CN1163613C - Human interleukin 6 core transcription factor expression plasmid series and its application in treating tumour - Google Patents
Human interleukin 6 core transcription factor expression plasmid series and its application in treating tumour Download PDFInfo
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Abstract
The present invention relates to a series of plasmid DNA-human interleukin 6 nuclear transcription factor expressing plasmid, which has the function of tumor inhibition in vivo. The series of the expression plasmid can be used for various kinds of tumorous treatment and prevention.
Description
The present invention relates to genetically engineered and field of gene, is about a kind of human interleukin 6 nuclear factor (NF-IL6) expression plasmid series and the application in oncotherapy thereof.
Existing anti-tumor medicine can not be controlled tumour and redemption patient's life satisfactorily because of toxicity and side effect big (as chemotherapeutics) and effect low (as herbal medicine and preparation thereof).
The inventor has found that in research work the human interleukin 6 nuclear factor of external source is pernicious at some Artificial expression in the cell causes the phenomenon of these malignant cell apoptosis, and this work is published in [Zhu Min-Sheng and Liu Ding-gan et al., DNA and Cell Biology, 1997,16 (2): 127-135]. State It is necessary to have announced also on the border that NF-IL6 is that macrophage is brought into play the function of its kill bacteria and malignant cell Result of study [Tanaka, T.et al., Cell, 1995,80 (2): 353-361]. These results of study show, NF-The IL6 gene may be used to treat malignant tumour.
For the NF-IL6 gene can be expressed in malignant cell and relevant effector cell, it need be connected with plasmid vector.Plasmid vector is a kind of instrument DNA that generally is used for molecular biology and genetically engineered research at present; They have the gene that makes artificial insertion and can express and (promptly be transcribed into messenger ribonucleic acid [mRNA], the genetic information that provides according to mRNA has been synthesized the protein of physiological action again) whole elements (as referring to [Sambrook et al., Molecular cloning:A Laboratory Manual.2nd ed., Cold Spring Harbor Laboratory, 1989]).Therefore, design construction contains NF-IL6 expression of gene plasmid series, and its application in oncotherapy is studied, and is highly significant with practical value.
The purpose of this invention is to provide NF-IL6 expression plasmid series, this plasmid can suppress the growth of in-vivo tumour, can be applicable to tumor treatment.
NF-IL6 expression plasmid series provided by the invention (being also referred to as " 14-6RS ") is that it has following basic structure according to the inventor with in the world about the design of the result of study of tumor phenotypes and genetic expression with make up:
The coding region of NF-IL6 coding region=human interleukin 6 nuclear transcription factor-2 gene;
The promotor of promotor=any eukaryotic gene, it is the element that starts its downstream next-door neighbour's expression of gene, for example SV40 virus early promoter; SV40 virus late promoter; Cytomegalovirus promoter; The promotor of metallothionein gene; The promotor of myosin gene; Adenovirus promoter; The vaccinia virus promotor; Herpes virus promoter; The influenza virus promotor; The promotor of actin gene; The promotor of collagen gene; The papillomatosis virus promoter; The polyomavirus promotor; The promotor of a-fetoprotein gene; The promotor of carcinoembryonic antigen; Mouse mammary tumor virus (MMTV) promotor etc.
The dna sequence dna between multiple clone site and polyA signal of any eucaryon plasmid carrier of dna sequence dna 1=generally contains the splicing signal of mRNA (messenger ribonucleic acid);
The polyA signal of polyA signal=any eucaryon plasmid carrier, it is the initiation site of polyA (polyadenous purine nucleotides) tail of mRNA;
The dna sequence dna between polyA signal and promotor of any eucaryon plasmid carrier of dna sequence dna 2=, the replication origin that generally contains plasmid vector DNA, also can contain antibiotics resistance gene, comprise and make bacterium obtain gene to certain antibiotic resistance (as aminobenzylpenicillin resistant gene amp, tetracycline resistance gene tet, or make eukaryotic cell obtain gene (as the gene neo of microbiotic G418 resistance etc.) kalamycin resistance gene kan etc.), to certain antibiotic resistance.
