CN1248733C - Tie2 receptor-induced gene transfer system for treating target tumor genes - Google Patents
Tie2 receptor-induced gene transfer system for treating target tumor genes Download PDFInfo
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Abstract
The present invention provides a gene transfer system mediated by an angiogenin promotion receptor Tie2, which comprises a ligand oligopeptide (a) specifically combined with the receptor Tie2, a polycation polypeptide (b), an optional endocytosis corpuscle released oligopeptide (c) and exogenous a DNA (d). The gene transfer system can effectively lead exogenous gene in endothelial cells of new vessels of tumor in a targeting manner and the tumor cells for expressing the receptor Tie2 in a human body or in vitro so as to inhibit the growth of the tumor.
Description
Technical field
The present invention relates to molecular biology and field of gene.Particularly, relate to a kind of based on the gene transfer system of the bonded part oligopeptide of angiogenesis hormone receptor Tie2 GA1.This transfer system imports the tumor cell of endothelial cells in tumor neogenetic blood vessels and expression Tie2 receptor by receptor mediated endocytosis with foreign DNA targeting ground, thereby reaches the purpose of treatment tumor.
Background technology
PCT application PCT/CN97/00106 (WO98/18951) discloses the novel receptor-mediated gene transfer system that is used for the targeting therapy of tumor, wherein discloses to comprise at EGFR, IGF I/II R and three kinds of receptor-mediated gene transfer systems of VEGFR and be used for oncotherapy.Yet because the receptor of different tumor cell surfaces is varied, expression has to be had less more, even does not express some receptor, therefore only has three kinds of systems can not solve whole problems.
Davis in 1996 etc. have found a kind of new albumen-Angiopoietin-1 (Ang-1), and it is the part of Tie2 receptor in the protein tyrosine kinase receptor family, and the Tie2 receptor is almost only expressed on endotheliocyte.Ang-1 is structurally different with the part of known angiogenesis factor or other protein tyrosine kinase receptor, can not directly promote the endothelial cells cultured growth external, so infer that it may be irrelevant with tumor neovasculature startup, and work in the later stage of angiogenesis, make blood vessel network stable.
After finding Ang-1, utilize the homology method for screening, Maisonpierre has found Angiopoietin-2 (Ang2) again, this albumen also can with the Tie2 receptors bind.Can not activate Tie2 receptor on the endotheliocyte at external Ang2, but can activate other cell, as the Tie2 receptor on the NIH-3T3 cell, so, the function of Ang2 may be and the Ang1 antagonism, make vasoganglion be in a kind of unsure state, help the division of endotheliocyte and rebuliding of vasoganglion.
Receptor-the Tie2 of these two kinds of parts is found in 1992.This albumen is made up of 1124 amino acid residues, after part and receptors bind, makes receptor form dimer, and 897 tyrosine autophosphorylation also activates kinase activity.The expression of Tie2 has relative specificity, mainly is expressed in new vessels endotheliocyte, part tumor cell and some hematopoietic cell surfaces in human body.Can't survive after the birth of the mice of Tie2 gene knockout, pathologic finding finds, vascularization is arranged in the mice body but can not constitute complicated blood vessel network.
In kinds of tumors, as breast carcinoma, pulmonary carcinoma, bladder cancer etc., and in the new vessels in these tumors, Ang-1 and Tie2 receptor expression all increase, so utilize the signal conduction of blocking-up Tie2 receptor, can suppress the growth of tumor neogenetic blood vessels and tumor cell simultaneously, reach directly and suppress indirectly the purpose of growth of tumour cell.
Bibliographical information is arranged, utilize adenovirus transduction soluble T ie2 receptor extracellular region gene, can suppress mouse breast cancer 4T1 and melanin tumour b16 F10.9 cell in vivo, suppression ratio reaches 64% and 47% respectively, and has suppressed the transfer of tumor effectively.This studies show that the signal transduction that suppresses the Tie2 receptor has certain depression effect to growth of tumor.
In sum, this area presses for exploitation gene transfer system new, the targeting therapy of tumor, especially utilizes the receptor-mediated gene transfer system of Tie2.
