CN106520708B - A kind of application of the oncolytic adenovirus of TGF-β targeting in kidney treatment - Google Patents
A kind of application of the oncolytic adenovirus of TGF-β targeting in kidney treatment Download PDFInfo
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Abstract
The present invention relates to biotechnologys and field of gene, in particular, provide a kind of oncolytic adenovirus and preparation method thereof of TGF-β targeting, and its verifying for replicating, killing and expressing in kidney cancer cell, and the application in kidney treatment.The oncolytic adenovirus expresses the fusion protein sTGF β RIIFc of TGF beta receptor soluble fragments and human IgG1 Fc by the duplication of telomerase promoter TERTp regulation virus.The oncolytic virus can in kidney cancer cell massive duplication and killing tumor cell, while express express target protein sTGF β RIIFc；Destination protein sTGF β RIIFc can target TGF-β signal, and the metastases for blocking TGF-β to mediate inhibit immunosupress caused by TGF-β, to achieve the purpose that treat tumour and its transfer.
Description
Technical field
The present invention relates to biotechnologys and field of gene, in particular, provide a kind of oncolytic adenopathy of TGF-β targeting
Poison and preparation method thereof, and its verifying for replicating, killing and expressing in kidney cancer cell, and the application in kidney treatment.
Background technique
In recent years, the disease incidence of China's kidney shows an increasing trend year by year, and is only second to prostate cancer, occupies male urinary system
The second for tumour of uniting.Currently, the essential therapeutic arsenals of kidney are surgical resection therapy, the effect of radiotherapy and chemotherapy
It is unsatisfactory.Therefore, it is badly in need of exploring a kind of significantly more efficient treatment means.
In recent years, oncolytic virus treatment is quickly grown, and is had multinomial oncolytic virus therapeutic agent and is entered the clinical II-III phase
Conceptual phase.In October, 2015, U.S. FDA, which has approved, carries granulocyte-macrophage colony stimutaing factor (Granulocyte-
Macrophage colony stimutaing factor, GM-CSF) herpe simplex oncolytic sick (T-VEC) enter market, be
Oncolytic virus treatment is filled with a cardiotonic.Currently, what is be most widely used in clinical test is oncolytic adenovirus, oncolytic
One of main construction strategy of adenovirus is to replace natural viral promotors with tissue-specific promoter to control virus again
The expression of promotor gene processed obtains the oncolytic adenovirus of transcriptional control type.
Theoretically, oncolytic adenovirus can replicate in tumour cell and crack tumour cell, releasing virus, further
Other tumour cells around infecting, and then Cascaded amplification effect is played, it is finally reached the purpose of tumors destroyed.However, practical
On, the curative effect of oncolytic adenovirus suffers from the restriction of many factors, and e.g., generally existing 5 type adenovirus antibody in human body can be clear
Except oncolytic oncolytic adenovirus；Anticipation etc. is not achieved in the tumor-specific promoters efficiency of starting virus replication.In recent years, by more
Kind mode modifies oncolytic adenovirus, can significantly improve the curative effect of oncolytic adenovirus, such as by carrying suicide gene, mentions
Killing-efficiency of the height to tumour cell；By carrying immunogene, the immune state of body and tumor by local is adjusted；Pass through carrying
Anti-angiogenesis gene blocks Nasopharyngeal neoplasms coherent signal activation etc..Therefore, by oncolytic adenovirus treatment and gene therapy
Use in conjunction has become the important directions and hot spot of oncolytic virus treatment.
Summary of the invention
The purpose of the invention is to prepare a kind of oncolytic adenovirus for targeting TGF-β signal, and it is applied to controlling for kidney
It treats, provides a kind of new strategy for the treatment of kidney.
Transforming growth factor β (Transforming growth factor β, TGF β) is used as multi-functional cell factor,
It plays an important role in the generation, development process of bone metastaes.Research has shown that, late the tumor by local of kidney patient,
TGF-β expression is obviously improved；And it can detecte high-caliber TGF-β in the peripheral blood of prostate cancer with osseous metastasis patient.
