CN107253986A - Integrin intracellular peptide sequence is suppressing the application during new vessels is formed - Google Patents

Abstract

The present invention relates to series containing integrin conserved sequence and with the polypeptide compound for blocking new vessels formation function in vivo and in vitro.Functional polypeptide compound is the amino acid sequence shown in claims Section 1.Those micromolecule polypeptide compounds can enter intracellular by being coupled with transmembrane polypeptide, and then by blocking the formation of new vessels with reference to common signal protein molecule, and with the effect of entity tumor growth in Inhibition test Mice Body, belong to biomedical sector.The present invention can suppress external Human umbilical vein endothelial cells formation vessel-like structure based on function micromolecule polypeptide compound and its effect protein molecule, suppress the generation that nude mice by subcutaneous is inoculated with matrigel medium vessels, and suppress nude mice by subcutaneous inoculation implanted solid tumor growth.Our research finds to provide not only a variety of lead compounds that there is resistance anti-angiogenesis to act on, and the new mechanism of a regulation and control new vessels formation is further disclosed, so as to be that exploitation anti-angiogenesis of new generation and antineoplastic provide new target site.

Description

Integrin intracellular peptide sequence is suppressing the application during new vessels is formed

Technical field

Can the present invention relates to three kinds of polypeptide compounds and its effector molecule based on integrin beta subunit intracellular conserved sequence Effectively extracorporeal blocking new vessels forms and had the effect for suppressing the growth of mouse subcutaneous solid tumors in vivo.

Background technology

Integrin is, positioned at the important cell adhesion molecule of a class of cell surface, to be passed through by two subunits of α and β non-covalent The cross-module heterodimeric glycoprotein acceptor that key is formed by connecting.Integrin is widely distributed in human body, nearly all different tissues All expression has one or more of integrin molecules on cell.In vertebrate, 18 α and 8 β subunits are had now been found that, α is sub- The various combination of base and β subunits forms 24 α/β integrin heterodimers altogether.The generation of integrin and tumour, develop close phase Close, it participates in the formation of new vessels and the structure of blood vessel network by adjusting the differentiation of endothelial cell, sticking, migrate and survive Build.Tumor Angiongesis be unable to do without vascular endothelial cell and sticked with extracellular matrix, and interstitial around intrusion blood vessel whereby, then The formation of blood vessel bar rope is promoted by the adhesion of cell-ECM.Connected as endothelial cell and extracellular matrix (ECM) Integrin in bridge, vascular endothelial cell plays an important role in tumor angiogenesis.

Integrin alpha v beta 3 is to study more deep integrin family member at present, and participates in tumor angiogenesis. Its low expression in the endothelial cell of dormancy, but expression quantity is obvious in kinds of tumor cells and endothelial cells in tumor neogenetic blood vessels Increase.Studies have shown that can induce neovascular endothelium Apoptosis by extracellular suppression α v β 3 function, suppress tumor neogenetic The formation of blood vessel, promotes apoptosis of tumor cells, reaches antineoplastic purpose, and this process can't normal tissue blood vessel Have undesirable effect.Therefore, integrin alpha v beta 3 turns into the target spot for preferably suppressing tumour and Tumor Angiongesis.

With block the anti-tumor neovascularization formation medicine that integrin and its extracellular aglucon are combined into target spot to be broadly divided into Under several classes：(1) integrin antibody class medicine；(2) the polypeptide micromolecular that high-affinity binding sequence is have with integrating is contained Compound, wherein the most typical with the micromolecule polypeptide containing RGD sequence (arginine-glycine-aspartic acid)；(3) integrin is Other class medicines of target spot etc..A portion medicine has manifested good anti-angiogenic rebirth and antitumor action, and has entered Enter clinical research critical stage (for example, antibody class Vitaxin [1,2], polypeptide Cilengitide [3-5]).

