CN104096238A

CN104096238A - Malignant tumor therapeutic vaccine and composition thereof

Info

Publication number: CN104096238A
Application number: CN201310111819.0A
Authority: CN
Inventors: 熊慧
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-01
Filing date: 2013-04-01
Publication date: 2014-10-15

Abstract

The invention relates to a malignant tumor therapeutic vaccine and a composition thereof. The malignant tumor therapeutic vaccine is a tumor cell line obtained by culture of a human tumor tissue's cancer cells. A TGF-beta2 antisense plasmid is transfected into the tumor cell line, and the tumor cell line is subjected to inactivation, thus obtaining the malignant tumor therapeutic vaccine. The invention also relates to a malignant tumor therapeutic vaccine composition, which includes the malignant tumor therapeutic vaccine and an immunopotentiator. The malignant tumor therapeutic vaccine provided by the invention can be used for treating the tumor cell sourced patients, and has the advantages of strong immune antigen specificity, repeated preparation and use and low price, and also can be prepared into a vaccine preparation for treatment of other patients suffering a same cancer. Animal experiments confirm that the malignant tumor therapeutic vaccine has a good treatment effect.

Description

A kind for the treatment of malignant tumor vaccine and compositions thereof

Technical field

The present invention relates to a kind for the treatment of malignant tumor vaccine and compositions thereof.

Background technology

At present, malignant tumor is still the No.1 killer who threatens human survival, severe jamming patient's health and affect life quality, and its Therapeutic Method mainly contains operation, chemotherapy, radiotherapy and four kinds of patterns of Biotherapeutics.Although can remove most of tumor cell and can make the state of an illness of most of patients be eased with operation, chemotherapy and treatment conventional therapy, chemotherapy, radiotherapy mostly exist serious toxic and side effects to patient.And these Therapeutic Method do not reach the effect of eradicating cancerous cell completely, most patient is finally died from the recurrence of tumor.Therefore after operation, chemotherapy, radiotherapy, Biotherapeutics has become the important means of combined therapy of tumour.Current, tumor biotherapy strategy mainly contains cytokine therapy, cell therapy, gene therapy, targeted therapy etc.But the effectiveness of most Biotherapeutics also reaches far away gratifying degree, its main cause is these treatments activating immune system removing tumor cells effectively.In recent years, along with molecular biology and engineered development, tumor vaccine becomes one of focus of research, and its principle is by activating patient's autoimmune system, to reach the object of removing or controlling tumor.Present stage is studied more tumor vaccine tumour-cell vaccine, tumor antigen vaccine, take DC(dendritic cell) be basic vaccine and nucleic acid vaccine etc.Although existing vaccine shows certain Graft Versus Tumor, but there is the shortcomings such as curative effect is not strong, poor stability, therefore, how to overcome the immunologic escape of immune system to tumor antigen, develop offer immunogenicity strong, effectively blocking immunity escape, the powerful immune phagocytic function of excitating organism and side effect little, the satisfied tumor vaccine of clinical efficacy is that can it obtain successful key.

Immunostimulant (immunopotentiator) is by different modes, reaches a para-immunity medicine of enhancing human body immunity power.Be usually used in clinically treatment disease and the immunodeficiency relevant with immunologic hypofunction.At present a lot of for the research of immunostimulant.

Modern pharmacology research shows, after medicine is worked into nanometer level, can significantly improve trap, diffusance, is conducive to reduce some side effect of medicine, improves drug effect.Conventionally the crushing technology of microbial cell is two kinds of Mechanical Method, on-mechanical methods.Mechanical Method comprises: high pressure homogenizer, high speed bead mill, ultrasonic wave concussion device, on-mechanical method comprises chemical method, enzymatic isolation method, osmotic pressure ballistic method, freeze-thaw method and seasoning.Although cytoclastic method is a lot, be all difficult to the nanometer level to homogeneous grain diameter by cell breakage.Medicine is worked into its granularity overwhelming majority to be all less than 500nm and to be technically difficult to realize.

Transforming growth factor β (transforming growth factor-β, TGF-β) is one group of adjusting albumen with multiple action, has the effect that regulates cell enlargement, assists immunosurveillance escape.Transforming growth factor β-2nd, topmost hypotype in TGF-'beta ' family.Generally, many raisings with TGF-β 2 levels in the immunosuppressant of cancer patient, the prognosis of TGF-β 2 levels and cancer patient also has substantial connection.2 pairs of natural killer cells of TGF-β, lymphocyte activator cell, dendritic cell have antagonism.Tumor cell vaccine contributes to the offer effect of enhancing body to tumor associated antigen, strengthens the removing of body to tumor cell.

RNA interference phenomenon, by first discoveries in 1998 such as FIRE, refers to by positive and negative adopted RNA fragment and forms double-stranded RNA, thereby suppress specifically the phenomenon that target gene is transcribed rear expression.The technology of expressing as efficient, special regulator gene, RNA disturbs the powerful that has become gene function and signal transduction system upstream and downstream molecule interrelationship study.Suppress the immunologic escape that the expression of TGF-β 2 can reversing tumor.

For this reason, the present invention proposes a kind for the treatment of malignant tumor vaccine and compositions thereof.

Summary of the invention

Primary goal of the invention of the present invention is to have proposed a kind for the treatment of malignant tumor vaccine.

The second goal of the invention of the present invention is to have proposed a kind for the treatment of malignant tumor vaccine combination.

In order to realize object of the present invention, the technical scheme of employing is:

The present invention relates to a kind for the treatment of malignant tumor vaccine, this treating malignant tumor vaccine is for to obtain tumor cell line by the cancerous cell of people's tumor tissues by cultivation, TGF-β 2 antisense plasmids are transfected in tumor cell line, and described tumor cell line obtains treating malignant tumor vaccine after deactivation.

The first optimal technical scheme of the present invention is: the cDNA of described TGF-β 2 is: NCBI Reference Sequence:NM_003238.3, fragment 1369bp-2613bp.

The second optimal technical scheme of the present invention is: the cancerous cell of people's tumor tissues is selected from pulmonary carcinoma tumor cell, tumor cells of hepatocellular carcinoma, ovarian cancer tumor cell, breast cancer tumour cell, gastric cancer tumor cell, pancreatic tumour cell, colon cancer tumor cell, esophageal cancer cell, cervical cancer cell or endometrial carcinoma cell.

The 3rd optimal technical scheme of the present invention is: described preparation method comprises the following steps:

(1) prepare people TGF-β 2 antisense plasmids:

(2), by the chopping of people's tumor tissues, digestion, cultivates and obtains tumor cell line;

(3) in tumor cell line people TGF-β 2 antisense plasmid transfections to cultivation being obtained;

(4) continue the amplification culture that goes down to posterity, collecting cell, after irradiating with 100Gy, subpackage is frozen.

The invention still further relates to a kind for the treatment of malignant tumor vaccine combination, described compositions comprises treating malignant tumor vaccine claimed in claim 1 and immunostimulant.