TGF-β can influence generation, development and the transfer of tumour by number of mechanisms: as TGF β passes through on PI3K/Akt/mTOR access
Hypoxia inducible factor (Hypoxia-Inducible Factors, HIFs) expression is adjusted, tumour growth is promoted；Pass through Scr/Fak/
Akt access raises vegf expression, promotes angiogenesis；By induce bone resorption correlation factor expression (IL-11, PTHrP,
CTGF), promote the occurrence and development etc. of bone metastaes.Studies have shown that in the Bone tumour model of breast cancer and prostate cancer,
TGF signal beta is blocked by sTGF β RIIFc (the soluble TGF-p receptor II of fusion people Fc), can inhibit SMAD2 and SMAD3 phosphorus
Acidification reduces calcium level etc. and plays therapeutic effect further by inhibiting bone resorption, promoting knitting.
In addition, TGF β is also important immune-regulating factor, the antitumor of tumor microenvironment can be inhibited by number of mechanisms
It is immune, comprising: enhancing derived from bone marrow inhibits the aggregation of cell (myeloid-derived suppressor cells, MDSCs)；
Induction CD4+T cell differentiation is CD4+Foxp3+ regulatory T cells (Regulatory T cells, Tregs)；Lower NK cell
With CD8+T cell surface NKG2D receptor, and then weaken its cellulotoxic effect etc..Research confirms, blocks TGF signal beta, can inhibit
MDSCs and CD4+Foxp3+Tregs promotes natural killer cells (Natural Killer cells, NK) and cytotoxic T leaching
The activity of bar cell (Cytotoxic T Lymphocytes, CTLs), enhancing interferon gamma (Interferon γ, IFN γ)
Expression and secretion, and then activate anti tumor immune response.However, the application of TGF beta inhibitor or sTGF β RIIFc albumen, it may
It will lead to the toxic side effects such as general immunity inhibition.
As described above, in addition to operative treatment, still lack effective therapeutic strategy for kidney at present, by oncolytic adenopathy
Poison and TGF-β signal target gene therapy use in conjunction, can effectively combine the excellent of oncolytic therapy, gene therapy and immunization therapy
Gesture is expected to provide a kind of significantly more efficient therapeutic strategy for the treatment of kidney, has highly important theory significance and real valence
Value.
Present invention finds the use in conjunction of oncolytic adenovirus and TGF-β signal target gene therapy, are not only able to pass through
In the duplication of tumor by local, promote the great expression of target gene, improves curative effect；The pair of poison caused by Formulations for systemic administration can also be reduced
Effect.In addition, the tumor lysis effect that oncolytic virus mediates, can discharge a large amount of tumour antigen, promote body effectively anti-swollen
The immune formation of tumor.
In the specific embodiment of the present invention, the present invention uses Adeasy system, by " telomerase promoter
TERTp regulates and controls the expression cassette of E1A, the expression cassette and cmv promoter regulation sTGF β RIIFc of E1B promoter regulation E1B-55KDa
Expression cassette " be building up to shuttle plasmid.Linearized using restriction enzyme PmeI, and in BJ5183 bacterial strain with
Adeasy-1 homologous recombination.Obtained recombinant adenovirus plasmid, after restriction enzyme PacI linearisation, transfection HEK293 is thin
Born of the same parents are packaged to be oncolytic adenovirus.And the table of target gene sTGF β RIIFc is identified in human renal carcinoma cell, mouse kidney cancer cell
It reaches；The oncolysis of oncolytic adenovirus is confirmed in vitro；Its clear therapeutic effect and machine in mouse Renca Transplanted tumor model
System.
In further preferred embodiment, method that the present invention uses specifically:
1, the building and preparation of oncolytic adenovirus rAd.sTGF β RIIFc:
Building is started by telomerase promoter TERTp starting adenoviral replication indispensable gene E1A expression by E1B promoter
The intermediate vector TE-TP-E1A of E1B55K expression；And functional sequence is obtained by the method for digestion, connection, and be cloned into carrying
On the shuttle plasmid of sTGF β RIIFc gene；Finally, using Adeasy system preparation and reorganization adenovirus vector pAd.sTGF β
RIIFc.Packaging obtains recombined adhenovirus in HEK293 cell.
Furthermore in preferred specific embodiment, in order to carry out function and Mechanism Study, three kinds of controls are also prepared for
Virus: (1) rAd.Null has the function of oncolytic, does not carry the control virus of target gene；(2) Ad.sTGF β RIIFc is carried
The replication defective sexual gland virus of sTGF β RIIFc gene；(4) Ad.Null does not carry the replication defective oncolytic gland of target gene
Virus.