Developed by blocking integrin signaling path to suppress tumour, because of some inconsistent researchs in research field Data, remain dispute.For example, the mouse of the type integrins of β 3 missing promotes the growth [6] of in-vivo tumour on the contrary；Further, low concentration Integrin inhibitors RGD peptidomimetics Cilengitide there is facilitation on the contrary to angiogenesis in Mice Body and tumour growth [7,8].In addition, the Cilengitide for coming from the research and development of EMD/Merck companies is based on blocking integrin alpha v beta 3 and α v β 5 The medicine of the novel therapeutic glioblastoma of extracellular ligand binding site exploitation, but it is after clinical trial and not up to pre- Phase effect [9].These discoveries add the FU that target drug is combined into integrin-aglucon.

Integrin can mediate two-way signaling to conduct, and integrin and extracellular aglucon combination by integrin intracellular sequence Accuracy controlling.Integrin α/β subunit intracellular sequence is shorter (in addition to β 4), and itself does not have enzymatic activity yet, but it is in integrin Played an important role in two-way signaling conduction, especially the conserved sequence of β subunits (β 4 and β 8 except) intracellular, such as two NxxY/F Region [10], they can be recognized with reference to a variety of intracellular signals and cytoskeletal protein molecule respectively.Such as, talin can be with β subunits Intracellular section membrane-proximal region NPxY sequences are combined, and are the key regulators of integrin activation；Other kindlin protein family molecules can It is be combined with each other with the remote film end NxxY/F of integrin beta subunit intracellular region, and cooperates with the activation [11-13] of talin induced integrins.This Outside, research is it has also been found that the remote film end NxxY sequences (NITY) of Integrin β3 intracellular can also be recognized with reference to β 3-endonexin.

Kindlin families include 3 family members, are Kindlin-1, Kindlin-2 and Kindlin-3 respectively.They Played a significant role in activation integrin, participated in different cells the cell migration of mediated by integrin, stick etc. it is multinomial Biological function.Kindlin protein abnormal expressions may further result in the generation of a variety of genetic diseases, in related human diseases and examination Test in mouse model and be all verified [14].β 3-endonexin are a polypeptides containing 111 amino acid, it is known that be that the types of β 3 are whole The binding factor of plain intracellular sequence is closed, but is not combined [15,16] with β 1, the type integrin intracellular sequences of β 2.At present, β 3- The activation regulation and control whether endonexin participates in the type integrins of β 3 are still not clear.Kindlin and β 3-endonexin are in the blood vessels [17-21] is expressed in chrotoplast.

Our early-stage Study finds that kindlin-2 participates in the adhesion of the vascular endothelial cell of integrin alpha v beta 3 mediation with moving Move, and there is the function [17,20] of promoting new vessels formation in zebra fish and Mice Body.There is a research to show, it is low β 3-endonexin play a part of angiogenesis inhibiting by HIF approach under the conditions of oxygen, and overexpression β 3-endonexin can Reduce the HIF-1 α mRNA level in-sites of hypoxia inducible, it is suppressed that HIF activation, and then suppress the mistake of endothelial cell formation new vessels Journey [22].Because kindlin-2 and β 3-endonexin be involved in modulating vascular it is newborn and with β integrin C-terminal NxxY sequences Row are combined, so it is presumed that the polypeptide compound comprising integrin beta intracellular section C-terminal NxxY sequences may have regulation and control newborn The function of vascularization.Accordingly, we devise 6 kinds of derivatives containing different integrin beta subunit intracellular conserved sequences to wear film more Peptide compounds (m β CTPs), each all includes the NxxY/F sequences of an integrin beta subunit C-terminal, and have evaluated it in body The ability of interior and external anti-angiogenetic therapy, finally found that some integrin beta subunit intracellular functional sequences can by with β 3- Endonexin interacts and suppresses new vessels and formed, so as to disclose the new of exploitation anti-angiogenesis of new generation Target site.

The content of the invention

An object of the present invention is the provision of the functional polypeptide compound based on integrin beta subunit intracellular conservative region Amino acid sequence.

