CN101744848B - Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors - Google Patents
Application of FHL3 (four and a half LIM domains 3) in preparation of medicines for treating tumors Download PDFInfo
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Abstract
The invention relates to an application of FHL3 (four and a half LIM domains 3) in the preparation of medicines for treating tumors, in particular to application of FHL3 in the preparation of medicines for adjusting the expression of oncogenes and/or cancer suppressor genes in mammal cells as well as an application of FHL3 in the preparation of medicines for inhibiting the growth of the tumor cells of the mammals. The invention also relates to an expression vector containing the nucleotide sequence of the code FHL3, application of the expression vector in the preparation of medicines for adjusting the representation of cancer genes and/or cancer-inhibiting genes in cells of mammals, as well as application of the expression vector in the preparation of medicines for inhibiting the growth of the tumor cells of the mammals. The expression vector in the invention can effectively inhibit the growth of various human tumor cells, and can adjust the expression of the oncogenes and/or cancer suppressor genes so as to inhibit the growth of various human tumor cells.
Description
Technical field
The present invention relates to the novel medical use of four and half LIM domain proteins, be specifically related to four and half LIM domain proteins 3 (FHL3) are used for treating the medicine of tumor in preparation purposes.
Background technology
Four and half LIM domain proteins (Four and a Half LIM domains protein, hereinafter referred FHL) family belongs to the LIM superfamily, and its family member is made up of 4 half LIM domains.The LIM domain is made up of about 50 aminoacid, is rich in cysteine, forms 2 zinc fingerses.The LIM domain mediates the interaction between many albumen and the albumen, regulates the effect of cytoskeletal protein, enzyme and transcription factor.Have been found that there are 5 members in FHL family, they are respectively FHL1, FHL2, FHL3, FHL4 and FHL5/ACT.FHL member's expression has tissue specificity, and FHL1, FHL2 and FHL3 mainly express in skeletal muscle and cardiac muscle, and FHL4 and FHL5/ACT mainly express in testis.Have now found that the function of FHL family mainly shows three aspects: first aspect, the FHL family protein has important function at myocyte's growth and atomization; Second aspect, the cofactor that the member of FHL family can be used as transcription factor plays a role; And the third aspect, the FHL family member possibly also have critical function in the incidence and development of tumor.
Tumor is the healthy common disease of one type of serious harm human life.In the doctor trained in Western medicine field, the traditional mode of oncotherapy mainly contains at present: operative treatment, chemotherapy, radiotherapy and treatment biology.Gene therapy is one type of new treatment that developed recently gets up; The main theory of gene therapy is one type " polygenic disease " according to being based on tumor, think at present tumor basic pathogeny since gene expression regulation unusually, not normal, the signal conduction abnormalities of somatomedin secretory function, cell generation malignant proliferation is taken place.Therefore, analyzing theoretically, is comparatively ideal means and approach of treatment tumor from the gene expression process of gene level regulating cell.Along with engineered develop rapidly, gene therapy has begun to be used for tumor treatment in recent years.But, nonetheless, still need new departure of safe and effective oncotherapy clinically.
At present, the expression of FHL3 in human tumor is not clear, and it is also unclear whether FHL3 can suppress human tumor cell's growth and how regulate the molecule mechanism of this growth.
Summary of the invention
The novel medical use that the purpose of this invention is to provide FHL3 for example is used for treating the purposes of the medicine of tumor in preparation, or is used to treat tumor through methods such as gene therapies.
The inventor finds unexpectedly under study for action; FHL3 can regulate oncogene and antioncogene expression in vivo; And further suppress the growth of the particularly human tumor cell of mammal thus, for the clinical treatment of tumor provides a kind of brand-new useful scheme.Based on this discovery, the inventor has accomplished the present invention.
On the one hand, the invention provides FHL3 is used for regulating the medicine that oncogene and/or antioncogene express in mammalian cell in preparation purposes.
Based on the purposes of first aspect present invention, second aspect present invention provides FHL3 to be used for suppressing the purposes of the medicine of mammalian tumor cell growth in preparation.
Third aspect present invention provides a kind of expression vector, and it contains the nucleotide sequence of the FHL3 that encodes.In one embodiment, described carrier is plasmid or virus.In another embodiment, described virus is adenovirus.
Fourth aspect present invention provides the described expression vector of third aspect present invention to be used for regulating the purposes of the medicine that oncogene and/or antioncogene express in preparation in mammalian cell.
Fifth aspect present invention provides the described expression vector of third aspect present invention to be used for suppressing the purposes of the medicine of mammalian tumor cell growth in preparation.
The present invention is described below in further detail.
First aspect present invention provides FHL3 to be used for regulating the purposes of the medicine that oncogene and/or antioncogene express in preparation in mammalian cell.
FHL3 of the present invention is a kind of known protein matter, and English full name is: four anda half LIM domains 3, it is numbered BC011697 at the given gene of NCBI (U.S. state-run biotechnology information centre).FHL3 can obtain through number of ways, and for example (Clontech, USA) middle amplification contains 280 amino acid whose complete coding region FHL3cDNA sequences from mammary gland cDNA library with PCR method.
