ± 2.97) %, (30.57 ± 2.64) %, (48.72 ± 3.45) % when E:T=5:1, E:T=20:1 and E:T=40:1.When E:T 〉=20:1, rAdv-hTERTC27-DCs activated T lymphocyte to the kill rate of Hepa1-6 tumor cell apparently higher than rAd-EGFP-DC and PBD-DC group ( p＜0.05), and rAd-EGFP-DC and PBS-DC group difference do not have significance (P〉0.05).

Fig. 9 be behind the injection rAdv-hTERTC27 to the figure that influences of rat liver cancer, as can be seen from the figure, injected the mice of rAdv-hTERTC27, its hepatocarcinoma has obtained good restraining.As shown in the figure, can see intuitively that by the cardinal principle model rAdv-hTERTC27 treatment group obviously dwindles (P＜0.05) than rAdv-EGFP and PBS treatment group gross tumor volume, measure gross tumor volume and be respectively (1012 ± 21.43) mm ³, (2567 ± 32.21) mm ³, (2789 ± 29.12) mm ³And rAdv-hTERTC27 treatment group obviously prolongs (P＜0.05) than rAdv-EGFP and PBS treatment group life cycle, is respectively 34.5 days and 31 days 68 days.This shows that rAdv-hTERTC27 can suppress the growth of rat liver cancer effectively, has prolonged the life cycle of tumor-bearing mice.Find simultaneously that in experimentation for mice, the injection volume of rAdv-hTERTC27 is 5 * 10 ⁷During the pfu/ Mus, its effect is best.

Antioncogene preparation rAdv-hTERTC27 of the present invention, the transfection efficiency height is regulated more effective to the increment and the apoptosis of tumor cell.Simultaneously, Antioncogene preparation of the present invention, can effectively the transfection of purpose peptide section be entered dendritic cell DCs, DCs is activated, secretion of gamma-IFN, IL-2, increase and the further T of stimulation lymphopoiesis, and impel the T lymphocyte that corresponding tumor cell is produced specific lethal effect CTL, tumor is carried out immunomodulating.By above approach, Antioncogene preparation of the present invention can be applicable to the treatment of kinds of tumors.

＜110〉Zhongshan University's sun yat-sen memorial hospital

 

＜120〉a kind of intravenous formulation of genetic immunization treatment tumor

 

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Claims (2)

1. the intravenous formulation of a genetic immunization treatment tumor is made up of replication-defective adenoviral loading human telomerase reverse transcriptase C-terminal polypeptide gene hTERTC27 and pharmaceutic adjuvant acceptable.

2. the intravenous formulation of a kind of genetic immunization treatment tumor according to claim 1, it is characterized in that: replication-defective adenoviral is Ad5.

Images