Above-described any eucaryon plasmid carrier, available following plasmid is example: pSV.SPORT1, pSPORT 1, pMSEx-1, pMSEx-2, pMSEx-3, pcDNA3, pSFV1, pCDV-1, pSVK3, pBPV, pMSG etc.
Basic structure 2 is seen Fig. 2, is by dna sequence dna 2 (part), promotor, the NF-IL6 coding region, dna sequence dna 1, polyA signal and dna sequence dna 2 (another part) be linked in sequence form the line style double chain DNA molecule, wherein:
The coding region of NF-IL6 coding region=human interleukin 6 nuclear transcription factor-2 gene;
The promotor of promotor=any eukaryotic gene, it is the element that starts its downstream next-door neighbour's expression of gene, for example SV40 virus early promoter; SV40 virus late promoter; Cytomegalovirus promoter; The promotor of metallothionein gene; The promotor of myosin gene; Adenovirus promoter; The vaccinia virus promotor; Herpes virus promoter; The influenza virus promotor; The promotor of actin gene; The promotor of collagen gene; The papillomatosis virus promoter; The polyomavirus promotor; The promotor of a-fetoprotein gene; The promotor of carcinoembryonic antigen; Mouse mammary tumor virus (MMTV) promotor etc.
The dna sequence dna between multiple clone site and polyA signal of any eucaryon plasmid carrier of dna sequence dna 1=generally contains the splicing signal of mRNA (messenger ribonucleic acid);
The polyA signal of polyA signal=any eucaryon plasmid carrier, it is the initiation site of polyA (polyadenous purine nucleotides) tail of mRNA;
Any eucaryon plasmid carrier of dna sequence dna 2=in polyA signal downstream and/or the dna sequence dna of promotor upstream, the replication origin that generally contains plasmid vector DNA, also can contain antibiotics resistance gene, comprise and make bacterium obtain gene to certain antibiotic resistance (as aminobenzylpenicillin resistant gene amp, tetracycline resistance gene tet, or make eukaryotic cell obtain gene (as the gene neo of microbiotic G418 resistance etc.) kalamycin resistance gene kan etc.), to certain antibiotic resistance.
Above-described any eucaryon plasmid carrier, available following plasmid is example: pSV.SPORT1, pSPORT 1, pMSEx-1, pMSEx-2, pMSEx-3, pcDNA3, pSFV1, pCDV-1, pSVK3, pBPV, pMSG etc.
NF-IL6 expression plasmid series of the present invention makes up with engineered method.To contain promotor, the polyA site, the plasmid replication starting point, the plasmid vector of antibiotics resistance gene and other regulating and controlling sequence is cut into line style with suitable restriction enzyme; The NF-IL6 coding region also has the plasmid of NF-IL6 gene to downcut with restriction enzyme from the clone, and purifying and warp are suitably handled; With dna ligase the two is linked to be ring-type then, after transform bacteria and the amplification, extracts plasmid and purifying in addition, promptly.Final product is pure plasmid DNA, does not contain any albumen, polysaccharide (comprising pyrogen).
Cut off with suitable restriction enzyme enzymolysis in the zone of above-mentioned dna sequence dna 2 as plasmid, promptly get the plasmid of line style purifying.
Will be among the following embodiment with pSN, pCN, pVN, pTN and pGN are example, describe the preparation method in detail.
" 14-6RS " of other structure all can be with reference to this example, with general gene engineering method preparation.
" 14-6RS " of the present invention is example with pSN through the treatment experiment to the human tumor of laboratory animal institute lotus, proves that it can suppress tumor growth effectively, even tumour is disappeared and fully recovers, and non-evident effect.The tumour that nude mouse subcutaneous vaccination human liver cancer cell is formed for example, every knurl single injection 1-10 microgram " 14-6RS " all can be observed tumour and dwindle gradually and be tending towards disappearing after 3 days, and the tumour of contrast is then increasing; Behind nude mouse abdominal cavity inoculation gastric carcinoma cells, every mouse abdominal cavity single injection 10 micrograms " 14-6RS " are behind the first quarter moon, collator's intraperitoneal is bled profusely, dissect and find that tumour is big and downright bad, show 1/3 no pathology and inject " 14-6RS " person dissection, 2/3 tumour is significantly less than contrast; To the tumour that nude mouse subcutaneous vaccination human lung carcinoma cell is formed, every knurl injection 1-10 microgram " 14-6RS " is observed the breeding ratio contrast of knurl and is significantly slowed down.
