CN105535993A - Use of recombinant oncolytic vaccinia virus in treatment of gastric cancer - Google Patents
Use of recombinant oncolytic vaccinia virus in treatment of gastric cancer Download PDFInfo
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Abstract
The invention belongs to genetic engineering and oncology, and relates to use of a recombinant oncolytic vaccinia virus carrying microRNA-101 in treatment of cancers. In treatment of gastric cancer, the recombinant virus has the beneficial effects that gene therapy and virus therapy of malignant tumors are effectively combined, and an oncolytic vaccinia virus capable of highly expressing miRNA-101 is prepared, so that the tumor-killing ability is enhanced in comparison to a simple gene therapy or virus therapy; 2, a tumor targeted therapy strategy is adopted, and tumor cells can be targeted effectively and proliferated specifically, thereby greatly enhancing the safety of an oncolytic vaccinia virus vector; 3, a virus replication relevant gene deletion method is adopted, and intratumoral specific replication of viruses is ensured, thereby greatly enhancing the safety of the oncolytic vaccinia virus vector; 4, a recombinant oncolytic vaccinia virus carrying small RNA microRNA-101 is constructed, so that a good anti-gastric-cancer effect is achieved.
Description
Technical field
The invention belongs to genetic engineering and oncology, relate to a kind of recombination oncolytic vaccinia virus of carrying Microrna microRNA-101 for the preparation of the purposes in the pharmaceutical composition for the treatment of gastric cancer, medicine or test kit.
Background technology
In all cancers in the whole world, the sickness rate of gastric cancer ranked fourth position, in women, ranked fifth position in male, and the mortality rate of gastric cancer occupies second, 5 years survival rates about 20%.Development of Novel safely and effectively Therapeutic Method has very important significance to improve survival periods of gastric carcinoma patients.
Viral therapy (Virotherapy) is the very fast a kind of oncotherapy new method of development in recent years, and has obtained some challenging achievements, and wherein some is just carrying out I phase and the clinical research of II phase in the world.The domestic New Drug Certificate having recombinant human adenovirus p 53 (Gendicine) to obtain State Food and Drug Administration to issue, is used for the treatment of solid tumor patient clinically.The report such as Pan1 oncolytic adenovirus (Oncolyticadenovirus) the combined radiotherapy treatment of advanced nasopharyngeal carcinima carrying p53 gene, result has 66.7% patient to obtain complete incidence graph (CR), and alone combination radiotherapy group CR is only 24.4%.Show that oncolytic virus is a kind of new method of promising Therapeutic cancer.Oncolytic vaccinia virus (OncolyticPoxvirus) is the novel carriers of viral therapy, and its advantage is: virus stability is good, pathogenic low, gene transfection effect is high, safety is good.Oncolytic vaccinia virus energy selectivity infected tumor's cell and in time multiplexed cell system, kill tumor cell, and the toxicity of normal tissue and cell is very little.
MicroRNA (miRNA) is that a kind of evolutionary process camber is conservative, endogenous non-coding tiny RNA, is distributed in widely in plant, animal and DNA viruses.MiRNA is at the encoding gene of the human protein of post-transcriptional level regulation and control at least 30%, and its function relates to many complicated life processes such as Growth of Cells, growth and apoptosis.The mode that miR-101 plays a role guides miRNA ribosome to be combined with the binding site of 3 ' untranslated region of target gene, suppresses the translation of target gene at post-transcriptional level.The direct acting target molecule of miR-101, comprises EZH2, Stathmin, DNA methylation transferring enzyme 3A (DNMT3A).These target molecules play the function promoting cell proliferation and inhibited apoptosis in cell, and miR-101 has mediated hyper-proliferative and the Apoptosis inhibitor of tumor cell by the Cumulate Sum cooperative effect of multiple target molecule.
He Xiaopu (2012) utilizes retroviral vector Lenti-virus to find as the transduction instrument of miR-101, miR-101 can cause the change of stomach cancer cell biological behaviour, and this change is likely by suppressing or reticent COX-2 expression realization.
Summary of the invention
The present inventor finds, the method effect of Therapeutic cancer of the prior art is more weak.For this reason, the technical problem to be solved in the present invention is to provide a kind of new method for the treatment of gastric cancer.Specifically, the new method being used for the treatment of cancer of the present invention comprises the recombination oncolytic vaccinia virus of carrying Microrna microRNA-101 is applied to experimenter.