In addition, use like this paper, term " oncogene " is meant the gene that the product of its coding is relevant with the neoplastic of cell, and it can promote the growth and the conversion of cell.Use like this paper, term " antioncogene " is the antonym of oncogene, and its normal function is cell growth inhibiting and tumor generation.
Through the inventor's further investigation, find that FHL3 can regulate the expression of oncogene in the mammalian cell, and/or can regulate the expression of antioncogene in the mammalian cell.More particularly, the inventor finds that FHL3 can suppress the expression of oncogene in the mammalian cell, and/or can promote the expression of antioncogene in the mammalian cell.Described mammalian cell can be the cell from any tissue of mammal or organ.Further, said mammalian cell includes but not limited to from the cell with undertissue or organ: mammary gland, liver, lungs, stomach, prostate, intestinal, kidney, uterus, ovary, skin.Further, said mammalian cell includes but not limited to from the cell with undertissue or organ: mammary gland, liver, lungs.On the other hand; Described mammalian cell can be in somatic cell; Also can be isolated cells, though the present invention is these results through adopting isolated cells to make an experiment and find; It will be apparent to those skilled in the art that can be easily at the mammiferous equifinality that in somatic cell, obtains through scheme of the present invention and result.In addition, based on industry purposes of the present invention, described mammalian cell is preferably mammiferous in somatic cell.These discoveries are to realize basis of the present invention.
Based on the purposes of first aspect present invention, second aspect present invention provides FHL3 to be used for suppressing the purposes of the medicine of mammal tumor and/or growth of cancer cells in preparation.
Be used for term " tumor " and " cancer " of this paper, its implication and both sides relation are well known to a person skilled in the art, and the two can exchange use in many cases.Described tumor and/or cancer can be medically known any tumors, comprise malignant tumor and/or cancer.Preferably, described tumor (comprising malignant tumor and/or cancer) includes, but are not limited to:
Malignant tumor includes but not limited to breast carcinoma, hepatocarcinoma, pulmonary carcinoma (comprising small cell lung cancer, nonsmall-cell lung cancer), colon cancer, renal carcinoma, ovarian cancer, gastric cancer, cervical cancer, carcinoma of prostate and skin carcinoma (comprising squamous cell carcinoma);
The tumor of maincenter and peripheral nervous system includes but not limited to astrocytoma and glioma; And
Other tumors include but not limited to melanoma.
More preferably, described tumor (comprising malignant tumor and/or cancer) is selected from breast carcinoma, hepatocarcinoma, pulmonary carcinoma (comprising small cell lung cancer, nonsmall-cell lung cancer), gastric cancer, colon cancer, renal carcinoma, ovarian cancer, cervical cancer, carcinoma of prostate and skin carcinoma.
Further preferably, described tumor (comprising malignant tumor and/or cancer) is selected from breast carcinoma, hepatocarcinoma, pulmonary carcinoma (comprising small cell lung cancer, nonsmall-cell lung cancer).
According to a second aspect of the invention; Described mammiferous tumor and/or cancerous cell can be in somatic cell; Also can be isolated cells, though the present invention has adopted isolated cells to make an experiment; It will be apparent to those skilled in the art that can be easily at the mammiferous equifinality that in somatic cell, obtains through scheme of the present invention and result.In addition, based on industry purposes of the present invention, described mammiferous tumor and/or cancerous cell are preferably suffered from the mammiferous in somatic cell of tumor and/or cancer.
The term " mammal " that is used for this paper is meant the experimenter who suffers from tumor according to the invention and/or cancer, and it includes but not limited to the domestic animal and the mankind such as pig, Canis familiaris L., cat, cattle, sheep.Preferably, described mammal is human.
Third aspect present invention provides a kind of expression vector, and it contains the nucleotide sequence of the FHL3 that encodes.Further, described carrier includes but not limited to can be used as any plasmid, virus and the phage that expression vector uses.Preferably, described carrier is plasmid and virus.Further, described virus can be the virus that can be used as carrier known in the art, preferred, described virus is adenovirus.
Term as herein described " expression vector "; Its implication is well known to a person skilled in the art; And typically refer to and contain an initiator sequence, can impel the genes of interest of insertion to transcribe effectively, and then translate the carrier of the protein product of this insertion gene code.
Fourth aspect present invention provides the described expression vector of third aspect present invention to be used for regulating the purposes of the medicine that oncogene and/or antioncogene express in preparation in mammalian cell.Described expression vector can be used as a kind of ingredient of said medicine.
Fifth aspect present invention provides the described expression vector of third aspect present invention to be used for suppressing the purposes of the medicine of mammalian tumor cell growth in preparation.Described expression vector can be used as a kind of ingredient of said medicine.