Summary of the invention
The objective of the invention is just to provide the gene transfer system of the receptor-mediated targeting therapy of tumor of a kind of Tie2, it is that a kind of part oligopeptide and endocytosis corpusculum with the Tie2 receptors bind discharges the constructed gene transfer system of oligopeptide.
In a first aspect of the present invention, the gene transfer system of the targeting gene therapy of a kind of growth factor receptors Tie2 mediation is provided, it comprises (a) specificity and is incorporated into the part oligopeptide of Tie2 receptor and (b) polycation polypeptide.
Preferably, described gene transfer system also contains (c) endocytosis corpusculum release oligopeptide.
In another preference, described gene transfer system also contains (d) foreign DNA.
In another preference, described part oligopeptide contains following aminoacid sequence: WKEYK VGFGNPSGEY WLGN (SEQ ID NO:1).
In another preference, described polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide (PEI), protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, above-mentioned albumen of modifying through Polyethylene Glycol (PEG) and composition thereof.
In another preference, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.More preferably, described foreign DNA is selected from down group:
(i) antioncogene: p53, Rb;
(ii) apoptosis gene: Caspase, p15, p16 and p21
WAF-1
(iii) cytokine gene: GM-CSF (CM-CSF) gene, TNF (tumor necrosis factor) α gene, INF (interferon) α, γ gene, IL (interleukin) 2, IL3, IL4, IL12, IL15, IL18 gene;
The (iv) antisense sequences of oncogene: the antisense sequences of oncogene ras, c-myc, Akt;
(v) recombinant eukaryotic expression plasmid: viral recombinant eukaryotic expression plasmid (retrovirus recombinant expression carrier, adenovirus recombinant expression carrier, adeno-associated virus recombinant expression carrier) and be the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40, CMV promoter.
In another preference, described component (a) part oligopeptide GA1, (b) polycation polypeptide and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
In another preference, described endocytosis corpusculum discharges the oligopeptide element and is selected from down group: HA20 or VP-1.
In a second aspect of the present invention, a kind of oligopeptide is provided, it is incorporated into the Tie2 receptor, and contains following aminoacid sequence: WKEYK VGFGN PSGEY WLGN (SEQ ID NO:1).
In a third aspect of the present invention, a kind of method of gene transfer system of the targeting gene therapy for preparing growth factor receptors Tie2 mediation is provided, comprise step: the part oligopeptide, (b) polycation polypeptide and optional (c) endocytosis corpusculum release oligopeptide that (a) specificity are incorporated into the Tie2 receptor mix, and form mixture.Preferably, for component (a) and (b), (c), can use any the two formation binary complex.
Description of drawings
Fig. 1 is the X-gal coloration result that GA1 gene transfer system of the present invention imports β-gal gene Lu-csf-1 SPC-A1.
A.GA1 gene transfer system mediation β-gal gene enters SPC-A1 (* 400)
B.GA1 gene transfer system mediation β-gal gene enters SPC-A1 (* 100)
C. normal saline contrast
D. empty plasmid β-gal contrast
Fig. 2 is in testing in the body, GA1 gene transfer system transduction external source β-gal gene distribution situation in vivo.Wherein, the A. heart; B. liver; C. spleen; D. lung; E. kidney; F. brain; G. stomach; H. intestinal.
Fig. 3 is in testing in the body, and GA1 gene transfer system transduction external source β-gal gene is at the expression of different time (1,2,3,7 day) in the lotus Humanmachine tumour A375 of the subcutaneous institute of nude mice.Wherein, A: first day; B: second day; C: the 3rd day; D: the 4th day.
Fig. 4 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus human lung adenocarcinoma SPC-A1 of the subcutaneous institute of nude mice.
Fig. 5 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus people of the subcutaneous institute of nude mice hepatocarcinoma SMMC-7721.
Fig. 6 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus people of the subcutaneous institute of nude mice adenocarcinoma of stomach SGC-7901.
Fig. 7 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus human breast carcinoma BCaP-37 of the subcutaneous institute of nude mice.The part of amplifying among the figure is a little blood vessel.
Fig. 8 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus people of the subcutaneous institute of nude mice hepatocarcinoma transplanted tumor.
