CN101596308A - ITGB4BP and derivant thereof are used to prevent and/or treat hypertrophic cicatrix and fibrosis lesion - Google Patents

Abstract

The present invention relates to β 4 and integrate plain conjugated protein (ITGB4BP) and derivant thereof, or the dna recombinant expression carrier of expressing ITGB4BP and derivant thereof, new application in preventing and/or treating hypertrophic cicatrix or fibrosis lesion, wherein the aminoacid sequence of ITGB4BP is SEQ ID NO 1, and described ITGB4BP derivant is to keep the binding site of ITGB4BP (SEQ ID NO 1) and the protein derivatives of functional part.Described application is by to the ITGB4BP or derivatives thereof of experimenter's administering therapeutic effective dose or express the recombinant gene expression vector of ITGB4BP or derivatives thereof or pharmaceutical composition of the present invention or test kit are realized.Wherein said pharmaceutical composition or test kit comprise ITGB4BP or derivatives thereof for the treatment of effective dose or recombinant gene expression vector and the pharmaceutical excipient or the carrier etc. of expressing the ITGB4BP or derivatives thereof.Preferably, described experimenter is the human or animal.

Description

ITGB4BP and derivant thereof are used to prevent and/or treat hypertrophic cicatrix and fibrosis lesion

Technical field

The present invention relates to the new drug purposes of known substance.Particularly, the present invention relates to β 4 and integrate plain conjugated protein (Integrin beta 4 binding protein, ITGB4BP) and derivant, express the dna recombinant expression carrier of ITGB4BP and derivant thereof, the new application in preventing and/or treating hypertrophic cicatrix and fibrosis lesion.

Background technology

Fibrotic disease is that (extracellular matrix ECM) causes hamartoplasia, hardening and a synulotic class pathological changes as over-deposit such as collagen because extracellular matrix.The chronic inflammatory disease long-time stimulus such as infection, autoimmune disease, anaphylaxis, chemical damage, radiation and tissue injury that continue cause fibroblast to break up to myofibroblast, cause a large amount of connective tissue deposition to form fibrosis, thereby cause the organ dysfunction forfeiture until dead (1-3).Hypertrophic cicatrix is a kind of of fibrotic disease, is because after deep corium was subjected to heating power or other form wound, the fibroplasia disorder caused, and usually causes the grievous injury (4) on the attractive in appearance and function of patient.We find that P311 gene (GenBank ID:hsu36189) has close getting in touch (5) with it in carrying out the Mechanism Study of hypertrophic cicatrix fibroblast differentiation, but its concrete mechanism how, and document but rarely has report.

The encoding gene of P311 (have another name called PTZ17, pentylenetetrazol 17) equals at first discovery (6) in the cerebral tissue of fetal rat in 1993 by Matthieu.P311 gene mapping is in No. 5 chromosome ORF13, its complete full length gene is about 2025bp, comprises 3 open reading frame, but only first reading frame coding contains 68 amino acid whose albumen P311, the molecular weight size is 8KD, and this reads frame high conservative between people and mice.In addition, the PEST domain (being rich in Pro, Glu, the zone of Ser and Thr) (7) that also has a high conservative at the N-terminal of people, mice and chicken P311.The P311 wide expression is in multiple tissue, high expressed in the cerebral tissue of cerebral tissue especially Late Embryogenesis and the cerebellum of manhood, Hippocampus and olfactory bulb wherein, also be expressed in simultaneously histiocytes (6) such as organ such as liver,spleen,kidney, eye, the heart and medulla mesenchyma, fibroblast, myofibroblast, and Pan and Fujitani, Taylor etc. confirm respectively that also this albumen can be expressed in the endochylema and karyon of myofibroblast, neurocyte.P311 is a kind of cytokine with important biomolecule effect of body, participates in the cells in vivo differentiation, regulates multiple normal or unusual activities biology (6-9) such as other albumen/genetic transcription, repair in trauma, tumor generation.

We utilize yeast-two hybrid technique screened among the one-tenth human liver cDNA library (U.S. BD Clontech company) can with the interactional proteic coded sequence of P311, obtained a genes of interest after the detection and localization altogether through bioinformatic analysis, recovery checking and laser co-focusing, its encoded protein is: β 4 integrates plain conjugated protein (Integrin beta 4 binding protein, ITGB4BP, SEQID NO 1) (10).This albumen of research prompting plays an important role in cytoskeleton formation and apoptosis, and may bring into play important function in hypertrophic cicatrix fibroblast atomization.Below summarize with regard to this albumen correlation function research both at home and abroad.

1.ITGB4BP basic feature

1.1 the encoding gene of ITGB4BP:

Francesca Sanvito equals 1998 by fluorescence in situ hybridization technique discovery ITGB4BP gene (SEQ ID NO 2, NCBI genebank registration number is NM-002212) be positioned chromosomal long-armed 20q11.2 district No. 20, its mRNA total length 1108bp, its open reading frame contains 735 nucleotide (11).The ITGB4BP gene has 7 exons and 6 introns, and 5 ' end of this gene lacks TATA promoter land, CpG island (12).ITGB4BP gene sequence in eukaryotic cell is conservative, and 72% homology is arranged in mammal and yeast, and 85% sequence similarity (13) is wherein arranged.The ITGB4BP gene is except finding in vertebratess such as people, mice, rat, Africa xenopus, on non-vertebratess such as fruit bat, sepiellae seu sepiae, molluscum, also find the existence of this gene, and the domain of this gene high conservative (14) between mammal and non-vertebrates.

1.2 ITGB4BP encoding proteins:

ITGB4BP albumen (SEQ ID NO 1, Swiss-Prot:P56537.1, [GI:3122258]) also claim EIF6, p27BBP, CAB, EIF3 etc., equal discovery in 1997 by Biffo, when getting in touch that plain β 4 and hemi desmosome form integrated in utilization yeast-two hybrid techniques such as Biffo research, find that albumen and β 4 have interaction, so called after β 4 integrate plain conjugated protein (Integrin beta 4binding protein, ITGB4BP).ITGB4BP albumen contains 245 aminoacid, and the molecular weight size is 27kd (15).This albumen is under the effect of Protein kinase C, and by the conduction of phosphorylation/dephosphorylation mediation signal, but its concrete mechanism it be unclear that (12,16).In the existing literature data research of ITGB4BP is mainly concentrated on ITGB4BP and cytoskeleton forms, the cancerous cell differentiation, albumen is synthetic and migration in.

