CN104098652A - Polypeptide and polypeptide compound for inhibiting tumor metastasis, as well as preparation methods and application of polypeptide and polypeptide compound - Google Patents
Polypeptide and polypeptide compound for inhibiting tumor metastasis, as well as preparation methods and application of polypeptide and polypeptide compound Download PDFInfo
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Abstract
The invention relates to a polypeptide-human serum albumin (HSA) compound and application thereof in the aspect of tumor metastasis inhibition, in particular to a method for improving the solubility of the polypeptide in a salt solution with HSA, and further provides a preparation method of the polypeptide-HSA compound and application of the polypeptide-HSA compound in tumor metastasis inhibition. The polypeptide-HSA compound is better to dissolve in the salt solution, and can greatly reduce the mobility of tumor cells. The polypeptide sequence and the polypeptide-HSA compound can provide a feasible curing method for tumor metastasis inhibition.
Description
Technical field
The present invention relates to biological medicine technology field, be specifically related to a peptide species and polypeptide complex, its preparation method and application thereof, especially the preparation method and the application aspect inhibition metastases thereof that relate to a peptide species and can improve polypeptide deliquescent polypeptide-human's serum albumin (human serum albumin, HSA) mixture in salts solution.
Background technology
Serum albumin is rich in protein the most in blood plasma, and its biological function comprises in conjunction with and transports a series of endogenous and ectogenic material, maintains normal osmotic pressure of blood etc.In recent years, human serum albumin (HSA) has been subject to paying close attention to widely as pharmaceutical carrier.HSA is comprised of 585 amino-acid residues, and molecular weight is about 66kD, has the advantages such as safety non-toxic, good biocompatibility.
Albuminous medicine carrying mode mainly contains two kinds: the one, between medicine and albumin, with molecular chain, be connected to form albumin medicine, i.e. chemical coupling albumin medicine carrying; The 2nd, rely on medicine and albuminous interaction to realize embedding medicine carrying, i.e. physical bond albumin medicine carrying, this combination can improve solvability and the stability of medicine.
At present the HSA pharmaceutical carrier of research is mainly in antitumor drug, the physically trapping effect of HSA medicine is physical action by HSA such as interaction embedding medicine carryings such as hydrophobic, static, can improve stability and the targeting of medicine, and there is slow releasing function, so HSA can be used as desirable pharmaceutical carrier for polypeptide drugs novel complexes.
Tumour is the healthy class diseases of serious harm people, and tumour is divided into innocent tumour and malignant tumour, and both key distinctions are the features that malignant tumour has transfer (metastasis) and recurrence.Metastases is the sign of prognosis mala, is also to cause tumour patient main causes of death.
Chemokine (chemokines) is a class strand small protein, by the interaction with g protein coupled receptor, causes the rearrangement of target cell support, attaches to securely endotheliocyte and directional migration.In recent years, research both at home and abroad is all pointed out, stroma cell derivative factor (stromal cell-derived factor-1, SDF-1), namely Chemokine CXCL12, and specificity Chemokine Receptors CXCR4 plays an important role in the organ specificity of kinds of tumors shifts.The Chemokine Receptors CXCR4 that tumor cell surface high expression level is special, and Chemokine CXCL12 high expression level in marrow, lymphoglandula and some organ.
The tumour cell of high expression level CXCR4 is under the chemotactic of CXCL12, and contrary concentration gradient is transferred to some organ that produces source as chemokine, such as lung, liver etc., form organ transspecific (Balkwill F., Semin Immunol, 2003,15,49-55).Therefore, use CXCR4 antagonist target inhibition CXCL12/CXCL4 interaction can block tumour cell and stroma cell sticks, and increases the susceptibility of tumour cell to chemotherapeutics, prevents transfer and the recurrence of tumour cell.
Because polypeptide is easy to synthesize, in human body, be easy to metabolism and can not bring toxic side effect and serious immune response, therefore for inhibition tumor cell, shift, the polypeptide that develops selectively targeted CXCR4 acceptor has very important significance.
Summary of the invention
The object of the present invention is to provide a peptide species and polypeptide complex, its preparation method and application, particularly the poor polypeptide of a kind of good water solubility, salt dissolubility and can improve polypeptide deliquescent polypeptide-HSA mixture, its preparation method and the application aspect inhibition tumor cell transfer thereof in salts solution.
