CN102335437A - Application of metadherin (MTDH) gene vaccine in preparation of breast cancer cell growth inhibitor - Google Patents

Application of metadherin (MTDH) gene vaccine in preparation of breast cancer cell growth inhibitor Download PDF

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CN102335437A
CN102335437A CN2010102330949A CN201010233094A CN102335437A CN 102335437 A CN102335437 A CN 102335437A CN 2010102330949 A CN2010102330949 A CN 2010102330949A CN 201010233094 A CN201010233094 A CN 201010233094A CN 102335437 A CN102335437 A CN 102335437A
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mtdh
vaccine
gene vaccine
gene
cell
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郭方
朱晗
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SHANGHAI YINUODI BIO-TECHNOLOGY CO LTD
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SHANGHAI YINUODI BIO-TECHNOLOGY CO LTD
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Abstract

The invention belongs to the field of biotechnologies, and relates to a gene vaccine capable of inhibiting cancer cell growth. The gene vaccine can express a cell membrane surface protein metadherin (MTDH) and thus being named as a MTDH gene vaccine. The MTDH gene vaccine is utilized for immunization in an oral way. The MTDH gene vaccine can activate an immune response of CD8+ cytotoxic T cells on breast cancer cells 4T1 expressing MTDH antigens. The MTDH gene vaccine can inhibit breast cancer cell growth and pulmonary metastasis. Through being combined with adriamycin, the MTDH gene vaccine can improve adriamycin effects of inhibiting breast cancer cell growth and pulmonary metastasis, and prolong a life of a mouse loaded with cancer. More significantly, the MTDH gene vaccine does not produce obvious damages on main organs of a human body. Therefore, the MTDH gene vaccine provides a novel approach for improvement of the existing breast cancer treatment method.

Description

MTDH gene vaccine and the application in preparation growth of breast cancers inhibitor thereof
Technical field
The invention belongs to biological technical field, relate to the MTDH gene vaccine, and the application of MTDH gene vaccine in preparation growth of breast cancers inhibitor.
Background technology
Breast carcinoma is one of cancer types very common among the women, it is reported, its mortality rate is first in all tumors.2009, there are 192370 people to be diagnosed as breast carcinoma approximately in the U.S., wherein 40170 people die from breast carcinoma [1].Also have report, diagnosed out neoplasm metastasis [2] above 40% breast carcinoma female patient.The therapeutic scheme of breast carcinoma comprises the excision primary lesion at present, regional lymph nodes cleaning art, hormone therapy and chemicotherapy or the like.Yet, even the successful excision primary lesion of operation and be aided with chemotherapy after, the relapse rate of breast carcinoma and mortality rate are still very high, research and analyse and think it possibly is to fail to excise fully MET and tumor causes reasons such as chemotherapeutics generation drug resistance owing to operation.Therefore, Metastasis in Breast Cancer and chemotherapy resistance property are to cause breast carcinoma morbidity and dead main reason.Means and effect based on present existing treatment breast carcinoma are limited, and this area researcher thinks that to press for exploitation new for Metastasis in Breast Cancer and drug-fast therapeutic strategy and medicine.
Prior art discloses gene vaccine (DNA vaccine) and has claimed nucleic acid vaccine or dna vaccination again; Be to encode certain antigenic gene fragment clone to eukaryon expression plasmid; This plasmid immune animal of reuse; Antigen encoding gene stimulates body to produce humoral immunization and cellullar immunologic response behind host's expression in vivo.Gene vaccine is after in the mouse muscle tissue, injecting DNA the earliest, and plasmid and the gene that carries thereof are absorbed by the mice myocyte, and in the myocyte, express, and mice can produce single-minded immunoreation to this foreign protein.Gene vaccine has received concern widely and has developed rapidly afterwards.Recent years, gene vaccine receives increasing attention in worldwide, becomes the focus of domestic and international research.Compare with the treatment of traditional antitumor immune, gene vaccine have safety, effectively, be prone in preparation, cost, the body immunological memory time lasting, preserve advantage such as convenient transport.Research shows that tumor gene vaccine can pass through activated T cell and/or the antibody-mediated immunoreation to the tumor autoantigen, thereby suppresses growth of tumor [3].According to this mechanism, developed at present some kinds to tumor specific antigen (tumor-specific antigens, TSA) or tumor associated antigen (tumor-associated antigens, gene vaccine TAA) [3].Common and the sub-coupling of immunostimulant of the tumor antigen that genetic vaccine vector is expressed through selecting different immunostimulant, helps the dissimilar immunoreation of antigenic activation.Gene vaccine to tumor has been in the news effective to several types of cancers; Comprise breast carcinoma, melanoma, pulmonary carcinoma, neuroblastoma or the like [4-7]; Also there are some vaccines to get into clinical experimental stage [3], as to vaccine of melanoma, colon cancer and carcinoma of prostate or the like.Yet known many potential tumor antigens generally all have expression in normal cell at present.Therefore, can only induce very faint immunoreation in vivo to these antigenic gene vaccines usually, perhaps cause body to produce to expressing these antigenic Normocellular autoimmune responses.Based on above reason, the selection of tumor antigen is most important concerning exploitation Antioncogene vaccine.
MTDH (Metadherin)/AEG-1 (Astrocyte Elevated Gene-1) is a kind of surface of cell membrane albumen.Brown etc. find this gene through the phage scanning technique, and find that this gene has lung positioning area (Lung Homing Domain), can promote the lung of breast tumor to shift [8] as shown in Figure 1.This kind of high expressed gene and can specificly be colonizated in lung in the breast cancer tissue, and after using MTDH antibody in advance in the body, breast cancer cell lung MET significantly reduces, and points out this gene in the lung of breast carcinoma shifts, to play an important role.