1.3 the expression of ITGB4BP and distribution:

ITGB4BP albumen is widely distributed, can be expressed in different tissues, the different cells of perhaps same tissue, perhaps same cell different cycles.Studies confirm that, the proteic expression of ITGB4BP is all arranged in epithelial cell, fibroblast, tumor cell, activated T cells, activatory mastocyte and muscular tissue fiber, also have to studies confirm that as long as the plain cell of expression alpha 6 beta 4 integration is all expressed ITGB4BP (17).The ITGB4BP protein expression is near endochylema (intermediate filament) and karyon (mainly being positioned at the kernel periphery) (18).In cellular proliferative stage, the up-regulated of ITGB4BP (19).Therefore can point out ITGB4BP albumen relevant with cytoskeleton and cell proliferation.

2.ITGB4BP biological function

2.1 it is synthetic to participate in modulin:

Eukaryotic cell is in growth and atomization, and the most genes in the genome are not transcribed, and have only the minority gene to express, these genes are through transcribing and transcribe post-treatment, become sophisticated mRNA, and translate into polypeptide chain on the ribosome in Cytoplasm, form the required albumen of cell.There are the regulation and control of multiple level in the translation of mRNA, and wherein the regulation and control to the eukaryotic translation initial period are very important steps.At the eukaryotic translation initial period, ITGB4BP gets off from the depolymerization of ribosome 60s subunit, promotes 40s subunit and 60s subunit in conjunction with formation 80s subunit, thereby starts proteinic translation (20-23).Discovery ribosome 60s subunit is lost after having laboratory to knock out the ITGB4BP gene, thereby proves that its formation to ribosome 60s has important effect (18).And to ITGB4BP in the reaction of extracellular signal is to regulate proteic translation (24) as eukaryotic cell translation initiation factor participation the earliest.On the other hand, the ITGB4BP modulin is synthetic also relevant with its adjusting to microRNA.ITGB4BP combines with RISC (RNA induces silencing complex), promote the effect of corresponding microRNA specifically, disturb mRNA to form from transcriptional level, suppress the expression of its corresponding target protein, then can remove microRNA behind the ITB4BP to the expression inhibiting effect of target protein and promote the expression (25 26) of target protein otherwise knock out.

2.2 participate in regulating the formation of cell adhesion and cytoskeleton:

Integrin family is the adhesion molecule receptor, participates in mediated cell and extracellular matrix, cell and intercellular interaction.The intercellular effect of sticking has conjugation, desmosome connections, a hemi desmosome connection etc., and it is mucoprotein that the extracellular matrix that hemi desmosome connects is mainly layer, and it is a layer mucoprotein receptor that β 4 integrates elements, thus itself and desmosome, hemi desmosome be formed with close getting in touch.It is plain conjugated protein that ITGB4BP is that β 4 integrates, and the functional areas of integrating plain born of the same parents' intracellular domain with intermediate filament and alpha 6 beta 4 combine, as both formation of bridge mediation hemi desmosome, mediated cell stick function (15).In addition, ITGB4BP also is present in the nuclear matrix, by with being connected of cytoskeletal structures such as nuclear matrix, intermediate filament, participated in the formation of cytoskeleton.There is experiment to prove ITGB4BP high expressed (17) on intermediate filament and nuclear matrix.And ITGB4BP can also regulate the expression of the beta-catenin in the Wnt signal path, thereby influences cytoskeleton (27).

2.3 take place and relations of metastasis with tumor:

The most tangible characteristics of tumor are that cell proliferation is unusual, and apoptosis suppresses, and shift easily.There is experiment confirm ITGB4BP to participate in the propagation of cell as the translation regulatory factor.Hepatoma carcinoma cell nuclear matrix composition before and after the induction is being carried out subcellular fraction protein science analysis discovery, high expressed in the cell of ITGB4BP after differentiation, prompting may participate in cancerous cell differentiation (28).ITGB4BP can detect at the normal mucosa cell, express but in cancerous cell, cross, as the up-regulated (19) of ITGB4BP in colorectal cancer, and particularly evident in the lymphatic metastasis kitchen range (29), so the high expressed of prompting ITGB4BP may participate in the propagation and the transfer of tumor.

As can be seen from the above, ITGB4BP is synthetic at ribosome, the proliferation and differentiation of cytoskeleton and tumor and shift in have important effect, but it is less for the proteic pertinent literature of ITGB4BP at present, yet there are no report for its effect and function in fibrillation related disease, so its function in fibrosis and effect are also needed further further investigation.

Summary of the invention

The inventor finds that in the Mechanism Study of carrying out the differentiation of hypertrophic cicatrix fibroblast the P311 gene has close getting in touch with it.It is unclear that for P311 gene function and Study on Mechanism at present, the inventor utilizes yeast-two hybrid technique screening in becoming human liver cDNA library (U.S. BD Clontech company) to obtain an energy and the interactional proteic coded sequence of P311, determine that through further identifying this coded sequence encoded protein is that β 4 integrates plain conjugated protein (Integrin beta 4binding protein, ITGB4BP, SEQ ID NO 1, Swiss-Prot:P56537.1, [GI:3122258]).On this basis, the inventor has carried out a series of researchs, proves that first ITGB4BP plays a significant role in hypertrophic cicatrix fibroblast atomization, and then has finished the present invention.

Especially, the present invention relates to β 4 and integrate plain conjugated protein (ITGB4BP) and derivant thereof, or the dna recombinant expression carrier of expressing ITGB4BP and derivant thereof, the application in preventing and/or treating hypertrophic cicatrix or fibrosis lesion.

Particularly, the invention provides the content of following each side:

In a first aspect of the present invention, the present invention's screening in becoming the human liver cDNA library has obtained carrying the recombinant vector pACT2-ITGB4BP of ITGB4BP gene, on this basis, utilize conventional gene clone technology, cloned the recombinant shuttle plasmid pShutlle-CMV-ITGB4BP-EGFP that carries ITGB4BP encoding gene and green fluorescence protein gene EGFP, utilize the dna homolog reorganization then, ITGB4BP-EGFP is recombinated among the adenovirus vector pAdEasy-1, obtain recombinant adenoviral expressing vector pAdEasy-ITGB4BP-EGFP, and be packaged into adenovirus particles, be used for the evaluation of ITGB4BP protein function and ITGB4BP gene function.And, further cloned slow virus interference carrier pFIV-H1/U6-copGFP-ITGB4BPRNAi about the ITGB4BP gene, carry out the RNA interference experiment, be used for the evaluation of ITGB4BP protein function and ITGB4BP gene function.