For reaching this goal of the invention, the present invention by the following technical solutions:
First aspect, the invention provides a kind of polypeptide can inhibition tumor cell shifting, and described polypeptide has and is selected from the aminoacid sequence of SEQ ID NO:1～7 shown in any one, as shown in table 1.
Polypeptide of the present invention can be synthesized and be made by artificial chemistry, has good water solubility, the poor feature of salt dissolubility.
As optimal technical scheme, described polypeptide is the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:3, and the name in corresponding table 1 is called E4 and E5.
Second aspect, the present invention also provides a kind of polypeptide deliquescent method in salts solution improving described in the invention described above, and described method comprises: described polypeptide is combined with human serum albumin.
Preferably, the combination of described polypeptide and human serum albumin is physical bond.
As optimal technical scheme, said method comprising the steps of:
1) obtain solution: described peptide molecule is mixed with to 1-5mg/mL solution, human serum albumin molecule is mixed with to 125-250mg/mL solution;
2) mix: human serum albumin molecular solution is joined in peptide molecule solution, fully mix.
Preferably, in step 1) in, the solvent of described polypeptide solution and human serum albumin is aseptic ultrapure water.
Preferably, in step 2) in, in described solution, the mol ratio of peptide molecule and human serum albumin molecule is 4:1-1:4, for example can 4:1,4:2,4:3,1:1,3:4,2:4,1:4, be preferably 1:1.
The third aspect, the present invention also provides a kind of polypeptide-human's serum albumin mixture, and this mixture comprises polypeptide and the human serum albumin described in the invention described above.
Preferably, described polypeptide combines by physical action with human serum albumin.
Preferably, the mol ratio of described polypeptide and human serum albumin is 4:1-1:4, for example can 4:1,4:2,4:3,1:1,3:4,2:4,1:4, be preferably 1:1.
Fourth aspect, the present invention also provides the application in the medicine shifting for the preparation of inhibition tumor cell of polypeptide-human's serum albumin mixture described in the invention described above.
As optimal technical scheme, described polypeptide-human's serum albumin mixture is for lateral transfer and/or the vertical migration of inhibition tumor cell.
The 5th aspect, the present invention also provides the application in the medicine for the preparation for the treatment of people metastases relative disease of polypeptide-human's serum albumin mixture described in the invention described above.
As optimal technical scheme, described metastases relative disease is the relevant tumour of Chemokine Receptors CXCR4 high expression level;
Preferably, the relevant tumour of described Chemokine Receptors CXCR4 high expression level is any one in mammary cancer, leukemia, lymphoma or bladder cancer.
Preferably, the relevant tumour of described Chemokine Receptors CXCR4 high expression level is mammary cancer, and wherein, SK-BR-3 and MDA-MB-231 are as breast cancer cell line; SK-BR-3 clone is hatched substratum containing 5%FBS; MDA-MB-231 clone is hatched substratum not containing FBS.
Compared with prior art, beneficial effect of the present invention is:
Polypeptide-HSA mixture of the present invention has the polypeptide of raising deliquescent ability in salts solution, the particle diameter of peptide molecule of the present invention 0h in PBS solution is all more than 300nm, and polypeptide-HSA molecule has obtained good dispersion in PBS solution, the particle diameter of 0h is all in 100nm left and right, and particle diameter does not have to occur very large change in 72h.Resulting polypeptide-HSA mixture has the effect that suppresses metastases simultaneously, and this peptide sequence and polypeptide-HSA mixture can provide for the inhibition of metastases feasible methods for the treatment of.
Accompanying drawing explanation
Fig. 1 a-1g is peptide molecule and polypeptide-HSA molecule dynamic light scattering experiment result figure in PBS solution
Fig. 2 is E4-HSA, the experimental result picture of E5-HSA molecule on the impact of SK-BR-3 cell viability
Fig. 3 is E4-HSA, the experimental result picture of E5-HSA molecule on the impact of MDA-MB-231 cell viability
Fig. 4 a-4b is E4-HSA molecule, the restraining effect result figure of E5-HSA molecule to the lateral transfer of CXCL12 molecule induction SK-BR-3 cell
Fig. 5 a-5d is E4-HSA molecule, the restraining effect result figure of E5-HSA molecule to CXCL12 molecule induction SK-BR-3 cell vertical migration
Fig. 6 a-6b is E4-HSA molecule, the restraining effect result figure of E5-HSA molecule to the lateral transfer of CXCL12 molecule induction MDA-MB-231 cell
Fig. 7 a-7d is E4-HSA molecule, the restraining effect result figure of E5-HSA molecule to CXCL12 molecule induction MDA-MB-231 cell vertical migration
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition, or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, is and can passes through the commercially available conventional products of regular channel.