The second object of the present invention is that providing polypeptide derivative compound (m β 3CTP, m β 5CTP, m β 6CTP) can suppress External Human umbilical vein endothelial cells formation vessel-like structure；Suppress the generation that nude mice by subcutaneous is inoculated with matrigel medium vessels；And to naked The growth of mouse subcutaneous vaccination solid tumor has different degrees of inhibitory action.Also, function test, which discloses these, has anti-new green blood The integrin beta subunit intracellular section sequence of pipe formation function can play work by combining effect protein factor-beta 3-endonexin With.

To illustrate above-mentioned purpose, the present invention is adopted the following technical scheme that：

Three kinds of polypeptide compounds based on integrin beta subunit intracellular conserved sequence, it is characterised in that the polypeptide compound is There are SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:Amino acid sequence shown in 3.

The above-mentioned polypeptide compound based on integrin beta subunit intracellular conserved sequence is preparing anti-angiogenesis medicine In application.

The above-mentioned polypeptide compound based on integrin beta subunit intracellular conserved sequence answering in antineoplastic is prepared With.

These three micromolecule polypeptide compounds of the present invention can be hindered by combining common effect signal protein molecular The formation of disconnected new vessels.It has entity tumor growth in Inhibition test Mice Body after coupling is with transmembrane ability polypeptide Effect, belongs to biomedical sector.The present invention can be suppressed based on function micromolecule polypeptide compound and its effect protein molecule External Human umbilical vein endothelial cells formation vessel-like structure, suppresses the generation that nude mice by subcutaneous is inoculated with matrigel medium vessels, and suppress Nude mice by subcutaneous is inoculated with the growth of solid tumor.Our research discovery provide not only a variety of with resistance anti-angiogenesis effect Lead compound, and the new mechanism of a regulation and control new vessels formation is further disclosed, so as to develop anti-new life of new generation Vascularization and antineoplastic provide new target site.

Brief description of the drawings

Fig. 1 is the amino acid sequence and composition and m β CTP series of small molecular polypeptide compound m β CTP of the present invention series Optionally suppress umbilical vein vascular endothelial cells (HUVEC) and form blood vessel sample tubule effect contrast figure in vitro.(A) m β CTP by Two parts are constituted：One is transmembrane polypeptide (CPP), for the sequence with 11 amino acid, the purpose being coupled can be mediated many Peptide molecule enters intracellular；Two be desired polypeptides (β CTP), for the polypeptide from different integrin beta subunit intracellular conserved sequences, Its own does not have transmembrane ability.Therefore, the serial polypeptides of m β CTP are the coupled complexes of transmembrane polypeptide and desired polypeptides, can be worn More cell membrane enters intracellular function.(B) it is can detect after being incubated by the m β 3CTP after FITC fluorescence labelings and HUVEC With the ability for wearing film.(C-D) the serial polypeptides of detection m β CTP suppress umbilical vein vascular endothelial cells (HUVEC) and form blood in vitro The ability of pipe sample tubule.

Fig. 2 is that small molecular polypeptide compound m β CTP of the present invention series suppresses subcutaneous implantation matrigel (Matrigel) group The ability of block medium vessels formation.(A) difference m β CTP are expelled to mouse subcutaneously after being mixed respectively with liquid matrix glue, and matrigel exists Solidify and induce rapidly the formation of new vessels in Mice Body.The situation that solid state substrate blob of viscose observes vascularization is taken out after 7 days. (B) blood vessel formed in histochemical stain (HE) observation matrigel.(C) hemoglobin content in matrigel is determined.

Fig. 3 is growth and the immuning tissue that small molecular polypeptide compound m β CTP of the present invention suppress nude mice by subcutaneous solid tumor Chemical analysis intratumoral vasculature distribution situation.(A) mouse is subcutaneously planted handles and observes it to swollen with different m β CTP respectively after knurl The influence of knurl growth.(B-C) the formational situation sum of blood vessel in knurl body and the observation of carry out group (CD31+) knurl in mouse is taken out Amount.

Fig. 4 for desired polypeptides (β 3CTP) in identification m β 3CTP in the present invention leitungskern sequence.(A) from β 3CTP two ends Deleting for amino acid sequence is carried out respectively, is built the m β 3CTP containing different aminoacids sequence and is derived polypeptide compound.(B-C) HUVEC cell seedings derive polypeptide compound in 24 orifice plates of matrigel have been completed, and with a variety of m β 3CTP shown in (A) Observation forms the ability of vascular canal spline structure after (20 μM) are incubated altogether.