Therefore, one aspect of the present invention provides recombination oncolytic vaccinia virus for the preparation of the purposes in the pharmaceutical composition of Therapeutic cancer, wherein operationally inserts the exogenous gene of the microRNA-101 that can encode in described recombination oncolytic vaccinia virus.
The exogenous gene of coding microRNA-101 can be SEQIDNO:1 or its complementary series.
On the other hand, recombination oncolytic vaccinia virus of the present invention can be modified further, with the protein active composition of expression treatment gastric cancer.Exemplarily, be p53 for the protein active composition of the Therapeutic cancer of expressing in the present invention.
Again on the one hand, pharmaceutical composition described in the present invention can containing other active component being used for the treatment of cancer.Exemplarily, other wherein said active component are paclitaxels.
Another aspect, pharmaceutical composition involved in purposes of the present invention contains pharmaceutically acceptable carrier.Exemplarily, described pharmaceutical composition is the form of solid or injection.Preferably, described pharmaceutical composition is packaged in medicine box; Preferred, the operation instructions of described medicine box also containing described pharmaceutical composition.
Specifically, the coded sequence of microRNA-101 of the present invention is any DNA sequence of any microRNA-101 that can encode, and preferably, this sequence is SEQIDNO:1 or its complementary series.On the other hand, the coded sequence of microRNA-101 of the present invention can be and suppresses stomach cancer cell to rise in value the polynucleotide of microRNA-101 of activity or its complementary series with being undertaken hybridizing by the nucleotide sequence of SEQIDNO:1 and encode to have under stringent condition;
" stringent condition " as herein described can be any one in low stringent condition, middle stringent condition, high stringent condition, is preferably high stringent condition.Exemplarily, " low stringent condition " can be 30 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, 52% Methanamide condition; " middle stringent condition " can be 40 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, 52% Methanamide condition; " high stringent condition " can be 50 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, 52% Methanamide condition.Those skilled in the art are to be understood that temperature gets over the polynucleotide that Gao Yueneng obtains high homology.In addition, those skilled in the art can select the synthesis result of the multiple factor formation of the temperature, concentration and probe concentration, probe length, ionic strength, time, salinity etc. of the stringency affecting hybridization to realize corresponding stringency.
In addition interfertile polynucleotide can also be, pass through FASTA, when the default parameters of the homology retrieval software defaults such as BLAST calculates, have about 60% or more with the polynucleotide of coded sequence numbers 6, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more polynucleotide of homogeneity.
The homogeneity of nucleotide sequence, can use algorithmic rule BLAST (Proc.Natl.Acad.Sci.USA87:2264-2268,1990 of Karlin and Altschul; Proc.Natl.Acad.Sci.USA90:5873,1993) determine.Program BLASTN, BLASTX based on BLAST algorithmic rule are developed (AltschulSF, etal:JMolBiol215:403,1990).When using BLASTN to analyze base sequence, be score=100, wordlength=12 as made parameter; When using BLASTX analysis of amino acid sequence in addition, be score=50, wordlength=3 as made parameter; When using BLAST and GappedBLAST program, the system of each program is adopted to set default parameter value.
Optionally, expression of recombinant virus of the present invention is known can suppress stomach cancer cell other albumen value-added, such as p53.Pharmaceutical composition of the present invention can also contain the active component of other treatment gastric cancer, such as paclitaxel.
The recombination oncolytic vaccinia virus of carrying Microrna microRNA-101 as active component of the present invention can use together with pharmaceutically acceptable carrier.In addition to the active ingredient (s, method of the present invention, purposes and product can also comprise the upper acceptable carrier of suitable pharmacy, comprise and promote that recombinant virus of the present invention is processed into excipient and the auxiliary agent of preparation.
Such as be suitable for injecting or the preparation of infusion comprises aqueous and non-aqueous sterile injection liquid and aqueous and non-aqueous sterile suspensions, described aseptic parenteral solution optionally comprises antioxidant, buffer agent, antibacterial and can make the solute of blood equipressure of preparation and object recipient, and described sterile suspensions can comprise suspending agent and thickening agent.Described preparation can be present in unit dose or multi-dose container, the ampoule such as sealed, and can be kept at freeze-dried (lyophilizing) condition, only needs to add sterile liquid carrier, such as water for injection before using immediately.