According to the present invention, contain the medicine of FHL3, perhaps contain the medicine of expression vector of the nucleotide sequence of the FHL3 that encodes, it can have the experimenter of treatment demand through any approach known in the art, and for example mammal is particularly human.Described route of administration includes but not limited to: whole body administration, local skin administration, focal zone topical etc.Especially, described route of administration includes but not limited to: intravenous, intra-arterial, intramuscular, per os, percutaneous, intraperitoneal, tumor focus district, through lung suck or be blown into, parenteral, through mucous membrane, per nasal, per rectum.More particularly, described route of administration includes but not limited to: intravenous and tumor focus district.For example, without limitation, according to the medicine of the FHL3 of containing of the present invention or contain the medicine of expression vector of the nucleotide sequence of the FHL3 that encode, it can perhaps pass through intravenous administration through tumor focus district local injection.Particularly, the experimenter of treatment demand can be arranged with reference to the administering mode well known in the art of relevant recombiant plasmid and adenovirus, for example with reference to Clayman GL et al.J Clin Oncol; 1998,16:2221-2232 and Martin B et al.Cancer Res, 1999; Method among the 59:1391-1399 is through the administration of tumor locus local injection; Perhaps with reference to Reid T et al.Cancer Gene Ther, 2002, the method among the 9:979-986 is passed through intravenous administration.Above document is incorporated this paper into by reference with its full content.
According to the present invention, described medicine can be any medicament forms that can be used for administration well known in the art, comprises pharmaceutical composition, pharmaceutical preparation etc.Specifically, for example be the formulation example such as the suppository that are used for topical, ointment, ointment etc.; For example be the preparation that is used for parenteral, for example local injection preparation and systemic injection are used preparation, and concrete dosage form can for example be injection solution agent, powder ampoule agent for injection etc.For enforcement of the present invention, medicine of the present invention preferably is used for the preparation of parenteral, includes but not limited to that local injection preparation and systemic injection use preparation, and concrete dosage form includes but not limited to injection solution agent and powder ampoule agent for injection.Preferred, described medicine is aseptic injection aqueous solution agent, or aseptic be used for before the clinical use redissolved the injectable powder, particularly lyophilization injectable powder of preparation with water for injection.
In addition, according to the result of study that hereinafter provides, effective dose when those skilled in the art can easily confirm in mammal, to use.Usually, the treatment effective dose for example can be 1 * 10 by experimenter's weighing machine
9To 1 * 10
14The VP/kg body weight/day, preferred 1 * 10
10To 1 * 10
13The VP/kg body weight/day, more preferably 1 * 10
11To 1 * 10
12The VP/kg body weight/day." VP " in the said dosage unit is meant the abbreviation of virion number (Viral Particle).Can with one day dosage in one day disposable all to the experimenter, also can be in one day the dosage of needs be divided into two, three, four or more a plurality of low dose with the suitable interval administration.Said low dose can be mixed with presented in unit dosage form, and for example each presented in unit dosage form contains the respective amount of the suitable number of times of TDD segmentation.Certainly, also can for example be administered once in one day, be administered once in two days with the administration of cycle regular hour, a week is administered once, be administered once January etc.
On the other hand; Medicine provided by the invention is in concrete clinical case; Their concrete use amount can possibly need to do corresponding the variation because of multiple factor, and these factors include but not limited to: the order of severity of experimenter's patient's condition, experimenter's age, sex, body weight; Route of administration, pharmaceutical dosage form or the like.
According to result of study of the present invention; Have been found that; Through carrier particularly the FHL3 expression vector that obtains of adenovirus can effectively suppress multiple human tumor cell's growth, thereby and can suppress multiple human tumor cell's growth through the expression of regulating oncogene and antioncogene.
Description of drawings
Fig. 1 shows FHL3 mammalian cell expression vector enzyme action qualification result, and wherein: 1 is the dna marker thing; 2 is pcDNA3; 3 is pcDNA3-FHL3.
Fig. 2 shows FHL3 recombinant adenoviral vector enzyme action qualification result, and wherein: 1 is the dna marker thing; 2 is pAdeasy-1; 3 is pAdeasy-FHL3.
Fig. 3 shows that the FHL3 recombinant adenovirus infects high expressed FHL3 behind the ZR75-1 cell, and wherein: 1 is pAdeasy-1; 2 is pAdeasy-FHL3.
Fig. 4 shows that FHL3 recombinant adenovirus infected tumor cell can suppress the growth of this tumor cell, and wherein: A is HepG2; B is MCF-7; C is H460.
Fig. 5 shows that the FHL3 recombinant adenovirus infects the HepG2 cell and suppresses its colony formation, and wherein: A is colony formation figure; B is that colony forms several comparison diagrams.
Fig. 6 shows that the injection of FHL3 recombinant adenovirus can suppress the tumor growth that hepatoma cell strain HepG2 implants.
Fig. 7 shows high expressed FHL3 albumen in the tumor piece of FHL3 recombinant adenovirus injection, and wherein: 1 is pAdeasy-1; 2 is pAdeasy-FHL3.
Fig. 8 shows that the FHL3 recombinant adenovirus infects the expression of HepG2 impact cell oncogene and antioncogene, and wherein: 1 is pAdeasy-1; 2 is pAdeasy-FHL3.
Fig. 9 shows the expression that influences oncogene and antioncogene in the tumor piece of FHL3 recombinant adenovirus injection, wherein: 1, pAdeasy-1; 2, pAdeasy-FHL3.
The specific embodiment
Further specify the present invention through concrete embodiment below, still, be to be understood that into, these embodiment are only used for the usefulness of explanation more in detail particularly, are used for limiting in any form the present invention and should not be construed as.