The prior art relevant with the present invention has following list of references:
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Summary of the invention
The purpose of this invention is to provide a kind of inhibition tumor, especially the gene vaccine of breast cancer cell growth.Be specifically related to MTDH gene vaccine and the application in preparation growth of breast cancers inhibitor thereof.
The present clinical treatment that has had multiple biotherapy to be used for breast carcinoma, for example cell vaccine [25], recombinant protein vaccine [26], cell transplantation immunization therapy [27] and gene vaccine [28].Cost is that gene vaccine is compared the clear superiority that has with the other biological therapy with body interior memory time of length.Anti-human papilloma virus gene vaccine Gardasil in 2006 by drugs approved by FDA.Also there are some gene vaccines that are directed against other cancers to get into clinical research [3] at present.Yet gene vaccine still is faced with the selection such as tumor specific antigen, the effectively selection of adjuvant, a series of challenge of caused side effect or the like in immunogenic raising and the body.In addition, vaccine is applied to prevent disease usually, and present most of tumor vaccines are applied to clinical treatment tumour patient not success as yet.Therefore, the therapeutic type vaccine is a research direction that clinical value is arranged very much of gene vaccine in future.In addition, existing report [29] before tumor vaccine and chemotherapy drugs in combination are used.
The recurrence of tumor and be present breast cancer relapse and dead major reason to the tolerance of chemotherapeutics.Generally believe that now tumor stem cell is one of tumor recurrence and extensive drug-fast major reason.Up to the present, there has been a series of tumor stem cell specificity marker thing to be identified out.The CD44+CD24-cell subsets belongs to the tumor stem cell [30] of human breast carcinoma.The CD133+ cell subsets has the character [31] of tumor stem cell in the human brain tumour.Aldehyde dehydrogenase (ALDH) is the class of enzymes of participating in stem cell self-protection, also is considered to belong to the tumor stem cell label, can improve the chemotherapy resistance property [32,33] of tumor cell.The aldehyde dehydrogenase of ALDH3A1 coding has the protection cell and avoids oxidative damage and the ability [34] of removing free radical.It is as a kind of important indicator [35] of confirming tumor to the sensitivity of cyclophosphamide one of (in breast cancer treatment traditional chemotherapeutics) clinically.People such as Hu Guohong think that MTDH raises ALDH3A1 and the expression of MET in breast cancer cell; After ALDH3A1 gene expression silence, can suppress the drug resistance ability [10] of MTDH mediation.Because MTDH raises the expression of ALDH3A1, and ALDH3A1 is the mark of tumor stem cell, so targeting MTDH possibly can eliminate the intravital tumor stem cell of cancer patient.
2009, people's such as Hu Guohong result showed that the MTDH gene has important function in processes such as breast carcinoma conversion, invasion and attack, transfer and drug resistance, and pointing out this gene is the target spot [9] of a potential oncotherapy.Verified, the MTDH gene is high expressed in tumors such as breast carcinoma, carcinoma of prostate, neuroblastoma, hepatocarcinoma, esophageal squamous cell carcinoma, and with its poor prognosis closely related [10-14].MTDH gene mapping is in human chromosome 8q22 district; Encoding proteins is 582 aminoacid; Be rich in lysine and serine residue, can be protein modified after the translation by other, like acetylation, ubiquitin modification and the serine of lysine residue, the phosphorylation modification of threonine residues.Through the PI3K-Akt signal path; Proto-oncogene Ha-Ras can strengthen the MTDH expression of gene; This adjusting mainly is (the Hu et al. that stablizes the c-Myc expression of gene and promote to realize on its promoter that is attached to MTDH through the phosphorylation inactivation of GSK3 β; Clinical Cancer Research, 2009).The MTDH gene is participated in the adjusting of many signal paths on this basis; Comprise PI3K-Akt, NF-κ B, Wnt/ β-catenin or the like signal path; Thereby MTDH can regulating cell propagation, cell survival, cellular infiltration and increase cell to the tolerance of chemotherapeutics etc. [9; 11,15-18].The activation of NF-κ B path is that the interaction through MTDH albumen and p65 and activating transcription factor CBP realizes.MTDH is through the ERK of enhancing MAPK family and the activity of p38, and phosphorylation GSK3 β also stablizes β-catenin, thereby activates Wnt/ β-catenin signal path; Simultaneously, MTDH can strengthen the expression of transcribing cofactor LEF-1 of β-catenin.In addition, MTDH strengthens the ability of tumor cell adhesion on endotheliocyte through combining with a kind of unknown endothelial cell receptor, thereby promotes the transfer of tumor.And MTDH can strengthen the drug resistance of tumor cell to chemotherapeutics through regulation and control a series of downstream drug resistant gene such as ALDH3A1 and MET.
The invention provides a kind of gene vaccine that suppresses tumor growth, this gene vaccine express cell film surface protein Metadherin.This gene vaccine contains the Metadherin encoding gene.MTDH (Metadherin)/AEG-1 (AstrocyteElevated Gene-1) is a kind of surface of cell membrane albumen.
Gene vaccine of the present invention can adopt MTDH gene and homology degree thereof to possess more than 70% and the nucleotide sequence of identical congenerous.Because the degeneracy of codon, so homology degree 85 even the 70% above DNA sequence same polypeptide of can encoding.For example, gene vaccine of the present invention can adopt the sequence of Gene ID 92140 in the ncbi database.
Gene vaccine of the present invention can attenuation salmonella be the attenuated strain carrier.