In a second aspect of the present invention, the present invention has separated normal skin fibroblast and hypertrophic cicatrix fibroblast, has set up the cicatrix cell model.And, identify the expression of ITGB4BP in above-mentioned two kinds of cells, find ITGB4BP low express (referring to Fig. 1) in the hypertrophic cicatrix fibroblast, show that the low expression of ITGB4BP has increased the weight of the fibrosis of hypertrophic cicatrix.

In a third aspect of the present invention, the present invention suppresses detection fibersization is relevant after the expression of ITGB4BP in the hypertrophic cicatrix fibroblast index TGF-β 1 (transforminggrowthfactor-), MMP9 (matrix metalloproteinase 9) and the expression of type i collagen by the RNAi technology.The result shows that after suppressing the ITGB4BP expression on the mRNA level, index TGF-β 1, the MMP9 that fibrosis is relevant and the expression of type i collagen raise (referring to Fig. 2), have obviously increased the weight of fibrosis.

In a fourth aspect of the present invention, the present invention by the expression that in normal skin fibroblast, increases ITGB4BP after relevant index TGF-β 1, the MMP9 of detection fibersization and the expression of type i collagen.The result shows that after increasing the ITGB4BP expression on the mRNA level, index TGF-β 1, the MMP9 that fibrosis is relevant and the expression of type i collagen significantly reduce (referring to Fig. 3), suppress fibrosis.

In a fifth aspect of the present invention, the present invention passes through to detect the myofibroblast surface marker α-SMA index TGF-β 1 relevant with fibrosis and the expression of type i collagen behind the high expressed ITGB4BP in the hypertrophic cicatrix fibroblast, and utilizes fibroblastic dimensional culture system (FPCL) to detect the inhibitory action that myofibroblast is shunk.The result shows, compares with negative control, and behind the high expressed ITGB4BP, α-SMA expression reduces (referring to Fig. 4), has suppressed the conversion of hypertrophic cicatrix fibroblast to myofibroblast in the hypertrophic cicatrix fibroblast; Index TGF-β 1, the MMP9 that fibrosis is relevant and the expression of type i collagen are all obviously suppressed (referring to Fig. 5 and 6) and the contractility of myofibroblast decline (referring to Fig. 7).

Therefore, the present invention proves that first ITGB4BP can suppress fibrocyte, fibroblast, myofibroblast and produce collagen protein and (comprise I type, III type etc. in the cell in vitro culture systems, this paper has shown about the proteic data of type i collagen, has not shown the data about the III collagen type); Prove that first ITGB4BP can suppress the contraction of myofibroblast; Find the expression that ITGB4BP can reduce TGF-β 1, MMP9.Therefore, the present invention draws such conclusion: in fibrotic disease, the low expression of ITGB4BP may be the important initiating agent of a regulation and control fibroblast phenotype conversion and changing function, and ITGB4BP may be that a fibrosis with strong effect forms relevant negative tonal signal.Therefore the high expressed of ITGB4BP then is to have tangible anti-fibrosis effect.Therefore, ITGB4BP and expression vector thereof can be used to prevent and/or treat hypertrophic cicatrix disease or fibrosis lesion.

On the other hand, because the sequence high conservative of ITGB4BP gene in eukaryotic cell, it has kept conservative functional domain with the binding site of ITGB4BP and the natural or engineered protein derivant of functional part performance biological action, have similar function, also can be used to prevent and/or treat hypertrophic cicatrix disease or fibrosis lesion.Especially, binding site and funtion part with ITGB4BP, and behind high expressed, can suppress the expression of the relevant index of fibrosiss such as TGF-β 1, MMP9 or type i collagen, or the ITGB4BP derivant that is suppressed to the expression of fibrocyte surface marker α-SMA comprises within the scope of the invention all.

Therefore, in a preferred embodiment of the invention, the invention provides a kind of pharmaceutical composition or test kit that is used to prevent and/or treat hypertrophic cicatrix disease or fibrosis lesion, described pharmaceutical composition or test kit comprise ITGB4BP or derivatives thereof for the treatment of effective dose or recombinant gene expression vector and the pharmaceutical excipient or the carrier etc. of expressing the ITGB4BP or derivatives thereof.

In another preferred embodiment of the present invention, described pharmaceutical composition or test kit also comprise the cicatrix that becomes known at present or the inhibitor of fibrosis lesion.

Therefore, the present invention also provides a kind of method that prevents and/or treats hypertrophic cicatrix and fibrosis lesion, described method comprises recombinant gene expression vector or aforementioned pharmaceutical compositions or the test kit to the ITGB4BP or derivatives thereof of experimenter's administering therapeutic effective dose or expression ITGB4BP or derivatives thereof, preferably, described experimenter is the human or animal.

It should be appreciated by those skilled in the art that described pharmaceutical composition or test kit can arrive cicatrix place or fibrosis place by topical, also can use by conventional methods of application such as oral, intravenous injection, intramuscular injections.Those skilled in the art can determine the ITGB4BP or derivatives thereof respectively or express the recombinant gene expression vector of ITGB4BP or derivatives thereof and the dosage level of pharmaceutical composition by routine test.Should be appreciated that, concrete dosage level about any concrete experimenter depends on multiple factor, comprises the seriousness etc. of the disease specific of experimenter's age, body weight, general health state, sex, diet, time of application, route of administration and excretion rate, medication combined and experience treatment.In using the situation of described pharmaceutical composition, the ITGB4BP or derivatives thereof or express the ITGB4BP or derivatives thereof recombinant gene expression vector can with other cicatrix or fibrosis lesion inhibitor simultaneously or at interval certain hour use.

The recombinant gene expression vector that the present invention also provides ITGB4BP albumen or derivatives thereof, express the ITGB4BP or derivatives thereof is used for preventing and/or treating the application of the medicine or the test kit of hypertrophic cicatrix disease or fibrosis lesion in preparation.

In the present invention, described hypertrophic cicatrix includes but not limited to burn, scald, incision, chemical damage, skin injuries such as frostbite.Described fibrosis lesion comprises the pathologic fibrosis of various histoorgans, for example, but is not limited to skin histology fibrosis, pulmonary fibrosis, hepatic fibrosis or renal fibrosis etc.; Also comprise the pathologic contraction that causes by various fibrosiss, for example, but be not limited to the pathologic contraction of body surface and internal organs of the body, tissue etc.