General description is carried out to the material and the test method that are used in the present invention's test in this part.Though for realizing that employed many materials of the object of the invention and operational approach are well known in the art, the present invention still does to describe in detail as far as possible at this.It will be apparent to those skilled in the art that hereinafter, if do not specify that material therefor of the present invention and operational approach are well known in the art.
(1), method
1, the structure of FHL3 mammalian cell expression vector
Complete coding region sequence by conventional method pcr amplification FHL3 gene from mammary gland cDNA library (available from U.S. Clontech company); The PCR primer is: 5 '-CGGGATCCATGAGCG AGTCATTTGAC-3 ' and 5 '-GCTCTAGATTAGGGCCCTGCCTGGAA-3 '; The PCR reaction condition is: behind 94 ℃ of 5min, and 94 ℃ of 1min, 60 ℃ of 1min; 72 ℃ of 1min carry out 30 circulations, last 72 ℃ of 7min.Use BamHI and Xba I enzyme (available from Britain Biolabs company) at 37 ℃ of following enzyme action PCR products and corresponding mammalian cell expression vector (pcDNA3 respectively; Available from American I nvitrogen company); PCR product behind the enzyme action is connected under 12-16 ℃ with T4DNA ligase (available from U.S. Promega company) with carrier; Obtain recombinant vector (it contains complete coding region sequence, BGH polyA termination signal, the ampicillin resistance gene of CMV promoter/enhancer, FHL3 gene); With this recombinant vector transformed into escherichia coli DH5 α, obtain transformant.Extract recombinant vector, identify recombinant vector with BamHI and Xba I enzyme enzyme action; Determined dna sequence confirms whether the complete encoding sequence of FHL3 gene is entirely true; Whether Western engram analysis reorganization FHL3 albumen expresses; After promptly albumen being carried out SDS-PAGE with conventional method; Albumen is transferred on the nitrocellulose filter; (available from U.S. ProteinTech company) reacts with the anti-people FHL3 of rabbit antibody, uses goat anti-rabbit igg antibody (available from U.S. Santa Cruz Biotech company) to react then and develops the color.
FHL3 is known in the art.Wherein, the aminoacid sequence of the complete coding region DNA sequence of FHL3 gene and the FHL3 gene code description of sequence table part that vide infra.
2, the structure of FHL3 recombinant adenoviral expressing vector
Complete coding region sequence by conventional method pcr amplification FHL3 gene; The PCR primer is: 5 '-GGGGTACCATGAGCGAGTCATTTGAC-3 ' and 5 '-CCCAAGCTT TTAGGGCCCTGCCTGGCT-3 '; The PCR reaction condition is: behind 94 ℃ of 5min, and 94 ℃ of 1min, 60 ℃ of 1min; 72 ℃ of 1min carry out 30 circulations, last 72 ℃ of 7min.Use Kpn I and Xbo I enzyme (available from Britain Biolabs company) difference enzyme action PCR product and corresponding adenovirus expression carrier (pShuttle-CMV under 37 ℃ respectively; Available from U.S. Stratagene company); PCR product behind the enzyme action is connected under 12-16 ℃ with T4DNA ligase (available from U.S. Promega company) with carrier; Connect product transformed into escherichia coli DH5 α, obtain transformant.Extract recombinant vector, identify with Kpn I and XhoI enzyme enzyme action whether recombiant plasmid makes up success; Determined dna sequence confirms whether the complete encoding sequence of FHL3 gene is entirely true; Whether Western engram analysis reorganization FHL3 albumen expresses.
After obtaining above-mentioned recombiant plasmid; The pShuttle-CMV carrier that contains the FHL3 gene complete encoding sequence with PmeI (available from Britain Biolabs company) at 37 ℃ of following enzyme action; Cut glue and reclaim linearizing fragment; Reclaim the conventional BJ5183 cell (available from U.S. Stratagene company) that contains pAdEasy-1 that transforms of product, that flat board of card-coating was hatched 16-20 hour for 37 ℃.Observe that flat board of card, grow large and small two kinds of bacterium colonies, less 5 bacterium colonies of picking are in the culture medium of that resistance of card; 37 ℃; Spend the night in the 200rpm shaking table, extract plasmid, PacI (available from Britain Biolabs company) is at 37 ℃ of following enzyme action; 0.7% agarose gel electrophoresis identifies, contains 3.0kb or 4.5kb size segmental promptly showing to recombinate successfully.Because BJ5183 cell purpose carrier output is lower; So the carrier routine that will successfully recombinate transforms XL-10 Gold competent cell (available from U.S. Stratagene company) with amplification vector; That flat board of card-coating; Hatched 16-20 hour for 37 ℃, picking monoclonal, gained bacterium liquid are the XL-10 Gold cell of high amplification purpose carrier.