The present invention has developed a kind of new gene vaccine, and administered through oral carries attenuation salmonella (Salmonellatyphimurium, the dam of MTDH gene -And AroA -) reach the effect that suppresses Metastasis in Breast Cancer.The dam of this attenuation salmonella -With AroA -Gene mutation causes its toxicity to reduce greatly, utilizes it to obtain extensive studies and obtained good immune effect as the various exogenous antigens of carrier prokaryotic expression, has become one of important immune mediator of new generation vaccine.Behind this attenuation salmonella administered through oral immune mouse, can pass through the enterocyte barrier, be positioned in the peyer's patch (Peyer patches).EGFP gene vaccine through the attenuation salmonella mediation can navigate to mice peyer's patch (Peyer patches).After the mice oral immunity carries the Salmonella of EGFP gene, in peyer's patch (Peyer patches), can be observed green fluorescence (Reisfeld et al., Immunological Reviews, 2004).
Generally speaking, after attenuation salmonella arrives peyer's patch, accumulate in this (antigen presenting cells; APCs) be activated after engulfing the Salmonella of intrusion; Activatory APC migrates to lymphoid tissues such as spleen, carrier bacterium cracking this moment, and the dna vector of antigen expressed gene gets into cytosol; Get into the nucleus of antigen presenting cell then, express purpose antigen.Afterwards; (cytotoxic T lymphcocyte's special cytotoxic T lymphocyte CTL) is activated, the antigen presenting cell of cracking antigen expressed; Antigenic activation helper T cell in the antigen presenting cell is also induced antibody response; So far cellular immunization and humoral immunization all be activated (Reisfeld et al., Immunological Reviews, 2004).In addition, the immunostimulatory sequence on the composition of attenuation Salmonella carrier bacterium such as lipopolysaccharide and the dna vaccine vector also can be used as the adjuvant booster immunization and replys.There is report to confirm in addition, when selecting ubiquitin, can activates the immunoreation of CD8+ cytotoxic T lymphocyte mediation specifically as the immunostimulatory sequence on the dna vaccine vector.
Experimental result shows that the MTDH gene vaccine can induce body to produce the immunoreation to breast carcinoma of CD8+ cytotoxic T lymphocyte mediation.This vaccine can not only shift by the effectively preventing breast carcinoma lung, and to after having transplanted the mouse inoculation of breast tumor, can strengthen the sensitivity of tumor to the chemotherapeutics amycin; This vaccine combines the treatment tumor-bearing mice with chemotherapy after, life cycle that can the significant prolongation mice.It should be noted that MTDH gene high expressed in Mouse Liver, thereby the MTDH gene vaccine possibly cause the damage of liver.Yet well-known, the regeneration capacity of liver is very strong, and after 1 week of immunity, liver possibly accomplished self-regeneration, has not compared obvious damage so observe the mouse liver of MTDH vaccine therapy group than matched group at last.Therefore, the MTDH gene vaccine is hopeful in the new selection that can be used as in the future prevention and treatment breast carcinoma.In sum, the MTDH gene vaccine combines with chemotherapy and can be used as a kind of new strategy and the means of prevention and treatment breast carcinoma.
The invention provides the method for preparing of MT reconnaissance DH gene vaccine, at first the Metadherin gene coded sequence is cloned into expression vector; Then, transform attenuated strain with recombinant expression carrier, screening obtains described gene vaccine.Concrete operation method can adopt the conventional operation in this area or carry out according to embodiment.
Described expression vector can adopt prokaryotic expression carrier, for example pcDNA3.1/Ub-Myc.Described attenuated strain can be attenuation salmonella strain S.typhimurium (dam-and AroA-) RE88.
Vaccine of the present invention can be used for preparation and suppresses the tumor growth medicament, and especially the lung to breast carcinoma and breast tumor shifts.
The present invention also provides a kind of compositions that suppresses tumor growth, and said composition comprises MT reconnaissance DH gene vaccine and amycin.MTDH gene vaccine and amycin coupling are shifted to suppress the breast carcinoma lung, also can be used to suppress the growth of breast tumor, prolong the individual survival phase.
The invention provides the MTDH gene vaccine, mice can significantly suppress the growth of 4T1 breast carcinoma at former position after inoculating the MTDH gene vaccine in advance, and can significantly suppress lung and shift; Behind tumor-bearing mice inoculation MTDH gene vaccine, can strengthen the drug susceptibility of tumor, thereby suppress growth of tumor and lung shifts, and can improve the time-to-live of mice after the chemotherapy amycin.
In sum, the invention provides a kind of MTDH gene vaccine of administered through oral immunity, and detect the effect that it suppresses Metastasis in Breast Cancer.This vaccine can activate the CD8+ cytotoxic T cell to expressing the immunoreation of the antigenic breast cancer cell 4T1 of MTDH.And the MTDH gene vaccine is after inoculation in advance, and the growth and the lung that can suppress breast tumor shift.The more important thing is that after MTDH gene vaccine and the amycin coupling, the lung that can suppress breast tumor shifts, and plays the effect of treatment, and can prolong the life-span of tumor-bearing mice.Therefore, immunotherapy combines with chemotherapy can provide new thinking and approach for the Therapeutic Method that improves present breast carcinoma.
Description of drawings
The lung positioning sequence of Fig. 1: MTDH.
Fig. 2-Fig. 3: the expression pattern analysis of MTDH gene in each organ of mice and mouse mammary carcinoma cell line 4T1.Fig. 2: protein immunoblot analyze the express spectra of MTDH gene in mice organ and 4T1 cell (on), β-actin contrasts (descending) as confidential reference items.Fig. 3: real-time quantitative PCR is analyzed the express spectra of MTDH gene in mice organ and 4T1 cell, and is corresponding with Fig. 2.
Fig. 4-Fig. 5: the detection of the structure of MTDH genetic vaccine vector and protein expression situation thereof.Fig. 4: with the collection of illustrative plates of pcDNA3.1/Ub-Myc carrier as MTDH gene vaccine expression vector.Fig. 5: behind the MTDH vaccine plasmid transfection HEK293T cell, cell lysis is through the antibody mediated immunity trace detection MTDH expression of anti-c-myc.
