CN101199862B

CN101199862B - B7-1, CD40L mass particle mixture

Info

Publication number: CN101199862B
Application number: CN 200710179849
Authority: CN
Inventors: 克晓燕; 赵灵芝; 王晶
Original assignee: Peking University Third Hospital
Current assignee: Peking University Third Hospital
Priority date: 2007-12-19
Filing date: 2007-12-19
Publication date: 2011-07-06
Anticipated expiration: 2027-12-19
Also published as: CN101199862A

Abstract

The invention discloses a plasmid compound of B7-1 and CD40L, with B7-1 accounting for 40-60% of the total volume, and CD40L accounting for 60-40% of the total volume. The compound has notable effect in inhibiting and eliminating large tumors bigger than or equal to 0.8cm, lymphomas in particular. The invention, which adopts injection of plasmid inside tumor, is of simple using method and few interfering factors, so that the invention is suitable for large-scale generalization.

Description

B7-1, CD40L plasmid mixture

Technical field

The present invention relates to the tumour immunity technical field, specifically, is a kind of plasmid mixture that is constituted jointly by plasmid B7-1 and plasmid CD40L.

Background technology

At present, immunotherapy of tumors is an important directions of lymphoma treating in recent years, and immunotherapy of tumors mainly comprises four aspects: 1. immunogene is treated 2. tumour-cell vaccine 3. based on the 4. therapeutic alliance of recombinant cytokine of immunization therapy of antibody.What the application of clinical trial at present was more is the cell vaccine treatment, promptly it is made as tumor vaccine feeds back in patient's body by external modification, cultivation to tumor cell, but because external modification and cultivation need condition higher, there are reasons such as easily pollution, clinical application is subjected to certain limitation, and the immunogene treatment is simple because of it, and increasing research is carried out at the method.The immunogene treatment is the vitro recombination gene, by recombinant virus, liposome, particle gun etc. it is directly imported in the intravital tumor tissues and expresses, and essence is the original position inducing antitumor immunity.The carrier that is used for carrying genes of interest has viral vector and non-virus carrier.Viral vector has the danger that causes latent infection or cause gene mutation, and only has in vivo and enter tumor cell on a small quantity, and immune effect is relatively poor.The non-virus carrier particularly application of plasmid vector is then very extensive.

Being that the immunogene treatment of carrier is main with the plasmid realizes by plasmid intramuscular injection, plasmid intratumor injection, particle gun bombardment technology.Plasmid can directly be absorbed by muscle cell after intramuscular injection, also can be by the picked-up of the full-time antigen presenting cell in the muscular tissue, and by antigenic processing, submission and active cell immunity and humoral immunization.The direct intratumor injection of plasmid, the gene order encoded protein molecule that tumor cells expression is imported.Particle gun bombardment technology is wrapped in genes of interest on the gold grain, makes gold grain with very high speed bombardment target cell, thereby changes exogenous gene over to cell.

Stimulate approach to make it become the new effective intervention point of therapy of tumor altogether as the important function of secondary signal path.Polygene combined treatment is the focus of present field of gene research, also is the pattern of following gene therapy.At present to the immunization therapy research of B7-1 mediation concentrate on cytokine therapeutic alliances such as GM-CSF, IL-2, IL-12 in, do not see as yet that both at home and abroad B7-1 and CD40L mixings plasmid are applied to the interior relevant report of mouse tumor.

Summary of the invention

The present invention has overcome above-mentioned shortcoming, has proposed a kind of B7-1, CD40L plasmid mixture that is used for the treatment of tumor.

The present invention solves the technical scheme that its technical problem takes:

A kind of B7-1, CD40L plasmid mixture, percentage ratio meter by volume, B7-1 accounts for 40～60%, and CD40L accounts for 60～40%.

In the above-mentioned mixture, preferred B7-1 and CD40L respectively account for 50% this ratio of cumulative volume.

The inventor has found above-mentioned B7-1, the purposes of CD40L plasmid mixture in preparation medicine for treating tumor thing.When the diameter of tumor during more than or equal to 0.8cm, effect is especially obvious.

B7-1 of the present invention, CD40L plasmid mixture are particularly useful for preparation treatment lymphoid tumor medicament.When lymphadenomatous diameter more than or equal to 0.8cm, effect is especially obvious.