3, the packing of FHL3 recombinant adenoviral vector and evaluation
PacI (available from Britain Biolabs company) enzyme action reorganization adenovirus vector well is to expose the left and right sides terminal repeat (ITR) on the pShuttle-CMV carrier, equal-volume phenol: the chloroform extracting is reclaimed DNA with impurity such as removal enzymes with ethanol precipitation; After measuring concentration, (the AD293 cell is a kind of mammalian cell strain available from U.S. Stratagene company to the description that provides by company, and is cell-derived by HEK293 with transfection reagent lipofectamine 2000 (available from American I nvitrogen company) transfection AD293 cell; Cultivate with conventional cell culture medium, adherent ability is strong, is used to produce infectious viral particle); Transfection the previous day is with AD293 cell inoculation to 24 orifice plate; Cell density is 70-80% during transfection, changes the culture medium of 5% serum in 4-6 hour after the transfection, continues to cultivate 7-10 days; Changed a subculture in every 3-4 days; Up to occur tangible cytopathic effect (cytopathic effect, CPE), promptly the cellular swelling, become circle, floating.Continue to cultivate; Results virus when treating complete CPE: piping and druming makes cell take off wall gently; Cell and supernatant are collected in the EP pipe in the lump, and multigelation 3 times is with releasing virus between-80 ℃ and 37 ℃, and centrifugal 15 minutes of 2500rpm is to remove cell debris and impurity; Supernatant is transferred in the new EP pipe, as seed virus be stored in-80 ℃ subsequent use.Simultaneously, the CPE method is measured the infection titer of seed disease venom fast, and concrete grammar is following: the AD293 cell inoculation in 24 orifice plates, was cultivated 24 hours, and cell density reaches at 90% o'clock, and cell number is 5 * 10
5Cells/well, with the culture fluid sucking-off in each hole, every hole adds 10 of 0.5ml
1To 10
9Doubly the viral liquid (diluting with complete medium) of dilution changes normal culture medium into after 2 hours, cultivates and observes the CPE situation after 48 hours, selects the highly diluted multiple that 100%CPE takes place, with following formula calculating virus titer:
Get above-mentioned viral liquid inductance and dye breast cancer cells such as ZR75-1; Infection intensity (multiplicityof infection; Be abbreviated as MOI) be 50 plaque forming units (plaque forming unit is abbreviated as pfu)/cell, the previous day with cell inoculation in 24 orifice plates; Cell density is 80-90% during infection; Infect changing virus-free fresh culture in back 2 hours, collecting cell after 48 hours is with specific antibody (available from U.S. ProteinTech company) the Western western blotting method detection FHL3 expression of gene situation of the anti-people FHL3 of rabbit.Express next step amplification and purification of successfully laggard row.
4, the amplification of FHL3 recombinant adenoviral vector and purification
The AD293 cell inoculation in the 15cm plate, when treating that cell density reaches 80-90%, is added viral liquid, and MOI is the 2-5pfu/ cell.When infecting about 48 hours cells and obvious CPE occurring, abandon supernatant, scrape with cell and scrape cell cryopreservation gently in-80 ℃.Unify purified virus when being accumulated to 50-60 plate.
With the AD293 cell of collecting multigelation 3 times between-80 ℃ and 37 ℃, 2500rpm removed cell debris in centrifugal 15 minutes.Preparation CsCl density gradient solution: get the CsCl of 30g, add PBS and (contain NaCl8g, KCl 0.2g, Na in every premium on currency solution
2HPO
41.44g, KH
2PO
40.4g pH7.4) to final volume 42.5ml, getting density is the CsCl solution of 1.5g/ml; Get the CsCl of 15ml1.5g/ml, add PBS to final volume 21ml, getting density is the CsCl solution of 1.35g/ml; Get the CsCl of the 1.5g/ml of 11ml, add PBS to final volume 20ml, getting density is the CsCl solution of 1.25g/ml.CsCl solution with the 1.25g/ml of the CsCl solution of the 1.35g/ml of the CsCl solution of the 1.5g/ml of 0.5ml, 2.5ml, 2.5ml joins in the 12ml ultracentrifugation pipe successively, viral liquid to be purified is added on respectively manages on the CsCl gradient liquid 35000rpm, 10 ℃; Centrifugal 1.5 hours, between the CsCl of the CsCl of 1.35g/ml solution and 1.25g/ml solution, can be observed the vaporific virus band of white, collect the virus band, and mix with the CsCl solution of 1.35g/ml; 35000rpm, 10 ℃, centrifugal 5.5 hours; Being further purified virus, is that dialysis solution is at 4 ℃ of viruses of dialysing down, dialysis solution of replacing in per 2 hours with Hanks liquid; Change altogether 5 times, take out the viral liquid of purification, promptly contain the solution of FHL3 recombinant adenoviral vector; Adding aseptic glycerol to final concentration is 10%, and portion package is preserved down at-70 ℃.
5, the mensuration of FHL3 recombinant adenovirus purity and titre
Get the virus of 24 μ l purification, add the PBS of 84 μ l and the 10%SDS of 12 μ l, mixing, the 0%SDS that adds 12 μ l with the PBS of 108 μ l is a blank, measures the absorbance value of 260nm and 280nm wavelength, i.e. OD
260nmAnd OD
280nmValue, the purity of estimation virus in view of the above.Purity=the OD of virus
260nmValue/OD
280nmValue, this Bi Zhi>1.3 the time, show that viral purity is higher.Calculate virion titre (VP/ml) then.With 1 OD
260nmValue is equivalent to 1.1 * 10
12Virion (VP) calculates the virion titre.Virion titre (VP/ml)=OD
260nmValue * 1.1 * 10
12* extension rate.