The first optimal technical scheme for the treatment of malignant tumor vaccine combination of the present invention is: described immunostimulant is selected from: short corynebacteria preparation, acellular short corynebacteria preparation, BCG-polysaccharide, nucleic acid preparation, Nocardia rubra cell skeleton, A group streptococcus preparation, acellular A group streptococcus preparation, bacillus pyocyaneus preparation, acellular bacillus pyocyaneus preparation, cloth Salmonella preparation, acellular cloth Salmonella preparation, acellular mycobacterium vaccae preparation, acellular smegmatis mycobacterium preparation, Thymopeptide, levamisole preparation, cholera mycin B subunit preparation, interleukin IL-1 preparation, interleukin IL-2 preparation, interleukin IL-12 preparation, interleukin IL-3 preparation, interleukin IL-4 preparation, interleukin IL-6 preparation, interleukin IL-11 preparation, interleukin IL-15 preparation, interleukin IL-18 preparation, interleukin IL-21 preparation, interleukin IL-23 preparation, heat shock protein white preparation, CpG oligodeoxynucleotide preparation, lyophilized bacillus calmette-guerin vaccine cell wall homogenate preparation, deactivation grass mycobacterium preparation, Pseudomonas aeruginosa MSHA pilus strain vaccine (PA-MSHA vaccine), ubenimex, Biostim, polyvalent bacterial lysate, interferon IFN-α preparation, interferon IFN-beta formulations, interferon IFN-γ preparation, colony-stimulating factor GM-CSF, colony-stimulating factor M-CSF, transfer factor preparation.

The second optimal technical scheme for the treatment of malignant tumor vaccine combination of the present invention is: described immunostimulant is selected from: acellular short corynebacteria preparation, acellular A group streptococcus preparation, acellular bacillus pyocyaneus preparation, acellular cloth Salmonella preparation, acellular mycobacterium vaccae preparation, acellular smegmatis mycobacterium preparation; Preferred acellular short corynebacteria preparation.

The 3rd optimal technical scheme for the treatment of malignant tumor vaccine combination of the present invention is: in described compositions, contain treating malignant tumor vaccine and immunostimulant and after subpackage, merge packing, treating malignant tumor vaccine combination respectively.

The 4th optimal technical scheme for the treatment of malignant tumor vaccine combination of the present invention is: in described compositions, contain treating malignant tumor vaccine 1 * 10 ⁷～1 * 10 ⁸cell, immunostimulant 0.5～5.0mg.

The 5th optimal technical scheme for the treatment of malignant tumor vaccine combination of the present invention is: in described compositions, contain treating malignant tumor vaccine 1 * 10 ⁷～5 * 10 ⁷cell, immunostimulant 0.5～2.5mg.

Tumor therapeutic vaccine of the present invention can be used for vein, subcutaneous or intradermal injection, and dosage is 1 * 10 ⁷～5 * 10 ⁷cell, usage is to inject once for every 10～60 days.

Treating malignant tumor vaccine of the present invention can be used for treating the patient in this tumor cell source, thereby has immunizing antigen high specificity, can repeated multiple times preparation uses, cheap advantage; Also can be prepared into bacterin preparation, be used for the treatment of the patient of other cancer of the same race.Of the present invention through transfection have the treating malignant tumor vaccine of people TGF-β 2 antisense plasmids can be subcutaneous or intravenous injection use.

The present invention relates to a kind for the treatment of malignant tumor vaccine combination, described compositions comprises treating malignant tumor vaccine of the present invention and immunostimulant, for treating malignant tumor vaccine and immunostimulant are combined use.By zoopery and clinical trial, confirm, treating malignant tumor vaccine is by the use of combining with immunostimulant, and its antitumor action strengthens greatly.When using treating malignant tumor vaccine, use immunostimulant, immunostimulant can be used for the mode of intramuscular injection.The cell-free preparation of immunostimulant of the present invention, as required, can be made into the suspension formulation of physiological saline solution, and solids content is 1.0mg/ml or 2.0mg/ml, also can make freeze-dried powder or be applicable to other medicinal dosage forms.Immunostimulant appropriate dose scope of the present invention is 0.001～0.2mg/Kg body weight, is preferably 0.01～0.08mg/Kg body weight, 1～7 day/time.

Described immunostimulant is selected from: short corynebacteria preparation, acellular short corynebacteria preparation, BCG-polysaccharide, nucleic acid preparation, Nocardia rubra cell skeleton, A group streptococcus preparation, acellular A group streptococcus preparation, bacillus pyocyaneus preparation, acellular bacillus pyocyaneus preparation, cloth Salmonella preparation, acellular cloth Salmonella preparation, acellular mycobacterium vaccae preparation, acellular smegmatis mycobacterium preparation, Thymopeptide, levamisole preparation, cholera mycin B subunit preparation, interleukin IL-1 preparation, interleukin IL-2 preparation, interleukin IL-12 preparation, interleukin IL-3 preparation, interleukin IL-4 preparation, interleukin IL-6 preparation, interleukin IL-11 preparation, heat shock protein white preparation, CpG oligodeoxynucleotide preparation (CpG-ODN), lyophilized bacillus calmette-guerin vaccine cell wall homogenate preparation (BCG-CW), deactivation grass mycobacterium preparation, Pseudomonas aeruginosa MSHA pilus strain vaccine (PA-MSHA vaccine), ubenimex (ubenimex), Biostim (biostim), polyvalent bacterial lysate (bacteriallysates, Broncho-Vaxom), interferon IFN-α preparation, interferon IFN-beta formulations, interferon IFN-γ preparation, colony stimulating factor preparation, granulocyte colony-stimulating factor preparation, granulocyte-macrophage colony stimutaing factor preparation, transfer factor preparation (transfer factor, TF).Described transfer factor preparation comprises that normal person is from cell transfer factor and placental transfer factor (placenta pepton).

Along with the development to the deep understanding of tumor invasion mechanism and molecular immune theory, the effect of nonspecific immunity in antitumor, is more and more valued by the people.Macrophage (M ф) not only plays an important role in body nonspecific immunity, and also plays very important effect in specific immunity.Mononuclear phagocyte system is except having phagocytic function, also can bring into play antigen presentation (antigen presentation) effect, also belong to antigen presenting cell (antigen-presentation cell, APC), can activate by helper T lymphocyte, and mutually stimulate its function with lymphocyte, thereby amplify specific immunity effect.Yet the M ф competence exertion Graft Versus Tumor of only having activation, and the antitumor action of the M ф of activation has selectivity, an i.e. killing tumor cell and do not kill and wound normal cell, the antigenic structure of its lethal effect and tumor and oncocyte hypertrophy cycle are irrelevant, and can kill and wound the tumor cell that chemotherapy, radiotherapy is to resistance.