2, the destination gene expression and oncolytic functional evaluation that oncolytic adenovirus mediates；
The effective expression of target gene is the guarantee of the proud performance of immune function.After recombined adhenovirus is infected kidney cancer cell
48h, Western-blot and ELISA detect the expression of sTGF β RIIFc respectively.
And oncolytic function is the essential characteristic of oncolytic adenovirus.In preferred specific embodiment, we use SRB
Decoration method has detected oncolytic adenovirus to oncolysis and oncolytic adenovirus the answering in kidney cancer cell of a variety of kidney cancer cells
Ability processed.
3, anti-tumor effect is evaluated in oncolytic adenovirus body
For anti-tumor effect in evaluation Mice Body, the mice-transplanted tumor model of Renca cell is established, by being administered in tumor
After mode is treated, oncolytic adenovirus is observed to the inhibiting effect of growth of transplanted human, specifies its therapeutic effect.
4, antitumor mechanism research in oncolytic adenovirus body
For the internal antitumor mechanism of clear oncolytic virus, tumor by local TGF-β target gene index of correlation, inflammation are had detected
Factor expression, immunocyte phenotype etc. tentatively illustrate its mechanism of action.
The innovation of the invention consists in that oncolytic adenovirus is effectively combined with TGF β targeted therapy.After tumor by local administration,
First by effective duplication in tumour cell, tumour cell is killed；The virus constantly replicated can continue to generate a large amount of mesh
Albumen sTGF β RIIFc.The sTGF β RIIFc of high local concentrations can block the TGF signal beta of abnormal activation in tumour cell, suppression
The expression and secretion of metastasis related protein processed.Meanwhile sTGF β RIIFc also can reduce the beta mediated immunosuppressive effect of TGF；It adjusts
Balance between antitumor immunity of organism and antiviral immunity plays the effect of oncolytic virus more longer.
Detailed description of the invention
1 oncolytic adenovirus rAd.sT of attached drawing and the genome structure for compareing virus rAd.Null, Ad.sT and Ad.Null
Schematic diagram.
Lethal effect of the 2 oncolytic adenovirus rAd.sT of attached drawing to renal carcinoma cell line: 1 is the lethal effect to 786-O cell；
3 be the lethal effect to Renca cell.
Replication capacity testing result of the 3 oncolytic adenovirus rAd.sT of attached drawing in renal carcinoma cell line.
4 oncolytic adenovirus rAd.sT of attached drawing mediates the ability of sTGF β RIIFc expression: 1 detects for Western-blotting
As a result；2 be ELISA testing result.
The therapeutic evaluation of 5 oncolytic adenovirus rAd.sT of attached drawing treatment Renca transplantable tumor: 1 is tumor growth curve；2 be to control
18 days tumour weight testing results after treatment.
18 days after 6 oncolytic adenovirus rAd.sT of attached drawing treatment, metastasis related gene is expressed real-time in tumor tissues
(real-time) testing result.
After 7 oncolytic adenovirus rAd.sT of attached drawing treatment, the testing result of T lymphocyte hypotype in blood: 1 is after treatment 7
It, 2 be the 18th day after treatment.
7 days after 8 oncolytic adenovirus rAd.sT of attached drawing treatment, cytokine-expressing is real-time (real-time) in blood cell
Testing result.
18 days after 9 oncolytic adenovirus rAd.sT of attached drawing treatment, real-time (real-time) that IFN-γ is expressed in spleen is detected
As a result.
Specific embodiment
The building and preparation of 1 recombined adhenovirus rAd.sT of embodiment
Method is gene chemical synthesis sTGF β RIIFc gene, is inserted it into pshuttle-cmv carrier, is obtained
pShuttle-cmv-sTGFβRIIFc(pSh.cmv.sT)；Using restriction enzyme MfeI digestion TE-TP-E1A, obtain multiple
Related gene processed is cloned into the corresponding site of pSh.cmv.sT, obtains shuttle plasmid pSh.cmv.sT.RE；Reference
Adeasy system obtains recombinant adenovirus plasmid pAd.sT.RE；And oncolytic adenovirus is prepared in HEK293 cell
rAds.sT.Specifically:
(1) building of pSh.cmv.sT
By gene synthesis technology, the complete sequence (as shown in sequence table 1) of sTGF β RIIFc gene is synthesized, and in upstream and downstream
It is separately added into the restriction enzyme site of BglII and KpnI, using above two restriction enzyme by after its digestion, is connected to
The corresponding site of pShuttle-cmv obtains pSh.cmv.sT carrier.