Fig. 6-Fig. 8: MTDH gene vaccine growth and lung to the 4T1 breast tumor after prophylactic immunization shifts inhibited.Female BALB/c mouse (6-8 age in week) is divided into two groups (n=6), uses 10 week about 8PUb-MTDH or pUb vaccine oral immunity, altogether immunity is 3 times.After the 3rd immunity finishes a week, give the interior injection 10 of fat pad of mice breast 5The 4T1 breast cancer cell brings out the breast carcinoma spontaneous lung and shifts.Inoculate after 28 days, put to death experimental group and control group mice, get lung, the interstitial content that statistics tumor cell lung shifts.Fig. 6: experimental implementation flow chart.Fig. 7: TIS weight statistics is a unit with the gram.Fig. 8: to the number statistical result of two groups of mouse lung transfering nodes.
Fig. 9-Figure 12: MTDH gene vaccine and amycin are united use can strengthen the curative effect of amycin to the breast carcinoma lung transfer of 4T1 tumor-bearing mice.Female BALB/c mouse (6-8 age in week) is divided into 4 experimental grouies (n=6) at random, with 10 5The 4T1 cell inoculation is designated as the 0th day in the fat pad of female BALB/c mouse.Afterwards, mice is in the oral salmonella vaccine immunity of difference in the 3rd, 7,11 day 3 times; In the 4th, 8,12 day dosage tail vein injection amycin by 5 milligrams/kg body weight; The 13rd day excision mammary gland TIS.Put to death mice on the 37th day, the interstitial content that statistics breast carcinoma lung shifts.Fig. 9: experimental implementation flow chart.Figure 10: the weight statistics after the excision of mice TIS.Figure 11: each organizes the lung preparation photo of mouse.Figure 12: each is organized the result of the number statistical of mouse lung transfering node.
Figure 13-Figure 14: MTDH gene vaccine and amycin are united the life-span that use can prolong the 4T1 tumor-bearing mice.Female BALB/c mouse (6-8 age in week) is divided into 4 experimental grouies (n=6) at random, with 10 5The 4T1 cell inoculation is designated as the 0th day in the fat pad of female BALB/c mouse.Afterwards, mice is in the oral salmonella vaccine immunity of difference in the 3rd, 7,11 day 3 times; In the 4th, 8,12 day dosage tail vein injection amycin by 5 milligrams/kg body weight; The 13rd day excision mammary gland TIS.Figure 13: experimental implementation flow chart.Figure 14: each organizes mice survival curve statistics (* P<0.05).
Figure 15-19:MTDH gene vaccine is through the cytotoxic T cell mediation anti tumor immune response of CD8+.Figure 15: the isolated splenocyte of MTDH vaccine immune mouse is compared significantly cracking 4T1 cell with matched group.Figure 16: behind the anti-CD 4 antibodies preincubate, the cytotoxicity of T cell does not receive obviously to influence.Figure 17: significantly suppressed the cytotoxic effect of T cell behind the anti-CD8 antibody preincubate to the 4T1 cell.Figure 18: the infiltration situation of CD8+T cell in the anti-CD8 antibody test primary tumo(u)r.Figure 19: TUNEL dyeing detects the inductive TIS apoptosis of cytotoxic T cell.
Figure 20: the MTDH gene vaccine does not cause significant side effects in the mice body.Female BALB/c mouse is with pUb-MTDH or pUb vaccine immunity 3 times.After the 3rd immunity finished for 1 week, take out following tissue of mice and organ, comprise heart (a), small intestinal (b), kidney (c), liver (d) lung (e) and spleen (f) are used FFPE, do haematoxylin-Yihong (H&E) dyeing after the section.
The specific embodiment
Embodiment 1
Materials and methods:
One, animal, bacterial strain and cell line source
6-8 week, female BALB/c mouse was purchased the Experimental Animal Center in Shanghai.The criterion operation that the raising of mice is used about laboratory animal according to Medical College, Shanghai Communication Univ..Attenuation salmonella strain (Salmonella typhimurium, dam -And AroA -) (The Scripps Research Institute, La Jolla CA) is so kind as to give by Ralph A.Reisfeld professor for RE88 and mice 4T1 breast cancer cell line.
Two, cell culture
(HyClone, Logan UT) add 10% hyclone (HyClone) to mouse mammary carcinoma cell line 4T1, and (Invitrogen, Carlsbad CA) cultivate for 100 units per ml penicillins and 100ug/mL streptomycin with the RMPI-1640 culture medium.To be HEK293T add 10% hyclone with DMEM (HyClone) to the human embryonic kidney cell, and penicillin and streptomycin be at 37 ℃, 5%CO 2Cultivate in the incubator.
Three, genetic vaccine vector structure, protein expression checking
(The Burnham Institute, La Jolla CA) is so kind as to give the plasmid of coding total length mice MTDH gene by Erkki Ruoslahti professor.After this gene pressed PCR method amplification, be cloned into [4,7] in the pcDNA3.1/Ub-Myc carrier, empty carrier is as contrast.Should express the proteic vaccine plasmid transfection of MTDH HEK293T cell, and receive cell protein and do the protein immunoblot analysis, with the antibody (Beyotime of anti-c-myc label; Haimen; Jiangsu China) detects MTDH protein expression situation, with β-actin (Santa Cruz Biotechnology; Santa Cruz CA) contrasts as confidential reference items.
Four, the conversion of attenuation salmonella
Press the method for bibliographical information, gene vaccine plasmid electricity is transformed among attenuation salmonella strain S.typhimurium (dam-and AroA-) RE88 [4,5].Concrete steps are following:
(1) have the LB plate of Salmonella to choose monoclonal from long, receive in the 3ml LB culture medium, 37 ℃, 250rpm is cultured to exponential phase.