Active component of the present invention optionally can be combined with solid excipient, and when needing, after adding suitable auxiliary agent, the mixture of processing granular, to obtain required dosage form.Suitable excipient particularly filler is such as sugared, comprises lactose, sucrose, mannitol or Sorbitol; Cellulose or starch formulation, gelatin, Tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).When needing, disintegrating agent can be added, such as crospolyvinylpyrrolidone, agar or alginic acid or its salt such as sodium alginate.
The effective dose of active component of the present invention can be treatment gastric cancer or alleviates any amount of gastric cancer symptom or suppression stomach cancer cell, and it can be equivalent to about 1x10
11-1x10
12v.p recombinant virus.Effective dose be determined in the ability of those skilled in the art, under the enlightenment particularly according to disclosure provided herein.
According to the present invention, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can use administration experimenter with any effective source strength.Preferably, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can with multidose administrations, such as from about 2 to about 15 dosage, more preferably from about 4-10 dosage, most preferably from about 6 dosage.In particularly preferred embodiment, in administration process, with administration in every three weeks frequency about once, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition are administered to experimenter, such as injection, infusion or oral.In particularly preferred embodiment, administration is for being applied to lotus tumor position by injection.
Be to be understood that pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can be prepared by the mode of any suitable for the administration by any suitable.
The dosage unit of pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition carries out administration experimenter based on routine.Such as, dosage unit can administration more than once a day, once in a week, monthly etc.Dosage unit can be by twice/week based on administration, namely weekly twice, such as every three days once.
As used herein, " comprising " and " comprising ", " containing " or " being characterised in that " synonym, and be included or opening, and do not get rid of the other element of not stating or method step.Term " comprises " any statement in this article, particularly when describing method of the present invention, purposes or product, be understood to include substantially by described component or element or step forms and those products, method and the purposes that are made up of described component or element or step.The present invention of description exemplified here suitably can put into practice when there are not concrete disclosed any one or Various Components, one or more restrictions herein.
The description relating to this pharmaceutical product comprised in pharmaceutical product of the present invention can containing, for example lower content: indication (such as gastric cancer), application dosage (such as above-mentioned exemplified property illustrates) and issuable side effect etc.
The term adopted herein and statement are used as descriptive instead of restricted term; and do not expect shown in getting rid of in the use of this kind of term and statement and any equivalent of described feature or its part, but will be appreciated that in the various scope of the present invention being modified at request protection be possible.Therefore, although be to be understood that the present invention is specifically disclosed by preferred embodiment and optional feature, but those skilled in the art can adopt modification and the change of concept disclosed herein, and this type of is modified and change is regarded as in the scope of the present invention such as defined by accessory claim.
For being illustrated more clearly in the present invention, be now described in detail in conjunction with following embodiment, but these embodiments are only to exemplary description of the present invention, can not be interpreted as the restriction to the application.
The invention has the beneficial effects as follows:
1, the gene therapy of malignant tumor effectively combines with viral therapy by the present invention, and preparing can the oncolytic vaccinia virus of high expression miRNA-101, enhances the kill capability to tumor relative to simple gene therapy or virus therapy.
2, present invention employs neoplasm targeted therapy strategy, can targets neoplastic cells effectively, and proliferated specifically wherein.Thus greatly strengthen the safety of oncolytic vaccinia virus carrier.
3, the present invention adopts the mode that virus replication related gene lacks, and guarantees that the tumor internal specific of virus copies, greatly strengthen the safety of oncolytic vaccinia virus carrier.
4, the present invention is by building the recombination oncolytic vaccinia virus of carrying Microrna microRNA-101, has played the effect of good anti-gastric cancer.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the recombination oncolytic vaccinia virus infection BGC-823 cell that flow cytomery carries GFP, the expression of GFP.
Fig. 2 is that RT-PCR detects the recombination oncolytic vaccinia virus infection BGC-823 cell carrying microRNA-101, the expression of microRNA-101.
Fig. 3 a and Fig. 3 b is that mtt assay detects cell survival rate analysis chart, and data represent with the percentage rate of dose gradient relative to PBS group.
Fig. 4 Apoptosis by Flow Cytometry rate.
