CN1510129A - Structure for recombinant adenovirus with double killer function and application in tumor treatment - Google Patents
Structure for recombinant adenovirus with double killer function and application in tumor treatment Download PDFInfo
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Abstract
A process for configuring the recombinant adenovirus with dual killing function includes such steps as providing E1-deficient rAdCD, rAdTK and fresh normal tissue close to human liver cancer, PCR amplifying to generate CD, CT and ES gene fragments, inserting CD-ES or TK-ES into shuttle plasmid pAdTrack-CMVCDglyES or pAdTrack-CMVTKglyES), homologous recombining with adenovirus 5 type skeleton plasmid pAdEasy-1 in bacterium to obtain recombinant adenovirus plasmid, and transfecting the cell 293. It can be used for genetic therapy of tumor by directly killing tumor cells, suppressing the generation of new blood vessel in tumor, and preventing transfer of tumor cells.
Description
Technical field
The present invention relates to the Molecular Virology technology, also relate to malignant tumour gene therapy or virus therapy.
Background technology
Malignant tumour has become the problem of a serious threat people of various countries health and lives at present.The World Health Organization (WHO) reports in world's healthy state of Geneva issue according in May, 1997, compared with the twentieth century tumor incidence seventies in 1997 and risen nearly 30%, in the period of the prediction following 25, the number of patients that tumour is suffered from by the most of country in the world may increase greatly.By 2005, European Union member countries women lung cancer will increase by 33%, and prostate cancer will increase by 40%, and the whole world will have 6,300,000 people to die from various tumours every year.
China is the same with world other countries, and the sickness rate of tumour has had bigger increase in recent years, and tumour has become the second deadly cause of disease that is only second to cardiovascular and cerebrovascular disease in China.The most of clinical treatment units of current China still continue to use traditional excision, put, amic therapy method treatment malignant tumour.These therapies can obtain satisfied effect to infantile tumour, but to the patient of middle and advanced stage with the far-end transfer, then are difficult to obtain satisfied curative effect.Therefore treatment for cancer has become the problem that world medical personnel presses for solution.
Over year, along with developing by leaps and bounds of immunology, molecular biology and genetic engineering technique, the novel method of the 4th kind of treatment cancer that be born again is a biotherapy surplus in the of nearly ten.This therapy is divided into three parts substantially, i.e. cytokine therapy, treated autologous cell and gene therapy, but tool potentiality surely belong to gene therapy.
The gene therapy of malignant tumour is meant the homology foreign gene is imported human body, finally reaches the purpose of direct or indirect killing tumor cell.The quality of the gene therapy curative effect of malignant tumour depends primarily on two aspects: the one, and carrier, the 2nd, the validity of goal gene.
The carrier that can be used for importing the human body goal gene has two kinds, first non-virus carrier, and it two is virus vector.Therapy of tumor is used virus vector more at present, the virus vector of having reported mostly is genetically modified virus, as retrovirus (Retrovirus), adenovirus (Adenovirus), adeno-associated virus (Adeno-associated Virus, AAV), hsv (Herpes Simplex Virus), Avian pneumo-encephalitis virus (Newcastle Disease Virus, NDV), poliovirus (Poliovirus), vesicular stomatitis virus (Vesicular Stomatitis Virus), reovirus (Reovirus), parvovirus (Parvoviruses), Alphavirus (Alphavirus) and sindbis alphavirus (Sindbis Virus, SIN) etc.When using virus vector, possess low toxicity, efficiently reach large vol as prerequisite with the gene therapy virus vector.It is carrier that the application selects Ad5 for use because adenovirus has been compared following advantage with other virus vector: 1)-host range is wide, but infected person and multiple Mammals, division stage and stationary phase cell all infect (cell surface has the CAR acceptor); 2) infection titer height, the rAd titre can reach 10 in 293 cells
7-10
9PFu/ml; 3) security is good, removes Ad4, and 7,11,21 and 37 types have pathogenic effects and Ad12 type that people's cell is had outside potential transformation and the animal oncogenic function to the people, equal non-evident sympton after other type infected person.Adenovirus vaccine used in septic yanks nearly 30 years, proved safely and effectively; 4) a large amount of foreign proteins of expressing are active in the horizontal mature protein in translation back, and good phosphorylation and glycosylation are promptly arranged; 5) good stability can be preserved for many years for-80 ℃, need not refrigerate after the freeze-drying; 6) use convenient, can be oral, collunarium tracheae, vein, abdominal cavity, subcutaneous and muscle multipath use.