Thereby, NF-IL6 expression plasmid series of the present invention be a kind of have tumour is dwindled and/or the plasmid DNA of the function that disappears.Use contains the plamid vector construction expression plasmid series of the present invention of different promoters, with making it may treat various malignant tumours in the different tissues, for example contains the expression plasmid of afp promoter, is specially adapted to treat liver cancer.It is expelled to lotus has near various human malignant lesions' the tumor locus of nude mouse or its, and compared with the control, tumour is dwindled significantly even disappeared.The effective dose that it uses is very low, and in tumor bearing nude mice, generally each knurl injection number microgram can suppress tumor proliferation.In observation so far, the animal of injection " 14-6RS " does not see overt toxicity reaction and drug side effect.Therefore, " 14-6RS " can be developed to a kind of efficient and safe genomic medicine, to be applied to various tumor treatment and prevention.
Description of drawings:
Fig. 1 is the basic structure 1 (ring-type) of NF-IL6 expression plasmid series.
Fig. 2 is the basic structure 2 (line style) of NF-IL6 expression plasmid series.
Fig. 3 is the schema of plasmid pSN construction process.
Fig. 4 is the schema of plasmid pCN construction process.
Fig. 5 is the schema of plasmid pVN construction process.
Fig. 6 is the schema of plasmid pTN construction process.
Fig. 7 is the schema of plasmid pGN construction process.
Fig. 8 is that plasmid pSN is to the treatment curve of people's liver cancer nude mice plantation knurl and the result curve figure of control group.
Fig. 9 is the treatment experiment (1) of plasmid pSN to people's cancer of the stomach nude mice plantation knurl, and in the mouse frame: the right side is the effective nude mice of treatment, and the left side is dying nude mice.
Figure 10 is the treatment experiment (2) of plasmid pSN to people's cancer of the stomach nude mice plantation knurl, the dissection of nude mice: the right side is the effective nude mice of treatment, and the left side is the nude mice that dies of illness.
Figure 11 is that plasmid pSN is to the treatment result curve of people's lung cancer nude mice plantation knurl and the graphic representation of control group.
The present invention is further described by following examples, but does not place restrictions on scope of the present invention.
The construction process of embodiment 1 plasmid pSN
A. the preparation of plasmid vector and NF-IL6 gene fragment
1. (German doctor Schuemann gives to get plasmid pMSEx-1, contain SV40 virus early promoter) 0.5 μ g (0.5 μ l), add 10X restriction enzyme StuI damping fluid 2 μ l, aqua sterilisa 17 μ l and restriction enzyme StuI 1 μ l, mix, enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Reaction solution with the phenol/chloroform extracting once, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HClpH8.0-1m mol/L EDTA of 10 μ l.
2. get above-mentioned dissolved pMSEx-1 9 μ l, 10X calf intestines alkaline phosphatase damping fluid 1 μ l and calf intestines alkaline phosphatase 0.5 μ l mix, and enzyme reaction was carried out in 37 ℃ of insulations in 30 minutes.Reaction solution adds 1 μ l 50m mol/L EDTA, and 75 ℃ of heating 10 minutes are used the phenol/chloroform extracting once then, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 10 μ l, is to be A liquid.
3. get plasmid pBlue610 (Japan examines good quiet male doctor and gives) 0.5 μ g (0.5 μ l), add 10X restriction enzyme SalI damping fluid 2 μ l, aqua sterilisa 17 μ l and restriction enzyme SalI 1 μ l mix, and enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Add 2 μ l in the reaction solution and contain dATP, dCTP, the solution of each 10m mol/L of dTTP and dGTP, and 1 μ l T4 archaeal dna polymerase continue insulation 20 minutes.Reaction solution separates with 0.7% low melting-point agarose gel electrophoresis, downcut 1.0kb district band, from gel, extract plasmid DNA, with phenol/chloroform extracting 1 time, the chloroform extracting once, the ethanol sedimentation plasmid, the 10mmol/L Tris.HCl pH8.0-1m mol/L EDTA that is dissolved in 10 μ l is standby, be to be B liquid, i.e. the NF-IL6 coding region of tack.