At last, the present invention also provides a kind of method of the ITGB4BP of obtaining or derivatives thereof, can be cloned in suitable prokaryotic expression carrier, carrier for expression of eukaryon such as Yeast expression carrier or the virus expression carrier by gene recombination technology by the nucleotide of the ITGB4BP or derivatives thereof of will encoding, make up suitable recombinant expression carrier, transform or transfection is carried out in the corresponding host recombinant expressedly, carry out protein purification step acquisition ITGB4BP or derivatives thereof at last.In a preferred embodiment of the invention, will the encode nucleotide of ITGB4BP of described method is cloned in the virus expression carrier, in adenovirus pAdEasy-1 carrier, carries out recombinant expressed.

Those skilled in the art should be noted that ITGB4BP of the present invention or its gene of encoding is mainly derived from the people.

Description of drawings

From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

The expression of Fig. 1: ITGB4BP in normal skin and hypertrophic cicatrix fibroblast.

Fig. 2: suppress the ITGB4BP expression in the hypertrophic cicatrix fibroblast after, the expression of TGF-β 1, MMP9 and type i collagen raises in the culture supernatant.2A: effective slow virus that screening ITGB4BP gene disturbs usefulness, 1#, 2# and 3# represent slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi1#/2#/3# respectively; 2B: after in the hypertrophic cicatrix fibroblast, suppressing the ITGB4BP expression, the detection of the expression of TGF-β 1, MMP9 and type i collagen in the culture supernatant, *: P=0.0001, * *: P=0.005, * * *: P=0.03.

Fig. 3: the detection of expression of TGF-β 1, MMP9 and type i collagen in the cells and supernatant behind the ITGB4BP high expressed in the normal skin fibroblast, *: P=0.038, * *: P=0.0005, * * *: P=0.007.

Fig. 4: ITGB4BP expresses the detection of expression of α-SMA in the back cell that raises in the hypertrophic cicatrix fibroblast.The 4A:mRNA horizontal detection; 4B: protein level detects; 4C: the protein level analysis, 1 is EGFP, 2 is ITGB4BP-EGFP.

Fig. 5: ITGB4BP expresses the detection of expression of TGF-β 1 in the back cell that raises in the hypertrophic cicatrix fibroblast.The 5A:mRNA horizontal detection; 5B: protein level detects; 5C: the protein level analysis, 1 is EGFP, 2 is ITGB4BP-EGFP.

Fig. 6: on the mRNA level, ITGB4BP expresses the detection of expression of type i collagen in the back cell that raises in the hypertrophic cicatrix fibroblast.

Fig. 7: myofibroblast contractile function detection behind the ITGB4BP high expressed in the hypertrophic cicatrix fibroblast.7A, the three dimensional gel model, left side figure is ITGB4BP-EGFP, right figure is EGFP; 7B, the shrinkage index analysis, P=0.008,1 is ITGB4BP-EGFP, 2 is EGFP.

Fig. 8: plasmid map.8A，pEGFP-N2；8B，p18MD-18；8C，pShuttle-CMV；8D，pAdEasy-1；8E，pFIV-H1/U6-copGFP。

The specific embodiment

Come further to illustrate the present invention by the following examples.But should be appreciated that described embodiment is illustrational purpose, and be not intended to limit the scope of the invention and spirit.

Experimental apparatus that the present invention is used and material and source thereof are presented in the following table 1.

Experimental apparatus that table 1. the present invention is used and material and their source

The structure of embodiment 1. shuttle vectors

1.1 obtaining of target gene sequences

This experiment uses pEGFP-N2 and pITGB4BP-EGFP plasmid to carry out, and wherein pEGFP-N2 purchases that (article No.: 6081-1), pITGB4BP-EGFP is the plasmid of this laboratory structure in clontech company.The construction method of pITGB4BP-EGFP is: according to the sequential design primer of recombinant vector pACT2-ITGB4BP (liver cDNA library, U.S. BD Clontech company):

Forward primer pITGB4BP-EGFP-EcoRI (inserting the EcoRI restriction enzyme site):

5′-CAGAATTCATGGCGGTCCGAGCTTCGTT-3′；

Downstream primer pITGB4BP-EGFP-BamHI (inserting the BamHI restriction enzyme site):

5′-CAGGATCCCGGTGAGGCTGTCAATGAGGGAAT-3′。

With pACT2-ITGB4BP is that template is carried out the PCR reaction, and reagent is TaKaRa company product.Reaction system is as follows:

PACT2-ITGB4BP 1 μ l (being about 1 μ g)

10 * PCR buffer, 5 μ l

dNTP(2.5mmol/L) 4μl

MgCl ₂(25mmol/L) 3μl

Forward primer 0.5 μ l

Downstream primer 0.5 μ l

RTaq enzyme (5U/ μ l) 0.5 μ l

ddH ₂O 35.5μl

Final volume 50 μ l

Reaction condition: 94 ℃ of degeneration after 4 minutes 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 60 seconds, totally 30 circulations, 72 ℃ were extended 10 minutes then, obtained the purpose fragment ITGB4BP of about 750bp size.At first connect test kit (TaKaRa company) operating procedure, the PCR product is connected in the pMD1 8-T carrier, obtain the pMD18-T-ITGB4BP recombinant according to pMD1 8-T carrier.Utilize PCR product and the green fluorescence protein expression carrier pEGFP-N2 of EcoRI and BamHI double digestion ITGB4BP then, reaction condition is as follows:

PEGFP-N2 (or pMD18-T-ITGB4BP) 40 μ l (being about 2 μ g)

10 * H buffer, 5 μ l

EcoRI 2.5μl

BamHI 2.5μl

Final volume 50 μ l

Reaction condition: 37 ℃ of reaction 2h.Then, use 0.6% agarose gel to carry out electrophoresis, the purpose band is cut glue and glue recovery.Glue reclaims and adopts the DNA purification to reclaim test kit (available from U.S. Omega company), carries out the purification recovery by the description that supplier provides.Finally obtain the ITGB4BP fragment and the pEGFP-N2 linear plasmid fragment of EcoRI and BamHI double digestion.Utilize T4DNA ligase (Dalian Takara company) to connect glue respectively then and reclaim product, reaction condition is as follows:

PEGFP-N2 3.5 μ l (being about 0.5 μ g) after the recovery

ITGB4BP 12 μ l (being about 1 μ g) after the recovery

10 * T4 DNA connects buffer 2 μ l

T4 dna ligase 2.5 μ l

Final volume 20 μ l

Reaction condition: 16 ℃ are spent the night.Recombinant vector called after pITGB4BP-EGFP behind the structure.Conversion connects product to the competence bacillus coli DH 5 alpha, utilizes the kalamycin resistance screening positive clone, and picking list bacterium colony increases, and extracts plasmid and uses EcoRI+BamHI double digestion connection product, and identify the enzyme action result with 0.8% agarose gel electrophoresis.So far the pITGB4BP-EGFP vector construction is finished.