Big quantity research previously proves that the generation of tumour and development are multistage complex processes, often relates to the unusual of several genes.The different patients' of the tumour of histological types or tumour of the same race gene unconventionality situation also is not quite similar.Therefore depend merely on a kind of gene and be difficult to reach extensively and effectively result of treatment.In this case, by in patient's body, importing generation, the development of several kinds of target gene, establish associating polygene therapeutic strategy and be only preferred plan from a plurality of link control tumours.Therefore aspect the validity of goal gene, except that the target of goal gene, what also emphasize a bit is to insert with identical carrier to be connected in series or to merge two or more genes of connection, so just reduced the usage quantity of carrier relatively, thereby reduced the immunne response of host to carrier, and can bring into play polygenic combined action, as 2000, American scholar Rogulshi KR etc. has made up the adenovirus carrier AdCDglyTK-5Fc/GCV system that contains two suicide genes, height when Kim etc. find target and radiation sensitivity all than single application with TK and CD gene combined utilization treatment tumour.And Adachi etc. have shown CD and two kinds of gene combined utilization of UPRT the synergy of antitumous effect in oncotherapy.
Summary of the invention:
Total design of the present invention is: use two suicide gene cytosine deaminase genes (CD gene) and thymidine kinase gene (TK gene) and endostatin gene (ES gene) to constitute CDglyES or TKglyES fusion gene as goal gene with the base sequence of nine glycine as linker.Can be after importing in the body by recombinant viral vector by the Isocytosine deaminase deamination of CD genetic expression, transforming avirulent chemotherapeutic precursor 5FC is cytotoxicity 5FU, directly kills and wounds oncocyte in the part; By hsv TK genetic expression thymidine kinase GCV/ACV is changed into triphosphoric acid GCV/ACV, it replaces guanosine triphosphate in cell DNA is synthetic, suppress the oncocyte dna polymerase activity, suppresses the extension of DNA chain, makes oncocyte death; Express Endostatin by endostatin gene, suppress the new vessel endotheliocyte (normal vascular endothelial cell is not almost had effect) of fast breeding thus suppressed tumor neogenetic blood vessels formation, cut off the oxygen and the nutrition supplement of tumour, make death of neoplastic cells, this is the starvation cure of tumour; Endostatin still can suppress subclinical metastasis formation and far-end transfer by suppressing new vessel formation in addition.
Expressing the suicide gene product simultaneously by recombinant adenoviral vector is that chemotherapeutic direct killing oncocyte and expression Endostatin pass through to suppress vascular endothelial cell by transforming the chemotherapeutic precursor, the oxygen and the nutrition supply that suppress vascularization blocking-up tumour kill and wound oncocyte indirectly, kill and wound oncocyte from two approach.
Transfection the oncocyte of this recombinant adenovirus can obviously increase susceptibility to radiotherapy, being easier to like this increases total effects greatly with the radiotherapy combined utilization.
The objective of the invention is to make up double play and hinder the recombinant adenovirus of fusion gene of function finally with two approach killing tumor cells.
The technical solution adopted in the present invention is as follows for achieving the above object:
One, makes up the recombinant adenovirus rAdCDglyES that contains the CDglyES fusion gene
1. from having inserted in E1 district the recombinant adenovirus rAdE1CMVCD of CD gene of the E1 that made up and E3 disappearance, be inserted into identical double digestion adenovirus shuttle plasmid pAdTrack-CMV after the purifying evaluation and be built into and contain CD adenovirus shuttle plasmid pAdTrackCMV-CD with PCR method amplification CD gene.
2. from people's fresh HCC tissue, clone endostatin gene:
At first from hepatic tissue, extract total RNA, by synthetic cDNA first chain of reverse transcription, then according to the synthetic upstream and downstream primer of the ES complete sequence of genebank, with cDNA first chain is that template obtains the human endostatin gene with the amplification of PCR method, cut and check order through agarose electrophoresis, enzyme, confirm that its sequence length is 573bp, be cloned into the pEZZ18 plasmid then and constitute the pEZZ18-ES plasmid.With the pEZZ18-ES plasmid that makes up is template, with the ES upstream and downstream primer (the ES upstream primer has 9 glysen sequences) that is added with corresponding restriction endonuclease sequence with the adenovirus shuttle plasmid multiple clone site, is PCR, obtains the glyES fragment that can insert plasmid.Then the glyES gene segment that obtains is inserted the CD gene downstream of the adenovirus shuttle plasmid pAdTrackCMV-CD that contains CD, constitute the adenovirus shuttle plasmid pAdE1CMVCDglyES that contains the CDglyES fusion gene.