Fig. 5 is integrin beta subunit intracellular sequence in the present invention and function m β CTP polypeptide compounds and β 3-endonexin Situation about being interacted with Kindlin-2.(A) using the integrin beta subunit intracellular sequence with GST labels of purifying with expressing The HUVEC cell pyrolysis liquids for having external source EGFP- β 3-endonexin and endogenous kindlin-2 are incubated, and determine different beta subunit born of the same parents Interior sequence and EGFP- β 3-endonexin and kindlin-2 binding ability.(B) determine different using yeast two-hybrid system Integrin beta subunit intracellular sequence and β 3-endonexin and kindlin-2 binding ability.(C) tested and surveyed using Competitive assays Determine function m β 3CTP and m β 5CTP and β 3-endonexin directly in conjunction with effect.

Fig. 6 is β 3-endonexin regulation and control huve cell vascular canals spline structure formation in the present invention and promotes The situation of mouse subcutaneous solid tumour growth.(A-C) endogenous β 3- in HUVEC are lowered respectively using two species specificity siRNA Endonexin expression simultaneously detects its influence to HUVEC formation blood vessel sample Minute Tubule Structures.(D-F) system is expressed using slow virus System up-regulation HUVEC in β 3-endonexin expression and detect its to HUVEC formation blood vessel sample Minute Tubule Structures influence.(G-H) Use can express EGFP- β 3-endonexin and control EGFP slow virus local injection mouse interior tumor respectively and to observe its right The influence of mouse interior tumor growth.

Embodiment

Embodiment one：Synthesize and purify the serial peptide molecules of m β CTP

The name of m β CTP series derivatives peptide molecule and amino acid sequence containing target polypeptides are referring to Fig. 1 (A).The series Peptide systhesis is in company, using manual solid-phase Fmoc methods, using Fmoc-Gly-Wang resins as initiation material, from C-terminal to N-terminal side To synthesis.Purifying uses HP1100 types (Agilent company of the U.S.) rp-hplc, and its purity is up to 99.62%.Should Method is micromolecule polypeptide conventional synthesis process.

Embodiment two：The serial polypeptides of umbilical vein vascular endothelial cells (HUVEC) blood vessel sample Tube formation assay research m β CTP Effect to new vessels formation:

Referring to Fig. 1, the human umbilical vein endothelial cell (HUVEC) of in vitro culture is in the basement membrane matrix containing growth factor The pipeline spline structure of similar blood vessel can be voluntarily assembled on glue.The different m β CTP (μ of final concentration 20 are added in cell culture fluid M), its ability for suppressing HUVEC cell assembling pipelines is observed.Referring to Fig. 1 (C-D), as a result show, compared with without medicine treatment group Compared with the derivative polypeptide m β 3CTP of addition, m β 5CTP, m β 6CTP can effectively suppress the formation of pipeline, illustrate these polypeptides pair HUVEC cells in vitro formation blood vessel sample pipeline configuration has significant inhibitory action.The experimental result also illustrates the suppression work(simultaneously Corresponding integrin beta intracellular sequence (β CTP) rather than coupled transmembrane polypeptide (CPP) can be come from, because equally having CPP M β 1CTP, m β 2CTP, the m β 7CTP of sequence form pipeline spline structure to HUVEC cells and had no significant effect.Meanwhile, it is not coupled The β CTP of transmembrane polypeptide simultaneously do not have suppression function, illustrate that they play a role in the cell.