Fig. 9 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus people of the subcutaneous institute of nude mice hepatocarcinoma transplanted tumor.
Figure 10 is in testing in the body, the expression of GA1 gene transfer system transduction β-gal gene in the lotus human cervical carcinoma of the subcutaneous institute of nude mice transplanted tumor.
Figure 11 is the comparison of Humanmachine tumour A375 tumor bearing nude mice Subcutaneous tumor in the long-pending size of injection GA1 gene transfer system transduction p53 each group tumor block of three week of gene back.
The specific embodiment
The inventor is through extensive and deep research, prepared that specificity is incorporated into the part oligopeptide of Tie2 receptor and based on the gene transfer system of this part oligopeptide.Utilize the characteristic of part oligopeptide identification Tie2 receptor of the present invention, endocytosis through cell, foreign DNA is optionally imported the endothelial cells in tumor neogenetic blood vessels or the tumor cell of expressing the Tie2 receptor by this carrier system, thereby angiogenesis inhibiting and tumor cell reach the purpose for the treatment of tumor simultaneously.Finished the present invention on this basis.
The part oligopeptide
The part oligopeptide is the key component of targeting non-virus carrier of the present invention.As used herein, " part oligopeptide (Ligand oligopeptide, LOP) " and " receptor identification oligopeptide " is used interchangeably, and refers to any identification and is incorporated into the oligopeptide that the surface contains the target cell of Tie2 receptor.As mentioned above, but part oligopeptide specific recognition of the present invention and be incorporated into the Tie2 receptor.
The particularly preferred part oligopeptide of one class is that length is 15-30 aminoacid, and contains the oligopeptide of elementary cell sequence WKEYK VGFGN PSGEY WLGN (SEQ ID NO:1).More preferred example is oligopeptide GA1, and its aminoacid sequence is WKEYK VGFGN PSGEY WLGN (SEQ ID NO:1).The aminoacid sequence of GA1 is to draw according to the bioinformatic analysis to the aminoacid sequence of angiogenesis hormone-1 and 2.
In addition, part oligopeptide of the present invention comprises that also the aminoacid in one or several (1-8 usually, preferably 1-5) site can replace formed part oligopeptide with the aminoacid of similar performance.
Part oligopeptide of the present invention can contain single elementary cell sequence, or a plurality of (as 2-10) elementary cell sequence.
Part oligopeptide of the present invention can be easily with synthetic or genetic engineering recombinant methods.About these synthetic oligopeptide method for makings, can be referring to such as WO98/18951 many documents such as (PCT/CN97/00106).
The polycation polypeptide
The polycation polypeptide be targeting non-virus carrier of the present invention another key component.
As used herein, " polycation polypeptide " refers to that a class is rich in the polypeptide of basic amino acid, and its basic amino acid (lysine, histidine and arginine) ratio is about more than 20%, thereby positively charged, can combine with electronegative DNA mat electrostatic attraction, make DNA more stable.
Can be used for polycation polypeptide of the present invention and be not particularly limited, as long as its basic amino acid (lysine and arginine) ratio is about more than 20%, preferably 30%, more preferably more than 40%, thereby positively charged getting final product.Representational example comprises: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, above-mentioned albumen of modifying through Polyethylene Glycol (PEG) and composition thereof.
The endocytosis corpusculum discharges the oligopeptide element
Targeting non-virus carrier of the present invention also can contain the endocytosis corpusculum and discharge the oligopeptide element, swallows the effect that the DNA of cell is degraded in it plays and prevents.
Any endocytosis corpusculum discharges oligopeptide and all can be used for the present invention, and representational example comprises (but being not limited to): HA20, VP-1.
The targeting non-virus carrier
As used herein, " targeting non-virus carrier " of the present invention refers to contain following component (a) and (b) or contain component (a) and (b) and mixture (c) or complex: (a) the specificity part oligopeptide, (b) polycation polypeptide, (c) endocytosis corpusculum that are incorporated into the Tie2 receptor discharges oligopeptide.