(2) building of pSh.cmv.sT.RE
Plasmid TE-TP-E1A contains TERTp promoter (as shown in SEQ ID NO:3) starting E1A expression and E1B promoter
Two expression cassettes of regulation E1B55K expression obtain this partial sequence by MfeI digestion, are then cloned into its forward direction
The corresponding site (MfeI) of pSh.cmv.sT obtains shuttle plasmid pSh.cmv.sT.RE.
(3) preparation of oncolytic adenovirus rAd.sT
Referring to Adeasy system preparation and reorganization adenoviral plasmid: PmeI digestion shuttle plasmid pSh.cmv.sT.RE, electricity turn
BJ5183 competent cell obtains recombinant adenovirus plasmid pAd.sT.RE with Adeasy-1 homologous recombination.PacI digestion recombinates gland
Virus particle, and by its transfected HEK 293, prepare oncolytic adenovirus rAd.sT (shown in attached drawing 1).It is reflected by sequencing
It after fixed correct, expanded, purified and titer determination；The results show that virus titer and purity meet the requirements (shown in table 1).
(4) building and preparation of control virus
According to oncolytic adenovirus rAd.sT building, preparation flow, pSh.cmv.sT, building are replaced using pShuttle-cmv
The oncolytic adenovirus rAd.Null of target gene is not carried with preparation.Meanwhile using Adeasy system, replication defect type pair is prepared
According to viral Ad.sT and Ad.Null.Three kinds of controls virus is expanded, is purified and titer determination after sequencing identification is correct；Knot
Fruit shows that virus titer and purity meet the requirements (shown in table 1).
1 virus titer of table, purity detecting result
The 2 external functional verification of oncolytic adenovirus rAd.sT of embodiment
Its method is, in kidney cancer cell, using Sulforhodamine B (SulforhodamineB, SRB) decoration method, detection
The lethal effect of oncolytic virus；Pass through the viral amplification from 3h to 48h after infection times of Adeno-X Rapid Titer Kit measurement
Number, the replication capacity of clear virus；By Western-blotting and ELISA testing goal gene sTGF β RIIFc in kidney
Expression in cell.Specifically:
(1) the killing ability detection of oncolytic adenovirus rAd.sT
By people's kidney cancerous cell line 786-O, people 769-P and mouse renal carcinoma cell line Renca respectively with 1 × 103Cells/well
It is inoculated with 96 orifice plates.Second day, oncolytic adenovirus rAd.sT and control virus rAd.Null, Ad.sT, Ad.Null were respectively with 5 times of ladders
Spend diluted mode infection cell (1.25 × 106Vps/ cell -80Vps/ cell).It 7th day (after infection the 6th day), uses
SRB dyeing detects the survival rate of cell in every hole, specifies the killing functions of immunocytes of oncolytic adenovirus.Calculation method are as follows: cell is deposited
OD value/control wells OD value under motility rate=corresponding infection titer.The results show that oncolytic virus rAd.sT and rAd.Null are not only
Human renal carcinoma cell 786-O and 769-P can effectively be killed, moreover it is possible to kill mouse kidney cancer cell Renca (as shown in Fig. 2).
(2) the replication capacity detection of oncolytic adenovirus rAd.sT
By the oncolytic virus rAd.sT of 25000VPs/ cell and control virus infection kidney cancer cell 786-O, 769-P and
3h after Renca washs cell removal virus with PBS.At once cell and supernatant are collected and after 48h, after multigelation, by it
Infect the HEK293 cell (1 × 10 in 24 orifice plates5Cells/well proposes the previous day inoculation).After 48h, using Adeno-X
RapidTiter Kit measures virus titer, and calculates virus amplification multiple.The results show that with replication-defective adenoviral phase
Than, oncolytic virus rAd.sT and rAd.Null can in human renal carcinoma cell massive duplication；And do not have in mouse kidney cancer cell
Embody apparent replication capacity (as shown in Fig. 3).
