CN102793914A - Novel vascular endothelial cell vaccine and preparation method thereof - Google Patents
Novel vascular endothelial cell vaccine and preparation method thereof Download PDFInfo
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- CN102793914A CN102793914A CN201210328481XA CN201210328481A CN102793914A CN 102793914 A CN102793914 A CN 102793914A CN 201210328481X A CN201210328481X A CN 201210328481XA CN 201210328481 A CN201210328481 A CN 201210328481A CN 102793914 A CN102793914 A CN 102793914A
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Abstract
The invention relates to a novel vascular endothelial cell vaccine containing an adjuvant and a preparation method thereof. The vascular endothelial cell vaccine is prepared by taking heterogenous animal, human being, mouse and cattle endothelial cells as a vascular endothelial cell source and combining the adjuvant OK432; and serving as an antigen immune substance, the vascular endothelial cell vaccine can be used for activating organism immune systems and generating immune response on tumor vessels. The preparation method comprises the following specific steps of: culturing human or other vascular endothelial cell strains with a DMEM (Dulbecco Modified Eagle Medium) containing 5 percent of fetal calf serum, 1 percent of an ECGS (Endothelial Cell Growth Supplement), penicillin and streptomycin; amplifying and collecting; washing to obtain a cell suspension; and mixing with the adjuvant OK432 in vitro to obtain the novel vascular endothelial cell vaccine. As proved by an animal experiment result, the endothelial cell vaccine can be used for inhibiting the growth of a plurality of mouse tumors. As proved by pathological section, the vascular endothelial cell vaccine is nontoxic to major organs of mice such as heart, liver, spleen, lung and kidney.
Description
Technical field
The invention belongs to the vaccine field, be specifically related to a kind of novel vascular endotheliocyte vaccine and preparation method thereof: promptly utilize vascular endothelial cell and adjuvant to prepare heteroimmune vaccine.
Background technology
The world today, tumor is one of main killer who threatens human health.At present, surgical operation, chemotherapy and radiotherapy are three kinds of main means of oncotherapy.Tumor be that a multistep is rapid, the process of too many levels, multi-level complicacy, the curative effect of these methods is restricted.Operative treatment although can effectively remove macroscopic tumor, can not guarantee that all tumor cells are all excised, so the problem of postoperative recurrence is serious as current efficacious therapy scheme.Chemicotherapy has serious toxic and side effects, and many tumors are insensitive to it, has drug resistance in various degree, so reduced the curative effect of chemicotherapy.
Depend on new vessels with transfer and form this hypothesis in theory since Judah Folkman proposes tumor growth, antineoplastic vascular generates treats the focus that has become oncotherapy.Anti-angiogenic therapy has medicine and reaches target cell easily, be difficult for to produce drug resistance, and antitumor spectra is extensive, and toxic and side effects is little, and has the advantage of synergy with chemotherapy, radiotherapy and radioimmunotherapy.The polypeptide drug of a large amount of target tumor angiogenesiss and monoclonal antibody have shown powerful antitumous effect before clinical and in the clinical experiment; And some target tumor angiogenesis medicaments are arranged; For example medicine such as Avastin, Endostar has gone through to go on the market, and becomes effectively replenishing of cancer clinical treatment.Yet the protein and peptide drugs biological half-life is short, and for reaching the effective antitumor effect, medicine needs to take in a large number at short notice, and this has caused toxic and side effects to a certain extent.In recent years along with the continuous development of immunotherapy of tumors; Application is made vaccine from the endotheliocyte of body or allosome; Utilize the mode of active immunity to induce body to produce the humoral immunization and the cellular immunization of target tumor blood vessel; Can reach good anti-tumor effect, and this scheme can be avoided taking the toxic and side effects that causes in a large number because of the protein and peptide drugs short-term.
Novel vascular endotheliocyte vaccine of the present invention; Be to originate as vascular endothelial cell with heterogenous animal people, Mus, cattle endotheliocyte; Parallel connection combination adjuvant OK432, preparation vascular endothelial cell vaccine is as cross-reacting antigen heteroimmune; The excitating organism immune system produces immunne response to the new vessels in the tumor.
OK432 has the effect that activates the panimmunity effector lymphocyte as the immunocompetence reinforcing agent, can improve the survival rate of malignant tumor patient safely and effectively, prolongs survival period.In Japan, OK432 has been widely used in clinical as the antineoplastic adjuvant drug, and clinical application shows to have good effect.