In the present invention, component (a) and (b), (c) can be non-types of attachment, also can be any both or three link together by mode covalently or non-covalently.Particularly preferred form comprises: part oligopeptide-polycation polypeptide binary complex, polycation polypeptide-endocytosis corpusculum discharge the oligopeptide binary complex, part oligopeptide-polycation polypeptide-endocytosis corpusculum discharges the oligopeptide ternary complex.For example HA20-poly-D-lysine, HA20-protamine, the covalently bound thing of HA20-histone etc.
Connection can be passed through chemical method (as coupling agents such as use SPDP), also can pass through recombination method, for example forms fusion rotein.
For fusion rotein, at (a) part oligopeptide, (b) polycation polypeptide, and (c) the endocytosis corpusculum discharges between the oligopeptide element, can randomly contain connection peptides, so that increase the flexibility and the stretching of complex polypeptide space structure, allow each element performance function separately.Can be used for connection peptides of the present invention and be not particularly limited, needed only interconnect function.One class connection peptides example is (Gly)
2-6Ser, for example (Gly)
4Ser.Connection peptides can be used in a plurality of series connection.
(a) of the present invention part oligopeptide, (b) the polycation polypeptide and (c) the endocytosis corpusculum discharge the oligopeptide element can be with the amino acid replacement of similar performance, as long as still keep separately similarly biological function.
The preparation method of targeting non-virus carrier
Usually, above-mentioned (a) and (b), (c) component or its complex are mixed, can obtain targeting non-virus carrier of the present invention.Wherein, the mixed proportion of (a) and (b), (c) component is generally 0.5-1: 1-2: 0-1 preferably is 0.8-1: 1-1.5: 0.5-1.
In addition, when with (a)-(b) complex with (b)-(c) when the form of complex is used, (a)-(b) complex: (b)-(c) mixed proportion of complex is generally 0.8-1.2: 0.8-1.2 preferably is 0.9-1.1: 0.9: 1.1.
Foreign DNA
Can be used for foreign DNA of the present invention is not particularly limited, can be various therapeutic or preventative DNA, for example genes of interest, antisense oncogene, antioncogene, suicide gene, natural death of cerebral cells gene, cytokine gene or its combination perhaps contains the carrier for expression of eukaryon DNA of said gene.The proto-oncogene antisense sequences comprises proto-oncogene (ras
H, ras
K, ras
N, c-myc, bcl-2, Akt), antisense dna sequence, antisense oligonucleotide and the antisense oligodeoxyribonucleotide of somatomedin and acceptor gene thereof; Antioncogene comprises p53, Rb, PTEN, and suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (coli cytosine deaminase) gene; Apoptosis gene comprises p15, p16, p21
WAF-1Cytokine gene comprises GM-CSF (granulocyte macrophage colony stimulating factor) gene, TNF α (tumor necrosis factor) gene, INF (interferon) α, γ gene, IL (interleukin) 2,3,4,12,15,18 genes etc.
Target gene entraining system
Targeting non-virus carrier of the present invention is mixed with foreign DNA, just constitute the gene import system of the receptor-mediated targeting therapy of tumor of Tie2.Depend on used foreign DNA, gene import system of the present invention can be used for treating diseases such as genetic diseases or tumor, in particular for gene therapy.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains one or more targeting non-virus carrier of the present invention and pharmaceutically acceptable carrier or excipient of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.
When using, also contain one or more above-mentioned foreign DNAs in the pharmaceutical composition.
Concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Major advantage of the present invention is,
(1) the receptor-mediated gene therapy non-virus carrier of Tie2 of the present invention, the characteristics of tumor tissues medium vessels endotheliocyte and tumor cell surface Tie2 receptor high expressed have been utilized, can effectively the relative targeting of exogenous gene ground be imported the tumor cell of tumor neovasculature endotheliocyte and expression Tie2 receptor, reach the purpose that suppresses growth of tumour cell.
(2) native system might import tumor cell and endothelial cells in tumor neogenetic blood vessels simultaneously with the external source therapeutic genes, by suppressing neonate tumour blood vessel and directly suppressing tumor cell, reaches the double inhibition effect to tumor growth.
(3) this system also might be applied to other and blood vessel hyperplasia diseases associated in addition, as diabetic ophthalmic, rheumatic arthritis etc.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The structure of embodiment 1 non-virus carrier
A. the acquisition of each component
(1) part oligopeptide GA1's is synthetic:
Adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide GA1, and its aminoacid sequence is WKEYK VGFGN PSGEY WLGN (SEQ ID NO:1).