(3) oncolytic adenovirus rAd.sT mediates the detection of target gene sTGF β RIIFc expression
Using 25000VPs/ cell oncolytic virus rAd.sT and control virus infection kidney cancer cell 786-O, 769-P and
16h after Renca, replaces with serum free medium for cell culture medium.Continue culture -32h for 24 hours, it is thin to collect supernatant respectively
Born of the same parents, using the expression of ELISA and Western-blotting difference testing goal gene sTGF β RIIFc.The results show that no matter
Replication-defective adenoviral Ad.sT or oncolytic adenovirus rAd.sT can efficiently express target gene in kidney cancer cell
(as shown in Fig. 4).
The therapeutic evaluation of 3 oncolytic adenovirus rAd.sT of embodiment treatment mouse kidney transplantable tumor
Its method is Transplanted tumor model to be established using mouse kidney cancer cell Renca, by the side for giving oncolytic virus in tumor
Formula is treated, and by the monitoring to its growing state, evaluates therapeutic effect of the oncolytic virus to mouse kidney transplantable tumor.Specifically
Are as follows:
Mouse kidney cancer cell single cell suspension is with 2 × 106Cell/mouse, subcutaneous lotus knurl 6-8 weeks Babl/c mouse are seen
The growing state for examining tumour, the 7d after lotus knurl detect the volume of tumour, and are divided into Buffer according to the grouping of volume stratified random
Group, Ad.Null group, Ad.sT group, rAd.Null and rAd.sT group.According to 2.5 × 1010The dosage of VPs/100 μ l, intratumor injection
Recombined adhenovirus is treated.After treatment, the health status of mouse, the growth volume of tumour are observed.Go out to Buffer group tumour
Existing ulceration (or volume is greater than 2000mm3) when, terminate experiment.After putting to death mouse, the weight of tumour is detected.The results show that
RAd.sT can effectively inhibit the growth (as shown in Fig. 5) of Renca cell transplantation tumor.
The Mechanism Study of 4 oncolytic adenovirus rAd.sT of embodiment treatment mouse kidney transplantable tumor
Its method is different time points after being treated by detection, the relevant TGF-β target gene of Bone tumour in tumor tissues
Expression, TGF-β signal can be blocked by specifying rAd.sT, to inhibit bone metastaes；Pass through T lymphocyte in blood and spleen
The analysis of hypotype and the expression of various cell factors specify rAd.sT for immune adjustment effect.Specifically:
(1) influence of the oncolytic adenovirus rAd.sT to bone metastaes index of correlation
18d after treatment, puts to death mouse, and separating mouse tumor tissues extract RNA.Detect TGF-β correlation target gene interleukin
11 (Interleukin 11, IL-11), Connective Tissue Growth Factor (connective tissue growth factor,
CTGF), CXCR4 (C-X-Cmotif receptor 4) and PTHrP (Parathyroid hormone related
) etc. protein the mechanism that rAd.sT adjusts kidney transfer (Bone tumour) is specified in expression.Our result show that with
Ad.Null group is compared, Ad.sT treatment the relevant TGF-β target gene of metastases can be significantly reduced, as IL-11, CTGF,
The expression of PTHrP, CXCR4 etc..Compared with replication-defective virus, oncolytic adenovirus itself can also lower tumour associated transitions base
The expression of cause, may be related to the destruction of tumour cell and splitting action to it (as shown in Fig. 6).
(2) regulating and controlling effect of the oncolytic adenovirus rAd.sT to antitumor immunity of organism
Immunological regulation is the important channel of TGF-β mediate tumor immunologic escape, and therefore, can rAd.sT by breaking TGF-
Beta mediated immunosupress is the key that rAd.sT treatment is succeeded.7d and 18d after treatment, we pass through detection blood respectively
With the hypotype of T cell in spleen, derived from bone marrow inhibit cell (Bone marrow derived suppressor cells,
MDSCs the expression etc. of cell factor, carrys out clear rAd.sT to the adjustment effect of mouse immune in ratio), haemocyte and spleen.
Our result show that rAd.sT treatment can change the ratio of CD4+T lymphocyte and CD8+T lymphocyte, and then activate
The anti tumor immune response of body.Compared with rAd.Null treatment, rAd.sT can promote the ratio of CD4+T lymphocyte.It controls
7d after treatment, cytokine profiles in blood cell, such as IL-2, TNF-α express up-regulation；And 18d after the treatment, mouse spleen
The expression of middle IFN-γ is obviously improved (as shown in attached drawing 7-9).