Summary of the invention
An object of the present invention is to provide a kind of endotheliocyte vaccine that contains adjuvant, comprise endotheliocyte antigen and adjuvant.
The present invention has increased the immunogenicity of endotheliocyte vaccine through introducing adjuvant OK432; Produce more effective immunoreation to bring out body; Thereby for prevention and the treatment behind the tumor operation clinically provides effective ancillary method, can effectively prolong survival time of patients, improve the quality of living.
Vascular endothelial cell vaccine of the present invention; Be to originate as vascular endothelial cell with heterogenous animal people, Mus, cattle endotheliocyte; Parallel connection combination adjuvant OK432, preparation vascular endothelial cell vaccine is as cross-reacting antigen heteroimmune; The excitating organism immune system is to the new vessels generation immunne response of tumor.The method for preparing of vascular endothelial cell vaccine: vascular endothelial cell can derive from people, Mus, the cattle blood vessel endothelial cell strain of In vitro culture; As with Human umbilical vein endothelial cells strain (HUVEC) preparation endotheliocyte vaccine,, place to contain 5%CO2 with ECM endotheliocyte special culture media; Cultivate in 37 ℃ of incubators; HUVEC after the collection amplification is in centrifuge tube, and pH 7.2 phosphate buffers wash three times, and 0.9% normal saline re-suspended cell is processed cell suspension; Behind the counting chamber counting, be diluted to 5 * 10 with 0.9% normal saline
7/ ml.Get 1ml cell suspension dissolving 0.5KE adjuvant OK432, fully mix, promptly process novel vascular endotheliocyte vaccine HUVEC-OK432.
Suppress the angiogenesis in the tumor, required oxygen and the nutrient substance supply of blocking-up tumor growth can suppress generation, development and the transfer of tumor.The endotheliocyte vaccine that we prepare.As cross-reacting antigen heteroimmune excitating organism immune system tumor vessel is produced immunne response; Destroy tumor neogenetic blood vessels through humoral immunization and cellular immunization number of ways; Suppress tumor growth; Cause the tumor neogenetic blood vessels pathologic damage, its antitumous effect is better than the medicine of angiogenesis inhibitor treatment greatly, and can overcome and take the toxic and side effects that the angiogenesis inhibitor medicine causes in a large number.
Description of drawings
Fig. 1 is preparation technology's flow chart of novel vascular endotheliocyte vaccine of the present invention.
Fig. 2 has shown the H&E coloration result; Demonstrate novel vascular endotheliocyte vaccine HUVEC-OK432 to the mice internal organs,, explain that this vaccine is safe like avirulences such as conscience spleen lung kidneys.Wherein, a:PBS; B:HUVEC-OK432; 1: heart; 2: liver; 3: spleen; 4: lungs; 5: kidney.
Fig. 3 novel vascular endotheliocyte of the present invention vaccine HUVEC-OK432 is to the zooprophylazis immunity test result of H22 hepatocarcinoma; Wherein, a:PBS; B:OK432; C:HUVEC; D:HUVEC-OK432.
Fig. 4 novel vascular endotheliocyte of the present invention vaccine HUVEC-OK432 is to the melanomatous zooprophylazis immunity test of B16F10 result; Wherein, a:PBS; B:OK432; C:HUVEC; D:HUVEC-OK432.
Fig. 5 novel vascular endotheliocyte of the present invention vaccine HUVEC-OK432 is to the zooprophylazis immunity test result of EAC breast carcinoma; Wherein, a:PBS; B:OK432; C:HUVEC; D:HUVEC-OK432.
The specific embodiment
The invention provides a kind of method for preparing of novel vascular endotheliocyte vaccine, preparation technology's flow process of this method is seen Fig. 1.
The effect of adjuvant OK432 in this novel endotheliocyte vaccine is: OK432 is as immune-active agent; Can strengthen the panimmunity cell activity; After stimulating lymphocyte; Demonstrate the LAK cytoactive, both obtained lymphocyte and can resist kinds of tumors, even the oncocyte of antagonism NK cell all shows active preferably.OK432 also participates in inducing generation CTL cell.Induce some cytokine expression and release, like IL-2, IFN-γ etc.