Utilize BglII and NotI (all available from Dalian Takara company) double digestion pEGFP-N2 and pITGB4BP-EGFP therefrom to obtain purpose fragment EGFP (the about 796bp of length) and ITGB4BP-EGFP (the about 1512bp of length) then, build up in the pShuttle-CMV (U.S. Abiogene company, the about 7.5kb of length) that same double digestion is handled.PEGFP-N2, pITGB4BP-EGFP and pShuttle-CMV enzyme action system separately is as follows:

PEGFP-N2 (or pITGB4BP-EGFP or

28 μ l (being about 2 μ g)

pShuttle-CMV

10 * H buffer, 4 μ l

0.1％BSA 4μl

Each 2 μ l of NotI, BglII

Final volume 40 μ l

Reaction condition: 37 ℃ of reaction 2h, 0.6% agarose gel carries out electrophoresis, and the purpose band is cut glue and glue recovery.Glue reclaims and adopts the DNA purification to reclaim test kit (available from U.S. Omega company), and method is undertaken by the description that supplier provides.Finally obtain EGFP, ITGB4BP-EGFP fragment and the pShuttle-CMV linear plasmid fragment of Bgl II and NotI double digestion.

1.2 the connection of shuttle vector

Utilize the T4 dna ligase to be connected among the same plasmid pShuttle-CMV after enzyme action, recovery respectively the purpose fragment EGFP and the ITGB4BP-EGFP that obtain after the previous step recovery, thereby obtain to contain the segmental shuttle vector of purpose.The two coupled reaction system separately is as follows respectively:

PShuttle-CMV 3.5 μ l (being about 0.5 μ g) after the recovery

EGFP after the recovery or ITGB4BP-EGFP 12 μ l (being about 1 μ g)

10 * T4 DNA connects buffer 2 μ l

T4 dna ligase 2.5 μ l

Final volume 20 μ l

Reaction condition: 16 ℃ are spent the night.

Connect the conversion and the amplification of product: according to normal intestinal bacteria method for transformation well known in the art, the competence e.colidh5 of product trans-utilization calcium chloride method preparation will be connected, utilize the kalamycin resistance screening positive clone, and positive colony is carried out plasmid amplification, evaluation and extraction recombiant plasmid.

The evaluation after 1.3 shuttle vector makes up

The connection product that previous step is obtained carries out the enzyme action evaluation with Xho I (available from Dalian Takara company), with difference called after pShuttle-CMV-ITGB4BP-EGFP of the recombinant shuttle plasmid carrier after successfully constructing and pShuttle-CMV-EGFP.The endonuclease reaction system of identifying is as follows:

PShuttle-CMV-ITGB4BP-EGFP or pShuttle-CMV-EGFP 5 μ l (being about 0.2 μ g)

10 * H buffer, 1 μ l

ddH ₂O 3.5μl

Xho?I 0.5μl

Final volume 10 μ l

Reaction condition: 37 ℃ of reaction 2h, 0.6% agarose gel electrophoresis identify whether structure is successful.

The homologous recombination of embodiment 2. adenoviruss

Reclaim after the shuttle vector that successfully constructs among the embodiment 1 utilized Pme I enzyme action, simultaneously it is converted into respectively among the competence escherichia coli BJ5183 that contains pAdEasy-1 plasmid (available from U.S. Qbiogene company) of Calcium Chloride Method preparation, finishes the homologous recombination of adenovirus.Concrete operations the contents are as follows:

2.1 the linearisation of shuttle plasmid:

Shuttle plasmid pShuttle-CMV-ITGB4BP-EGFP and pShuttle-CMV-EGFP that success is made up carry out enzyme action, recovery with Pme I (available from Britain NEB company).The two enzyme action system separately is as follows respectively:

PShuttle-CMV-ITGB4BP-EGFP or pShuttle-CMV-EGFP 18 μ l (being about 0.5 μ g)

NEB buffer 45 μ l

100×BSA 0.5μl

ddH ₂O 25.5μl

Pme?I 1μl

Final volume 50 μ l

Reaction condition: behind 37 ℃ of reaction 2h, 0.6% agarose gel electrophoresis is cut glue and glue and is reclaimed (method with among the embodiment 1 1.1).

2.2 the homologous recombination of adenovirus:

Utilize normal intestinal bacteria method for transformation well known in the art, the shuttle plasmid after the linearisation is converted into respectively among the escherichia coli BJ5183 of calcium chloride preparation of gland-containing virus pAdEasy-1 carrier.The positive bacterium colony of screening in the solid medium of kalamycin resistance.Petite in the picking from the large, medium and small three kinds of bacterium colonies that grow, after increasing, extract plasmid, utilize Pac I (available from Britain NEB company) that it is carried out enzyme action and identify that the adenovirus vector that reorganization is successful is called after pAdEasy-ITGB4BP-EGFP and pAdEasy-EGFP respectively.The enzyme action system of identifying is as follows respectively:

PAdEasy-ITGB4BP-EGFP or pAdEasy-EGFP 4 μ l (being about 0.1 μ g)

NEB buffer 12 μ l

100×BSA 0.2μl

ddH ₂O 1?3.5μl

Pac?I 0.3μl

Final volume 20 μ l

Reaction condition: behind 37 ℃ of reaction 1h, 0.6% agarose gel electrophoresis, Goldview nucleic acid staining dye manifest the band of 23kb and 3.0kb or 23kb and 4.5kb respectively, show that adenovirus vector recombinates successfully.

The packing and the amplification of embodiment 3. adenoviruss

3.1 adenovirus vector transfection HEK293 cell encapsidated adenovirus virus

3.1.1 the linearisation of adenovirus vector:

With Pac I enzyme action recombinant adenoviral vector pAdEasy-ITGB4BP-EGFP and pAdEasy-EGFP and recovery.The enzyme action system is as follows:

PAdEasy-ITGB4BP-EGFP or pAdEasy-EGFP 48 μ l (being about 1.0 μ g)

NEB buffer 18 μ l

100×BSA 0.8μl

ddH ₂O 21.7μl

Pac?I 1.5μl

Final volume 80 μ l

Reaction condition: behind 37 ℃ of reaction 1h, 0.6% agarose gel electrophoresis, the Goldview nucleic acid staining dye manifests the band of 23kb and 3.0kb or 23kb and 4.5kb respectively, two bands of above-mentioned 23kb (mainly comprising reorganization genes of interest pAdEasy-ITGB4BP-EGFP and pAdEasy-EGFP respectively) is carried out glue reclaim (method with among the embodiment 1 1.1).