Unless specialized, breast cancer cell line SK-BR-3, MDA-MB-231 used in following examples are all purchased from the Chinese Academy of Medical Sciences.
Unless specialized, in following examples, the solvent of the aqueous solution used is aseptic ultrapure water solution.
Unless specialized, reagent used in following examples is analytical reagent.
Unless specialized, PBS solution used in following examples is 1 * PBS solution.
Embodiment 1: polypeptide synthetic
According to the synthetic polypeptide of the sequence shown in table 1 (synthetic by Ke Tai bio tech ltd, Shanghai, purity is 98%), before experiment, be mixed with the mother liquor of suitable concn.
Wherein, the aminoacid sequence of N8, E4, E5, Pep11, Pep12, Pep15, Pep16 polypeptide corresponds respectively to SEQ ID NO:1～7 in summary of the invention.
The effect of vitamin H (Biotin) is and Streptavidin (SA) combination, belongs to conventional technique means, and the effect of polypeptide of the present invention is not had to substantial effect.
Table 1
| Title | Sequence |
| N8 | NNNNNNNN |
| E4 | Biotin-GGRSFILLRIIQGCRRRNTVDD |
| E5 | Biotin-GGRSFFLLRRIQGCRFRNTVDD |
| Pep11 | SRVILRNGDLSRGVTISYDSYCDN |
| Pep12 | GRRVQFRISITSNDCPDDRSRSFDIDG |
| Pep15 | YCDDRNSSRICSNNGRCVCGQCVCDDDRN |
| Pep16 | QCVCDDDRNTNRIYSGDFCRCRNFNCRDS |
Embodiment 2: the gathering of peptide molecule in PBS solution and the solvability of polypeptide-HSA molecule
The preparation of 10 * PBS solution: NaCl80.00g, KCl2g, Na
2hPO
412H
2o35.8g or Na
2hPO
414.2g, KH
2pO
42.7g, is settled to 1000mL with ultrapure water, and regulating its pH value is 7.2～7.4, autoclaving.The preparation of 1 * PBS solution: by 10 times of aseptic ultrapure water dilutions for 10 * PBS solution.
Peptide molecule is mixed with to 1mg/mL solution with aseptic ultrapure water, HSA molecule is mixed with to 125mg/mL solution with aseptic ultrapure water, get a certain amount of HSA aqueous solution and add in the peptide molecule aqueous solution, making the mol ratio of peptide molecule and HSA molecule in solution is 1:1.After solution fully mixes, add 10 * PBS solution of solution 1/9 volume, making it dilution is 1 * PBS solution of polypeptide-HSA molecule.The solution preparing is placed in to 4 ℃ of refrigerators.With only containing 1 * PBS solution of peptide molecule in contrast.
At 0h, after being shaken up, the test soln preparing gets 1mL, with the liquid-transfering gun of 1mL, shifted in 1cm * 1cm plastic sample pond of standard configuration is used in test, carry out dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, Britain) test.24h, carries out DLS test after solution is shaken up.Test solution is placed in to whizzer (TGL-16B, Anke, China) with the centrifugal 3min of speed of 5000rpm, get supernatant liquor and be placed in centrifuge tube, after solution in centrifuge tube is shaken up, carry out DLS test.After test, solution is placed in to 4 ℃ of refrigerators.48h, carries out DLS test after solution is shaken up.Test and solution centrifugal is got to supernatant liquor be placed in centrifuge tube, after solution in centrifuge tube is shaken up, carry out DLS test.After test, solution is placed in to 4 ℃ of refrigerators.72h, carries out DLS test after solution is shaken up.Test and solution centrifugal is got to supernatant liquor be placed in centrifuge tube, after solution in centrifuge tube is shaken up, carry out DLS test.
The particle diameter that dynamic light scattering has reflected molecules in solution over time, as shown in Fig. 1 a～1g, the particle diameter of peptide molecule 0h in PBS solution is all more than 300nm, and in every 24h, the particle diameter of polypeptide in PBS solution has obvious increase, centrifugal rear particle diameter can significantly reduce, and illustrates that polypeptide has the trend of gathering in PBS solution.And polypeptide-HSA molecule has obtained good dispersion in PBS solution, and in 72h, particle diameter does not have very large change occurs, and after 72h, tends towards stability.Illustrate that HSA molecule can increase the solvability of polypeptide in salts solution significantly.