Fig. 1：M β CTP series polypeptides optionally suppress the shape of huve cell (HUVEC) vascular canal spline structure Into.(A) the serial polypeptides of m β CTP include two parts：One is transmembrane polypeptide (CPP)；Two be target polypeptides, includes integrin beta subunit Intracellular NxxY/F containing C-terminal sequence (β CTP).β CTP itself are without film ability is worn, and it can after being coupled with transmembrane polypeptide CPP Cell membrane is passed through into intracellular function.(B) N-terminal mark fluorescent element FITC m β 3CTP and HUVEC is incubated altogether, and it wears film Characteristic passes through fluorescence microscope.(C) HUVEC is layered on matrigel, and the serial polypeptide drugs (20 μM) of m β CTP are added respectively； It is control without group.It is incubated the formational situation for observing (5 times of object lens) vascular canal spline structure after 12h under inverted microscope.(D) The number for forming pipeline spline structure is counted, statistical result is represented with means standard deviation (Mean ± SD), experiment is independent Sample average uses independent samples t test, and illustrating data, there were significant differences in statistical analysis by P ﹤ 0.01 (* *).

Embodiment three：Basement membrane matrix medium vessels formation experiment in Mice Body：

Referring to Fig. 2, experiment material is nude mice (BALB/C), 6 week old, male (every group of 7 nude mices), basement membrane matrix glue (Matrigel).First basement membrane matrix glue is placed on and melts after liquid, to add 600ng/ml bFGF and 100U/ml on ice Heparin and different m β CTP polypeptides (50 μM).Then in the μ l of mouse bare subcutaneous injection 500 basement membrane matrix glue mixture. Basement membrane matrix glue enters rapid solid state in Mice Body and induces the growth of new vessels, lasts after 7 days, by solid-state Matrigel blocks carefully subcutaneously peel off taking-up from mouse, determine the content of hemoglobin in basement membrane matrix glue；Simultaneously to fixation Basement membrane matrix glue afterwards carries out tissue chemical analysis' (HE dyeing).As a result find in the matrix of identical weight, m β 3CTP, m β 5CTP, m β 6CTP treatment groups are substantially reduced relative to without content of hemoglobin contained by medicine control group.Also, m β 3CTP and m β 5CTP The new vessels quantity for the treatment of group without the control group that medicine is handled than substantially reducing.The experimental result, which also illustrates to have, simultaneously suppresses work( Can m β CTP polypeptides in effect sequence is from corresponding integrin beta intracellular sequence (β CTP) rather than coupled to wear film more Peptide (CPP), because equally with the m β 1CTP of CPP sequences, m β 2CTP, m β 7CTP to the formation of blood vessel in matrigel without obvious shadow Ring.

Fig. 2：The serial polypeptide compounds of mouse subcutaneous implantation matrigel experiment detection m β CTP suppress the ability of vascularization. (A) mouse bare subcutaneous injection matrigel Matrigel (500 μ l) (bFGF containing 600ng/ml and 100U/ml heparin), and simultaneously Add the serial polypeptide drugs of (50 μM) of m β CTP；Dosing group is not used as control.Blob of viscose takes out and taken pictures in careful after 7 days.(B) it is solid Through HE dyeing and microexamination after matrigel Matrigel FFPEs after fixed.Dark red color part is contaminated for blood vessel (red blood cell), Lightpink background dyes for matrigel Matrigel.(C) in Matrigel samples hemoglobin total content measure, statistical result Represented with means standard deviation (Mean ± SD), experiment independent sample average uses independent samples t test, P ﹤ 0.05 (*) or P ﹤ Illustrating data, there were significant differences in statistical analysis by 0.01 (* *).

Example IV：The effect that function m β CTP polypeptides grow to mouse entity knurl：

Referring to Fig. 3 (A), experiment material is nude mice (BALB/C), 6 week old, male, is randomly divided into 2 groups, control group and m β CTP treatment groups (every group 6).Every is subcutaneously injected mouse prostate cancer cell RM1 (1.2x10⁶/ only), and add 1% matrix Glue.After lotus knurl 5 days, treatment group (50 μM of Local subdermal injection function m β CTP every other day；100 μ l PBS solutions)；Control group is subcutaneously noted Equivalent PBS is penetrated, while measuring mouse tumor size (V) (V=L × W × 0.52；Wherein L is measurement of tumor length, and W is Measurement of tumor width).Relative to control group it can be seen from Fig. 3 (A), m β 3CTP, m β 5CTP treatment groups gross tumor volume it is obvious It is smaller；And growth of the m β 6CTP treatment groups to tumour has no significant effect.This difference is probably because desired polypeptides and its intracellular (seeing below example) that the combination power of effect protein molecule is caused.