(2) poly-L-lysine (Poly-L-lysine is abbreviated as P.L.)
Available from U.S. SIGMA company, molecular weight distribution is 15000-30000 dalton.
(3)HA20
Adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide HA20, and its aminoacid sequence is GLFEA IAEFI EGGWE ELIEG (SEQ IDNO:2).
B. the preparation of binary complex
At first, GA1, HA20 and poly-L-lysine (Poly-L-lysine is abbreviated as P.L.) are dissolved among the PSB (0.1M sodium phosphate buffer, pH7.4 contain 0.1M NaCl), then, follow these steps to react.
(1)P.L.+SPDP→P.L.-PDP
Annotate: SPDP is N-butanimide-3-(2-pyridine dithio)-propionic ester.
P.L. the mol ratio with SPDP (N-) is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Reaction finishes the back by the unreacted SPDP of dialysis removal.The P.L.-PDP lyophilization.
(2) P.L.-PDP dry powder is dissolved among the PSB
P.L.-PDP+DTT→P.L.-SH
Annotate: DTT is a dithiothreitol, DTT.
DTT is excessive in the reaction, and the response time is 1 hour, and temperature is 25 ℃.Reaction finishes the back by the unreacted DTT of dialysis removal.The product lyophilization.
(3)GAl+SPDP→GAl-PDP
HA20+SPDP→HA20-PDP
The mol ratio of GA1, HA20 and SPDP is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Unreacted SPDP is removed in bag filter dialysis by MWCO1000 after reaction finishes.The product lyophilization.
(4) GA1-PDP, HA20-PDP and P.L.-SH dry powder all are dissolved among the PSB.
GA1-PDP+P.L.-SH→P.L.-GA1
HA20-PDP+P.L.-SH→P.L.-HA20
The mol ratio of GA1-PDP, HA20-PDP and P.L.-SH is 3: 1, and the response time is 24 hours, and temperature is 25 ℃.Reaction finishes the back by dialysis unreacted GA1-PDP of removal and HA20-PDP.
C. the preparation of targeting non-virus carrier
Mix P.L.-GA1 and P.L.-HA20 in 1: 1,0.8: 1.2,1.2: 0.8 with mass ratio respectively, promptly get required non-virus carrier.
Perhaps, GA1, polylysine and HA20 are mixed, form required non-virus carrier by mass ratio 1: 1.5: 1.
The extracting and the purification of embodiment 2 plasmid DNA
The isolation and purification of used recombinant eukaryon expression vector plasmid DNA all utilizes the Maxi plasmid kit of QIAGEN company and the pure product of DNA that obtain in the present embodiment.
Select monoclonal from the LB flat board and be inoculated in the 5mlLB culture medium that contains ampicillin, 37 ℃, the 250rpm jolting is spent the night.Transfer at 1: 1000 in the 200ml LB culture medium that contains ampicillin, 37 ℃, 250rpm jolting 16 hours.Shift bacterium liquid in the 500ml centrifuge tube, 4,000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, collect thalline.Precipitation is resuspended in 10ml Buffer P1 (50mM Tris.Cl, pH8.0,10mM EDTA pH8.0,100 μ g/ml Rnase A).(200mM NaOH puts upside down 4-6 time after 1%SDS) gently, and mixing left standstill under the room temperature 5 minutes to add 10ml Buffer P2.Add 10ml Buffer P3 (3.0M NaAc, pH5.5), put upside down mixing after, be transferred to the 50ml centrifuge tube, 10,000rpm, 4 ℃ centrifugal 30 minutes, get supernatant.Supernatant is aseptically filled in the QIAGEN Maxi plasmid extraction post through four layers and (uses 10ml Buffer QBT balance in advance), allow under its spontaneous current, enter in the post fully Deng supernatant, add Buffer QC 30ml washing secondary again, add BufferQF 15ml at last, collect effluent, add 0.7 times of volume isopropyl alcohol, 10,000rpm, 4 ℃ centrifugal 30 minutes, abandon supernatant, 75% washing with alcohol precipitation 1 time, natural drying under the room temperature is dissolved among the TE (pH 8.0) of an amount of volume the quality of the firm DNA of 1% agarose gel electrophoresis, its purity of UV spectrophotometer measuring is also quantitative ,-20 ℃ of preservations.