Embodiment 3:E4-HSA molecule, the test experience of E5-HSA molecule to SK-BR-3 cell proliferation-toxicity
Using SK-BR-3 as the model system of studying breast cancer cell line.In Corning96 orifice plate, every hole is used 100 μ L RPMI-1640 substratum (containing 10% foetal calf serum FBS and 1% mycillin) to cultivate 1 * 10
4individual cell, by 96 orifice plates at 37 ℃, 5%CO
2preculture 24h in the incubator of condition, to the PBS solution that adds 10 μ L different concns polypeptide-HSA molecules in culture plate, the ultimate density that makes polypeptide is 10nM, 100nM, 200nM, 400nM, 1 μ M, 2 μ M, 4 μ M.Blank is for only adding 10 μ L PBS solution.Culture plate is hatched in incubator to 48h, to every hole, add 10 μ L CCK solution (Beijing Fanbo Biochemicals Co., Ltd.).Culture plate is hatched in incubator to 2h, by the multi-functional microplate reader of continuous spectrum (ecan infinite M200, TECAN, Switzerland), be determined at the absorbance (OD value) under 450nm wavelength, calculate cell survival rate (cell survival rate=OD
450nm(polypeptide-HSA molecule)/(OD
450nm(blank) * 100%).
In preliminary experiment, in SK-BR-3 cell, add the cell survival rate of the HSA molecule of 4 μ M to compare with independent SK-BR-3 cell, there is no significant difference.As shown in Figure 2, add polypeptide-HSA molecule of 10nM～4 μ M in SK-BR-3 cell, its cell survival rate is compared with independent SK-BR-3 cell, there is no significant difference.In this concentration range, the polypeptide-HSA molecule adding neither can promote the propagation of cell, also can not affect cell survival rate.
Embodiment 4:E4-HSA molecule, the test experience of E5-HSA molecule to MDA-MB-231 cell proliferation-toxicity
Using MDA-MB-231 as the model system of studying breast cancer cell line.In Corning96 orifice plate, every hole is used 100 μ L DMEM in high glucose substratum (containing 10% foetal calf serum FBS and 1% mycillin) to cultivate 1 * 10
4individual cell, by 96 orifice plates at 37 ℃, 5%CO
2preculture 24h in the incubator of condition, to the PBS solution that adds 10 μ L different concns polypeptide-HSA molecules in culture plate, the ultimate density that makes polypeptide is 10nM, 100nM, 400nM, 1 μ M, 4 μ M.Blank is for only adding 10 μ L PBS solution.Culture plate is hatched in incubator to 48h, to every hole, add 10 μ L CCK solution.Culture plate is hatched in incubator to 2h, by the multi-functional microplate reader of continuous spectrum, be determined at the OD value under 450nm wavelength, calculate cell survival rate.
In preliminary experiment, in MDA-MB-231 cell, add the cell survival rate of the HSA molecule of 4 μ M to compare with independent MDA-MB-231 cell, there is no significant difference.As shown in Figure 3, add polypeptide-HSA molecule of 10nM～4 μ M in MDA-MB-231 cell, its cell survival rate is compared with independent MDA-MB-231 cell, there is no significant difference.In this concentration range, the polypeptide-HSA molecule adding neither can promote the propagation of cell, also can not affect cell survival rate.
Embodiment 5:E4-HSA molecule, the restraining effect of E5-HSA molecule to the lateral transfer of CXCL12 induction SK-BR-3 cell
In Corning six orifice plates, every hole is used 2mL RPMI-1640 substratum (containing 10% foetal calf serum FBS and 1% mycillin) to cultivate 40 * 10
4individual SK-BR-3 cell, by six orifice plates at 37 ℃, 5%CO
2preculture 24h in the incubator of condition, SK-BR-3 Growth of Cells is when closing on 90% fusion, with the aseptic rifle head of 10 μ L in the hole of culture plate along straight line cut, then use PBS solution by the soft washing of cell 3 times, rear every hole adds the RPMI-1640 substratum containing 5%FBS, add the induction of the CXCL12 aqueous solution simultaneously, the CXCL12 molecular conecentration that makes every hole is 100ng/mL, and add the PBS solution of polypeptide-HSA molecule, the ultimate density that makes peptide molecule is 10nM, 100nM, 1000nM, and blank adds PBS solution.Microscope (IX71, OLYMPUS, Japan) the observation of cell situation of movement of 10 * camera lens and the variation of cut width L for 0h after cut, 24h, calculate cell migration rate (cell migration rate=polypeptide-HSA molecular cell mobility (L
0h-L
24h/ L
0h)/blank cell migration rate (L
0h-L
24h/ L
0h) * 100%).