Immunostimulant is if acellular short corynebacteria preparation antitumor action is mainly by the stimulation powerful and lasting to mononuclear phagocyte system, cause macrophage ( ) hypertrophy, activation, phagocytic function enhancing, secretion inducing tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL-2), active oxygen (H ₂o ₂, NO) etc. cancerous cell killer factor and strengthen NK cell killing activity, reach and suppress and the object of killing tumor cell, and can be divided into mononuclear phagocyte by promotion hematopoietic pluripotential stem cell, further transfer body immune system and contain anticancer potentiality.

Due to the gene order of TGF-β 2 and the gene order height homology of people TGF-β 2 of mice, so the present invention also adopts mice as animal model, carry out zoopery, confirm that treating malignant tumor vaccine of the present invention has good therapeutical effect.

Accompanying drawing explanation:

Fig. 1 is the structure chart of pIRESpuro3 plasmid.

The specific embodiment of the present invention only limits to further explain and explanation the present invention, not to Composition of contents restriction of the present invention.

The specific embodiment

Embodiment 1: the preparation method of lung cancer vaccine:

1. the preparation of people TGF-β 2 antisense plasmids:

By the cDNA(NCBI Reference Sequence:NM_003238.3 of people TGF-β 2, fragment 1369bp-2613bp) be cloned in the multiple clone site MCS of pIRESpuro3 carrier in the other direction, through order-checking, confirm.Promoter and enhancer because pIRESpuro3 carrier contains CMV can copy in mammal; The internal ribosome entry site that contains encephalomyocarditis virus (internal ribosome entry site, IRES) and express the gene of anti-puromycin, can make antisense mRNA single expression in cell of people TGF-β 2, and under puromycin screening, only has the cell ability stable growth of the antisense mRNA of expressing people TGF-β 2.The structure of pIRESpuro3 plasmid as shown in Figure 1.

2. the preparation of people's pulmonary carcinoma tumor tissues cancerous cell line

2.1 chopping tissues

Get people's pulmonary carcinoma tumor tissues, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 2～3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

Cold digestion is spent the night: in the tissue of chopping, add the trypsin of 0.25% fresh configuration, put 4 ℃ and spend the night after shaking up, next day, the centrifugal rear abandoning supernatant of the slow-speed of revolution, washed 2～3 times again with PBS washing; Add a small amount of culture fluid piping and druming, disperse to make cell suspension, by suitable concentration sub-bottle, cultivate, thoroughly cell suspension is not made after dispersion treatment as stated above by the little block organization of digestion, after cell counting, by suitable concentration sub-bottle, cultivates;

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid of the RPMI-1640 that contains 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtain pulmonary carcinoma tumor cell line;

3. transfection

By centrifugal (200g, 5min) under cell room temperature, at electroporation buffer (10% glycerol, 1Mm MgCl ₂) middle re-suspended cell, concentration is adjusted to 2 * 10 ⁶/ ml, adds people TGF-β 2 antisense plasmid 25ug/ml, and 800ul cell suspension is moved to 4mm electric shock cup, and shock parameters is voltage 620v, persistent period 60us, pulse number 1 time.

Cell strain after transforming is added to the fresh RPMI1640/10% hyclone culture medium containing the puromycin of 10 μ g/ml, within every 3 days, change once new screening culture medium, until the cell of stably express transfection plasmid reaches after some, after about transfection about 15 days, continue to cultivate after 10 days, subpackage is frozen.

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCL, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the normal lung JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Pulmonary carcinoma tumor cell cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 2: the preparation of breast cancer tumour therapeutic vaccine

1. the preparation of people TGF-β 2 antisense plasmids:

2. HBT organizes the preparation of cancerous cell line

2.1 chopping tissues

Get human breast carcinoma tumor tissues, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

Cold digestion after first heat digestion: by piece of tissue first with digesting 20min in 37 ℃ of water-baths of trypsin, after washing, with culture fluid, disperse to make cell suspension, thoroughly the small tissue blocks of digestion is not spent the night with 4 ℃ of trypsin after washing, and extract cell again and be dispersed into cell suspension next day, and sub-bottle is cultivated;

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid of the RPMI-1640 that contains 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtain breast cancer tumour cell line;

3. transfection

Cell strain after transforming is added to the fresh RPMI1640/10% hyclone culture medium containing the puromycin of 10 μ g/ml, within every 2～3 days, change once new screening culture medium, until the cell of stably express transfection plasmid reaches after some, after about transfection about 15 days, continue to cultivate after 5～10 days, subpackage is frozen.

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCL, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the normal breast JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Breast cancer tumour cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 3: the preparation of oophoroma tumor therapeutic vaccine

1. the preparation of people TGF-β 2 antisense plasmids:

2. human ovarian tumor is organized the preparation of cancerous cell line

2.1 chopping tissues

Get human oophoroma tumor tissue, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid of the RPMI-1640 that contains 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtain ovarian cancer tumor cell system;

3. transfection

By centrifugal (200g, 5min) under cell room temperature, at electroporation buffer (10% glycerol, 1mMMgCl ₂) middle re-suspended cell, concentration is adjusted to 2 * 10 ⁶/ ml, adds people TGF-β 2 antisense plasmid 25ug/ml, and 800ul cell suspension is moved to 4mm electric shock cup, and shock parameters is voltage 620v, persistent period 60us, pulse number 1 time.

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCl, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the normal ovarian JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Ovarian cancer tumor cell cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 4: the preparation method of Hepatoma Vaccine:

1. the preparation of people TGF-β 2 antisense plasmids:

2. the preparation of people's hepatocarcinoma tumor tissues cancerous cell line

2.1 chopping tissues

Get people's hepatocarcinoma tumor tissues, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 2～3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

Cold digestion is spent the night: in the tissue of chopping, add the trypsin of 0.25% fresh configuration, put 4 ℃ and spend the night after shaking up, next day, the centrifugal rear abandoning supernatant of the slow-speed of revolution, washed 3 times again with PBS washing; Add a small amount of culture fluid piping and druming, disperse to make cell suspension, by suitable concentration sub-bottle, cultivate, thoroughly cell suspension is not made after dispersion treatment as stated above by the little block organization of digestion, after cell counting, by suitable concentration sub-bottle, cultivates;

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid in RPMI-1640 of containing 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtaining hepatocarcinoma tumor is cell;

3. transfection

Cell strain after transforming is added to the fresh RPMI1640/10% hyclone culture medium containing the puromycin of 10 μ g/ml, within every 2～3 days, change once new screening culture medium, until the cell of stably express transfection plasmid reaches after some, after about transfection about 15 days, continue to cultivate after 5-10 days, subpackage is frozen.