Fig. 3 (A)：M β 3CTP, m β 5CTP and m β 6CTP suppress the situation of nude mice tumor growth in vivo.(A) nude mice by subcutaneous is noted RM1 cells are penetrated, the local β 3CTP of administration m respectively subcutaneous every other day, m β 5CTP and m β 6CTP (50 μM)；Dosing group is not used as control. Tumour length (L) and wide (W) are recorded, and calculates volume V (V=L*W*W*0.52) and draws tumor growth curve.Statistical result with Means standard deviation (Mean ± SD) represents that experiment independent sample average uses independent samples t test, and P ﹤ 0.01 (* *) illustrate number According in statistical analysis, there were significant differences.

Embodiment five：Using immunohistochemical analysis tumor group sample, m β CTP are to the formation of blood vessel in solid tumor for research Influence：

Referring to Fig. 3 (B-C), Prostate Carcinoma of Mice RM-1 cell inoculation nude mice is taken out after 20 days after different treatments and formed Tumour, through PBS, neutral formalin is fixed, and is then manufactured paraffin section and is carried out CD31 SABC detections.After processing Group sample microexamination each group sample vessel chamber formational situation.There is obvious blood vessel in control group tumor tissues without drug-treated Generation, and majority has formed complete vascular canal spline structure；Although m β 3CTP and m β 5CTP (Fig. 3, B) administration group is swollen Tumor tissue has CD31 positive cells through SABC, but the number of blood vessel formed is significantly reduced, and the vascular canal formed is not advised Whole, there were significant differences compared with control group for its vessel size and form.The results show m β 3CTP and m β 5CTP are to tumour The formation of interior new vessels has significant inhibitory action, with the effect for suppressing solid tumor growth.Comparatively speaking, m β The inhibitory action that 6CTP is formed to intratumoral vasculature is weaker.

Fig. 3 (B-C)：M β 3CTP, m β 5CTP and m β 6CTP suppress the situation of Tumor angiogenesis in nude mouse.(B) nude mice RM1 cells are subcutaneously injected, the local β 3CTP of administration m respectively subcutaneous every other day, m β 5CTP and m β 6CTP (50 μM)；Dosing group is not made For control.Groupization is cut into slices and formed in CD31 immunostainings (brown part) (C) each group tumor tissues after solid tumor takes out Blood vessel number statistics, statistical result represents with means standard deviation (Mean ± SD), and experiment independent sample average is using independence Sample t-test, illustrating data, there were significant differences in statistical analysis by P ﹤ 0.01 (* *).

Embodiment six：Identify the Core Feature sequence in m β 3CTP desired polypeptides：

Referring to Fig. 4, to the ammonia in the target polypeptides (β CTP) from Integrin β_3 intracellular sequence in functional polypeptide m β 3CTP The sequence of base acid is truncated from N- ends and C- ends and synthesizes corresponding derivative polypeptide respectively sees Fig. 4 (A), then uses throat aspirate Endothelial cell (HUVEC) blood vessel sample Tube formation assay come verify these derive polypeptide anti-angiogenesis ability.Knot Fruit shows that at least two amino acid residue of NITY sequences and its N- ends in m β 3CTP target polypeptides forms leitungskern sequence.

Figure -4：In m β 3CTP in desired polypeptides function core sequence identification.(A) amino is carried out respectively from β 3CTP two ends Deleting for acid sequence, builds the m β 3CTP containing different aminoacids sequence and derives polypeptide compound.(B-C) HUVEC cell seedings Derive polypeptide compound (20 μM) incubation altogether in 24 orifice plates of matrigel have been completed, and with a variety of m β 3CTP shown in (A)；No It is control to add polypeptide group.(5 times of object lens) are observed under inverted microscope to take pictures, and the pipeline spline structure of formation is counted, Statistical result represents that experiment independent sample average uses independent samples t test, P ﹤ 0.01 with means standard deviation (Mean ± SD) (* *) illustrating data, there were significant differences in statistical analysis.