(2) ddH2O with ice washes 2 times, and is resuspended with 200ul ddH2O.
(3) change electric revolving cup over to, 10 minutes on ice after getting the 100ul bacterium and 2ug DNA mixing.
(4) electricity changes: 2.5kV, 25uF, 200 Ω.
(5) add the 200ul culture medium, recovered 30 minutes for 37 ℃, coated plate is cultivated.
Five, avoidance mode vaccine and tumor inoculation
In avoidance mode, female BALB/c mouse (6-8 age in week) is divided into two experimental grouies (n=6), uses 10 week about 8The attenuation salmonella vaccine that has pUb-MTDH or pUb plasmid is irritated stomach, and immunity is 3 times altogether.After the 3rd immunity finishes a week, give the interior inoculation 10 of fat pad of all mice breast 5The 4T1 breast cancer cell brings out the breast carcinoma spontaneous lung and shifts.Inoculate after 28 days, put to death experimental group and control group mice, get lung, soak, under anatomic microscope, add up the interstitial content of tumor cell lung transfer afterwards with Bo Enshi solution (Bouin ' s solution).
Bo Enshi solution compound method:
Picric acid saturated aqueous solution 75ml;
Formalin (40% formaldehyde) 25ml;
Glacial acetic acid 5ml.
Six, treatment pattern tumor and vaccination and chemotherapy drugs in combination use
In the treatment pattern, female BALB/c mouse is divided into 4 experimental grouies (n=14) at random, then with 10 5The 4T1 cell inoculation is designated as the 0th day in the fat pad of all mices.Afterwards, mice is in the oral salmonella vaccine immunity of difference in the 3rd, 7,11 day 3 times; In the 4th, 8,12 day dosage tail vein injection amycin by 5 milligrams/kg body weight; The 13rd day excision mammary gland TIS.The 37th day, put to death 6 mices for every group, the interstitial content that statistics breast carcinoma lung shifts; Add up all the other mices (8 s' every group) life span.
Seven, preparation mouse boosting cell
(1) female BALB/c mouse (6-8 age in week) is divided into two experimental grouies, uses 10 week about 8The attenuation salmonella vaccine that has pUb-MTDH or pUb plasmid is irritated stomach, and immunity is 3 times altogether.
(2) the 3rd immunity finished after five days, and tail vein injection 4T1 cell is with further activating immune system.
(3) mice is put to death in dislocation of cervical vertebra, puts in 70% ethanol and soaks 5 minutes, gets spleen and shreds.
(4) 70 μ m cell strainer place the plate that contains RPMI-1640, grind spleen with piston, and it is cotton-shaped to be white in color to spleen, the splenocyte of RPMI-1640 washing and filtering, collecting cell suspension in aseptic centrifuge tube, 1500rpm, centrifugal 10 minutes.
(5) wash 1 time with RPMI-1640, reuse 1ml erythrocyte cracked liquid is resuspended, room temperature 1 minute.
(6) add 10 times of RPMI-1640 with upper volume, mixing, centrifugal 10 minutes of 1500rpm abandons supernatant, washes altogether 3 times.
(7) add 10% hyclone (HyClone) with RMPI-1640 culture medium (HyClone); 100 units per ml penicillins, 100ug/mL streptomycin (Invitrogen) and 100 unit of activity/milliliter interleukin II (R&D Systems; Inc., Minneapolis MN) cultivates.
(8) simultaneously; The Tissue Culture Dish bottom surface is covered with 25 μ g/ milliliter ametycins (Roche Applied Science, Mannheim, Germany) 20 minutes 4T1 cell of pretreatment; With isolated splenocyte at 37 ℃, the incubator that contains 5% carbon dioxide was cultivated 5 days altogether.
Eight, the T cytotoxicity detects
Press document said [22]; With
Figure BSA00000199835400121
on-radiation cytotoxicity detection kit (Promega; Madi son; WI) detect the cytotoxicity of splenocyte to the 4T1 cell, the concrete operations step is following:
(1) 96 hole analysis plates is provided with (100ul/ hole):
Figure BSA00000199835400122
After application of sample was accomplished, 250g was imitated target cell with assurance in centrifugal 4 minutes and is fully contacted.
(2) put Sptting plate in incubator 4 hours.In the time of 3 hours 15 minutes, add in 10ul lysate to the maximum release aperture.After cracking was accomplished, centrifugal 4 minutes of 250g got supernatant.
(3) LDH measures: analysis buffer 12ml is added to one bottle of substrate, put upside down mixing.Change supernatant 50ul to 96 orifice plate, every hole adds 50ul substrate mixed liquor, and the room temperature lucifuge was hatched 30 minutes.
(4) every hole adds the 50ul stop buffer, on ELIASA, surveys the OD490 absorbance in 1 hour.
(5) calculate the cell killing rate: experimental port, the spontaneous release aperture of target cell and the spontaneous release aperture absorbance of effector lymphocyte all deduct culture fluid background absorbance; The maximum release aperture absorbance of target cell deducts volume control hole absorbance.
Figure BSA00000199835400131
(6) at CD8 +Or CD4 +The T cell suppresses in the experiment, and the anti-CD8 antibody (2.43) of splenocyte elder generation and 10 μ g/ milliliters or anti-CD 4 antibodies (GK1.5) preincubate 0.5 hour are done the T cytotoxicity then and detected.
Nine, protein immunoblotting analysis
(1) collecting cell, PBS are washed twice.
(2) with an amount of volume SDS protein lysate re-suspended cell, 100 ℃, 10 minutes.
(3) cell pyrolysis liquid is used the 10%SDS-PAGE gel electrophoresis.
(4) behind the electrophoresis transfer protein to nitrocellulose filter (Amersham Bioscience, Buckinghamshire, UK) on.