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCL, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the normal hepatocytes JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Tumor cells of hepatocellular carcinoma cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 5: the preparation method of gastric cancer vaccine:

1. the preparation of people TGF-β 2 antisense plasmids:

2. the preparation of people's gastric cancer tumor tissues cancerous cell line

2.1 chopping tissues

Get people's gastric cancer tumor tissues, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 2～3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid of the RPMI-1640 that contains 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtaining gastric cancer tumor is cell;

3. transfection

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCL, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the normal gastric JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Gastric cancer tumor cell cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 6: the preparation method of cancer of pancreas vaccine:

1. the preparation of people TGF-β 2 antisense plasmids:

2. the preparation of human pancreas cancer tumor tissues cancerous cell line

2.1 chopping tissues

Get human pancreas cancer tumor tissues, fully remove blood, fat, connective tissue and downright bad part, in plate, with PBS liquid, clean 2～3 times, tissue is cut into 1～2mm left and right, with PBS, clean 3 times;

2.2 digestion

2.3 cell culture

Cell is with 5 * 10 ⁸～10 * 10 ⁸/ L cell concentration, in the full culture fluid of the RPMI-1640 that contains 10% hyclone in 37 ℃, 5%CO ₂constant incubator in cultivate, obtaining pancreatic tumour is cell;

3. transfection

Cell strain after transforming is added to the fresh RPMI1640/10% hyclone culture medium containing the puromycin of 10 μ g/ml, within every 3 days, change once new screening culture medium, until the cell of stably express transfection plasmid reaches after some, after about transfection about 15 days, continue to cultivate after 5 days, subpackage is frozen.

3. the detection of stable cell line people TGF-β 2 expressions

Collector TGF-β 2 antisense stable cell lines, add protein lysate (PH8.0,20MmTris-HCL, 2%triton-X-100) cell lysis, with anti-human TGF-β 2 antibody, carry out western blot detection, people TGF-β 2 expressions should decline and surpass 40% than the Normal Pancreas JEG-3 that does not proceed to people TGF-β 2 antisense plasmids.

4. the preparation of tumor vaccine

Pancreatic tumour cell strain by above-mentioned stably express people TGF-β 2 antisensenucleic acidses through evaluation, continues the amplification culture that goes down to posterity, and collecting cell, after irradiating with 100Gy, gets 1 * 10 ⁷cells/ml, subpackage is frozen.

Embodiment 7: the preparation of acellular cloth Salmonella preparation

One, the preparation of cloth Salmonella preparation

With reference to the method for describing in 2000 editions < < Products in China rules > >, carry out foundation, calibrating and other calibrating of antibacterial seed lot.

1. production seed: will work after seed lot strain breakdown, and be inoculated on LIA inclined-plane or other appropriate medias, 37 ℃ of cultivations 44～48 hours are 1st generation strain.1st generation strain can be preserved 15 at 2～8 ℃; By on 1st generation strain subcultivation liver agar or other appropriate medias, put 37 ℃ and cultivate 44～48 hours, be 2nd generation strain, after the pure bacterium passed examination of naked eyes, with sterilizing physiological sodium chloride solution, wash lower lawn and make suspension.Using this as production seed, for the production of.

2. production culture medium: use LIA culture medium.

3. strain inoculation and cultivation: produce with after seed in inoculation of medium, put 37 ℃ and cultivate 44～48 hours, naked eyes, by bottle inspection, have miscellaneous bacteria person discarded.

Two, the preparation of acellular cloth Salmonella preparation

(1) described preparation heated and boiled is obtained to inactivated bacterial liquid for 15 minutes;

(2) the bacterium liquid that sterility test is qualified, after normal saline washing 3 times, uses superhigh pressure water jet collider at 1000kgf/cm under aseptic condition ²under pressure, broken thalline is 2 times;

(3) suspension after bacterial cell disruption is through 10000rpm collecting precipitation after centrifugal 0.5 hour, and suspension is made in washing precipitation, and described washing is by physiological saline solution centrifuge washing 5 times for precipitate;

(4) steriling test (2000 editions < < Products in China rules > >) and pyrogen test (2000 editions < < the People's Republic of China pharmacopeia > >) qualified after by suspension subpackage, at 0.5～2 hour heat sterilization of 60～65 ℃ of heating, obtain the cell-free preparation of described preparation;

(5), by after the cell-free preparation subpackage obtaining, cillin bottle heats 15～30 minutes 60 ± 2 ℃ of heating for 30 minutes or 121 ℃.

Embodiment 8: the preparation of BCG-polysaccharide, nucleic acid preparation

BCG-polysaccharide, nucleic acid preparation system extract polysaccharide, nucleic acid with bacill calmette-guerin through hot phenol method, are equipped with sterilizing physiological sodium chloride solution and make, for preventing and treat the diseases such as chronic bronchitis, flu, asthma.

One, the preparation of BCG-polysaccharide, nucleic acid preparation

Source: adopt the strain of national drug homologation to produce, forbid to use strain by animal for the production of.

Freeze-drying lactobacillus every biography in the logical potato culture of Soviet Union, the logical culture medium of bile potato culture and liquid Soviet Union is once a generation.The strain of cultivating on potato culture is put Refrigerator store and is no more than 2 months.For the production of total algebraically of culture be no more than for 12 generations.

Microorganism collection: culture should be by bottle inspection, if there are the situations such as pollution, muddiness to discard.Collect the logical surperficial culture of culture or liquid Soviet Union in potato culture, press dry, add appropriate distilled water, with standby after high speed pulverization crusher machine thalline.

Extract: get broken bacteria suspension and mix with waiting calorimetric phenol, with settled process or centrifugation thalline, draw supernatant, with dialysis, except phenol, add appropriate ethanol precipitation polysaccharide, nucleic acid, collecting precipitation thing.With ethanol, ether, mix washing respectively, be drying to obtain refining polysaccharide, nucleic acid after centrifugal.

Embodiment 9: the preparation of acellular A group streptococcus preparation

A group streptococcus preparation is the dried frozen aquatic products of being made after cultivation, potentiation and penicillin are processed by the less-virulent strain of A group streptococcus, for the immunization therapy of cancerous hydrothorax and malignant tumor.

One, the preparation of A group streptococcus preparation

Strain is CMCC32235 strain or SIPI722 strain, is seeded in Todd-Hewitt meat soup or yeast extract or other suitable solid or fluid medium.

To work after seed lot strain breakdown, be seeded on suitable solid or fluid medium, 36 ± 1 ℃ of cultivations, the amplification of 2～4 generations, prepares suitable work seed liquor thus.

Strain inoculation and cultivation: adopt large bottle or large tank liquid culture.In inoculation of medium production labor, do after seed liquor, in incubation and before sterilization, pure bacterium test is carried out in sampling, and smear is done gram stain microscopy, as discovery pollution microbes should be discarded.

Sampling and processing: the centrifugal or membrane filtration of normal culture, thalline is washed twice with appropriate physiological sodium chloride solution, according to thalline weight in wet base, add appropriate benzylpenicillin, at 660nm place, measure A value, calculate content or thalline is suspended in appropriate BBM solution, at 660nm place, measuring after A value is calculated content and add appropriate benzylpenicillin.Bacterium liquid after multistep is processed is stock solution to be diluted.