3, the pAdTrackCMVCDglyES plasmid is cut the back with the Pmel enzyme and shift host bacterium Bj5183 with adenovirus skeleton plasmid pAdEasy-1 by electroporation, homologous recombination constitutes the recombinant adenovirus plasmid pAdCMVCDglyES that contains the CDglyES fusion gene in thalline.
4, the pAdCMVCDglyES plasmid is cut into linearity with the Pacl enzyme,, monitors transfection, results recombinant adenovirus rAdCDglyES after 7 days with GFP with liposome transfection 293 cells.
Two, making up the recombinant adenovirus rAdTKglyES method contain the TKglyES fusion gene and structure, to contain the method for recombinant adenovirus rAdCDglyES of CDglyES fusion gene identical, only the CD gene is changed to the HSV-I-TK gene.
Be described further below in conjunction with the single-gene function of specific embodiment fusion gene among the recombinant adenovirus Ad5 of the present invention
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 contains the structure of CDgene adenovirus shuttle plasmid
Fig. 2 pAdTrackCMV-CDglyES makes up schema
Fig. 3 contains the recombination adenovirus construction of CDglyES fusion gene
Fig. 4 effect 48 hours, different concns AdlacZ is to the comparison of Hela cell inhibitory rate
Fig. 5 effect 48 hours, different concns rAdCDglyES is to the comparison of Hela cell inhibitory rate
The tumor growth situation of Fig. 6 administration separate groups of mice in the time of 6 days
The tumor growth situation of Fig. 7 administration separate groups of mice in the time of 6 days
Fig. 8 separate groups of mice knurl heavily changes comparison diagram
A (I group) is the DMEM negative control; B (II group) is the AdLacZ virus control; C (III group) tests one group for rAdCDglyES; D (IV group) tests two picture groups, 9 contrast virus of A dLacZ, GCV effect back Hela inhibitory rate of cell growth figure for rAdECDglyES
A: virus concentration is 0 TCID
50/ 0.1ml; B: virus concentration is 10
4TCID
50/ 0.1ml;
C: virus concentration is 10
5TCID
50/ 0.1ml; D: virus concentration is 10
6TCID
50/ 0.1ml.GCV effect 48 hours.
The Hela cell inhibitory rate relatively after the different prodrug GCV of Figure 10, ACV acted on transfection rAdTKglyES
Figure 11 rAdCDglyES 293 cells and supernatant are to the comparison of the growth inhibition ratio of ECV-304 cell
Embodiment
CD gene function checking among the embodiment 1.rAdCDglyES
CD biological activity in the fusion gene of the recombinant adenovirus rAdCDglyES that contains the CDglyES fusion gene has been carried out the inside and outside checking of body, confirmed in the fusion gene of expressing, still to show independently CD enzymic activity.
A. experiment in vitro:
Method:
1) well-grown Hela cell digests with 0.02%EDTA; Add in 96 orifice plates 5000 cells/well, 37 ℃ of 5%CO then
2Cultivate into individual layer in the environment.
2) the contrast recombinant adenovirus AdLacZ and the rAdCDglyES that will only contain Lac Z is diluted to 10 respectively
5, 10
6With 10
7TCID
50/ 0.1ml joins in 96 orifice plates of individual layer tumour cell every hole 0.1ml respectively.Every kind of virus concentration of each extent of dilution is done 6 holes, cultivates 12 hours in 37 ℃ of 5%CO2 environment.
3) abandon viral liquid, wash cell twice, continue and cultivated 12-18 hour with the full nutrient solution of cell with 1640 substratum.
4) 5-FC is diluted to 40,400,4000 μ mol/L with the full nutrient solution of cell, joins in above-mentioned 96 orifice plates, each fixed concentration is done 6 multiple holes, cultivates 48 hours in 37 ℃ of 5%CO2 environment.
5) every hole adds 10 μ l 0.01M MTT, cultivates 45 minutes in 37 ℃ of 5%CO2 environment.