3.1.2 the packing of adenovirus:

With 3 * 10 ⁵Cell HEK293 cell (purchasing the cell bank in the Chinese Academy of Sciences) is seeded in six orifice plates (U.S. Corning company), treats that cell begins transfection when reaching 80% fusion.Transfection method is as follows: get 8 μ g and mix room temperature, 5min with 250 μ l nonreactive DMEM (that is, not containing antibiotic DMEM) (available from U.S. Hyclone company) respectively in the adenoviral plasmid of 3.1.1 neutral lineization; Get 20 μ l liposomees and softly mix with 480 μ l nonreactive DMEM, room temperature, 5min is divided into 2 equal portions; To go up two step mixed liquors and mix, room temperature leaves standstill 20min; PBS adds plasmid liposome mixture after cleaning twice in 293 cells in six orifice plates; CO ₂Change fresh nonreactive DMEM (containing 10% calf serum), every hole 2ml behind 37 ℃ of cultivation 4hr in the incubator.Begin luciferase expression situation in the observation of cell after continuing to cultivate 24h.

3.2 the collection of adenovirus and amplification:

Behind the HEK293 cell transfecting the 6th day, fluorescence is more intense, and cell is suspended state fully, with aseptic suction nozzle cell is blown and beaten, be collected in the EP pipe (available from U.S. Axygen company), multigelation is 3 times in-80 ℃ and 37 ℃, 12, the supernatant that obtains behind the 000rpm, 4 ℃ of centrifugal 15min is former generation virus liquid, called after Ad-ITGB4BP-EGFP and Ad-EGFP respectively.Subsequently viral liquid is continued to infect 293 cells, profit uses the same method, and its 3-4 is standby for viral liquid in collection.

The structure and the packing of embodiment 4. slow virus interference carriers

The slow virus interference carrier pFIV-H1/U6-copGFP-ITGB4BPRNAi of required ITGB4BP gene is available from company of Chongqing Jin Mai Bioisystech Co., Ltd (address: TianXing, Shapingba District, Chongqing City bridge converge 3-11) goldenly in the present embodiment, as the interference sequence of negative control also available from the said firm.Slow virus difference called after: slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi1#/2#/3# and negative control with said two devices acquisition after transfection 293FT cell packing.

The candidate segment that RNA selects for use when disturbing screening is respectively:

ITGB4BP?RNAi1#-1：AAAG?GGGCTGGTGCATCCCAAGA

ITGB4BP?RNAi1#-2：AAAA?TCTTGGGATGCACCAGCCC

ITGB4BP?RNAi2#-1：AAAG?GGCTCAGAGAACTTCTACA

ITGB4BP?RNAi2#-2：AAAA?TGTAGAAGTTCTCTGAGCC

ITGB4BP?RNAi3#-1：AAAG?CAGCCTCCCAGACACAGTG

ITGB4BP?RNAi3#-2：AAAA?CACTGTGTCTGGGAGGCTG

Negative control RNAi-1:AAAG TCGACTCAGTCGGGTGGCA

Negative control RNAi-2:AAAA CTGCTATCGAGCCTGGCGA

The structure of slow virus carrier is undertaken by the pFIV--H1/U6-copGFP siRNA of SBI company test kit (Cat:#SI111A-1) operating instruction.

4.1 the structure of slow virus interference carrier pFIV-H1/U6-copGFP-ITGB4BPRNAi

With above-mentioned candidate segment according to the connection of annealing of following ratio.Reaction system is as follows:

Upstream fragment 2.5 μ l (being about 2.5 μ g)

Downstream fragment 2.5 μ l (being about 2.5 μ g)

2 * annealing buffer, 25.0 μ l

ddH ₂O 20.0μl

Final volume 50.0 μ l

Reaction condition: 5 minutes postcooling of 95 ℃ of reactions are to room temperature.

Fragment with successful connection is connected with pFIV-H1/U6-copGFP then.Reaction system is as follows:

Candidate segment 1 μ l (being about 0.01 μ g)

PFIV-H1/U6-copGFP 2.5 μ l (being about 0.5 μ g)

10 * T4 DNA connects buffer 1 μ l

T4 dna ligase 1 μ l

ddH ₂O 4.5μl

Final volume 10 μ l

Reaction condition is as follows: 16 ℃ are spent the night.

Connect the conversion and the amplification of product: according to normal intestinal bacteria method for transformation well known in the art, the competence e.colidh5 of product trans-utilization calcium chloride method preparation will be connected, utilize the amicillin resistance screening positive clone, and positive colony is carried out plasmid amplification, evaluation and extraction recombiant plasmid.

4.2 the packing of slow virus interference carrier pFIV-H1/U6-copGFP-ITGB4BPRNAi

With 4 * 10 ⁵Cell 293FT cell (purchasing the cell bank in the Chinese Academy of Sciences) is seeded to 75cm ²In the culture bottle (Shanghai City ten thousand red glass instrument plants), treat that cell begins transfection when reaching 70% fusion.Transfection method is as follows: get 2.5 μ g envelope protein plasmid pVSV-G, 7.5 μ g packaging plasmid pFIV34N and the above-mentioned expression plasmid that successfully constructs of 2 μ g add 155 μ l2M CaCl again at ddH2O (cumulative volume 1095 μ l) mixing ₂And then slowly add 1250 μ l, 2 * PBS mixing.Behind twice in the PBS cleaning 293FT cell, add said mixture; CO ₂Change fresh nonreactive DMEM (10% calf serum), every bottle of 10ml behind 37 ℃ of cultivation 7hr in the incubator.Green fluorescence appears in cell after continuing to cultivate 48h.The collecting cell supernatant, 3000rpm behind 4 ℃ of centrifugal 5min, is our needed slow virus liquid after getting the pvdf membrane sucking filtration of supernatant with 0.45 μ m.

The separation and the cultivation of embodiment 5. skin flbroblast of former generation

The foreskin normal fibroblast is from Xinan Hospital, Chongqing Urology Surgery (through patient's written consent), and the hypertrophic cicatrix fibroblast is from the Xinan Hospital, Chongqing Department of B urn, picks up from the patient's (through patient's written consent) in back 1 year of the burn.