In conclusion we successfully construct by telomerase promoter regulation oncolytic (TERTp starts E1A expression), expression
The oncolytic adenovirus rAd.sT of sTGF β RIIFc albumen.Our result of study confirms, which being capable of kidney in vitro
Massive duplication and kidney cancer cell is killed in cancerous cell line, high efficient expression of the sTGF β RIIFc in kidney cancer cell can be mediated.?
In the Transplanted tumor model of mouse kidney cancer cell Renca, rAd.sT can effectively inhibit the growth and progress of tumour；Meanwhile it is bright
Really rAd.sT can be pressed down in tumor by local by adjusting the relevant TGF-β target gene of Bone tumour, EMT related gene and VEGF etc.
The occurrence and development of metastases processed.In addition, we specify the adjustable TGF-β mediated immunity tolerance of rAd.sT.
Therefore, which can not only realize the inhibition and scavenging effect of local tumor by oncolysis, also
It can inhibit the transfer of tumour by inhibiting metastasis related gene and adjusting the modes such as immune.
Sequence table
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gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300
ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360
tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420
gcacgtggga agccctggcc ccggccaccc ccgcg 455
Claims (5)
1. a kind of oncolytic adenovirus for kidney treatment, it is characterised in that with the Adeasy carrier system of stratagene company
Based on, it is inserted into the expression cassette of adenoviral replication controlling gene sequence and soluble T GF β RIIFc (sTGF β RIIFc) albumen,
It is inserted in the area adenoviral gene group E1；The adenoviral replication controlling gene sequence is by telomerase promoter TERTp and adenopathy
The malicious area E1 replication initiation gene E1A and E1B55K composition, wherein the Adenovirus E1A gene is by telomerase promoter TERTp
Regulation, for E1B55K by natural E1B promoter regulation, the nucleotide sequence of telomerase promoter TERTp is SEQ ID No:3 institute
Show；The adenoviral replication controlling gene sequence is from 5 ' to 3 ' directions of sequence are as follows: TERTp- Adenovirus E1A gene-natural E1B
Promoter-E1B55K, wherein the gene order of coding sTGF β RIIFc albumen is nucleotide sequence shown in SEQ ID No:1,
Amino acid sequence with sTGF β RIIFc albumen is shown in SEQ ID NO:2.
2. oncolytic adenovirus according to claim 1, it is characterised in that from 5 ' to 3 ' directions of sequence are as follows: soluble T GF β
The expression cassette of RIIFc albumen-adenoviral replication controlling gene sequence.
3. oncolytic adenovirus according to claim 1, it is characterised in that the expression cassette of soluble T GF β RIIFc albumen by
Cmv promoter and sTGF β RIIFc gene composition, sTGF β RIIFc albumen are TGF beta receptor soluble fragments and human IgG1 Fc
Fusion protein.
4. the purposes of oncolytic adenovirus according to any one of claim 1-3 in medicine preparation, the drug are used for
Kidney treatment, including the insensitive kidney treatment of TGF-β.
5. the purposes of oncolytic adenovirus according to any one of claim 1-3 in medicine preparation, the drug are used for
Kidney cancer cell is replicated and killed in kidney cancer cell, and mediates high efficient expression of the sTGF β RIIFc in kidney cancer cell.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008067025A2 (en) * | 2006-09-29 | 2008-06-05 | Evanston Northwestern Healthcare | Oncolytic adenoviruses and uses thereof |
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| WO2008067025A2 (en) * | 2006-09-29 | 2008-06-05 | Evanston Northwestern Healthcare | Oncolytic adenoviruses and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| Potent antitumor effects of combined therapy with a telomerasespecific, replication-competent adenovirus (OBP-301) and IL-2 in a mouse model of renal cell carcinoma;P Huang等;《Cancer Gene Therapy》;20100219;第17卷;484-491页 * |
| TGFβ1及其I、II型受体在肾癌中的表达;林天歆 等;《中华泌尿外科杂志》;19990731;第20卷(第7期);393-395页 * |
| 双重靶向条件复制型腺病毒的包装及抗肿瘤作用;王宏芳 等;《西安交通大学学报（医学版）》;20130531;第34卷(第3期);摘要 * |
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