Embodiment seven：Using Pull down experiment and yeast two-hybrid assay study integrin beta subunit intracellular sequence and The situation that function m β CTP polypeptides are combined with β 3-endonexin and Kindin-2：

Referring to Fig. 5, first, with Pull down identify the intracellular sequence of integrin beta subunit and β 3-endonexin and Kindin-2 interaction.HUVEC cells expression external source EFGP- β 3-endonexin and endogenous Kindlin-2 respectively with It is incubated with reference to the intracellular sequence of the integrin beta subunit that GST is merged on medium.Wash away after non-specific binding albumen, use Western blot experimental identifications combine EFGP- β 3-endonexin and Kindlin-2 up.Result is shown in Fig. 5 (A), β 3-endonexin and Integrin β_3, β 5 and β 6 intracellular peptide sequence is combined, but wherein combined with β 6 intracellular peptide sequence compared with It is weak.Compare, another intracellular integrin associated proteins Kindlin-2 and β 1, β 3 and β 7 intracellular peptide sequence are combined.By This, the intracellular sequence of β 3-endonexin and kindlin-2 combination integrin beta subunits has different selectivity.Then, ferment is used Female two-hybrid system (Matchermarker^TMGold the intracellular sequence and β 3- of integrin beta subunit) are further identified Endonexin and Kindlin-2 interaction.β 3-endonexin and Kindlin-2 are cloned on pGBDT7 carriers, are built BD (DNA binding structural domains) fusion protein；Meanwhile, the intracellular section sequence of β subunits is cloned on PGADT7 carriers, is built AD and (is turned Record activation structure domain) fusion protein.If two kinds of albumen have interaction, it is possible to form functional transcription factor (AD- BD), and the transcription of downstream reporter gene is activated, causes yeast strain to be grown on Selective agar medium (SD-3).Such as Fig. 5 B Shown, the testing result of yeast two-hybrid is consistent with Pull down experimental results.Finally, we are further tested with competitive assay Having demonstrate,proved m β 3CTP and m β 5CTP can specifically bind with β 3-endonexin, see Fig. 5 (C).These experimental results functions m β CTP polypeptides are to the inhibitory action of new vessels and tumour growth possibly via with reference to effect signal protein molecular β 3- Endonexin is realized.

Figure -5：The detection that the intracellular section of integrin beta subunit interacts with β 3-endonexin and Kindin-2.(A) Pull down are tested：EFGP- the β 3-endonexin and Kindlin-2 of HUVEC cells expression are respectively with combining on medium The intracellular sequence of the integrin beta subunit of GST fusions is incubated.Wash away after non-specific binding albumen, it is real with Western Blot Authenticate and determine EFGP- β 3-endonexin and Kindlin-2 that combination is got on.(B) yeast two-hybrid system (Matchermarker^TM Gold yeast two-hybrid system) further intracellular sequence and the β 3-endonexin of identification integrin beta subunit and Kindlin-2 interaction.β 3-endonexin and Kindlin-2 are cloned on pGBDT7 carriers, and building BD, (DNA is combined Domain) fusion protein；Meanwhile, the intracellular section sequence of β subunits is cloned on PGADT7 carriers, builds AD (transcriptional activation structures Domain) fusion protein.If two kinds of albumen have interaction, it is possible to form functional transcription factor (AD-BD), and activate The transcription of downstream reporter gene, causes yeast strain (SD-3) can be grown on Selective agar medium.(C) m β 3CTP and m β 5CTP Can be with the intracellular of integrin beta subunit section competition binding EFGP- β 3-endonexin.The EFGP- β 3- of HUVEC cells expression Endonexin is incubated with combining the intracellular sequence for the Integrin β3 that the GST on medium is merged, while being separately added into 20 μM M β 3CTP and m β 5CTP.Wash away after non-specific binding albumen, combined up with Western Blot experimental identifications EFGP-β3-endonexin。