B. being connected of tack NF-IL6 coding region and plasmid vector pMSEx-1, bacterium transforms and identifies
Get A liquid 5 μ l, B liquid 10 μ l, 10X T4 dna ligase damping fluid 2 μ l, 10m mol/L ATP2 μ l and T4 dna ligase 2 μ l mix 4 ℃ of incubated overnight.Next day, get 2 μ l reaction solution transformed into escherichia coli DH5 α, be coated on the LB solid medium that contains 100 μ g/ml penbritins, overnight incubation, picking colony at random, with 1ml LB liquid nutrient medium amplification bacterium, the extracting plasmid is used restriction enzyme EcoRI enzymolysis respectively, and reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, selection 1.3kb occurs and inserts segmental plasmid, is plasmid pSN.It is standby to preserve and increase.
Above process as shown in Figure 3.
The construction process of embodiment 2 plasmid pCN
A. the preparation of plasmid vector and NF-IL6 gene fragment
1. get plasmid pcDNA3 and (be derived from Invitrogen company, contain SV40 virus early promoter) 0.5 μ g (0.5 μ l), 10X restriction enzyme EcoRI damping fluid 2 μ l, aqua sterilisa 16 μ l, restriction enzyme EcoRI 1 μ l and restriction enzyme XhoI 1 μ l, mix, enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Reaction solution with the phenol/chloroform extracting once, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 10 μ l, is to be A liquid.
2. get plasmid pBlue610 0.5 μ g (0.5 μ l), 10X restriction enzyme EcoRI damping fluid 2 μ l, aqua sterilisa .17 μ l, restriction enzyme EcoRI 1 μ l and restriction enzyme XhoI 1 μ l mix, and enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Reaction solution separates with 0.7% low melting-point agarose gel electrophoresis, downcuts 1.0kb district band, extracts plasmid DNA from gel, and the 10m mol/L Tris.HCl pH8.0-1mmol/L EDTA that is dissolved in 10 μ l is standby, is to be B liquid, i.e. NF-IL6 coding region.
B.NF-IL6 coding region and plasmid vector pcDNA3 are connected and the bacterium conversion
Get A liquid 5 μ l, B liquid 10 μ l, 10X T4 dna ligase damping fluid 2 μ l, 10m mol/L ATP2 μ l and T4 dna ligase 1 μ l mix, and 16 ℃ of incubated overnight are carried out enzyme reaction.Next day, get 1 μ l reaction solution transformed into escherichia coli DH5 α, be coated on the LB solid medium that contains 100 μ g/ml penbritins, overnight incubation, picking colony at random, with 1ml LB liquid nutrient medium amplification bacterium, extracting plasmid, each plasmid are all used restriction enzyme EcoRI and XhoI double enzymolysis, and reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, select to occur about 1.0kb and insert segmental plasmid (needn't identify separately), be plasmid pCN.It is standby to preserve and increase.
Above process as shown in Figure 4.
The construction process of embodiment 3 plasmid pVN
A. the preparation of plasmid vector and NF-IL6 gene fragment
1. get plasmid pSV.SPORT1 (available from GibcoBRL company, contain SV40 virus early promoter) 0.5 μ g (0.5 μ l), 10X restriction enzyme SalI damping fluid 2 μ l, aqua sterilisa 17 μ l and restriction enzyme SalI 1 μ l, mix, enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Reaction solution with the phenol/chloroform extracting once, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/ITris.HCl pH8.0-1mmol/L EDTA of 10 μ l.