Detailed description of the invention
The present invention is further described with example by reference to the accompanying drawings.Unless otherwise indicated, the present invention can adopt this area routine techniques.
The preparation method of embodiment 1, recombination oncolytic vaccinia virus, is characterized in that carrying out following steps successively:
1), by method for synthesizing gene, the primary transcript pri-miRNA-101 of microRNA-101 is obtained:
tgcctcctcacgtctccaaccagaaggtgatcttttagtccttcacttcatggggagccttcagagagagtaatgcagccaccagaaaggatgccgttgaccgacacagtgactgacaggctgccctggctcagttatcacagtgctgatgctgtctattctaaaggtacagtactgtgataactgaaggatggcagccatcttaccttccatcagaggagcctcaccgtacccaggaagaaagaaggtgaaagaggaatgtgaaacaggtggctgggacccagaaaccctcttaccctgcacctctgtcat
(SEQIDNO：1)；
2), pri-miRNA-101 sequence is loaded on shuttle plasmid pCB, obtain pCB-pri-miRNA-101, wherein said shuttle plasmid pCB is according to such as Publication about Document preparation: Fang Yourong, Li Hongyu, the structure of the two selection markers vaccinia virus recombinant carrier of Chen Keda, Zhou Wenshuo, Yan Hui .Zeocin and GFP, international epidemic lemology magazine, volume the 3rd phase June the 39th in 2012; It should be noted that, the present invention with other pCB, such as, can not have the shuttle plasmid pCB of selected marker yet.
3), in HEK293 cell, plasmid pCB-pri-miRNA-101 and Wild-type vaccinia strain generation homologous recombination, pri-miRNA-101 sequence is inserted in Wild-type vaccinia strain thymidine kinase (TK) gene;
4), after mycophenolic acid drug screening, the recombination oncolytic vaccinia virus VACV-miRNA-101 of purification is obtained.
Embodiment 2: flow cytomery carries the recombination oncolytic vaccinia virus infection BGC-823 cell (SGC-7901, purchased from Chinese Academy of Sciences's cell bank) of GFP (green fluorescent protein), the expression of GFP.
5 × 10 are inoculated in six orifice plates
5the BGC-823 cell of individual/mL, adds the recombination oncolytic vaccinia virus (VACV-GFP) of carrying GFP of 1MOI, 5MOI respectively, adds the PBS of equivalent, 37 DEG C, 5%CO in matched group
2cultivate.Collecting cell after 24h, adopts flow cytometer (BDAccuriC6) to analyze the GFP expression rate of each group of sample.The results are shown in Figure 1, along with the increase of VACV-miR-101 dosage, GFP expression increases thereupon, shows that recombination oncolytic vaccinia virus can effectively be attacked stomach cancer cell.
Embodiment 3:RT-PCR detects the recombination oncolytic vaccinia virus infection human gastric cancer cells BGC-823 carrying microRNA-101, the expression of microRNA-101.
The recombination oncolytic vaccinia virus (VACV-miR-101) of carrying microRNA-101 infects RPMI-8226 and U266 cell with the dosage of 4MOI, 37 DEG C, 5%CO
2cultivate 24 hours under condition.Cell total rna is extracted by TRIZOL method, with the total serum IgE of extracting for template reverse transcription becomes cDNA, application Primer5.0 software design primer.CDNA carries out quantitative fluorescent PCR by SYBRqPCRMix, and amplification system is 20 μ L.
Amplification is: 95 DEG C of 1min, 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.Result is by AppliedBiosystems7300real-timePCR instrument software analysis.After the results are shown in Figure 2, VACV-miR-101 treatments B GC-823 cell, in mRNA level in-site, microRNA-101 expression significantly increases, show VACV-miR-101 can in stomach cancer cell stably express microRNA-101.
Embodiment 4: carry the recombination oncolytic vaccinia virus of microRNA-101 to the impact of stomach cancer cell BGC-823 proliferation activity.