Limiting dilution assay is measured the infection titer (IU/ml) of virus: get the virus of 10 μ l purification, add in the DMEM culture medium that 990 μ l contain 5%FBS (10
2Dilution), takes out 100 μ l behind the mixing, be diluted in the DMEM culture medium that 900 μ l contain 5%FBS (10
3Dilution), by that analogy up to 10
12Dilution.Simultaneously with the AD293 cell inoculation in 96 orifice plates, 50 μ l/ holes, cell number is 1 * 10
4Cells/well.Add 10 of 50 μ l then
5To 10
12The viral liquid of dilution is done 8 dilution gradients altogether, and each gradient is done 10 multiple holes.Infect the back and observed CPE on the the 10th to 12 day, the highly diluted multiple CPE hole count down of CPE takes place in counting, calculates viral infection titer according to following formula:
6, the transfection of mammalian cell
(the HEK293 cell is the HEKC available from a kind of immortalization of U.S. ATCC cell bank at the HEK293 cell; Cultivate with conventional cell culture medium; Adherent ability) 24h before the transfection, with the DMEM culture medium that contains 10% NBCS with cell inoculation in Tissue Culture Dish.Inoculating cell density during with transfection cell density reach 90% and be advisable.Description by company provides is carried out transfection with cell transfecting reagent Lipofectamine 2000 (available from American I nvitrogen company).
7, Western immunoblotting assay
(1) SDS-PAGE electrophoresis: the centrifugal back total protein with every hole 20 μ g of the total protein of cell of degeneration is carried out the SDS-PAGE electrophoresis; Voltage 120V, about 1.5h.
(2) change film: electrophoresis is transferred to albumen on the nitrocellulose filter after finishing; Voltage 15V shifts about 2h.
(3) sealing: (with the preparation of TBST solution, TBST solution is to contain Tris2.42g, NaCl8g, Tween-20 10ml in every premium on currency solution to 5% defatted milk powder, and pH7.6) room temperature sealing nitrocellulose filter 1h or 4 ℃ of sealings are spent the night.
(4) one resistive connections close: on nitrocellulose filter, add one anti-(the anti-people FHL3 of rabbit antibody is available from the U.S. ProteinTech company) of diluting by a certain percentage with 5% defatted milk powder, room temperature jog 1h, TBST solution wash film 3 times, each 7min.
(5) two resistive connections close: on nitrocellulose filter; The IgG (goat anti-rabbit igg antibody is available from U.S. Santa Cruz Biotech company) that adds the horseradish peroxidase of diluting by a certain percentage with 5% defatted milk powder, room temperature jog 1h; TBST solution is washed film 3 times, each 7min.
(6) develop: the explanation (U.S. Pierce company) that provides by company, with the chemoluminescence method 5min that develops the color, tabletting develops.
(2) result
1, the evaluation of FHL3 mammalian cell expression vector and FHL3 recombinant adenoviral vector
FHL3 mammalian cell expression vector and FHL3 recombinant adenoviral vector show through enzyme action, determined dna sequence; The complete coding region sequence that is inserted into the FHL3 gene in the carrier is entirely true; Be that FHL3 mammalian cell expression vector (pcDNA3-FHL3) is behind BamHI and XbaI enzyme cutting; Can cut out the FHL3 complete coding region sequence of the about 840bp of size, and corresponding empty carrier (pcDNA3) can not cut out this band (referring to Fig. 1); Similarly, FHL3 recombinant adenoviral vector (pAdeasy-FHL3) can cut out the band (referring to Fig. 2) of the about 4.5kb of size behind the PacI enzyme action, and corresponding empty carrier (pAdeasy-1) can not cut out this band, shows the success of recombinating.
2, the purity of FHL3 recombinant adenovirus and titer determination
The titre of CPE method mensuration FHL3 recombinant adenovirus and corresponding contrast recombinant adenovirus is respectively 4.2 * 10 fast
8Pfu/ml and 4.0 * 10
8Pfu/ml.Purity after FHL3 recombinant adenovirus and the corresponding contrast recombinant adenovirus poisons amplification purification is respectively 1.33 and 1.34, explains that purity is higher.The granule titre of recombinant adenovirus behind the purification and corresponding contrast recombinant adenovirus is respectively 1.0 * 10
12VP/ml and 1.2 * 10
12VP/ml, the infection titer of measuring with limiting dilution assay is respectively 3.2 * 10
10IU/ml and 4.3 * 10
10IU/ml.
3, the FHL3 albumen of FHL3 mammalian cell expression vector or mediated by recombinant adenovirus
Expression
With FHL3 mammalian cell expression vector transfection HEK293 cell or with 50MOI recombinant adenovirus infected person breast carcinoma ZR75-1 cell; Carry out Westernblot with FHL3 antibody; The result shows that transfection or metainfective cell all can express FHL3 albumen (referring to Fig. 3; Showing typically wherein that the FHL3 recombinant adenovirus infects behind the ZR75-1 cell can high expressed FHL3, behind the FHL3 mammalian cell expression vector transfection HEK293 cell similar results is arranged, but data is unlisted).