B7-1 is the most deep, the most sophisticated molecule of application of research in the present costimulatory molecules.B7-1 is the part of CD28 and CTLA-4, is expressed in multiple antigen presenting cell surface.By combining the immunne response that raises body with the CD28 and the CTLA-4 molecule of T lymphocytic cell surface, and can be by breaking the tumour immunity tolerance, T lymphocyte performance antitumor action is assisted in the inducing T cell activation.CD40L can raise the expression of B7-1 molecule, increases the secretion of IL-12.The immunne response that they separately can activated T cell is simultaneously broken the immune tolerance state of tumor.So both mixture have broad prospects in the treatment of lymphoma immunogene.

The invention has the advantages that: plasmid intratumor injection when the present invention uses, using method is simple, and interference factor is few, is fit to large-scale promotion.Be specially adapted to size diameter more than or equal to 0.8cm tumor lymphoma particularly.

The specific embodiment

(1) material

Animal: male BALB/c mouse, age in 6-8 week.

Cell strain: lymphoma cell strain A20 is derived from BALB/c mouse.37 ℃ are incubated in 1640 culture medium that contain 10% hyclone.

Plasmid and bacterial strain: mice B7-1 total length plasmid, plasmid vector is pCDM8, the amplification bacterial strain is P3E.coli, mice CD40L total length plasmid, plasmid vector is pCDNA3.1, the amplification bacterial strain is DH5 α E.coli.

The RPM RPMI-1640: by the preparation of RPMI 1640 powder, U.S. Gibco company produces.

Hyclone (FBS): produce Tianjin ,-20 ℃ of preservations.

(2) method

The amplification of plasmid DNA, purification and evaluation: E.coli bacterium amplification plasmid also uses QingenPlasmid Maxi test kit to extract and plasmid purification, and concrete steps are seen the test kit explanation.Ultraviolet spectrophotometer is surveyed plasmid DNA concentration: get 100 times of 2ul plasmid dilutions, survey the OD value and estimate DNA concentration.Plasmid DNA is stored in-80 ℃.Plasmid mB7-1 identifies that with Xho I restriction enzyme enzyme action plasmid mCD40L identifies with EcoR I restricted enzyme and Nhe I restriction enzyme enzyme action.

Make up bearing mouse model: the 2*10 of the growth of taking the logarithm ⁶The A20 cell makes up bearing mouse model at back, BALB/c mouse right side subcutaneous injection.

(3) to be used for the treatment of lymphadenomatous experiment as follows for B7-1 and CD40L expression vector.

3.1. embodiment 1 and Comparative Examples 1～4 are used for little tumor

Little tumor group: treat that mouse tumor grows to diameter 3-4mm, random packet is got three for every group and is carried out intratumor injection as follows, injects tumor growth situation of survey in per 2 days after 7 days for the second time with same dosage.Tumor size by formula a*b2/2 is calculated (a is the tumor maximum gauge, and b is and its vertical diameter).Inject for the second time and got mice spleen in back 5 days and make the CTL killing experiments.

Comparative Examples 1 (blank): every injection PBS 100ul.

Comparative Examples 2 (empty plasmid contrast): transferring empty plasmid concentration with PBS is 1ug/ul, every injection 100ul.

Comparative Examples 3, B7-1 (plasmid is injected separately): with the modified grain of PBS concentration is 1ug/ul, every injection 100ul.

Comparative Examples 4, CD40L plasmid (injection separately): with the modified grain of PBS concentration is 1ug/ul, every injection 100ul.

Embodiment 1 (B7-1, CD40L plasmid mixture): with the modified grain of PBS concentration is 1ug/ul, and plasmid mixture is made up of 50ul B7-1 plasmid and 50ul CD40L plasmid.This mixture is injected in the mice body.

3.2. embodiment 1 and Comparative Examples 1～4 are used for big tumor

Big tumor group: when treating mouse tumor length to diameter 7-8mm, same by little tumor group mode random packet, get three for every group and carry out intratumor injection, inject for the second time with same dosage after 7 days, inject for the second time and got mice spleen in back 4 days and make the CTL killing experiments, and get tumor tissue and make pathological section and carry out morphological observation.

Comparative Examples 1 (blank): every injection PBS 100ul.