(5) transfer film that takes a turn for the better was with the TBST solution room temperature sealing that contains 5% skim milk and 1 ‰ tween 20s 1 hour.
(6) with the antibody of anti-MTDH (Invitrogen) and β-actin (Santa Cruz Biotechnology) 4 ℃ of incubated overnight.
(7) TBST washes 3 times, each 5 minutes.
(8) with horseradish peroxidase (HRP) labelling two anti-(Jackson ImmunoResearch Laboratories, West Grove PA) are at room temperature hatched 1 hour.
(9) TBST washes 3 times, each 5 minutes.
(10) (IL USA) develops the color and is exposed on the X-ray sheet to detect protein expression and changes for Pierce Biotechnology, Rockford with chemical luminous substrate SuperSignal West Pico Chemi luminescent Substrate.
Ten, the extracting of cell RNA
Extract tissue and cell RNA with Invitrogen company's T RIzol test kit.Concrete steps are following:
(1) collects about 0.1mg flesh tissue, be ground to powdery with liquid nitrogen on ice.Add 1ml TRIzol, continue to be ground to no obvious piece of tissue and get final product.Cell directly adds 1ml TRIzol.Concuss 15 seconds, room temperature was placed 15 minutes.
(2) add the 0.2ml chloroform according to every 1mL TRIzol, concuss 15 seconds, room temperature was placed 2-3 minute.
(3) 12000g, 4 ℃, centrifugal, 15 minutes.
(4) carefully shift the upper strata water to another centrifuge tube, every 1ml TRIzol adds the 0.5mL isopropyl alcohol, slow mixing, and room temperature was placed 10 minutes.12000g, 4 ℃ centrifugal, 10 minutes.
(5) abandon supernatant, add 1mL 75% washing with alcohol RNA deposition.7400g, 4 ℃ centrifugal, 5 minutes.
(6) abandon supernatant, dry RNA deposition.Add an amount of DEPC water dissolution ,-80 ℃ of refrigerators are preserved.
11, real-time quantitative polymerase chain reaction (real-time quantitative PCR) detects
Reverse transcription system with Promega becomes cDNA with the RNA reverse transcription.Real-time quantitative RT-PCR carries out quantitative PCR detection on LC480 real-time quantitative PCR appearance (Roche Applied Science).Reaction system is used
Figure BSA00000199835400141
Premix Ex Taq TM(Perfect Real Time) (Takara Biotechnology, Dalian, Liaoning; China) preparation, 10 μ l reaction systems are following: 2 * SYBR Green PCR Master Mix, 5 μ l, each 1 μ l of the forward and reverse primer of genes of interest or β-actin; CDNA0.2 μ l, ddH 2O 3.8 μ l.Response procedures is following: 95 ℃ 30 seconds; 95 ℃ 5 seconds, 60 ℃ 30 seconds, circulate 50 times; Set up solubility curve (dissociation curve or melting curve) at last.Each sample is done 3 secondary holes, the standard deviation of experiment with computing.Calculate the value of Δ Ct, correct the amount of the RNA of differential responses with β-actin.The primer of quantitative PCR with reference to the website ( Http:// pga.mgh.harvard.edu/primerbank/index.html), sequence is following:
5’-AGCCCAAACCAAATGGACG-3’(SEQ?ID?NO?1)
With 5 '-AACTGTTTTGCACTGCTTTAGC-3 ' (SEQ ID NO 2) be the MTDH primer;
5’-GGCTGTATTCCCCTCCATCG-3’(SEQ?ID?NO?3)
With 5 '-CCAGTTGGTAACAATGCCATGT-3 ' (SEQ ID NO 4) be β-actin primer.
12, histologic analysis
Female BALB/c mouse (6-8 age in week) is divided into two groups (n=4), irritates stomach with the vaccine that has pUb-MTDH or pUb plasmid week about, and immunity is 3 times altogether.After the 3rd immunity finished for 1 week, take out the heart, spleen, kidney, liver, lung and the small intestine of mice, use FFPE, do haematoxylin-Yihong dyeing after the 5 μ m section, and observe down in mirror.
13, immunohistochemical analysis
Tumor tissues with O.C.T.compound (Tissue-Tek, Sakura Finetechnical, Tokyo, frozen section is done in Japan) embedding then.
(1) section is fixed 20 minutes with cold acetone, and PBS washes 3 times afterwards, each 5 minutes.
(2) 0.3%H 2O 2-methanol treatment 20 minutes is washed 3 times with PBS afterwards, each 5 minutes.
(3) with confining liquid room temperature sealing 1 hour, with anti-CD8 or anti-CD 4 antibodies (eBioscience, Inc., San Diego, CA) 4 ℃ of incubated overnight.
(4) PBS washes 3 times, each 5 minutes.
(5) with horseradish peroxidase (HRP) labelling two anti-(Jackson ImmunoResearch Laboratories, West Grove PA) are at room temperature hatched 2 hours.
(6) PBS washes 3 times, each 5 minutes.
(7) with DAB colour reagent box DAB Substrate Kit (Vector Laboratories, Inc., Burlingame, CA) colour developing.
(8) washing is 5 minutes; Brazilwood extract dyeing 5 minutes; Washed 5 minutes.
(9) dehydration, mounting.
14, TUNEL staining analysis
Apoptosis of tumor cells employing In Situ Cell Death Detection Kit test kit (Roche Applied Science, Mannheim Germany) detect, and concrete steps are following:
(1) section washes twice with PBS, each 5 minutes.
(2) at room temperature fix 20 minutes with 4% paraformaldehyde.
(3) PBS washes twice, each 5 minutes.
(4) preparation TUNEL reaction mixture: 50 μ l Enzyme solution+450 μ l Label Solution.
(5) processed group adds 50 μ l TUNEL reaction mixtures, and negative control group adds 50 μ l Label Solution, and 37 ℃ of lucifuges were reacted 1 hour in dark wet box.