2. get above-mentioned dissolved pSV.SPORT1 9 μ l, 10X calf intestine alkaline phosphatase damping fluid 1 μ l and calf intestine alkaline phosphatase 0.5 μ l mix, and enzyme reaction was carried out in 37 ℃ of insulations in 30 minutes.Reaction solution adds 1 μ l 50m mol/L EDTA, and 75 ℃ of heating 10 minutes are used the phenol/chloroform extracting once then, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 10 μ l, is to be A liquid.
3. get plasmid pBlue610 0.5 μ g (0.5 μ l), 10X restriction enzyme SalI damping fluid 2 μ l, aqua sterilisa 17 μ l and restriction enzyme SalI 1 μ l mix, and enzyme reaction was carried out in 37 ℃ of insulations in 1 hour.Reaction solution separates with 0.7% low melting-point agarose gel electrophoresis, downcuts 1.0kb district band, extracts plasmid DNA from gel, and the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA that is dissolved in 10 μ l is standby, is to be B liquid, i.e. NF-IL6 coding region.
The b.NF-IL6 coding region is connected with plasmid vector pSV.SPORT1's, and bacterium transforms and identifies
Get above-mentioned A liquid 5 μ l, B liquid 10 μ l, 10X T4 dna ligase damping fluid 2 μ l, 10m mol/LATP 2 μ l and T4 dna ligase 1 μ l mix, and 16 ℃ of incubated overnight are carried out enzyme reaction.Next day, get 1 μ l reaction solution transformed into escherichia coli DH5 α, be coated on the LB solid medium that contains 100 μ g/ml penbritins, overnight incubation, picking colony at random, with 1ml LB liquid nutrient medium amplification bacterium, the extracting plasmid is used restriction enzyme SalI enzymolysis respectively, reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, select to occur about 1.0kb and insert segmental plasmid, further purify, so that identify.
C. the evaluation of the recombinant plasmid of forward insertion
Be taken at the recombinant plasmid 9 μ l that the b step obtains, add 10X EcoRI enzyme buffer liquid 1 μ l and EcoRI enzyme 4 units, mix, enzyme reaction was carried out in 37 ℃ of insulations in 2 hours, reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, selects the segmental recombinant plasmid of about 1.0kb not occur, and this is plasmid pVN.It is standby to preserve and increase.
Above process as shown in Figure 5.
The structure of embodiment 4. plasmid pTN
A. the preparation of plasmid vector and NF-IL6 gene fragment
1. (professor Shen Xiaozhou of Institute of Zoology, Academia Sinica gives to get plasmid pSMT, this plasmid contains the metallothionein gene promotor) 0.5 μ g (0.5 μ l), 10X restriction enzyme buffer A (Boehringer-Mannheim company) 2 μ l, aqua sterilisa 17 μ l and Restriction enzyme Sma I 1 μ l, mix, 25 ℃ of insulations were carried out enzyme reaction in 2 hours, added restriction enzyme KpnI 1 μ l then, and 37 ℃ are continued insulation 2 hours.Reaction solution is with phenol/chloroform extracting 1 time, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 10 μ l, is to be A liquid.
2. get plasmid pBlue610 0.5 μ g (0.5 μ l), 10X restriction enzyme buffer A 2 μ l, aqua sterilisa 17 μ l and Restriction enzyme Sma I 1 μ l, mix, 25 ℃ of insulations were carried out enzyme reaction in 2 hours, added restriction enzyme KpnI 1 μ l then, and 37 ℃ are continued insulation 2 hours.Reaction solution separates with 0.7% low melting-point agarose gel electrophoresis, downcuts 1.0kb district band, extracts plasmid DNA from gel, and the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA that is dissolved in 10 μ l is standby, is to be B liquid, i.e. NF-IL6 coding region.
B.NF-IL6 coding region and plasmid vector pSMT are connected and the bacterium conversion
Get A liquid 5 μ l, B liquid 10 μ l, 10X T4 dna ligase damping fluid 2 μ l, 10m mol/L ATP2 μ l and T4 dna ligase 1 μ l mix, and 4 ℃ of incubated overnight are carried out enzyme reaction.Next day, get 1 μ l reaction solution transformed into escherichia coli DH5 α, be coated on the LB solid medium that contains 100 μ g/ml penbritins, overnight incubation, picking colony at random, with 1ml LB liquid nutrient medium amplification bacterium, extract plasmid, each plasmid is all used Restriction enzyme Sma I and KpnI double enzymolysis, and reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, select to occur about 1.0kb and insert segmental plasmid (needn't identify separately), be plasmid pTN.It is standby to preserve and increase.