Former generation, skin separated with cultural method as follows with hypertrophic scar-derived fibroblasts: piece of tissue is put into sterile petri dish, and PBS flushing three times is removed epidermis and subcutaneous tissue with sterile scissors, shreds dermal tissue; Tissue after shredding is put into 25ml taper culture bottle, adds 0.5% trypsin available from U.S. Hyclone company) 10ml, room temperature, 2h slightly vibrates; Add the DMEM (available from U.S. Hyclone company) that 10ml contains 10% calf serum (available from Chengdu Harris Corp) and stop digestion, suspension is discarded fragment of tissue after by aseptic strainer filtering; Centrifugal, 800rpm 10min abandons supernatant, and the DMEM that adding 10ml contains 10% calf serum washes recentrifuge two times; After abandoning supernatant, re-suspended cell contains among the DMEM of 10% calf serum in 10ml, moves to 75cm ²In the culture bottle (available from Shanghai City ten thousand red glass instrument plants), 37 ℃, 5%CO ₂Cultivate in the incubator, change culture medium behind the 24h, remove not attached cell simultaneously.After treating that cell covers with fully, the cultivation of can going down to posterity.Select for use 6-10 for fibroblast during experiment.

5.1 the extraction of normal skin and hypertrophic cicatrix fibroblast RNA

Concrete steps are: get the 8th generation fibroblast (cultivation of six orifice plates), discard culture medium; Every hole adds 200 μ l Tripure, and (available from Switzerland Luo Shi (Roche) company, catalog number (Cat.No.) is: 11667157001), scrape all cells and collection on the ice chest behind the placement 5min; (chloroform: Tripure, volume ratio v/v) added 40 μ l chloroform, and thermal agitation 15sec places 10min on the ice chest by 1: 5; Every then hole adds 200 μ l Tripure, places 5min on the ice chest; 4 ℃, 12000g, centrifugal 15min; Shift the upper strata water, add isopyknic isopropyl alcohol, mixing is placed 10min on the ice chest; 4 ℃, 12000g, centrifugal 10min; Abandon supernatant, add 75% ethanol (dilution of 1 ‰ DEPC water), 500 μ l washing precipitations; 4 ℃, 7500g, centrifugal 5min; After treating that ethanol is evaporated completely under the room temperature, the 1 ‰ DEPC water dissolutioies precipitation that adds 10 μ l is RNA; RNA is carried out concentration determination, and-80 ℃ frozen.

5.2 extract the reverse transcription reaction behind the RNA

The RNA that utilizes previous step to extract carries out reverse transcription reaction for template.The reverse transcription reaction system is as follows:

5.1 the about 1 μ g of the RNA of middle extraction

AMV reverse transcription 1 μ l

RNA enzyme inhibitor 0.5 μ l

DNTP mixed liquor 2 μ l

10 * RT buffer, 2 μ l

Oligo dT-junctional complex primer 1 μ l

MgCl ₂ 4μl

RNA 1μg

DdH ₂O completion to 20 μ l

Final volume 20 μ l

Reaction condition: 50 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min, 4 ℃ of preservations;

5.3 real-time fluorescence quantitative PCR detects (Real-time PCR)

The real-time PCR reactions system is as follows:

RT reactant 2 μ l

SYBR Green PCR in real time Master Mix 10 μ l

Forward primer (10nM) 0.4 μ l

Reverse primer (10nM) 0.4 μ l

ddH ₂O 7.2μl

Final volume 20 μ l

Primer sequence used herein is:

ITGB4BP: forward primer: 5 '-CCCAACAATACCACCGACCAGGA-3 '

Reverse primer: 5 '-GCCCTCCCTGATTGCTGAAGACA-3 '

GAPDH: forward primer: 5 '-GGGGAAGGTGAAGGTCGGAGTC-3 '

Reverse primer: 5 '-TCGCTCCTGGAAGATGGTGATG-3 '

Reaction condition: 95 ℃ of degeneration 2min; 95 ℃ of degeneration 15 seconds, 55 ℃ of annealing 35 seconds, 72 ℃ were extended totally 40 circulations 31 seconds.

Carry out signals collecting and utilize 7500 systems soft wares to analyze at 72 ℃, the result is presented among Fig. 1.

Embodiment 6.ITGB4BP gene disturbs the screening with effective slow virus

Because constructed slow virus interference carrier is not all to have interference effect, must select interference and experimentize for the most effective group through screening.

Screening uses 24 orifice plates (U.S. Coming company) to experimentize, and every hole fibroblasts from hypertrophic scars number is about 5 * 10 ³Individual, every hole is added in the slow virus 200 μ l that obtain among the embodiment 4, and pair cell utilizes the real-time fluorescence quantitative PCR technology for detection to infect the mRNA expression of ITGB4BP in slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi1#/2#/3# and the negative control infected group after four days.

Used primer sequence is (with embodiment 5):

ITGB4BP: forward primer: 5 '-CCCAACAATACCACCGACCAGGA-3 '

Reverse primer: 5 '-GCCCTCCCTGATTGCTGAAGACA-3 '

GAPDH: forward primer: 5 '-GGGGAAGGTGAAGGTCGGAGTC-3 '

Reverse primer: 5 '-TCGCTCCTGGAAGATGGTGATG-3 '

Concrete steps are: observing fibroblasts from hypertrophic scars, to infect behind the slow virus luciferase expression about 50%, and cellular morphology is spindle shape, and negative control group is not seen fluorescence, discards culture medium; Use Tripure, be extracted into fibrocellular RNA by 5.1 method among the embodiment 5, carry out reverse transcription reaction with the RNA that extracts as template, the primer of reverse transcription, reaction system and reaction condition are all identical with 5.2 reverse transcription reaction among the embodiment 5.Then, utilize resulting reverse transcription product as template, carry out PCR in real time according to 5.3 method among the embodiment 5 and detect, 5.3 used primers are identical among used primer and the embodiment 5, and reaction system is also identical with reaction condition.

Carry out signals collecting and utilize 7500 systems soft wares to analyze at 72 ℃, the result is presented among Fig. 2 A, and the effective slow virus of interference that screening at last obtains is slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi2#, is used for subsequent experimental.

Embodiment 7.ITGB4BP is the low detection of expression of expressing back fibrosis index of correlation in the hypertrophic cicatrix fibroblast

According to previous studies show that, cell will be in cells and supernatant eccrine fiber associated protein such as TGF-β 1, type i collagen and MMP-9, by detecting these proteic secreting, expressings, the expression that can understand them changes.Utilize the changes of expression level of these fibrosis associated protein, can infer the formation situation of cicatrix.