(6) the PBS rinsing is 3 times.Nucleus dyes with DAPI, washes twice with PBS at last, and under fluorescence microscope, observes.
15, experimental data statistics
Compare the difference between two groups of data with Student ' s t-test check, the P value is considered to difference less than 0.05 has statistical significance.Survival curve is used the Kaplan-Meier methods analyst.
Embodiment 2, the expression pattern analysis of MTDH in each tissue of mice and 4T1 breast cancer cell line
Existing document confirms that MTDH is at most of breast cancer disease philtrum expressions higher [10], thereby it maybe be as the target spot of treatment breast carcinoma.Because the tumor vaccine target spot will possess high expressed and two primary conditions of the low expression of normal structure in tumor; Whether feasible in order to verify that the MTDH gene vaccine is used to treat breast carcinoma, at first the express spectra of MTDH in mouse tissue and mouse mammary carcinoma cell line 4T1 analyzed.The result shows that the expression of MTDH albumen in 4T1 cell line and liver is higher, at the heart, spleen, kidney, lung, brain and small intestinal expression very low (Fig. 2).Quantitative PCR is the result also show, the MTDH mrna expression in mouse liver and 4T1 cell is apparently higher than its hetero-organization (Fig. 3).These results show that MTDH can be used as the potential target spot of treatment breast carcinoma.
The detection of the structure of embodiment 3, MTDH genetic vaccine vector and protein expression situation thereof
Select the pcDNA3.1/Ub-Myc carrier as the gene vaccine expression vector, mice MTDH full length gene cDNA sub-clone is arrived this expression vector (Fig. 4).Previous studies shows, behind the N end coupling ubiquitin protein of target protein, can effectively improve the I type MHC angtigen presentation approach [4,7] of target protein.Based on the protein degradation mechanism of ubiquitin mediation, ubiquitin coupling target protein can improve the degradation efficiency of albumen through proteasome, and the vaccine of this type coupling ubiquitin can be induced the cell-mediated immunoreation of stronger CD8+T than the vaccine that lacks ubiquitin.With MTDH vaccine plasmid or empty carrier transfection HEK293T cell, detect its expression (Fig. 5) through immunoblotting.
After confirming that MTDH vaccine plasmid can be expressed, the vaccine plasmid is transformed, inserted a termination codon, make the MTDH albumen of expression not have the c-myc label at the N of c-myc epi-position end.So doing is because the c-myc label has strong immunogenicity, can induce very strong immunoreation in vivo, and influence is to the evaluation of vaccine effect.After vaccine intelligence process sequence verification sequence was correct, electricity transformed the Salmonella of attenuation, prepares the administered through oral immunized mice.
Embodiment 4, MTDH gene vaccine can suppress the 4T1 breast tumor after prophylactic immunization growth and lung shift
In order to observe the effect of MTDH gene vaccine inhibition breast carcinoma, two groups of female BALB/c mouses (n=6) use 10 week about 8The attenuation salmonella vaccine that has pUb-MTDH or pUb plasmid is irritated stomach, and immunity is 3 times altogether.After last immunity finishes a week, give the interior injection 10 of fat pad of all mice breast 5The 4T1 breast cancer cell brings out the breast carcinoma spontaneous lung and shifts.Inoculate after 28 days, put to death mice, the interstitial content that statistics tumor cell lung shifts.Experiment flow is as shown in Figure 6.Experimental result shows that the MTDH gene vaccine is compared with control vaccine pUb, and the growth (P<0.05) that after 28 days, can obviously suppress 4T1 breast tumor in the BALB/c mouse body (Fig. 7).In addition, to the interstitial content statistical results show that lung shifts, the MTDH gene vaccine is compared with control vaccine also can suppress lung transfer (Fig. 8, P<0.05) more significantly.Above result shows that under the situation of inoculating the MTDH gene vaccine in advance, the growth of 4T1 breast tumor and lung shift and can be suppressed significantly.
Embodiment 5, MTDH gene vaccine and amycin are united use can strengthen the curative effect of amycin to the breast carcinoma lung transfer of 4T1 tumor-bearing mice
At present, exploitation faces two very big challenges to the gene vaccine of cancer: the one, and most of vaccine efficient is very low, can only evoke very weak immunoreation; The 2nd, present vaccine can only by the vaccine of the treatment cervical cancer of FDA approval, still not have the listing of other treatment property tumor vaccine except that at present in that the inoculation back is effective in advance.In order to detect the therapeutic effect of this MTDH gene vaccine, the present invention unites use with vaccine and chemotherapeutics amycin, and the latter is one of chemotherapeutics of widely used anti-breast cancer.With 10 5The 4T1 cell inoculation is designated as the 0th day in the fat pad of female BALB/c mouse.Afterwards, mice is in the oral salmonella vaccine immunity of difference in the 3rd, 7,11 day 3 times; In the 4th, 8,12 day dosage tail vein injection amycin by 5 milligrams/kg body weight; The 13rd day excision mammary gland TIS.Put to death mice on the 37th day, the interstitial content that statistics breast carcinoma lung shifts.Experiment flow is as shown in Figure 9.Shown in figure 10, the tumor weight of amycin treatment back excision in the 13rd day has been compared remarkable decline with MTDH gene vaccine processed group, but the tumor weight of amycin individual processing and combined vaccine processing back excision does not have significant difference.It is shown in figure 11 to put to death the lung tissue of taking out behind the mice on the 37th day.Lung shifts the node count results and shows, after MTDH gene vaccine or amycin were treated separately, the transfer of 4T1 breast carcinoma lung is compared with matched group can be by obvious inhibition.And amycin associating MTDH gene vaccine treatment breast carcinoma lung transfer ratio uses the better effects if (Figure 12) of treatment separately.These results show that under the situation of inoculating the 4T1 breast tumor in advance, the MTDH gene vaccine can suppress 4T1 breast carcinoma lung to be shifted, and after uniting use with the chemotherapeutics amycin, can further improve curative effect.