Two, the preparation of acellular A group streptococcus preparation

(1) described preparation heated and boiled is obtained to inactivated bacterial liquid for 15～60 minutes;

(2) the bacterium liquid that sterility test is qualified, after normal saline washing 3 times, uses superhigh pressure water jet collider at 1500kgf/cm under aseptic condition ²under pressure, broken thalline is 2 times;

(3) suspension after bacterial cell disruption is through 20000rpm collecting precipitation after centrifugal 10 minutes, and suspension is made in washing precipitation, and described washing is by physiological saline solution centrifuge washing 4 times for precipitate;

Embodiment 10: the preparation of Nocardia rubra cell skeleton

Lyopgized Nocardia rubra-cell Wall Skeleton (N-CWS) is that nocardia rubra is through fermentation, broken, extraction acquisition cell wall skeleton, after adding appropriate emulsifying agent, lyophilizing is made, main component mycolic acid, arabinogalactan and mucopeptide etc. containing this bacterial cell wall, for pernicious ascites pleural fluid and cancer patient's immunization therapy.

One, the preparation of Nocardia rubra cell skeleton

Nocardia rubra PO-8 strain for strain for manufacture;

Production culture medium: use glucose-yeast extract medium.

Production seed: will be seeded on Nutrient agar inclined-plane 28 ℃ of cultivations after work seed lot strain breakdown.In 2～3 generations, adopted the liquid oscilaltion of glucose-yeast extract to cultivate 36 hours.Pure bacterium is tested pollution-free miscellaneous bacteria person can be made for seed.

Inoculation and cultivation: in glucose-yeast extract culture fluid, inoculate seed liquor, vibration aerobic culture 5 days, sampling smear for microscopic examination in incubation and before sterilization, as discovery pollution microbes, should discard.

Microorganism collection: collect culture fluid, centrifugal results wet thallus.

Breaking cellular wall: wet thallus adding distil water, with ultrasonic disruption thalline.Add respectively trypsin, Chymotrypsin and chain protease and be hydrolyzed 16～24 hours, centrifugal, obtain cell wall crude extract.

Extract: respectively with ether-ethanol, chloroform and chloroform-methanol extraction 16 hours.

Dry: the cell wall skeleton after solvent extracts is in 60 ℃ of drying under reduced pressure to till without solvent taste.The cell wall skeleton porphyrize in agate mortar obtaining, obtains the former powder of N-CWS.Put-20 ℃ of kept dry, effect duration is 2 years.

Embodiment 11: the preparation of acellular bacillus pyocyaneus preparation

This strain Pseudomonos aeruginosa MSHA fimbria strain is got lawn and is made suspension after cultivating, and adds formaldehyde sterilization, with PBS, is diluted to every 1ml containing bacterium 1.8 * 10 ⁹make, for the auxiliary treatment of malignant tumor.

One, the preparation of bacillus pyocyaneus preparation

With reference to the method for describing in 2000 editions < < Products in China rules > >, carry out foundation, calibrating and other calibrating of antibacterial seed lot.To work after seed lot strain breakdown, be seeded in Nutrient agar or other appropriate medias, put 35～37 ℃ of cultivations, generally be no more than 18 hours, and must not go down to posterity and surpassed for 5 generations, and prepare thus the production labor of suitable number and make seed, for the production of.

Production culture medium: by the Nutrient agar of pH7.2～7.4 or other culture medium of national drug administrative authority approval.In culture medium, should not contain harmful or other anaphylactogen materials.

Strain inoculation and cultivation: adopt Kolle flask to be coated with kind of a method, put 37 ℃ and cultivate 18～20 hours.In incubation and before sterilization, pure bacterium test is carried out in sampling, and smear is done gram stain microscopy, and discovery pollution microbes should be discarded.

Results and sterilization: after cultivation finishes, in the culture gathering, add the PBS that is no more than 1% formaldehyde, the stock solution after adding of germicide is placed room temperature (20～26 ℃), holds time 14～16 days, is stored in 2～8 ℃ after centrifuge washing.

Two, the preparation of acellular bacillus pyocyaneus preparation

(1) described preparation heated and boiled is obtained to inactivated bacterial liquid for 45 minutes;

(2) the bacterium liquid that sterility test is qualified, after normal saline washing 3 times, uses superhigh pressure water jet collider at 2000kgf/cm under aseptic condition ²under pressure, broken thalline is 2 times;

(3) suspension after bacterial cell disruption is through 6000rpm collecting precipitation after centrifugal 1.5 hours, and suspension is made in washing precipitation, and described washing is by physiological saline solution centrifuge washing 3 times for precipitate;

Embodiment 12: the preparation of acellular pseudomonas preparation

One, the preparation of pseudomonas preparation

The seed lot strain of working is inoculated in meat extract basal medium after opening, and puts 30 ℃ and cultivates 48 hours, must not go down to posterity and surpasses for 5 generations.Prepare thus the production labor of suitable number and make seed.

Culture medium: meat extract basal medium.

Strain inoculation and cultivation: adopt kind of the method that is coated with, by planting rearmounted 30 ℃, cultivate 72 hours.Before incubation and sterilization, pure bacterium test is carried out in sampling, and smear for microscopic examination, as found pollution microbes, should be discarded.

Gather and sterilization: after cultivation finishes, scraping lawn is suspended in the bottle of physiological sodium chloride solution.The bacteria suspension of results is put to 70 ℃ of water-bath discontinuous sterilizations 2 times, each 2 hours, then in 3500r/min centrifugal 30 minutes, and abandon supernatant, and adding physiological sodium chloride solution suspendible, bacteria suspension is put 2～8 ℃ of preservations.

Two, the preparation of acellular pseudomonas preparation

(3) suspension after bacterial cell disruption is through 8000rpm collecting precipitation after centrifugal 1 hour, and suspension is made in washing precipitation, and described washing is by physiological saline solution centrifuge washing 1～5 time for precipitate;

Embodiment 13: the preparation of mycobacterium vaccae preparation

One, the preparation of mycobacterium vaccae preparation

The seed lot strain of working is inoculated in Russell medium after opening, and puts 30 ℃ and cultivates 48 hours, must not go down to posterity and surpasses for 5 generations.Prepare thus the production labor of suitable number and make seed.

Culture medium: Russell medium.

Two, the preparation of acellular mycobacterium vaccae preparation

(2) the bacterium liquid that sterility test is qualified, after normal saline washing 3 times, uses superhigh pressure water jet collider at 1600kgf/cm under aseptic condition ²under pressure, broken thalline is 2 times;

(3) suspension after bacterial cell disruption is through 18000rpm collecting precipitation after centrifugal 1 hour, and suspension is made in washing precipitation, and described washing is by physiological saline solution centrifuge washing 1～5 time for precipitate;

Embodiment 14: the preparation of smegmatis mycobacterium preparation

One, the preparation of smegmatis mycobacterium preparation

Culture medium: Russell medium.