In preliminary experiment, added the SK-BR-3 cell of the HSA molecule of 1000nM to compare and there is no significant difference with the mobility of blank.As shown in Fig. 4 a, 4b, after having added the E4-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability of SK-BR-3 cell has been reduced by 31.38%, 54.39%, 47.70% successively, after having added the E5-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability of SK-BR-3 cell has been reduced by 44.25%, 55.75%, 47.7% successively, i.e. polypeptide-HSA molecule lateral transfer effect that inhibition tumor cell is induced by Chemokine CXCL12 effectively.
Embodiment 6:E4-HSA molecule, the restraining effect of E5-HSA molecule to CXCL12 induction SK-BR-3 cell vertical migration
The SK-BR-3 cell of results logarithmic phase, the RPMI-1640 substratum suspension cell with containing 5%FBS, contains 15 * 10 by 200 μ L
4the PBS solution of the suspension of individual cell and different concns E4-HSA molecule adds the upper chamber of transwell cell (diameter is the PET millipore filtration of 8 μ m), the ultimate density that makes peptide molecule is 10nM, 100nM, 1000nM, and blank adds PBS solution.The RPMI-1640 substratum that lower chamber (Corning24 orifice plate) adds 800 μ L to contain 5%FBS contains the CXCL12 aqueous solution (100ng/mL) induction simultaneously.Culture plate is at 37 ℃, 5%CO
2after cultivating 24h in the incubator of condition, take out transwell cell, with cotton swab, carefully wipe the not migrating cell of chamber face on filter membrane, with crystal violet solution, (A liquid is that 2g Viola crystallina is dissolved in 20mL95% alcohol, B liquid is that 0.8g ammonium oxalate is dissolved in 80mL distilled water, after A, B liquid hybrid filtering, use) fixedly move to the cell of cell film lower surface the 20min that dyes, after rinsing, observes in clear water the cell of migration with 10 * microscope (DMI3000B, LEICA, Germany).Each cell film is got 5 visuals field according to the orientation up and down, counts the cell count N of migration in each visual field, calculates cell migration rate (cell migration rate=N after averaging
polypeptide-HSA molecule/ N
blank* 100%).Indirectly, 800 μ L33% acetic acid solution wash-out Viola crystallinas for the cell film after dyeing, get 100 μ L elutriants and are determined at the OD value under 570nm wavelength by microplate reader.
In preliminary experiment, added the SK-BR-3 cell of the HSA molecule of 1000nM to compare and there is no significant difference with the mobility of blank.As shown in Fig. 5 a～5d, after having added the E4-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability that cell counting by cell film lower surface calculates SK-BR-3 cell has been reduced by 52.50%, 85.00%, 60.00% successively, and the Viola crystallina after dyeing by wash-out show that the OD value of elutriant is reduced to 0.25,0.11,0.21 successively by 0.37 of control group indirectly; After having added the E5-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability that the cell counting by cell film lower surface calculates SK-BR-3 cell has been reduced by 65.79%, 76.32%, 52.63% successively; Viola crystallina after dyeing by wash-out show that the OD value of elutriant is reduced to 0.24,0.18,0.33 successively by 0.48 of control group indirectly.It is the vertical migration effect that inhibition tumor cell is induced by Chemokine CXCL12 effectively of polypeptide-HSA molecule.
Embodiment 7:E4-HSA molecule, the restraining effect of E5-HSA molecule to the lateral transfer of CXCL12 induction MDA-MB-231 cell
In Corning six orifice plates, every hole is used 2mL DMEM in high glucose substratum (containing 10% foetal calf serum FBS and 1% mycillin) to cultivate 20 * 10
4individual cell, by six orifice plates at 37 ℃, 5%CO
2preculture 24h in the incubator of condition, MDA-MB-231 Growth of Cells is when closing on 80% fusion, with the aseptic rifle head of 10 μ L in the hole of culture plate along straight line cut, then use PBS solution by the soft washing of cell 3 times, rear every hole adds serum-free DMEM in high glucose substratum, add the induction of the CXCL12 aqueous solution simultaneously, the CXCL12 molecular conecentration that makes every hole is 100ng/mL, and add the PBS solution of polypeptide-HSA molecule, the ultimate density that makes peptide molecule is 10nM, 100nM, 1000nM, and blank adds PBS solution.The variation of microscope observing cell situation of movement and cut width for 0h after cut, 24h, calculates cell migration rate.