Embodiment 6, MTDH gene vaccine and amycin are united the life cycle that use can prolong the 4T1 tumor-bearing mice
Result before shows that the MTDH gene vaccine can suppress the growth and the transfer of 4T1 breast tumor effectively.Therefore infer that the MTDH gene vaccine possibly prolong the life-span of breast carcinoma tumor-bearing mice.In order to verify this hypothesis; The present invention arrived the 4T1 cell inoculation in the fat pad of female BALB/c mouse (n=8) in the 0th day; Mice is in the 3rd, 7,11 day oral immunity 3 times afterwards; In the 4th, 8,12 day dosage tail vein injection amycin by 5 milligrams/kg body weight, the 13rd day excision mammary gland TIS.Experiment flow is shown in figure 13.The survival curve result shows; The mice of MTDH vaccine and amycin therapeutic alliance group has 62.5% (5/8) the still survival up to the 73rd day; Be superior to the effect that MTDH vaccine or amycin use separately, and the mice of control vaccine processed group within 50 days all dead (Figure 14).Above presentation of results after amycin associating MTDH gene vaccine uses, can prolong the life-span of 4T1 tumor-bearing mice significantly.
Embodiment 7, MTDH gene vaccine pass through CD8 +Cytotoxic T cell mediation anti tumor immune response
People's such as Luo Yunping experiment is verified, and gene vaccine can activate CD8 +The anti tumor immune response [23,24] of cytotoxic T cell mediation.Because vaccine possibly activate CD8 +T cell and CD4 +Which crowd the T cell belongs in order to confirm the inductive GVT cell of MTDH gene vaccine, and the present invention is with pUb-MTDH or pUb vaccine immune mouse, and immunity is 3 times altogether.The 3rd immunity finished after five days, and tail vein injection 4T1 cell is with the immunoreation of further activating immune system to the 4T1 cell.Separate mouse boosting cell afterwards and cultivated 5 days, detect the toxicity of splenocyte the 4T1 cell; At CD8 +Or CD4 +The T cell suppresses in the experiment, the anti-CD8 antibody of splenocyte elder generation and 10 μ g/ milliliters or anti-CD 4 antibodies preincubate 0.5 hour, and then do the T cytotoxicity and detect.Experimental result shows that the vaccine-induced immune t-cell of MTDH is compared significantly cracking 4T1 cell (Figure 15) with control vaccine.In addition, behind CD8 antibody preincubate, the cytotoxicity of T cell is significantly suppressed (Figure 17); By contrast, behind the anti-CD 4 antibodies preincubate, the cytotoxicity of T cell does not receive obviously to influence (Figure 16).In order further to confirm cytotoxicity CD8 +The effect in vivo of T cell, the present invention is with CD8 in the antibody test primary tumo(u)r of anti-CD8 +The infiltration situation (Figure 18) of T cell, and detect the inductive apoptosis of tumor cells of cytotoxic T cell (Figure 19) with TUNEL dyeing, result show that the MTDH gene vaccine is compared with control vaccine and can bring out the stronger immunoreation to tumor cell in vivo.Above result shows, the activated CD8 of MTDH gene vaccine +Cytotoxic T cell can kill breast tumor cell in vitro and in vivo, reaches the effect of prevention and treatment tumor.
Embodiment 8, MTDH gene vaccine do not cause significant side effects in the mice body
One of side effect of gene vaccine possibly cause expressing the immunoreation of antigenic self normal cell and tissue exactly.Before result shows, the MTDH gene except that expression in mouse liver is higher, expression lower (Fig. 2-3) in other major organs.With pUb-MTDH or pUb vaccine immune mouse 3 times; After the 3rd immunity finished for 1 week; Take out the heart, spleen, kidney, liver, lung and the small intestine of mice, with doing haematoxylin-Yihong (H&E) dyeing after the specimens paraffin embedding slices, the histology that mirror is observed each internal organs down changes.The result shows; MTDH gene vaccine processed group is compared with matched group by mice; Do not show tangible histopathology difference (Figure 20); Explain that the MTDH gene vaccine to the not obviously damage of each major organs of mice, does not cause significant side effects yet, thereby can be used for the breast carcinoma clinical treatment in the mice body.
Figure ISA00000199835600011
Figure ISA00000199835600021

Claims (10)

1. a MTDH gene vaccine is characterized in that, this gene vaccine express cell film surface protein Metadherin.
2. gene vaccine as claimed in claim 1 is characterized in that this gene vaccine contains the Metadherin encoding gene.
3. gene vaccine as claimed in claim 1 is characterized in that, this gene vaccine is the attenuated strain carrier with the attenuation salmonella.
4. the method for preparing of the said gene vaccine of claim 1 is characterized in that, it comprises step: at first the Metadherin gene coded sequence is cloned into expression vector; Then, transform attenuated strain with recombinant expression carrier, screening obtains the described gene vaccine of claim 1.
5. method for preparing as claimed in claim 4 is characterized in that, described attenuated strain is attenuation salmonella strain S.typhimurium (dam-and AroA-) RE88.
6. the application of the said gene vaccine of claim 1 in preparation breast tumor growth inhibitor.
7. the application of the said gene vaccine of claim 1 in preparation breast tumor lung transfer inhibitor.
8. a compositions that suppresses tumor growth is characterized in that, said composition comprises described gene vaccine of claim 1 and amycin.
9. the described compositions of claim 8 suppresses the application in the breast carcinoma lung diversion medicaments in preparation.
10. the application of the described compositions of claim 8 in the growth medicine of preparation inhibition breast tumor.
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