Two, the preparation of acellular smegmatis mycobacterium preparation

Embodiment 15: the preparation of acellular short corynebacteria preparation

After continuous the going down to posterity for 2 times of 1.CP work seed lot strain, be inoculated in large bottle or nutrition tank, low pyrogen, not containing in the sulphur glycollate culture medium of agar 37 ℃ cultivate 7 days: CP formal name used at school is propionibacterium acne, and bacterium number is 65101(76-27 or 7627), 65102(H-84) or 65103(77-1 or 771);

2. collect the thalline without living contaminants, heated and boiled obtains bacterium liquid for 15 minutes:

3. the bacterium liquid that sterility test is qualified is after washing, with the broken thalline of superhigh pressure water jet collider; Described thalline is inactivated bacteria, and cell concentration is 10,000,000,000/ml; Wherein said bacterial cell disruption is to carry out under aseptic condition.Described washing is by precipitate physiological saline solution centrifuge washing at least 2 times;

4. the collecting precipitation after centrifugal of the suspension after bacterial cell disruption, suspension is made in washing precipitation: described washing is by physiological saline solution centrifuge washing 2 times for precipitate; After bacterial cell disruption, the centrifugal condition of suspension is centrifugal 50 minutes of 4 ℃, 12000rpm;

5. by the sterilizing of suspension subpackage post-heating, 65 ℃ are heated 0.5 hour, obtain acellular short corynebacteria preparation.

Embodiment 16:

Acellular short corynebacteria preparation difference subpackage by the lung cancer vaccine of embodiment 1 preparation, embodiment 13 preparations, merges packing, prepares lung cancer vaccine with the compositions of immunostimulant.

Can prepare according to the method the compositions of various cancer vaccines and immunostimulant.

Embodiment 1: the vaccine zoopery of lung cancer therapy vaccine

1. preparation experiment animal

The cultivation of 1.13LL cell

Lewis lung cancer cells strain is purchased from Shanghai Chest Hospital's breast tumor institute.

RPMI-1640 complete medium with containing 10% hyclone, adds dual anti-(100U/mL penicillin, 100 μ g/mL streptomycins), is placed in 37 ℃, 5%CO ₂in mist incubator, cultivate, every 1-2 days changes liquid, within every 3 days, goes down to posterity once, goes down to posterity and uses 0.25% trypsinization.Get 1 * 10 ⁴cell is inoculated in 24 orifice plates, and every 24h counting, repeats 3 holes, continues 8 days, calculates every porocyte mean, draws growth curve.

The preparation of 1.2 cell suspension

The take the logarithm Lewis lung cancer cells of trophophase, trypsinization with 0.25%, collecting cell, the centrifugal supernatant that goes, use physiological saline solution washed twice, by cell suspension, in normal saline, Trypan Blue cell viability is measured and is greater than 90%, and carry out cell counting, adjust cell concentration and be respectively 1 * 10 ⁵/ mL, 1 * 10 ⁶/ mL, 1 * 10 ⁷/ mL, carries out subcutaneous vaccination and lung in-situ inoculating to mice immediately.

1.3 laboratory animals and inoculation method

48 of laboratory animal C57BL/6 mices, male and female half and half, age in 8-10 week, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..Adopt in-situ inoculating method, specific as follows:

(1) grouping: mice is divided into 3 groups at random, and 16 every group, a group is used for periodic monitor tumor formational situation, tests for oncotherapy for other 2 groups; The 1st group of Mice Inoculated Lewis lung cancer total cellular score is 1 * 10 ⁵.

(2) anesthesia: pentobarbital sodium solution 50mg/kg intraperitoneal injection, pentobarbital sodium is made into the working solution of 10mg/mL, by 0.05mL/10g body weight dosage, mice is anaesthetized.

(3) inoculation: after mouse anesthesia, lain on the back and be fixed on operating board, thoracic wall before mice is carried out to alcohol disinfecting, in the arcus costarum of left anterior axillary line, about 1.5cm place makes the little otch of an about 5mm, separate skin and subcutaneous tissue are exposed to thoracic wall, 50 μ L cell suspension and 50 μ L Matrigel are mixed, before solidifying soon with insulin injection syringe by Lewis lung cancer cells and Matrigel suspension totally 100 μ L inject the left lung of mice, the about 3mm of inserting needle, after having injected, stop pin 5s, sew up the incision after pulling out pin.

The observation of 1.4 growth of xenografted and measurement

The situations such as the spirit of routine observation C57BL/6 mice, diet, defecation, body weight and activity, subcutaneous vaccination group starts to measure tumor size on the 3rd day after tumor forms, with vernier caliper measurement tumor major diameter and minor axis, with formula V=a ²b pi/2 (minor axis that a is tumor, the major diameter that b is tumor) calculates tumor size.Orthotopic transplantation group after inoculation the 3rd, 6,9,12,20 days respectively, puts to death 2 mices, observes tumor and forms and transfer case.

2 results: 1 * 10 ⁵the mice of dosage level has into tumor sign at the 9th day when dissected, and tumor formation rate is 100%.Be 35d life cycle.

3. treatment

Prepare as stated above experimental animal, what employing was prepared according to the method for embodiment 1 is transfected into Lewis lung cancer cells by people TGF-β 2 antisense plasmids, obtains the therapeutic vaccine of pulmonary carcinoma.

First group gives tumor therapeutic vaccine when inoculating in position, intravenous injection, and dosage is 1 * 10 ⁵cell/only, life span is weekly, and the mice mean survival time (MST) of first group is 86.3 days;

Second group with in-situ inoculating after within 2 weeks, give tumor therapeutic vaccine, intravenous injection, dosage is 1 * 10 ⁵cell/only, once, the mice mean survival time (MST) of second group is 73.3 days to every 3 days of life span;

Because tumor is disliked liquid reason, the size of tumor can change to some extent, so gross tumor volume is not added up.

Experimental example 2: the therapeutic alliance of lung cancer therapy vaccine

Method by experimental example 1 is prepared experimental animal, adopts the tumor therapeutic vaccine of embodiment 1 preparation to carry out therapeutic test:

The 3rd group gives tumor therapeutic vaccine when inoculating in position, intravenous injection, and dosage is 1 * 10 ⁵cell/only, life span is weekly; Adopt immunostimulant-acellular short corynebacteria preparation simultaneously, 0.5mg/0.1ml intramuscular injection, life span is weekly.The mice mean survival time (MST) of the 3rd group is 128.3 days;

The 4th group with in-situ inoculating after within 2 weeks, give tumor therapeutic vaccine, intravenous injection, dosage is 1 * 10 ⁵cell/only, every 3 days of life span is once; Adopt immunostimulant-acellular short corynebacteria preparation simultaneously, 0.5mg/0.1ml intramuscular injection, life span is weekly.The mice mean survival time (MST) of the 3rd group is 113.2 days;

Embodiment 3: the vaccine zoopery of breast cancer treatment vaccine

1. materials and methods

1.1 estrogen receptor (estrogen receptor, ER) SABC test kit is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.