In preliminary experiment, added the MDA-MB-231 cell of the HSA molecule of 1000nM to compare and there is no significant difference with the mobility of blank.As shown in Fig. 6 a, 6b, after having added the E4-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability of MDA-MB-231 cell has been reduced by 18.54%, 40.49%, 30.73% successively, after having added the E5-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability of MDA-MB-231 cell has been reduced by 50.87%, 59.09%, 51.52% successively, i.e. polypeptide-HSA molecule lateral transfer effect that inhibition tumor cell is induced by Chemokine CXCL12 effectively.
Embodiment 8:E4-HSA molecule, the restraining effect of E5-HSA molecule to CXCL12 induction MDA-MB-231 cell vertical migration
The MDA-MB-231 cell of results logarithmic phase, with serum-free DMEM in high glucose substratum suspension cell, contains 10 * 10 by 200 μ L
4the PBS solution of the suspension of individual cell and different concns E4-HSA molecule adds the upper chamber of transwell cell, and the ultimate density that makes peptide molecule is 10nM, 100nM, 1000nM, and blank adds PBS solution.Lower chamber adds 800 μ L serum-free DMEM in high glucose substratum, contains the CXCL12 aqueous solution (100ng/mL) induction simultaneously.Culture plate is at 37 ℃, 5%CO
2after cultivating 24h in the incubator of condition, take out transwell cell, with cotton swab, carefully wipe the not migrating cell of chamber face on filter membrane, with crystal violet solution, fixedly move to cell the dyeing of cell film lower surface, the cell moving with microscopic examination after clear water rinses.Each cell film is got 5 visuals field according to the orientation up and down, counts the cell count of migration in each visual field, calculates cell migration rate after averaging.Indirectly, 800 μ L33% acetic acid solution wash-out Viola crystallinas for the cell film after dyeing, get 100 μ L elutriants and are determined at the OD value under 570nm wavelength by microplate reader.
In preliminary experiment, added the MDA-MB-231 cell of the HSA molecule of 1000nM to compare and there is no significant difference with the mobility of blank.As shown in Fig. 7 a～7d, after having added the E4-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability that cell counting by cell film lower surface calculates MDA-MB-231 cell has been reduced by 46.15%, 88.46%, 80.77% successively, and the Viola crystallina after dyeing by wash-out show that the OD value of elutriant is reduced to 0.37,0.16,0.19 successively by 0.52 of control group indirectly; After having added the E5-HSA molecule of 10nM, 100nM, 1000nM and cell to hatch, the transfer ability that cell counting by cell film lower surface calculates MDA-MB-231 cell has been reduced by 71.43%, 89.29%, 82.14% successively, and the Viola crystallina after dyeing by wash-out show that the OD value of elutriant is reduced to 0.28,0.17,0.23 successively by 0.55 of control group indirectly.It is the vertical migration effect that inhibition tumor cell is induced by Chemokine CXCL12 effectively of polypeptide-HSA molecule.
Above-mentioned experiment shows: polypeptide-human's serum albumin of the present invention (HSA) molecule is compared and in salts solution, had good solvability with polypeptide, and has the ability that suppresses metastases, and feasible methods for the treatment of can be provided for the inhibition of metastases.
Applicant's statement, the present invention illustrates processing method of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to the selection of the interpolation of the equivalence replacement of the selected raw material of the present invention and ancillary component, concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (10)
1. the polypeptide that inhibition tumor cell shifts, is characterized in that,
Described polypeptide has and is selected from the aminoacid sequence of SEQ ID NO:1～7 shown in any one.
2. the polypeptide that inhibition tumor cell according to claim 1 shifts, is characterized in that,
Described polypeptide is the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.
3. polypeptide deliquescent method in salts solution described in raising claim 1 or 2, is characterized in that, described method comprises: described polypeptide is combined with human serum albumin;
Preferably, the combination of described polypeptide and human serum albumin is physical bond.