(4) conclusion

1, for diameter 0.3cm with interior little tumor, act on B7-1 separately, the CD40L gene can make it reduce, embodiment 1 is the tumor complete obiteration in 13 days, and PBS and empty plasmid matched group tumor growth are rapid, interior diameter reached 10cm in 13 days.Statistical analysis shows that for little tumor, Comparative Examples 3, Comparative Examples 4 all have obvious tumor-inhibiting action with embodiment 1, but embodiment 1 there is no obvious improvement.

The little tumor group volume of table 1 (mm ³)

1d

2d

4d

6d

8d

9d

11d

13d

Comparative Examples 1 Comparative Examples 2 Comparative Examples 3 Comparative Examples 4 embodiment 1

18.00 ±0 16.50 ±2.60 16.50 ±2.60 18.00 ±0 16.50 ±2.60

34.67± 4.62 22.67± 8.08 34.67± 4.62 34.67± 10.10 26.83± 11.62

57.67± 8.37 50.17± 11.41 31.50± 8.76 31.50± 8.76 24.17± 7.15

107.17± 19.26 90.17± 16.66 31.50± 8.76 31.50± 8.76 15.00± 2.60

149.33± 40.42 126.00± 0 16.50± 2.60 19.50± 2.60 13.50±0?

223.33± 47.34 187.33± 14.15 16.50± 2.60 16.50± 2.60 4±0?

309.83 ±60.40 270.67 ±12.70 5.33± 1.16 5.33± 1.16 1.33± 2.31

480.33 ±60.33 405.00 ±0 2.67± 2.31 2.67± 2.31 0±0?

2, for the tumor of diameter 0.8cm, embodiment 1 tumor in 13 days disappears substantially, and tumor body diameter reaches below the 3cm.Comparative Examples 3 and Comparative Examples 4 tumor growths are inhibited, and the matched group tumor growth is rapid.Statistical analysis shows that embodiment 1 effect is better than Comparative Examples 3 and Comparative Examples 4.

The big tumor group volume (mm of table 2 ³)

	1d	2d	4d	5d	6d
						Comparative Examples 1 Comparative Examples 2 Comparative Examples 3 Comparative Examples 4 embodiment 1	164.50±35.52 156.33±26.27 164.50±35.52 164.50±35.52 156.33±26.27	227.83±48.79 224.17±30.17 216.00±42.43 204.17±14.15 204.17±14.15	306.83±49.94 320.83±43.256 306.00±24.25 281.00±37.59 270.67±12.70	498.50±52.27 570.33±112.96 432.00±23.83 381.50±78.44 278.00±0	705.83±34.93 824.00±193.99 588.50±66.68 533.17±141.50 204.17±14.15

The big tumor group volume (mm of continuous table 2 ³)

	8d	9d	11d	13d
					Comparative Examples 1 Comparative Examples 2 Comparative Examples 3 Comparative Examples 4 embodiment 1	1100.67±142.61 1311.33+416.85 688.00±152.42 632.17±217.04 114.00±10.39	1278.67±165.70 1570.17±713.51 944.50±210.15 765.33±359.76 66.67±7.22	1762.50±64.95 2263.83±1134.47 1220.33±233.52 951.67±547.04 26.83±11.62	2456.50±144.50 3243.50+1939.73 1435.17±153.25 1185.50±771.27 8.50±4.80

3, spleen CTL lethal effect and tumor tissue pathology all support The above results.

The little tumor spleen of table 3 CTL kill rate

The big tumor spleen CTL kill rate of table 4

More than B7-1 provided by the present invention, CD40L plasmid mixture are described in detail, used specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof; Simultaneously, for one of ordinary skill in the art, according to thought of the present invention, the part that all can change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.

Claims

1. the mixture of a B7-1 expression plasmid, CD40L expression plasmid, it is characterized in that: B7-1 expression plasmid that the quality volumetric concentration is identical and CD40L expression plasmid be the percentage ratio meter by volume, the B7-1 expression plasmid accounts for 40～60%, and the CD40L expression plasmid accounts for 60～40%.

2. mixture according to claim 1 is characterized in that: described B7-1 expression plasmid and CD40L expression plasmid respectively account for 50% of cumulative volume.

3. claim 1 or the 2 described mixture purposes in preparation medicine for treating tumor thing.

4. purposes according to claim 3 is characterized in that: the diameter of described tumor is more than or equal to 0.8cm.

5. purposes according to claim 3 is characterized in that: described tumor is a lymphoma.

6. purposes according to claim 5 is characterized in that: described lymphadenomatous diameter is more than or equal to 0.8cm.