1.2 animals: Balb/c mice, female, in 6～8 week age, body weight 18～22g,, purchased from Guangdong Medical College's Experimental Animal Center, raises under SPF level condition totally by 30.Be divided into 3 groups and test, 4 every group.

1.3 origins of cell, in the breast cancer cell MCF-7 of Balb/c mice, are preserved by Guangdong Medical College's Biochemistry and Molecular Biology institute.

1.4MCF-7 cell strain cellar culture, went down to posterity 1 time every 2 days, and the trophophase cell of taking the logarithm after the sufficient amount that goes down to posterity is made into cell suspension, Trypan Blue, and blood cell counting plate counting, cell concentration is adjusted to 1 * 10 ⁶/ ml.At Balb/c mice thoracic wall 5～6 intercostals or the 4th couple of mammary gland place subcutaneous vaccination tumor cell 0.1ml of stomach wall, be equivalent to 1 * 10 ⁶a cell/mice, observes the growing state of tumor every day.

1.5 according to the method for embodiment 2, prepare people TGF-β 2 antisense plasmids are transfected into the breast cancer tumour therapeutic vaccine that mouse mastopathy cell MCF-7 obtains mice.

2. tumor growth situation: breast cancer cell is inoculated latter 10 days, and tumor nodule appears in place at inoculation position breast pad, rounded, ellipse or lobulated growth, quality is harder.Tuberosity is cumulative large, the first group of Balb/c mice-transplanted tumor average external volume 108.1mm that inoculate rear 17 days ³.

3. treatment

Prepare as stated above experimental animal, adopt the tumor vaccine of embodiment 2 preparations to carry out therapeutic test:

The 2nd group gives tumor vaccine when inoculating in position, intravenous injection, and dosage is 1 * 10 ⁵cell/only, weekly, the 2nd group of Balb/c mice-transplanted tumor average external volume 55.2mm that inoculate rear 17 days ³.

The 3rd group with in-situ inoculating after within 2 weeks, give tumor vaccine, intravenous injection, dosage is 1 * 10 ⁵cell/only, every 3 days once, the 3rd group of Balb/c mice-transplanted tumor average external volume 62.1mm that inoculate rear 17 days ³.

Experimental example 4: the therapeutic alliance of breast cancer treatment vaccine

By the method for experimental example 3, prepare 2 groups of experimental animals, adopt the breast cancer tumour vaccine of embodiment 2 preparations to carry out therapeutic test:

The 4th group gives tumor vaccine when inoculating in position, intravenous injection, and dosage is 1 * 10 ⁵cell/only, life span is weekly; Adopt immunostimulant-acellular A group streptococcus preparation simultaneously, 0.5mg/0.1ml intramuscular injection, weekly.Inoculate latter 17 days Balb/c mice-transplanted tumor average external volume 38.1mm ³.

The 5th group with in-situ inoculating after within 2 weeks, give tumor vaccine, intravenous injection, dosage is 1 * 10 ⁵cell/only, every 3 days of life span is once; Adopt immunostimulant-acellular A group streptococcus preparation simultaneously, 0.5mg/0.1ml intramuscular injection, weekly.Inoculate latter 17 days Balb/c mice-transplanted tumor average external volume 44.1mm ³.

Experimental example 5: the treatment experiment of gastric cancer tumor therapeutic vaccine

1.1 32 of material B alb/c mices, Beijing dimension tonneau China Experimental Animal Center provides, quality certification SCXK (capital) 2002-0003.Female, in 6～8 week age, body weight 18～20g, raises under SPF level condition.Mouse Gastric Cancer cell MFC, purchased from Chinese Academy of Sciences's Shanghai cell biological institute.DMEM culture fluid is purchased from Sigma company, and hyclone is purchased from Ilex purpurea Hassk.[I.chinensis Sims biological product company limited.

The Mouse Gastric Cancer cell MFC subcutaneous vaccination that 1.2 methods are bought: inoculating cell number is 1 * 10 ⁸the 21st day tumor tissues of Mus grow to 1.0cm/0.8cm size, central authorities occur hemorrhage and downright bad, tumor tissues average external volume is 0.32cm ³.

3. treatment

Prepare as stated above experimental animal, what adopt embodiment 5 preparations is transfected into Mouse Gastric Cancer cell MFC by people TGF-β 2 antisense plasmids, and the gastric cancer tumor vaccine preparing carries out therapeutic test:

The 2nd group gives tumor vaccine in subcutaneous vaccination, intravenous injection, and dosage is 1.5 * 10 ⁵cell/only, weekly, inoculate latter 21 days Balb/c mice-transplanted tumor average external volume 0.17mm ³.

The 3rd group with in-situ inoculating after within 2 weeks, give tumor vaccine, intravenous injection, dosage is 1.5 * 10 ⁵cell/only, every 3 days once, inoculates latter 21 days Balb/c mice-transplanted tumor average external volume 0.20mm ³.

Experimental example 6: the therapeutic alliance of curing gastric cancer vaccine

Prepare as stated above 2 groups of experimental animals, adopt the gastric cancer tumor vaccine of experimental example 6 preparations to carry out therapeutic test:

The 4th group gives tumor vaccine when inoculating in position, intravenous injection, and dosage is 1.5 * 10 ⁵cell/only, life span is weekly; Adopt immunostimulant-acellular cloth Salmonella preparation simultaneously, 0.5mg/0.1ml intramuscular injection, weekly.Inoculate latter 21 days Balb/c mice-transplanted tumor average external volume 0.15mm ³.

The 5th group with in-situ inoculating after within 2 weeks, give tumor vaccine, intravenous injection, dosage is 1.5 * 10 ⁵cell/only, every 3 days of life span is once; Adopt immunostimulant-acellular cloth Salmonella preparation simultaneously, 0.5mg/0.1ml intramuscular injection, weekly.Inoculate latter 21 days Balb/c mice-transplanted tumor average external volume 0.15mm ³.

The zoopery of embodiment 7 treatment of pancreatic cancer vaccines

1. animal model is prepared:

1.1 laboratory animals and reagent:

Balb/c mice, body weight 18～22g,, is divided into 4 groups by 40; Mice pancreatic adenocarcinoma cell MPC-83 is purchased from Chinese Academy of Sciences's Shanghai cell biological institute.

1.2 mice pancreatic adenocarcinoma cell MPC-83 transplantability cancer of pancreas modellings:

After being cultivated, mice pancreatic adenocarcinoma cell MPC-83 is made into 1 * 10 ⁷/ ml, getting 0.2mL, to be inoculated in mice subcutaneous, becomes tumor after 4wk.Respectively within 20,30,40 days, using vernier caliper measurement tumor size.

According to formula V=ab ²/ 2 calculate gross tumor volumes [V=gross tumor volume, the major diameter of a=tumor body, the minor axis of b=tumor body].The transfer of perusal tumor and ascites production.