(1) method
With the influence of crystal violet measuring FHL3 to the kinds of tumor cells growth; Step is following: with 0.25% pancreatin (available from U.S. Sigma-Aldrich company) digestion kinds of tumor cells, comprise breast carcinoma cell strain (MCF7, ZR75-1), hepatoma cell strain (HepG2, SMMC7721), lung cancer cell line (H460).Behind the cell counting, be diluted to suitable concentration, the 0.5ml Cell sap is put into 24 orifice plates, make the cell number in every hole be about 10,000.The method described in the embodiment 1 of pressing infects these tumor cells with these tumor cells of FHL3 mammalian cell expression vector transfection or with the FHL3 recombinant adenovirus of 20-100MOI; Cultivate after 1-4 days; Remove culture medium; Every hole adds 1% glutaraldehyde (fixative) of 0.5ml, and static 15-20min removes glutaraldehyde.Every hole adds 0.5ml crystal violet (available from U.S. Sigma-Aldrich company), places 15min (crystal violet is stored in room temperature), also washes several times with the tap water submergence; Distilled water room temperature submergence 15min; Abandon most distilled water, in air drying 24 holes (spending the night when needing), every hole adds sorenson ' the s solution (containing in every premium on currency solution: sodium citrate 8.967g, 0.1N HCl 0.195L, 90% ethanol 0.5L) of 0.5ml; On horizontal shaking table, shake 30min; 100 μ l to 96 orifice plates are got in every hole, measure the OD value in the wavelength of 590nm, represent the cell relative growth rate with the OD value.
(2) result
The result shows; The growth that the expression of FHL3 can suppress above-mentioned all tumor cells is (referring to Fig. 4; It shows that typically the FHL3 recombinant adenovirus infects the growth that breast carcinoma cell strain MCF7, hepatoma cell strain HepG2 and lung cancer cell line H460 can suppress these cells; Other cell strain has similar results, but data are unlisted).
The grappling that embodiment 3, FHL3 suppress kinds of tumor cells does not rely on growth
(1) method
The tumor cell grappling is not relied on the influence of growth with soft agar measuring FHL3.At first; The method described in the embodiment 1 of pressing is with FHL3 mammalian cell expression vector transfection tumor cell or with the FHL3 recombinant adenovirus infected tumor cell of 20-100MOI; Comprise breast carcinoma cell strain (MCF7), hepatoma cell strain (HepG2), lung cancer cell line (H460), adopt 60mm then
2Tissue Culture Dish; In culture dish, add the DMEM culture medium (available from American I nvitrogen company) that 3ml contains 0.7% agar; Add 3ml after solidifying above that and contain above-mentioned 20; The DMEM culture medium of 000 tumor cell and 0.25% agar, every group of cell established 3 parallel appearance, cultivate 3 week the back colony of under inverted microscope, counting every group of cells form number.
(2) result
The result shows; The colony that the expression of FHL3 can suppress above-mentioned all tumor cells forms (referring to Fig. 5; It shows that typically the FHL3 recombinant adenovirus infects the grappling that hepatoma cell strain HepG2 can suppress cell and do not rely on growth, and other cell strain has similar results, but data are unlisted).
Embodiment 4, FHL3 suppress the growth of nude mice tumor cell
(1) method
The method described in the embodiment 1 of pressing is with FHL3 mammalian cell expression vector transfection hepatoma cell strain HepG2.
With 1 * 10
7The cell of individual above-mentioned cells transfected or untransfected be inoculated in respectively 5 age in week female BALB/c nude mice back right lateral side, be divided into 4 groups, i.e. the tumor cell group of the tumor cell group of FHL3 mammalian cell expression vector transfection and corresponding empty carrier transfection; The tumor cell group of the corresponding contrast virus of the tumor cell group of injection FHL3 recombinant adenovirus with injection.Every group of each 10 nude mice.For the adenovirus group, treat tumor growth when being about 1cm, at tumor locus direct injection adenovirus, ID is 1 * 10
10VP.Observe inoculation position have or not ooze out, tumor growth and nude mice situation.Measure the maximum gauge L (cm) and the minimum diameter D (cm) of tumor, calculate by following formula and respectively organize gross tumor volume V (cm
3) size: gross tumor volume (cm
3)=L
2* D * 1/2.Treat about 10 weeks of cell transplantation, put to death each treated animal, get part tumor piece; 0.9% normal saline flushing with ice-cold is clean, puts into the 5ml centrifuge tube, adds the lysate (1 * PBS of 1ml pre-cooling to 0 ℃ immediately; 1%NP-40,0.5% NaTDC, 0.1%SDS; 1/1000 protease inhibitor), homogenate in ice bath, the Western western blotting method of pressing among the embodiment 1 is analyzed the proteic expression of FHL3 in the tumor piece.
(2) result
After nude mice begins to occur the tumor piece, measure the tumor block size weekly, it is long-pending to calculate the tumor block.The result shows that FHL3 can suppress the growth (referring to Fig. 6, it shows that typically the injection of FHL3 recombinant adenovirus can suppress the tumor growth that hepatoma cell strain HepG2 implants) of above-mentioned tumor cell in the nude mice.The Western immunoblotting assay shows, high expressed FHL3 albumen in the tumor piece of injection FHL3 recombinant adenovirus, and inject the low FHL3 albumen (referring to Fig. 7) of expressing in the tumor piece of corresponding contrast adenovirus.