The method for preparing of novel endotheliocyte vaccine is following: vascular endothelial cell can derive from people, Mus, the cattle blood vessel endothelial cell strain of In vitro culture; As with Human umbilical vein endothelial cells strain (HUVEC) preparation endotheliocyte vaccine,, place to contain 5%CO2 with ECM endotheliocyte special culture media; Cultivate in 37 ℃ of incubators; HUVEC cell after the collection amplification is in centrifuge tube, and pH 7.2 phosphate buffers wash three times, and 0.9% normal saline re-suspended cell is processed cell suspension; Behind the counting chamber counting, be diluted to 5 * 10 with 0.9% normal saline
7/ ml.Get 1ml cell suspension dissolving 0.5KE adjuvant OK432, fully mix, promptly process novel vascular endotheliocyte vaccine HUVEC-OK432.
In second aspect of the present invention is the purposes that contains the endotheliocyte vaccine of adjuvant, as stated, clinically, is expected to tumor patient is carried out effective auxiliary treatment with the adjuvant endotheliocyte vaccine that contains of the present invention, prolongs patient's life cycle, the quality of making the life better.
The existing combination more detailed description of following instance novel endotheliocyte vaccine production method of the present invention and application thereof.Provide the purpose of these instances only to be exemplary explanation the present invention, can not it be interpreted as it is the restriction to scope of the present invention and essence.
Material:
(1) cell strain
Human umbilical vein endothelial cells strain (HUVEC) is available from the rich Science and Technology Ltd. of the abundant perseverance in Beijing.
(2) enzyme and reagent
Pancreatin is given birth to the worker available from Shanghai.OK432 is available from Paekche antitumor pharmacy, Guangzhou.
(3) culture medium
ECM endotheliocyte special culture media is available from the rich Science and Technology Ltd. of the abundant perseverance in Beijing.
Embodiment 1:
The HUVEC-OK432 vaccine production
Vascular endothelial cell can derive from people, Mus, the cattle blood vessel endothelial cell strain of In vitro culture, as preparing the endotheliocyte vaccine with Human umbilical vein endothelial cells strain (HUVEC), with ECM endotheliocyte special culture media; Place to contain 5%CO2, cultivate in 37 ℃ of incubators, the HUVEC cell after the collection amplification is in centrifuge tube; PH 7.2 phosphate buffers washing three times; 0.9% normal saline re-suspended cell is processed cell suspension, behind the counting chamber counting, is diluted to 5 * 10 with 0.9% normal saline
7/ ml.Get 1ml cell suspension dissolving 0.5KE adjuvant OK432, fully mix, promptly process novel vascular endotheliocyte vaccine HUVEC-OK432.
Embodiment 2:
The HUVEC-OK432 vaccine safety is estimated
Select 12 of 6-7 BALB/c mouses in age in week for use, vaccine group and matched group are 6, body weight 20-25g.According to Immunization programme in 0 the week every mouse subcutaneous injection 100 μ L vaccine and PBS.Whenever once, carry out booster immunization altogether 2 times later at a distance from two all booster immunizations.Two weeks of immunity back are put to death mice for the third time, get heart, liver, spleen, lungs and the kidney of respectively organizing mice, the formalin fixed with 10%; The conventional method FFPE, tissue slice, 4 μ m thickness; Hematoxylin and eosin dyeing, simple microscope is observed and is taken pictures.Vaccine immunity group and matched group organs and tissues section result observe, and do not find that vaccine has overt toxicity (Fig. 2) to each organ of animal.
Embodiment 3:
The HUVEC-OK432 vaccine is to the pharmacodynamics evaluation of H22 hepatocarcinoma
Select 24 of 6-7 BALB/c mouses in age in week for use, experimental group is 6, body weight 20-25g with each matched group.According to Immunization programme in 0 week every mouse subcutaneous injection 100 μ L vaccine with each control drug.Whenever once, carry out booster immunization altogether 2 times later at a distance from two all booster immunizations.The 6th week is inoculation H22 tumor in the oxter, right side.Inoculum density is 2 * 10
7/ ml, every 100 μ L.The 8th week was put to death mice, got tumor, the observation of taking pictures.Can find out that from the result after the HUVEC-OK432 immunity, the vaccine group tumor is significantly less than matched group, show that the HUVEC-OK432 vaccine can effectively suppress the growth (Fig. 3) of H22 hepatocarcinoma.