4. method according to claim 3, is characterized in that, said method comprising the steps of:
1) obtain solution: described peptide molecule is mixed with to 1-5mg/mL solution, human serum albumin molecule is mixed with to 125-250mg/mL solution;
2) mix: human serum albumin molecular solution is joined in peptide molecule solution, fully mix.
5. method according to claim 4, is characterized in that,
In step 1) in, the solvent of described polypeptide solution is aseptic ultrapure water; Preferably, described human serum albumin solution's solvent is aseptic ultrapure water;
Preferably, in step 2) in, in described solution, the mol ratio of peptide molecule and human serum albumin molecule is 4:1-1:4, is preferably 1:1.
6. polypeptide-human's serum albumin mixture, is characterized in that, comprises polypeptide and human serum albumin described in claim 1 or 2;
Preferably, described polypeptide combines by physical action with human serum albumin;
Preferably, the mol ratio of described polypeptide and human serum albumin is 4:1-1:4, is preferably 1:1.
7. the application of polypeptide-human's serum albumin mixture according to claim 6 in the medicine shifting for the preparation of inhibition tumor cell.
8. application according to claim 7, is characterized in that, described polypeptide-human's serum albumin mixture is for lateral transfer and/or the vertical migration of inhibition tumor cell.
9. the application of polypeptide-human's serum albumin mixture according to claim 6 in the medicine for the preparation for the treatment of people metastases relative disease.
10. application according to claim 9, is characterized in that, described metastases relative disease is the relevant tumour of Chemokine Receptors CXCR4 high expression level;
Preferably, the tumour that described CXCR4 high expression level is relevant is any one in mammary cancer, leukemia, lymphoma or bladder cancer.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410353810.5A CN104098652B (en) | 2014-07-23 | 2014-07-23 | A kind of polypeptide suppressing metastases and polypeptide complex, its preparation method and application thereof |
| EP15824964.9A EP3173420B1 (en) | 2014-07-23 | 2015-07-01 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| PCT/CN2015/083021 WO2016011878A1 (en) | 2014-07-23 | 2015-07-01 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| DK15824964.9T DK3173420T3 (en) | 2014-07-23 | 2015-07-01 | POLYPEPTID AND POLYPEPTID COMPLEX FOR SUPPRESSING TUMOR METASTASE AND TREATMENT OF LEUKEMIA AND PROCEDURE FOR MANUFACTURING ITS AND USING IT |
| CN201580008736.6A CN106029683B (en) | 2014-07-23 | 2015-07-01 | Polypeptide and polypeptide compound for inhibiting tumor metastasis and treating leukemia, and preparation method and application thereof |
| US15/328,355 US10258669B2 (en) | 2014-07-23 | 2015-07-01 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN105175503A (en) * | 2015-09-08 | 2015-12-23 | 苏州普罗达生物科技有限公司 | Factor VIII inhibitory polypeptide and application thereof |
| WO2016011878A1 (en) * | 2014-07-23 | 2016-01-28 | 国家纳米科学中心 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| CN106432424A (en) * | 2016-10-18 | 2017-02-22 | 国家纳米科学中心 | Polypeptide capable of inhibiting tumor metastasis, and application of polypeptide |
| CN110841056A (en) * | 2018-08-03 | 2020-02-28 | 国家纳米科学中心 | PAMAM-polypeptide complex, preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016011878A1 (en) * | 2014-07-23 | 2016-01-28 | 国家纳米科学中心 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| US10258669B2 (en) | 2014-07-23 | 2019-04-16 | National Center For Nanoscience And Technology, China | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| CN104231050A (en) * | 2014-09-25 | 2014-12-24 | 国家纳米科学中心 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method for polypeptide complex and application of polypeptide and polypeptide complex |
| CN105175503A (en) * | 2015-09-08 | 2015-12-23 | 苏州普罗达生物科技有限公司 | Factor VIII inhibitory polypeptide and application thereof |
| CN106432424A (en) * | 2016-10-18 | 2017-02-22 | 国家纳米科学中心 | Polypeptide capable of inhibiting tumor metastasis, and application of polypeptide |
| CN106432424B (en) * | 2016-10-18 | 2019-11-12 | 国家纳米科学中心 | It is a kind of inhibit metastases polypeptide and its application |
| CN110841056A (en) * | 2018-08-03 | 2020-02-28 | 国家纳米科学中心 | PAMAM-polypeptide complex, preparation method and application thereof |
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