Embodiment 5, FHL3 can suppress the expression of oncogene in the tumor cell and promote antioncogene
Expression
(1) method
With 1 * 10
6Individual cell inoculation is in Tissue Culture Plate; Then with the transfection tumor cell's strain of FHL3 mammalian cell expression vector or with the FHL3 recombinant adenovirus infected tumor cell strain of 100MOI, these tumor cell lines comprise breast carcinoma cell strain MCF7 and ZR75-1, hepatoma cell strain HepG2 and SMMC7721, lung cancer cell line H460.With these tumor cell inoculations in Tissue Culture Plate; Cultivate after 24 hours; Collecting cell is used FHL3 antibody (the anti-people FHL3 of rabbit antibody is available from U.S. ProteinTech company), p21 (the anti-people p21 of rabbit antibody respectively by the method among the embodiment 1; Available from U.S. Santa Cruz Biotech company) and p15 cancer suppressor protein antibody (the anti-people p15 of rabbit antibody; Available from U.S. Santa Cruz Biotech company), c-myc cancer protein antibody (mouse-anti people c-myc antibody is available from U.S. Santa Cruz Biotech company) carries out the Western immunoblotting assay, detects the expression of corresponding protein in the above-mentioned tumor cell.
In addition, get the nude mice tumor tissues among the embodiment 4, detect p21 and the proteic expression of c-myc in the tumor tissues with the Western immunoblotting assay.
(2) result
The result shows; FHL3 all expresses in above-mentioned tumor cell; The overexpression of FHL3 has suppressed the expression of c-myc cancer protein, but the expression of raise p21 and p15 cancer suppressor protein (referring to Fig. 9, it shows that typically the FHL3 recombinant adenovirus infects FHL3 behind the hepatoma cell strain HepG2, c-myc, p21 and the proteic expression of p15; Other cell strain has similar results, but data are unlisted).
In addition, the nude mice tumor tissues is detected p21 and the proteic expression of c-myc, result and above-mentioned experiment in vitro result consistent (referring to Figure 10) with the Western immunoblotting assay.
Although the present invention has carried out description to a certain degree, significantly, under the condition that does not break away from the spirit and scope of the present invention, can the suitable variation of each condition of carrying out.Be appreciated that to the invention is not restricted to said embodiment, and belong to the scope of claim, it comprises the replacement that is equal to of said each factor.
Sequence table
< 110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
< 120>FHL3 is used for treating the purposes of the medicine of tumor in preparation
<210>1
<211>843
<212>DNA
<220>
< 223>complete coding region of FHL3 gene
<400>1
<210>2
<211>280
<212>PRT
<220>
< 223>aminoacid sequence of FHL3 gene code
<400>2
Claims (11)
1.FHL3 be used for regulating the purposes of the medicine that oncogene and/or antioncogene express in preparation in mammalian cell, wherein said FHL3 suppresses the expression of oncogene in the mammalian cell, and/or promotes the expression of antioncogene in the mammalian cell; Said oncogene is the c-myc gene, and said antioncogene is p21 gene or p15 gene; Said mammalian cell is HCC HepG2.
2. purposes according to claim 1, wherein said mammalian cell are selected from somatic cell or isolated cells; Wherein, in the HCC HepG2 at body, FHL3 suppresses the c-myc expression of gene, and/or promotes the p21 expression of gene; In the HCC HepG2 that exsomatizes, FHL3 suppresses the c-myc expression of gene, and/or promotes p21 gene or p15 expression of gene.
3.FHL3 be used for suppressing the purposes of the medicine of mammalian tumor cell growth in preparation; Wherein, said mammalian tumor cell is breast cancer cell MCF7, HCC HepG2 or lung carcinoma cell H460.
4. the expression vector that contains the nucleotide sequence of the FHL3 that encodes is used for regulating the purposes of the medicine that oncogene and/or antioncogene express in mammalian cell in preparation; The wherein said expression that is adjusted to oncogene in the inhibition mammalian cell, and/or the expression of the interior antioncogene of promotion mammalian cell; Said oncogene is the c-myc gene, and said antioncogene is p21 gene or p15 gene; Said mammalian cell is HCC HepG2.
5. purposes according to claim 4, wherein, described carrier is selected from plasmid, virus and phage.
6. purposes according to claim 5, wherein, described carrier is a virus.
7. purposes according to claim 6, wherein, described carrier is an adenovirus.
8. the expression vector that contains the nucleotide sequence of the FHL3 that encodes is used for suppressing the purposes of the medicine of mammalian tumor cell growth in preparation; Wherein, said mammalian tumor cell is breast cancer cell MCF7, HCC HepG2 or lung carcinoma cell H460.
9. purposes according to claim 8, wherein, described carrier is selected from plasmid, virus or phage.
10. purposes according to claim 9, wherein, described carrier is a virus.
11. purposes according to claim 10, wherein, described carrier is an adenovirus.
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Non-Patent Citations (3)
| Title |
|---|
| Xun Li.Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate.《CANCER SCIENCE》.2008,1326-1333. * |
| 丁丽华.FHL家族研究进展.《生物技术通讯》.2007,第18卷(第6期),全文. * |
| 丁丽华等.FHL家族的克隆及其融合蛋白的纯化.《生物技术通讯》.2006,第174页. * |
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