6) abandon supernatant, every hole adds DMSO 0.1ml, jolts 5-10 minute gently, surveys the OD570 value, and calculates inhibitory rate of cell growth by following formula:
The result: compare with AdLacZ and to observe that the CD/5FC system is to human cervical carcinoma Hela cell's vitro inhibition effect among the AdCDglyES, the result is shown in Fig. 4, Fig. 5 and following table 1:
Table 1.AdlacZ and rAdCDglyES effect after 48 hours to the comparison sheet of Hela cell inhibitory rate
The prodrug concentration unit is μ mol/L in the last table of ※; The virus unit is TCID
50/ 0.1ml.
From the graph, can find out that the AdLacZ virus-4 that does not have goal gene only was about 11% to the Hela cell inhibitory rate in 8 hours in the table, its with preceding concentration and virus concentration increase change not obvious; And rAdCDglyES group 48 hours to the Hela cell inhibitory rate near 60%, inhibiting rate increases with preceding concentration and recombinant virus concentration.This result has confirmed that the contained fusion gene of recombinant virus has the CD gene activity.
B. experiment in the body:
Method:
(1) sets up lotus knurl T739 inbreeding mouse inbred lines model (strain of inoculation L795 mouse adenocarcinoma cell).
(2) observe the interior antitumor activity of CD/5FC body in the rAdCDglyEs system
1. grouping: mouse is divided into 4 groups at random, 10 every group
The I group: DMEM trains basic negative control group
II group: AdLacZ virus control group
III group: for rAdCDglyES/5Fc tests one group
IV group: for rAdCDglyES/5Fc tests two groups
Other establishes 20 mouse is normal control (promptly the lotus knurl is not treated group)
2. the 3rd day beginning treated after the lotus knurl
1) I group: every mouse tumor body injection DMEM 0.1ml.
2) II group: (concentration is 10 to every mouse tumor body injection AdLacZ 0.1ml
6TCID
50/ 0.1ml).
3) III, IV group: every mouse tumor body is all injected rAdCDglyES 0.1ml, and (concentration is 10
6TCID
50/ 0.1ml).
3. after the lotus knurl, began to the oral precursor medicine of mouse in the 3rd day
1) I group: every mouse oral normal saline 0.2ml/ days
2) II, III the group: every the oral 5Fc 500mg/kg/ of mouse days
3) IV group: every the oral 5Fc 1000mg/kg/ of mouse days
Each group was all taken medicine 11 days continuously
4. rose in the 4th day after the lotus knurl, measure every day, calculate the knurl body weight of respectively organizing mouse, method of calculation are: knurl weight=A * B
2/ mg (A: major diameter, B: minor axis), continuously measured 11 days.
5. observe every group of mouse continuously, until its death.And write down every mouse existence fate.
The result:
(1) comparative result of respectively organizing mouse tumor volume morphing and weight (average) after the medication on the 6th day is seen Fig. 6, Fig. 7 and Fig. 8.
From Fig. 6-8 as seen, contrast I and II group mouse are administered at ten one o'clock, and mouse tumor body weight in average reaches 7000-8000mg, and two test group mouse tumor body weight only are 3700-4200mg, and experimental group is compared with control group, shows that the knurl bulk-growth has extremely evident difference.
(2) comparison of survival time of mice
After the lotus knurl the 15th day, control group mice began death, and each is organized the mouse mean survival time (MST) and sees the following form 2, can find out from table 2, and the control group mice mean survival time (MST) is about 18 days, and the treatment group is 23 days.This result shows that tumor-bearing mice has obviously prolonged the lifetime of tumor-bearing mice behind the rAdCDglyES/5Fc systematic treating, has confirmed that fusion gene CDglyES has the CD activity in body.
Table 2 is respectively organized the comparison of survival time of mice
TK gene function checking among the embodiment 2.rAdTKglyES
| Lifetime (my god) | ????F | ????P | |
| ????I | ????18.11±1.62 | ||
| ????II | ????17.67±1.87 | ????27.43 | ????0.000 |
| ????III | ????23.00±1.66 | ||
| ????IV | ????23.11±1.68 |
Purpose is to confirm that the recombinant adenovirus rAdTKglyES fusion gene TKglyES that contains fusion gene has the TK biological activity.
Method:
The experiment in vivo and vitro method is with embodiment 1, and only the chemotherapeutic precursor is replaced 5Fc with GCV or ACV.The result:
Experimental result is shown in Fig. 9, Figure 10 and table 3 in the body.