The structure of embodiment 3 quaternary complex gene import systems
1) after .GA1-P.L. and HA20-P.L. were dissolved in water, through 0.22 μ m filter filtration sterilization ,-20 ℃ stored for future use.All plasmids extract and purification through the QIAGEN post, the ethanol of 2 times of volumes of reuse and the 3M NaAc of 1/3 volume precipitation, and after 75% ethanol is washed once, air drying.With an amount of MilliQ water dissolution, make concentration become 0.2 μ g/ μ l.
2). the optimum quality ratio when non-virus carrier and DNA form complex between the two.
0.2 μ g CMV-β-gal plasmid DNA was mixed with mass ratio with non-virus carrier in 1: 0.5,1: 1,1: 1.5,1: 2,25 ℃ left standstill 30 minutes, with the retardance situation of 1% agarose gel electrophoresis identification of dna, determined optimum quality ratio.
3). prepare quaternary complex gene transfer system by above-mentioned definite proportioning.
Embodiment 4GA1 gene import system transduction CMV-β-gal gene in vitro imports experiment
1). cell culture
The SPC-A1 passage is cultivated: the SPC-A1 cell with 0.25% trypsinization after, be inoculated in the Tissue Culture Flask, adding contains the DMEM complete culture solution of 10% calf serum, at 37 ℃, 5%CO
2Cultivate in the incubator.
2). external importing experiment
Cultured cell with 0.25% trypsinization after, be inoculated in six orifice plates every hole 1.5 * 10
5, the cell full scale was about 60% o'clock in second day, added the quaternary complex gene transfer system that is equivalent to 2 μ g DNA in the 1ml culture fluid.After the transfection 24 hours, renew the bright DMEM complete culture solution that contains 10% calf serum, continue to cultivate 24 hours.Cell is washed 2 times with PBS then, add freshly prepared fixative and fix 10 minutes in room temperature, reuse PBS rinsing 3 times, each 15 minutes, with thorough removal fixative, blot PBS on tumor piece and the internal organs with filter paper, add freshly prepared dyeing liquor in 37 ℃ of insulations after 24 hours, the situation that the observation exogenous gene imports (form: 1mg/ml X-gal, 5mM K by dyeing liquor
4[Fe (CN)
6], 5mMK
3[Fe (CN)
6], 2mM MgCl
2).
The result as shown in Figure 1.As can be seen from Figure, the cell of transduction β-gal gene is blue, the matched group that adds normal saline and the simple plasmid respectively importing of source gene of then not regarding sb. as an outsider.
Embodiment 5GA1 gene import system transduction pCMV-β-gal gene distribution in vivo
Get Humanmachine tumour A375 tumor bearing nude mice, wrapped up the GA1 gene import system 100 μ l of 0.2 μ g DNA in the injection of subcutaneous tumors week.Sacrificed nude mice after the 2nd day, the internal organs such as the heart, liver, spleen, lung, kidney, brain, stomach, intestinal of getting nude mice dye with said method and carry out frozen section and redye.
The results are shown in shown in Figure 2.The result shows that the heart, liver, lung, kidney, brain there is no indigo plant and dye, and has exogenous gene to import in spleen.And in stomach, intestinal mucosa, though also there is indigo plant to dye, we dye to nude mice stomach, the intestinal of not doing any processing by laboratory, and glandular epithelium has found that also indigo plant dyes, and illustrates to have nonspecific galactosidase activity in these tissues.
Embodiment 6GA1 gene import system transduction pCMV-β-gal gene is at the expression of different time
Get Humanmachine tumour A375 tumor bearing nude mice, the GA1 gene import system 100 μ l of 0.2 μ g DNA have been wrapped up in the injection of subcutaneous tumors week, the the 1st, 2,3 and 7 day sacrifice nude mice after injection, take out Subcutaneous tumor, carry out tissue staining and section by method 6, observed result and taking pictures under optical microscope.