Embodiment eight：The formation of β 3-endonexin controllable HUVEC cellular vascular pipeline spline structures and the life of tumour It is long：

Referring to Fig. 6, we are by targetting β 3-endonexin siRNA (001,002) to HUVEC cell transfectings come silence Endogenous β 3-endonexin expression；Simultaneously with non-target tropism siControl as control, the endogenous β 3- of downward are as a result shown The ability of HUVEC formation capillary structure is substantially deteriorated (see Fig. 6, A-C) after endonexin expression.And then, we use slow virus Expression system is overexpressed EGFP- β 3-endonexin in HUVEC cells, finds to be overexpressed after EGFP- β 3-endonexin The ability of HUVEC formation vascular canal spline structures is remarkably reinforced (see Fig. 6, D-F).Finally, we are to mouse subcutaneous solid tumors point High expression EGFP- β 3- Zhu She not be found containing EGFP- β 3-endonexin and control EGFP slow virus can be expressed Endonexin can remarkably promote the growth of tumour.These experimental results illustrate that β 3-endonexin are likely to a kind of new green blood Pipe formation and the promotive factor of tumour growth.With reference to above m β CTP function and its binding ability with β 3-endonexin, It is concluded that functional polypeptide m β CTP anti-angiogenesis and neoplasm growth effect is possibly via combining and block β 3-endonexin function is realized.

Figure -6：β 3-endonexin can promote formation and the tumor growth in vivo of HUVEC cellular vascular pipeline spline structures. (A) HUVEC cell transfectings targeting β 3-endonexin siRNA (001,002) carrys out silencing endogenous β 3-endonexin table Reach；Simultaneously control is used as with non-target tropism siControl.Transfect 48h after siRNA and collect cell, and detected by qRT-PCR β 3-endonexin mRNA expressions in HUVEC cells.(B-C) after transfection siRNA, HUVEC cells is collected and carry out blood vessel The formation experiment of pipeline spline structure, and the number for forming pipeline spline structure is counted.(D) slow disease is passed through in HUVEC cells Malicious system infections expression external source EGFP and EGFP- β 3-endonexin.After 48h, metainfective HUVEC cells are collected, and pass through Western Blot test to detect EGFP expressions.(E＆F) expression EGFP and EGFP- β 3-endonexin HUVEC is thin Born of the same parents form vascular canal spline structure, and count number of tubes.(G＆H) experiment material is nude mice (BALB/C), and 6 week old are male, with Machine is divided into 2 groups, and every group 5, every is subcutaneously injected mouse prostate cancer cell RM1 (1.2x10⁶/ only).Two components from the 5th day Slow virus (the titre=2x10 of expression EGFP and EGFP- β 3-endonexin respectively is not injected⁷IU/ml, is injected 2 times for one week, The μ l/ of per injection 100 are only).After 15 days, take out tumour and measure.Statistical result represents with means standard deviation (Mean ± SD), Experiment independent sample average uses independent samples t test, and illustrating data, there were significant differences in statistical analysis by P ﹤ 0.01 (* *).

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<110>Shanghai University

<120>Integrin intracellular peptide sequence is suppressing the application during new vessels is formed

<160> 3

<210> 1

<211> 12

<212>Polypeptide

<213>Amino acid sequence

<400> 1

ATSTFTNITYRG 12

<210> 2

<211> 20

<212>Polypeptide

<213>Amino acid sequence

<400> 2

PISTHTVDFTFNKFNKSYNG 20

<210> 3

<211> 12

<212>Polypeptide

<213>Amino acid sequence

<400> 3

STSTFKNVTYKH 12

Claims (3)

1. three kinds of micromolecule polypeptide compounds based on integrin beta subunit intracellular conserved sequence, it is characterised in that the peptide Thing is by SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:Amino acid sequence shown in 3.

2. the polypeptide compound according to claim 1 based on integrin beta subunit intracellular conserved sequence is preparing anti-new life Application in vascularization medicine.

3. the polypeptide compound according to claim 1 based on integrin beta subunit intracellular conserved sequence prepare it is antitumor Application in medicine.