Table 3.Hela cell is through AdLaCZ (TCID
50/ 0.1ml), GCV (μ mol/l) and rAdTKglyES
(TCID
50/ 0.1ml) effect 48 hours after inhibitory rate of cell growth (%)
| ??GCV | ????AdLacZ ????0??????10 4?????10 5?????10 6 | ????rAdTKglyES ????0????????10 4??????10 5?????10 6 |
| ??0 | ????0??????12.61????12.76????12.94 ???????????±1.55???±0.86???±0.96 | ????0????????13.72?????13.84????14.08 ?????????????±2.34????±2.26???±2.27 |
| ??10 | ????10.22??12.77????12.86????12.95 ????±1.52?±1.63???±1.29???±1.22 | ????10.81????27.50?????33.85????36.39 ????±2.30???±1.71????±2.13???±2.11 |
| ??100 | ????10.44??12.58????12.87????13.17 ????±1.35?±1.05???±1.64???±1.77 | ????11.28????29.79?????37.34????39.92 ????±2.42???±1.51????±1.01???±4.92 |
| ??1000 | ????10.96??12.66????12.90????13.32 ????±1.03?±1.72???±1.30???±11.67 | ????11.60????41.50?????48.09????49.46 ????±1.17???±3.34????±4.55???±1.22 |
| ??10000 | ????11.19??12.78????12.79????13.54 ????±0.89?±1.92???±11.61??±1.12 | ????11.62????78.11?????78.54????82.59 ????±1.90???±2.27????±3.11???±5.70 |
| ??F | ????????????0.133 ????????????1.000 | ?????????????????72.557 ?????????????????0 |
| ??P |
From table 3, Fig. 9 and Figure 10 as seen, the same with the CD expression product, up to 82%, AdLacZ is then still about 13% to the growth inhibition ratio of Hela cell for rAdTKglyES/ACV or rAdTKglyES/GCV systemic effect Hela cell 48 hours.The external fusion gene that confirmed has the TK activity.
In vivo test: result and embodiment 1 are very similar, are not repeated in this description at this.
ES gene function checking among the embodiment 3.rAdCDglyES
The endotheliocyte inhibition test
Method:
1. well-grown Human umbilical vein endothelial cells (ECV-304) is digested to monolayer cell with 0.02%EDTA, is diluted to 2 * 10 with 1640 nutritive mediums that contain endothelial cell growth factor (ECGF) (ECGF) 5mg/L
8Cell/L inoculates 96 orifice plates, and except that first a row Ensure Liquid liquid, every hole, surplus hole adds 0.2ml.Cultivate into individual layer in 37 ℃ of 5%CO2 environment.
2. inhale and remove liquid, the first row blank and secondary series cell contrast Ensure Liquid liquid, the 3rd～5 row add the recombinant adenovirus rAd-CD original content cell culture supernatant that only contains the CD gene, the 6th～8 row add 6 times of spissated rAdCDglyES cell culture supernatants, the 9th～11 row add the rAdCDglyES cell culture supernatant stoste of original content, every kind of every hole adds 0.2ml, cultivates 48 hours in 37 ℃ of 5%CO2 environment.
3. every hole adds MTT solution 20 μ l, continues to cultivate 4 hours.
4. inhale and go liquid, every hole to add 200 μ l DMSO, vibration mixing 10 minutes.
5. microplate reader is surveyed OD automatically
570, calculate inhibiting rate as follows:
The result:
With the rAd-CD recombinant adenovirus is contrast, and the ECV-304 cell of the fast breeding after handling with ECGF is a target, observe rAdCDglyES to the restraining effect result of the endotheliocyte of fast breeding shown in Figure 11 and table 4.
Table 4.rAd-CD and rAdCDglyTK cells and supernatant press down the growth of ECV-304 cell
The comparison of system rate
n??????????X???????????SD
rAd-CD?supemant??????????????9??????????24.2％??????9.7％
rAdCDglyTK?supemant??????????9??????????28.8％??????6.3％
6×rAdCDglyTK????????????????9??????????78.7％??????1.8％
F＝180.277???????????P＜0.01
From table 4 and Figure 11 as seen, rAd-CD is about 24% to the ECV-304 endotheliocyte inhibiting rate of the fast breeding handled through ECGF, and through 6 times of spissated rAdCDglyES supernatants to this cell inhibiting rate up to 78%.This result has confirmed that the ES gene activity among the rAdCDglyES is uninfluenced, and expressed fusion protein has the Endostatin activity.