The results are shown in Figure 3.The result showed that the gene that GA1 gene import system imports begins promptly that at the 1st day expression is arranged, and expression was maximum in the 2nd day, begins later on to reduce, and still had a certain amount of expression to the 7th day.
Import experiment in embodiment 7 bodies
Get nude mice (the SPC-A1 adenocarcinoma of lung of the different tumors of lotus, SMMC-7721 hepatocarcinoma, the SK-OV-3 ovarian cancer, the SGC stomach cancer cell line, the A375 Humanmachine tumour, the BCaP-37 human breast carcinoma, lotus human cervical carcinoma transplanted tumor nude mice), the GA1 gene import system 100 μ l that are equivalent to 0.2 μ g β-gal DNA in the injection of subcutaneous tumors week, sacrificed nude mice in back 48 hours in injection, tumor under the bark fetching, with PBS rinsing 2 times, add freshly prepared fixative and fix 20 minutes in room temperature, reuse PBS rinsing 3 times, each 15 minutes, with thorough removal fixative, blot PBS on tumor piece and the internal organs with filter paper, (dyeing liquor is formed: 1mg/ml X-gal, 5mM K to add freshly prepared dyeing liquor
4[Fe (CN)
6], 5mMK
3[Fe (CN)
6], 2mM MgCl
2), after 24 hours, observe the situation that the tumor piece imports exogenous gene in 37 ℃ of insulations, carry out paraffin section after 48 hours, with the nuclear fast red pathological section is redyed then, observe exogenous gene and import situation (whether having blue spot to produce).
The result is shown in Fig. 4-10.The expression of reporter gene is arranged in the part blood vessel in tumor peplos and tumor as can be seen.Owing to also have the Tie2 receptor expression in the part surface of tumor cells, so in tumor cell, also can be observed expression of exogenous gene.
This shows, gene transfer system of the present invention, and mediate foreign gene targeting ground imports part blood vessel and the tumor cell in tumor peplos and the tumor effectively.
Effectively experiment in embodiment 8 bodies
1) preparation of tumor bearing nude mice
With the strain of A375 human melanoma cell be inoculated in 5 the week age nude mice subcutaneous, treat that the tumor diameter grows to 0.5cm after, as the tumor source.Sacrifice nude mice, take out the tumor piece and cut into the about 1mm of size
3Fragment, with puncture needle be inoculated into 6 the week age nude mice subcutaneous.
After one week, treat that tumor is long when diameter is about 0.3-0.4cm, pick out the uniform animal of tumor body, be divided into 5 groups at random, 6 every group standby.
2) effective experiment of GA1 system mediation pCEP-p53 gene
Experiment divides five groups, is respectively:
1. normal saline group,
2. non-virus carrier group (1 μ g GA1-PL),
3. simple plasmid pCEP-p53 organizes (1 μ g pCEP-p53 plasmid),
4. low dose group (every injection is equivalent to the quaternary complex gene transfer system of 0.2 μ g plasmid),
5. high dose group (every injection is equivalent to the quaternary complex gene transfer system of 1 μ g plasmid).
Injection once continues 1 month weekly.Use the length (a) of vernier caliper measurement nude mice subcutaneous tumors body, short (b) weekly directly, according to V=π ab
2/ 6 calculate the tumor volume.
Experimental result such as table 1, shown in Figure 11.The result shows that the meansigma methods of five groups is respectively: 1. 1.498 ± 0.600cm
32. 1.734 ± 0.811cm
33. 1.727 ± 0.526cm
34. 1.169 ± 0.381cm
35. 0.731 ± 0.328cm
3High dose group with 1., 2., 3. the group compare, suppression ratio is respectively 51.2%, 57.8%, 57.7%, and with the equal significance of the difference of three matched groups (P＜0.05), and low dose group only with the difference significance (P＜0.05) of simple plasmid group.This shows, the present invention is based on the gene transfer system of the part oligopeptide of Tie2 receptor, can mediate the growth that the pCEP-p53 gene enters cell and effectively suppresses Humanmachine tumour A375 effectively.