Stomach cancer cell BGC-823 (purchased from Chinese Academy of Sciences's cell bank) cultivates (37 DEG C, 5%CO2, saturated humidity) in containing the DMEM (purchased from Gibco) of 10% hyclone (FBS), gets forth generation cell by 1 × 10
4the concentration of/ml inoculates 100 μ l in 96 well culture plates.Setting matched group, recombination oncolytic vaccinia virus group (VACV), carry the recombination oncolytic vaccinia virus group (VACV-miR-101) of microRNA-101, often organize and all do 3 multiple holes.Treat that cell density reaches 60-70%, add VACV (1MOI, 2MOI, 4MOI, 8MOI), Ad-miR-101 (Tang Fuxiang, Zheng Quan respectively, Huang Zonghai, Zhang Qingguo, Che little Yan. the AdEasy system of application enhancements prepares recombinant adenovirus. No.1 Military Medical Univ.'s journal, 2003; 23 (5): 501-503) (1MOI, 2MOI, 4MOI, 8MOI) and VACV-miR-101 (1MOI, 2MOI, 4MOI, 8MOI), continue cultivation after 72 hours, add tetramethyl azo azoles salt (MTT) the 20 μ l solution of 5mg/ml, continue cultivation after 4 hours, stop cultivating, centrifugal segregation culture fluid, every hole adds 150 μ lDMSO, after vibration l0min, the full-automatic microplate reader of ThermoVarioskanFlash is selected wavelength 490nm measure each hole light absorption value (A value), above-mentioned experiment weighs 3 times.Calculate cell inhibitory rate according to A value, computing formula is: cell inhibitory rate (%)=(negative control group A value-dosing group A value)/negative control group A value × 100%.
The results are shown in Figure 3b, along with the increase of dosage, VACV, Ad-miR-101 and VACV-miR-101 are further remarkable to stomach cancer cell BGC-823 inhibitory effects on proliferation, and VACV-miR-101 is significantly better than VACV and Ad-miR-101 to stomach cancer cell BGC-823 inhibitory effects on proliferation.
Embodiment 5: carry the recombination oncolytic vaccinia virus of microRNA-101 to the impact of human normal cell line proliferation activity.
People's normal cell lines of human liver QSG-7701 (purchased from Chinese Academy of Sciences's cell bank) cultivates (37 DEG C, 5%CO2, saturated humidity) in containing the RPMI1640 (purchased from Gibco) of 10% hyclone (FBS), gets forth generation cell by 1 × 10
4the concentration of/ml inoculates 100 μ l in 96 well culture plates.Setting matched group, recombination oncolytic vaccinia virus group (VACV), carry the recombination oncolytic vaccinia virus group (VACV-miR-101) of microRNA-101, often organize and all do 3 multiple holes.Treat that cell density reaches 60-70%, add VACV (1MOI respectively, 2MOI, 4MOI, 8MOI), Ad-miR-101 (1MOI, 2MOI, 4MOI, 8MOI) with VACV-miR-101 (1MOI, 2MOI, 4MOI, 8MOI), continue cultivation after 72 hours, add tetramethyl azo azoles salt (MTT) the 20 μ l solution of 5mg/ml, continue cultivation after 4 hours, stop cultivating, centrifugal segregation culture fluid, every hole adds 150 μ lDMSO, after vibration l0min, the full-automatic microplate reader of ThermoVarioskanFlash is selected wavelength 490nm measure each hole light absorption value (A value), above-mentioned experiment weighs 3 times.Calculate cell inhibitory rate according to A value, computing formula is: cell inhibitory rate (%)=(negative control group A value-dosing group A value)/negative control group A value × 100%.
The results are shown in Figure 3a, along with the increase of dosage, VACV, Ad-miR-101 and VACV-miR-101 to people's normal cell lines of human liver QSG-7701 without obvious depression effect.
Embodiment 6: carry the recombination oncolytic vaccinia virus of microRNA-101 to the apoptosis-promoting effect of stomach cancer cell BGC-823.
5 × 10 are inoculated in six orifice plates
5the BGC-823 cell of individual/mL, add the recombination oncolytic vaccinia virus (VACV-miR-101) of carrying microRNA-101 and the unloaded recombination oncolytic vaccinia virus (VACV) of 4MOI respectively, the PBS of equivalent is added, 37 DEG C, 5%CO in matched group
2cultivate.With reference to AnnexinV/PI cell apoptosis detection kit operating instruction after 48h: collecting cell, with the PBS washed cell twice of pre-cooling, with 1 × BindingBuffer re-suspended cell of 100 μ L, add the Annexin-V of 5 μ L and the PI of 1 μ L, on ice after lucifuge 15min, add 400 μ L1 × BindingBuffer, adopt flow cytometry analysis.