1.3. transplanted tumor change in volume monitoring

Latter the 20th, 30,40 days of inoculation, measures gross tumor volume meansigma methods and is respectively 84.1 ± 21.9mm ³, 413.7 ± 208.4mm ³, 2187.3 ± 1882.8mm ³.Before result shows the latter and 10d, comparing difference all has statistical significance (P<0.01).Anatomical results finds wherein to have tumor interior occur liquefying kitchen range and the area increase of 3 lotus tumor Balb/c mices, simultaneously with bloody ascites.

3. treatment

Prepare as stated above experimental animal, the method for employing embodiment 6 is transfected into mice pancreatic adenocarcinoma cell MPC-83 by people TGF-β 2 antisense plasmids and obtains pancreatic tumour therapeutic vaccine, carries out therapeutic test:

The 3rd group gives tumor therapeutic vaccine in Mice Inoculated pancreas adenocarcinoma cell MPC-83, intravenous injection, and dosage is 1.5 * 10 ⁵cell/only, weekly, the 20th, 30,40 days, measure gross tumor volume meansigma methods and be respectively 65.3 ± 14.6mm ³, 379.3 ± 164.8mm ³, 1068.7 ± 1202.5mm ³.

The 4th group gives tumor therapeutic vaccine at Mice Inoculated pancreas adenocarcinoma cell MPC-83 after two weeks, intravenous injection, and dosage is 1.5 * 10 ⁵cell/only, every 3 days once, the 20th, 30,40 days, measures gross tumor volume meansigma methods and be respectively 72.1 ± 10.8mm ³, 358.6 ± 143.2mm ³, 1308.5 ± 1230.1mm ³.

Experimental example 8: the therapeutic alliance for the treatment of of pancreatic cancer vaccine

Prepare as stated above 2 groups of experimental animals, adopt the treatment of pancreatic cancer vaccine of experimental example 7 preparations to carry out therapeutic test:

The 3rd group gives therapeutic vaccine in Mice Inoculated pancreas adenocarcinoma cell MPC-83, intravenous injection, and dosage is at 1.5 * 105 cells/only, once in a week, adopt immunostimulant-acellular cloth Salmonella preparation simultaneously, 0.5mg/0.1ml intramuscular injection, weekly.20th, 30,40 days, measure gross tumor volume meansigma methods and be respectively 45.7 ± 16.2mm ³, 265.3 ± 134.9mm ³, 975.7 ± 838.4mm ³.

The 4th group gives therapeutic vaccine at Mice Inoculated pancreas adenocarcinoma cell MPC-832 after week, intravenous injection, and dosage is 1.5 * 10 ⁵cell/only, every 3 days once, adopts immunostimulant-acellular cloth Salmonella preparation simultaneously, and 0.5mg/0.1ml intramuscular injection is weekly.20th, 30,40 days, measure gross tumor volume meansigma methods and be respectively 53.8 ± 20.7mm ³, 280.6 ± 102.1mm ³, 1033.5 ± 954.1mm ³.

Claims

1. a treating malignant tumor vaccine, it is characterized in that, described treating malignant tumor vaccine is for to obtain tumor cell line by the cancerous cell of people's tumor tissues by cultivation, TGF-β 2 antisense plasmids are transfected in tumor cell line, and described tumor cell line obtains treating malignant tumor vaccine after deactivation.

2. treating malignant tumor vaccine according to claim 1, is characterized in that, the cDNA of described TGF-β 2 is: NCBI Reference Sequence:NM_003238.3, fragment 1369bp-2613bp.

3. treating malignant tumor vaccine according to claim 1, it is characterized in that, the cancerous cell of described people's tumor tissues is selected from pulmonary carcinoma tumor cell, tumor cells of hepatocellular carcinoma, ovarian cancer tumor cell, breast cancer tumour cell, gastric cancer tumor cell, pancreatic tumour cell, colon cancer tumor cell, esophageal cancer cell, cervical cancer cell or endometrial carcinoma cell.

4. treating malignant tumor vaccine according to claim 1, is characterized in that, described preparation method comprises the following steps: (1) prepares people TGF-β 2 antisense plasmids:

5. a treating malignant tumor vaccine combination, is characterized in that, described compositions comprises treating malignant tumor vaccine claimed in claim 1 and immunostimulant.

6. described treating malignant tumor vaccine combination according to claim 5, it is characterized in that, described immunostimulant is selected from: short corynebacteria preparation, acellular short corynebacteria preparation, BCG-polysaccharide, nucleic acid preparation, Nocardia rubra cell skeleton, A group streptococcus preparation, acellular A group streptococcus preparation, bacillus pyocyaneus preparation, acellular bacillus pyocyaneus preparation, cloth Salmonella preparation, acellular cloth Salmonella preparation, acellular mycobacterium vaccae preparation, acellular smegmatis mycobacterium preparation, Thymopeptide, levamisole preparation, cholera mycin B subunit preparation, interleukin IL-1 preparation, interleukin IL-2 preparation, interleukin IL-3 preparation, interleukin IL-4 preparation, interleukin IL-6 preparation, interleukin IL-11 preparation, interleukin IL-12 preparation, interleukin IL-15, interleukin IL-18, interleukin IL-21, interleukin IL-23, heat shock protein white preparation, CpG oligodeoxynucleotide preparation, lyophilized bacillus calmette-guerin vaccine cell wall homogenate preparation, deactivation grass mycobacterium preparation, Pseudomonas aeruginosa MSHA pilus strain vaccine (PA-MSHA vaccine), ubenimex, Biostim, polyvalent bacterial lysate, interferon IFN-α preparation, interferon IFN-beta formulations, interferon IFN-γ preparation, colony-stimulating factor GM-CSF, colony-stimulating factor M-CSF, transfer factor preparation.

7. described treating malignant tumor vaccine combination according to claim 5, it is characterized in that, described immunostimulant is selected from: acellular short corynebacteria preparation, acellular A group streptococcus preparation, acellular bacillus pyocyaneus preparation, acellular cloth Salmonella preparation, acellular mycobacterium vaccae preparation, acellular smegmatis mycobacterium preparation; Preferred acellular short corynebacteria preparation.

8. treating malignant tumor vaccine combination according to claim 5, is characterized in that, contains treating malignant tumor vaccine and immunostimulant and after subpackage, merge packing, treating malignant tumor vaccine combination respectively in described compositions.

9. treating malignant tumor vaccine combination according to claim 5, is characterized in that, contains treating malignant tumor vaccine 1 * 10 in described compositions ⁷～1 * 10 ⁸cell, immunostimulant 0.5～5.0mg.

10. treating malignant tumor vaccine combination according to claim 9, is characterized in that, contains treating malignant tumor vaccine 1 * 10 in described compositions ⁷～5 * 10 ⁷cell, immunostimulant 0.5～2.5mg.