Above process as shown in Figure 6.
The structure of embodiment 5. plasmid pGN
A. the preparation of plasmid vector and NF-IL6 gene fragment
1. get plasmid pMSG (Pharmacia Biotech company, this plasmid contains mouse mammary tumor virus [MMTV] promotor) 0.5 μ g (0.5 μ l), 10X restriction enzyme buffer A (Boehringer-Mannheim company) 2 μ l, aqua sterilisa 17 μ l and Restriction enzyme Sma I 1 μ l, mix, 25 ℃ of insulations were carried out enzyme reaction in 2 hours, added restriction enzyme XhoI 3 μ l then, and 37 ℃ are continued insulation 2 hours.Reaction solution is with phenol/chloroform extracting 1 time, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 10 μ l, is to be A liquid.
2. get plasmid pBlue610 0.5 μ g (0.5 μ l), 10X restriction enzyme buffer A 2 μ l, aqua sterilisa 17 μ l and Restriction enzyme Sma I 1 μ l, mix, 25 ℃ of insulations were carried out enzyme reaction in 2 hours, added restriction enzyme XhoI 3 μ l then, and 37 ℃ are continued insulation 2 hours.Reaction solution separates with 0.7% low melting-point agarose gel electrophoresis, downcuts 1.0kb district band, extracts plasmid DNA from gel, and the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA that is dissolved in 10 μ l is standby, is to be B liquid, i.e. NF-IL6 coding region.
B.NF-IL6 coding region and plasmid vector pMSG are connected and the bacterium conversion
Get A liquid 5 μ l, B liquid 10 μ l, 10X T4 dna ligase damping fluid 2 μ l, 10m mol/L ATP2 μ l and T4 dna ligase 1 μ l mix, and 16 ℃ of incubated overnight are carried out enzyme reaction.Next day, get 1 μ l reaction solution transformed into escherichia coli DH5 α, be coated on the LB solid medium that contains 100 μ g/ml penbritins, overnight incubation, picking colony at random, with 1ml LB liquid nutrient medium amplification bacterium, extract plasmid, each plasmid is all used Restriction enzyme Sma I and XhoI double enzymolysis, and reaction solution separates with the gel electrophoresis of 0.7% plain agar sugar, select to occur about 1.0kb and insert segmental plasmid (needn't identify separately), be plasmid pGN.It is standby to preserve and increase.
Above process as shown in Figure 7.
The structure of embodiment 6 linear plasmid pVN
Get the plasmid pVN 100 μ g (100 μ l) that embodiment 3 makes up, add 10X restriction enzyme PvuI damping fluid 20 μ l, aqua sterilisa 70 μ l and restriction enzyme PvulI 10 μ l (100 unit) mix, and enzyme reaction was carried out in 37 ℃ of insulations in 2 hours.Reaction solution with the phenol/chloroform extracting once, the chloroform extracting once, the ethanol sedimentation plasmid is dissolved in the 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA of 100 μ l, promptly gets linear plasmid pVN.Can preserve standby.
Embodiment 7: plasmid pSN is to the treatment experiment of people's liver cancer nude mice plantation knurl
15 of nude mouses, subcutaneous every injection point inoculation 10
7People's liver cancer SMMC7721 cell.After 1 week, all form tumour.Per 4 is one group (control group of not injecting any medicine is 3), and each group is injected following material at tumor locus respectively:
0.1 group ... ... ... ..pSN 0.1 μ g adds liposome 50 μ l
1 group ... ... ... ..pSN 1 μ g adds liposome 50 μ l
10 groups ... ... ... ..pSN 10 μ g add liposome 50 μ l
Control group ... ... ... ..0
After the injection, measured once major diameter of each tumour (a) and minor axis (b) every 3 days, and calculate gross tumor volume V by following formula:
V＝0.4a?b
2。
The result shows that the tumour of having injected pSN all stops to breed and being tending towards dwindling, and the part tumour disappears at last.As table 1 and shown in Figure 8.