Embodiment 4:
The HUVEC-OK432 vaccine is to the melanomatous pharmacodynamics evaluation of B16F10
Select 24 of 6-7 C57BL/6 mices in age in week for use, carry out the HUVEC-OK432 vaccine to the melanomatous pharmacodynamics evaluation of B16F10 with quadrat method with embodiment 3.Can find out that from the result after the HUVEC-OK432 immunity, the vaccine group tumor is significantly less than matched group, show that the HUVEC-OK432 vaccine can effectively suppress the melanomatous growth of B16F10 (Fig. 4).
Embodiment 5:
The HUVEC-OK432 vaccine is to the pharmacodynamics evaluation of EAC breast carcinoma
Select 24 of 6-7 BALB/c mouses in age in week for use, carry out of the pharmacodynamics evaluation of HUVEC-OK432 vaccine with quadrat method EAC breast carcinoma with embodiment 3.Can find out that from the result after the HUVEC-OK432 immunity, the vaccine group tumor is significantly less than matched group, show that the HUVEC-OK432 vaccine can effectively suppress the growth (Fig. 5) of EAC breast carcinoma.
Claims (7)
1. novel vascular endotheliocyte vaccine that contains adjuvant, it is characterized in that: comprise endotheliocyte antigen and adjuvant, as cross-reacting antigen heteroimmune, the excitating organism immune system produces immunne response to tumor vessel.
2. according to the described vascular endothelial cell vaccine of claim 1, it is characterized in that: vascular endothelial cell is the endothelial cell strain that derives from heterogenous animal people, Mus, cattle.
3. according to the described vascular endothelial cell vaccine of claim 1, it is characterized in that: adjuvant is OK432.
4. according to the described vascular endothelial cell vaccine of claim 1, it is characterized in that: can be used for the immunization therapy of various tumors.
5. according to the described vascular endothelial cell vaccine of claim 1, it is characterized in that: not only can use separately but also can unite use with the other medicines active component.
6. the method for preparing of a vascular endothelial cell vaccine, it is characterized in that: vascular endothelial cell can derive from people, Mus, the cattle blood vessel endothelial cell strain of In vitro culture, as with Human umbilical vein endothelial cells strain (HUVEC) preparation endotheliocyte vaccine; With ECM endotheliocyte special culture media; Place to contain 5%CO2, cultivate in 37 ℃ of incubators, the HUVEC cell after the collection amplification is in centrifuge tube; Wash three times; 0.9% normal saline re-suspended cell is processed cell suspension, behind the counting chamber counting, is diluted to 5 * 10 with 0.9% normal saline
7/ ml gets 1ml cell suspension dissolving 0.5KE adjuvant OK432, fully mixes, and promptly processes novel vascular endotheliocyte vaccine HUVEC-OK432.
7. according to right 6 described method for preparinies: washing is a phosphate buffer with cleaning mixture.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379566A (en) * | 2018-02-01 | 2018-08-10 | 郑州大学 | Anti-tumor Human umbilical vein endothelial cells vaccine and its application in anticancer |
| WO2021093864A1 (en) * | 2019-11-13 | 2021-05-20 | 中国医药大学 | Use of heterologous tissue cell composition for treating cancers |
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| CN1887296A (en) * | 2006-07-31 | 2007-01-03 | 南京大学生物制药工程研究中心 | Method of inducing antitumor immunity and its application in preparing medicine |
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| CN1276245A (en) * | 1999-06-04 | 2000-12-13 | 华西医科大学 | Vascular endothelial cell heteroimmune vaccine and its preparing process |
| CN1406629A (en) * | 2001-08-13 | 2003-04-02 | 四川大学华西医院 | Vaccin of heterogenous blood vessel endothetial cell growth factor and preparing process thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379566A (en) * | 2018-02-01 | 2018-08-10 | 郑州大学 | Anti-tumor Human umbilical vein endothelial cells vaccine and its application in anticancer |
| WO2021093864A1 (en) * | 2019-11-13 | 2021-05-20 | 中国医药大学 | Use of heterologous tissue cell composition for treating cancers |
| TWI776276B (en) * | 2019-11-13 | 2022-09-01 | 中國醫藥大學 | Use of xenogeneic tissue cell composition for treating cancer |
| JP2023501433A (en) * | 2019-11-13 | 2023-01-18 | 中國醫藥大學 | Methods of Using Heterologous Tissue Cell Compositions |
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Application publication date: 20121128 |