The checking result of ES gene activity is no longer repeated at this with embodiment 3 among the rAdTKglyES.
Note:
Double play of the present invention was hindered the recombinant adenovirus of function, had handed over Chinese typical culture collection center preservation on December 19th, 2002.
Preserving number: CCTCC-V202009
Strain number: 0106
Symbol: rAd5CDglyES
Claims (6)
1, a kind of double play is hindered the structure of the recombinant adenovirus of function, it is characterized in that: this recombinant adenovirus is the recombinant adenovirus that contains CDglyES fusion gene or TKglyES fusion gene, it is that rAdCD and rAdTK with disappearance E1 district is material, performing PCR increase CD and TK gene fragment; With the other normal liver tissue of liver cancer is material, row RT-PCR amplifies the ES gene fragment, order is inserted pAdTrack-CMV with CD, ES or TK, ES and is worn the rib plasmid construction and become the purpose shuttle plasmid then, two kinds of purpose shuttle plasmids are recombinated with adenovirus pAdEasy-1 skeleton plasmid homology in bacterium respectively, obtain two kinds of recombinant adenovirus plasmids, rotaring redyeing 293 cell respectively, can obtain double play hinder function two kinds of recombinant adenovirus.
2, double play according to claim 1 is hindered the structure of the recombinant adenovirus of function, it is characterized in that: CD and ES gene and TK and ES gene all are that the form with fusion gene is present in adenovirus shuttle plasmid and the recombinant adenovirus.
3, double play according to claim 1 is hindered the structure of the recombinant adenovirus of function, it is characterized in that: CD and ES gene Fusion and TK and ES gene Fusion all are to be linker with nine amino acid.
4, double play according to claim 1 hinder function the structure of recombinant adenovirus, it is characterized in that: used adenovirus shuttle plasmid has the GFP reporter gene, its adenovirus left arm partly contains ITR and Ψ.
5, a kind of double play as claimed in claim 1 is hindered the application of recombinant adenovirus in oncotherapy of function, it is characterized in that: the fusion gene expression product of insertion had both shown the CD enzymic activity: but the catalysis cytosine(Cyt) is converted into the deamination reaction of uridylic, and the 5Fc of no cytotoxicity is converted into Cytotoxic 5Fu.5Fu kills oncocyte by suppressing the synthetic extension of oncocyte dna polymerase activity and DNA.Simultaneously expression product has the TK enzymic activity again: HSV-TK catalysis GCV form single phosphoric acid-GCV once more catalysis make it form triphosphoric acid-GCV, in DNA is synthetic, replace guanosine triphosphate, the DNA chain after adding, triphosphoric acid-GCV is no longer prolonged, it also suppresses dna polymerase activity simultaneously, and acting in conjunction causes tumor cell growth to suppress and be dead.It is inhibition of endothelial cell proliferation that the fusion gene that inserts has the ES gene activity again, and then suppresses new vessel formation and cut off tumour oxygen and nutrition supplement, kills oncocyte, stops the far-end of oncocyte to shift.
6, double play according to claim 5 is hindered the application of recombinant adenovirus in oncotherapy of function, it is characterized in that: two kinds of recombinant viruses all can increase the susceptibility of radiotherapy, are easy to and radiotherapy combined utilization treatment malignant tumour.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286433A (en) * | 2010-12-26 | 2011-12-21 | 马丁 | Obtainment and application of novel oncolytic adenovirus-thymidine kinase genetic construct |
| CN103357024A (en) * | 2012-03-27 | 2013-10-23 | 曾桥 | Liver cancer suicide gene therapy medicine based on aminated silicon dioxide nanoparticle-CD/TK (cytosine deaminase-thymidine kinase) fused gene compound |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103623B (en) * | 2013-03-01 | 2014-03-19 | 山东维真生物科技有限公司 | Adenovirus chip |
-
2002
- 2002-12-24 CN CN 02158022 patent/CN1219054C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286433A (en) * | 2010-12-26 | 2011-12-21 | 马丁 | Obtainment and application of novel oncolytic adenovirus-thymidine kinase genetic construct |
| CN103357024A (en) * | 2012-03-27 | 2013-10-23 | 曾桥 | Liver cancer suicide gene therapy medicine based on aminated silicon dioxide nanoparticle-CD/TK (cytosine deaminase-thymidine kinase) fused gene compound |
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