Table 1 plasmid pSN is to the treatment experiment of people's liver cancer nude mice plantation knurl
0.1 group of 1 group of 10 groups of control group of injection back fate
0 1 1 1 1
3 0.49+-0.41 0.53+-0.11 0.66+-0.10 0.94+-0.21
6 0.21+-0.20 0.21+-0.08 0.24+-0.14 1.7+-0.56
9 0.06+-0.04 0.11+-0.08 0.04+-0.05 1.9+-0.67
The ratio of this gross tumor volume when data are term day gross tumor volume and injection in the table (mean value+-standard deviation), make graphic representation 8 with this implementation data.
Embodiment 8 plasmid pSN are to the treatment experiment (1) of people's cancer of the stomach nude mice plantation knurl
12 of nude mouses, each abdominal injection 6 * 10
6Individual cancer of the stomach MKN-45 cell, one week the back 8 each abdominal injection 10 μ g pSN are wherein added liposome 50 μ l and physiological saline 250 μ l, all the other 4 are contrast.Observe after 8 days, the result is as follows:
A) 4 contrast mouse all are in moribund condition, and abdominal tympanites is dissected and found a large amount of intraperitoneal hemorrhage (see figure 9)s;
B) treat in the mouse for 12,5 also are in moribund condition, and abdominal tympanites is dissected and found big tumour and a large amount of intraperitoneal hemorrhages; 4 performances are active, dissect and find that also tumour and internal hemorrhage are arranged, but the degree comparison is according to significantly light; 3 are as good as with normal mice, dissect and also do not find tumour, illustrates by the healing (see figure 10).Therefore the injection of the pSN tumor proliferation speed that can significantly slow down, even cure tumour.
Embodiment 9 plasmid pSN are to the treatment experiment (2) of people's lung cancer nude mice plantation knurl
6 of nude mouses, each subcutaneous injection 10
7Individual's lung cancer SPC-A-1 cell after one week, all forms tumour.Mice with tumor is equally divided into 3 groups, and 1 group is contrast, and all the other 2 groups respectively at tumor locus injection pSN0.1 μ g and 1 μ g.Measured once major diameter of each tumour (a) and minor axis (b) every 2-3 days, and calculate gross tumor volume V by following formula:
V＝0.4ab
2。
The result shows that the rate of propagation of having injected the tumour of pSN contrasts and significantly slows down, as table 2 and shown in Figure 11.
Table 2 plasmid pSN is to the treatment experiment of people's lung cancer plantation knurl
0.1 group of 1 group of control group of injection back fate
0 1 1 1
4 2.19+-1.11 0.80+-0,48 3.53+-1.11
6 2.15+-0.16 1.40+-0.58 6.18+-1.63
8 6.45+-3.45 2.63+-0.76 9.78+-4.47
12 9.38+-3.76 5.48+-1.74 16.2+-6.44
The ratio of this gross tumor volume when data are term day gross tumor volume and injection in the table (mean value+-standard deviation), make graphic representation 11 with this experimental data.
Claims (1)
1, human interleukin 6 core transcription factor expression plasmid series is used to prepare the purposes of the genomic medicine of treatment and prophylaxis of tumours.
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| CNB981220223A CN1163613C (en) | 1998-11-20 | 1998-11-20 | Human interleukin 6 core transcription factor expression plasmid series and its application in treating tumour |
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| ATE469972T1 (en) | 2003-12-23 | 2010-06-15 | Medimmune Llc | MULTIPLASMID SYSTEM FOR GENERATING THE FLU VIRUS |
| CN100402648C (en) * | 2005-09-08 | 2008-07-16 | 中国科学院遗传与发育生物学研究所 | Human Nanog gene and its encoded protein and application |
| US8673613B2 (en) | 2007-06-18 | 2014-03-18 | Medimmune, Llc | Influenza B viruses having alterations in the hemaglutinin polypeptide |
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