The results are shown in Figure 4, VACV-miR-101 and the VACV processed group cell death inducing that all energy is strong, and VACV-miR-101 processed group apoptosis-promoting effect is more remarkable.
Embodiment 7: carry microRNA-101 recombination oncolytic vaccinia virus (VACV-miR-101) and on the impact of stomach cancer cell BGC-823 proliferation activity and carry microRNA-101 retroviral vector Lenti-virus (Len) on the impact of stomach cancer cell BGC-823 proliferation activity.
What knows uncut jade .miR-101 to the impact of gastric cancer COX-2 overexpression and biological effect thereof according to: [D]. Nanjing: Nanjing Medical University, 2012 have prepared the retroviral vector Lenti-virus (Len-miR-101) of carrying microRNA-101, and compare itself and impact of carrying the recombination oncolytic vaccinia virus stomach cancer cell BGC-823 proliferation activity of microRNA-101 of the present invention according to embodiment 5.Found that, along with the increase of dosage, Len-miR-101 and VACV-miR-101 is further remarkable to stomach cancer cell BGC-823 inhibitory effects on proliferation, and VACV-miR-101 is significantly better than Len-miR-101 (figure slightly) to stomach cancer cell BGC-823 inhibitory effects on proliferation.
Although with above embodiments describing the present invention, should be understood that, under the prerequisite not deviating from spirit of the present invention, the present invention can further modify and change, and these are modified and variation all belongs within protection scope of the present invention.
Claims (10)
1. recombination oncolytic vaccinia virus is for the preparation of the purposes in the pharmaceutical composition of Therapeutic cancer, wherein operationally inserts the exogenous gene of the microRNA-101 that can encode in described recombination oncolytic vaccinia virus.
2. purposes according to claim 1, wherein said exogenous gene is SEQIDNO:1 or its complementary series.
3. the purposes according to any one of claim 1-2, wherein said recombination oncolytic vaccinia virus is modified further, with the protein active composition of expression treatment gastric cancer.
4. purposes according to claim 4, the protein active composition of wherein expressed treatment gastric cancer is p53.
5. purposes according to claim 4, wherein said pharmaceutical composition contains other active component being used for the treatment of cancer.
6. purposes according to claim 5, other wherein said active component are paclitaxels.
7., according to the purposes of claim 1 or 2, wherein said pharmaceutical composition contains pharmaceutically acceptable carrier.
8., according to the purposes of claim 1 or 2, wherein said pharmaceutical composition is the form of solid or injection.
9., according to the purposes of claim 1 or 2, wherein said pharmaceutical composition is packaged in medicine box.
10. purposes according to claim 9, the operation instructions of wherein said medicine box also containing described pharmaceutical composition.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564700A (en) * | 2018-06-06 | 2019-12-13 | 杭州功楚生物科技有限公司 | Oncolytic vaccinia virus carrying limulus lectin gene, construction method and application |
| CN111150748A (en) * | 2019-12-27 | 2020-05-15 | 杭州荣谷生物科技有限公司 | Application of recombinant oncolytic virus in preparation of medicine for treating digestive tract cancer |
| CN112261951A (en) * | 2018-05-02 | 2021-01-22 | 通尼克斯制药控股有限公司 | Stem cells comprising synthetic chimeric vaccinia virus and methods of use thereof |
| CN113583979A (en) * | 2021-08-03 | 2021-11-02 | 杭州荣谷生物科技有限公司 | Recombinant oncolytic vaccinia virus, preparation method and application thereof |
| CN114340649A (en) * | 2019-08-26 | 2022-04-12 | 拜耳诺克斯有限公司 | Pharmaceutical composition for treating cancer comprising vaccinia virus and hydroxyurea as active ingredients |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130071430A1 (en) * | 2010-04-09 | 2013-03-21 | The University Of Tokyo | Microrna-controlled recombinant vaccinia virus and use thereof |
-
2015
- 2015-12-25 CN CN201510995340.7A patent/CN105535993A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130071430A1 (en) * | 2010-04-09 | 2013-03-21 | The University Of Tokyo | Microrna-controlled recombinant vaccinia virus and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 何晓璞: "miR-101对胃癌COX_2过度表达的影响及其生物学效应", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 郝明强 等: "溶瘤病毒的研究进展", 《生物技术通讯》 * |
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