The long-pending comparison of tumor block in effectively testing in table 1 body
| 1. normal saline | 2. empty carrier | 3. simple plasmid | 4. high dose | 5. low dosage | |
| 0.849 0.998 1.094 1.735 2.0036 2.306 | 0.697 0.934 1.752 2.037 2.081 2.901 | 1.218 1.277 1.286 1.979 2.353 2.248 | 0.315 0.421 0.558 0.719 1.120 1.255 | 0.928 0.941 1.000 1.115 1.232 1.795 | |
| Mean+SD | 1.498± 0.600 | 1.734± 0.811 | 1.727± 0.526 | 0.731± 0.381 | 1.169± 0.328 |
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Tumor
＜120〉gene transfer system of the receptor-mediated targeting therapy of tumor of Tie2
<130>025446
<160>2
<170>PatentIn version 3.1
<210>1
<211>19
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(19)
＜223〉be incorporated into the part oligopeptide of Tie2 receptor
<400>1
Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu Tyr Trp
1 5 10 15
Leu Gly Asn
<210>2
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(20)
＜223〉the endocytosis corpusculum discharges oligopeptide HA20
<400>2
Gly Leu Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Glu
1 5 10 15
Leu Ile Glu Gly
20
Claims (10)
1. the gene transfer system of the targeting gene therapy of growth factor receptors Tie2 mediation is characterized in that, it comprises (a) specificity and is incorporated into the part oligopeptide of Tie2 receptor and (b) polycation polypeptide.
2. gene transfer system as claimed in claim 1 is characterized in that, it also contains (c) endocytosis corpusculum and discharges oligopeptide.
3. gene transfer system as claimed in claim 1 or 2 is characterized in that, it also contains (d) foreign DNA.
4. gene transfer system as claimed in claim 1 is characterized in that, described part oligopeptide contains following aminoacid sequence: WKEYK VGFGN PSGEY WLGN.
5. gene transfer system as claimed in claim 1, it is characterized in that, the polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, and through polyethyleneglycol modified above-mentioned albumen and composition thereof.
6. gene transfer system as claimed in claim 3 is characterized in that, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.
7. gene transfer system as claimed in claim 3 is characterized in that, described foreign DNA is selected from down group:
(i) antioncogene: p53, Rb;
(ii) apoptosis gene: Caspase, p15, p16 and p21WAF-1;
(iii) cytokine gene: GM-CSF gene, TNF α gene, INF α, γ gene, IL2, IL3, IL4, IL12, IL15, IL18 gene;
The (iv) antisense sequences of oncogene: the antisense sequences of oncogene ras, c-myc, Akt;
(v) recombinant eukaryotic expression plasmid: viral recombinant eukaryotic expression plasmid and be the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40, CMV promoter.
8. gene transfer system as claimed in claim 1, it is characterized in that, described gene transfer system also contains (c) endocytosis corpusculum and discharges oligopeptide, and described component (a) specificity be incorporated into the part oligopeptide, (b) polycation polypeptide of Tie2 receptor and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
9. gene transfer system as claimed in claim 2 is characterized in that, described endocytosis corpusculum discharges the oligopeptide element and is selected from down group: HA20 or VP-1.
10. an oligopeptide is characterized in that, it is incorporated into the Tie2 receptor, and contains following aminoacid sequence: WKEYK VGFGN PSGEY WLGN.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02150902 CN1248733C (en) | 2002-12-02 | 2002-12-02 | Tie2 receptor-induced gene transfer system for treating target tumor genes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02150902 CN1248733C (en) | 2002-12-02 | 2002-12-02 | Tie2 receptor-induced gene transfer system for treating target tumor genes |
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| Publication Number | Publication Date |
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| CN1504235A CN1504235A (en) | 2004-06-16 |
| CN1248733C true CN1248733C (en) | 2006-04-05 |
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| CN 02150902 Expired - Fee Related CN1248733C (en) | 2002-12-02 | 2002-12-02 | Tie2 receptor-induced gene transfer system for treating target tumor genes |
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| CN107033249B (en) * | 2017-05-16 | 2020-07-14 | 成都开乐药业有限公司 | sTie2 fusion protein, carrier thereof and pharmaceutical composition containing sTie2 fusion protein |
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| Publication number | Publication date |
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