ITGB4BP in the employing ELISA method detection hypertrophic cicatrix fibroblast is suppressed the expression of extracellular matrix protein TGF-β 1, type i collagen and MMP-9 in the culture medium supernatant of back on the mRNA level.

In brief, proved that in embodiment 6 slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi2# can effectively suppress the expression of the ITGB4BP mRNA in the fibroblasts from hypertrophic scars.Fibroblasts from hypertrophic scars gone down to posterity to experimentize in 24 orifice plates (U.S. Corning company), and every hole fibroblasts from hypertrophic scars number is about 5 * 10 ³Individual, every hole adds the slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi2#200 μ l that is packaged to be according to embodiment 4 methods, adds the DMEM 800 μ l that contain 10% calf serum, and 37 ℃, 5%CO ₂Cultivate in the incubator, collect the fibroblasts from hypertrophic scars supernatant of cultivating after four days, utilize ELISA to detect the content of TGF-β 1, type i collagen and MMP-9 in the supernatant.

ELISA detects the experimental procedure (description of enclosing with reference to the people TGF-β 1ELISA detection kit of U.S. RB company) of TGF-β 1: add 100 μ l doubling dilutions (promptly, serial dilution, the high concentration standard substance serial dilution of 2000ng/ml is become 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.6ng/ml, standard substance 0ng/ml) are in corresponding Sptting plate, and are duplicate; Add 100 μ l samples (i.e. experiment grouping: fibroblasts from hypertrophic scars+ITGBRBP RNAi, and fibroblasts from hypertrophic scars+negative control) successively, totally 4 holes; Mixing is 30 seconds gently, seals plate hole with preservative film, 37 ℃ of incubation 60min; Wash plate, get rid of liquid in the most plate,, and remove water droplet (in thick folded absorbent paper, patting dry) with cleaning mixture washing reaction plate (adding 350 μ l cleaning mixture in every hole); Cyclic washing 5 times; Every hole adds 100 μ l, 1 * biotin (test kit is with).Mixing is 30 seconds gently, seals plate hole, 37 ℃ of incubation 60min; Wash plate, get rid of liquid in the most plate,, and remove water droplet (in thick folded absorbent paper, patting dry) with cleaning mixture washing reaction plate (adding 350 μ l cleaning mixture in every hole); Cyclic washing 5 times; Every hole adds 100 μ l, 1 * HRP (test kit is with).Mixing is 30 seconds gently, seals plate hole, 37 ℃ of incubation 30min; Wash plate, get rid of liquid in the most plate,, and remove water droplet (in thick folded absorbent paper, patting dry) with cleaning mixture washing reaction plate (adding 350 μ l cleaning mixture in every hole); Cyclic washing 5 times; Every hole adds 100 μ l TMB colour developing liquid, and mixing is 10 seconds gently, 37 ℃ of dark place incubations 25 minutes (15 ± 10 minutes); Every hole adds 100 μ l stop buffers (test kit is with).Mixing is 30 seconds gently; Utilize 680 type microplate reader to read the OD value in 30 minutes at the 450nm place.Determine the concentration of TGF-β 1 according to standard substance.

Here should be noted that, people TGF-β 1 detection kit, people's type i collagen detection kit and the people MMP-9 detection kit of U.S. RB company can be used for detecting the different indexs in the same sample, particularly, in this experiment, with a sample classify in three categories a part a, b and c, a is used for TGF-β 1 detection kit and detects TGF-β 1, and b is used for type i collagen detection kit detection type i collagen and c is used for MMP-9 detection kit detection MMP-9.Three kinds of used reagent of detection are test kit and are with, and the step of detection is identical with the above-mentioned step of utilizing people TGF-β 1 detection kit to detect TGF-β 1.

The expression of fibrosis index of correlation mensuration behind the ITGB4BP high expressed in embodiment 8. normal fibroblasts

Use 24 orifice plates to experimentize, every hole normal skin fibroblast number is about 5 * 10 ³Individual, every hole is added in adenovirus Ad-EGFP and each the 200 μ l of Ad-ITGB4BP-EGFP that obtain among the embodiment 3, adds the DMEM 800 μ l that contain 10% calf serum, and 37 ℃, 5%CO ₂Cultivate in the incubator, utilize ELISA to detect the content (method uses people TGF-β 1 detection kit, people's type i collagen detection kit and the people MMP-9 detection kit of U.S. RB company to carry out respectively with embodiment 7) of TGF-β 1, type i collagen and MMP-9 in the supernatant after five days.

The expression of fibrosis index of correlation mensuration behind the ITGB4BP high expressed in the embodiment 9. hypertrophic cicatrix fibroblasts

9.1mRNA the expression of horizontal detection α-SMA, TGF-β 1 and type i collagen:

Use six orifice plates to experimentize, every hole hypertrophic cicatrix fibroblast number is about 8 * 10 ⁴Individual, every hole is added in adenovirus Ad-EGFP and each the 600 μ l of Ad-ITGB4BP-EGFP that obtain among the embodiment 3, adds the DMEM 2400 μ l that contain 10% calf serum, and 37 ℃, 5%CO ₂Cultivate in the incubator, utilize real-time fluorescence quantitative PCR to detect the expression (experimental technique of RNA extraction, reverse transcription reaction and real-time fluorescence quantitative PCR is with embodiment 5) of α-SMA, TGF-β 1 and type i collagen in the cell after five days.

Used primer sequence is:

TGF-β 1: forward primer: 5 '-TGGAAACCCACAACGAAATCTATGA-3 '

Reverse primer: 5 '-TGGAAACCCACAACGAAATCTATGA-3 '

Type i collagen: forward primer: 5 '-tcccaccaatcacctgcgtaca-3 '

Reverse primer: 5 '-cgccggtggtttcttggtcg-3 '

α-SMA: forward primer: 5 '-cggctttgctggggacgat-3 '

Reverse primer: 5 '-caggggcaacacgaagctcat-3 '

9.2 protein level detects the expression of TGF-β 1 and α-SMA:

Use six orifice plates to experimentize, every porocyte number is about 8 * 10 ⁴Individual, every hole is added in adenovirus Ad-EGFP and each the 600 μ l of Ad-ITGB4BP-EGFP that obtain among the embodiment 3, adds the DMEM 2400 μ l that contain 10% calf serum, and 37 ℃, 5%CO ₂Cultivate in the incubator, utilize western blotting to detect the expression of TGF-β 1 and α-SMA in the cell after five days.

9.2.1 the proteic extraction of hypertrophic cicatrix fibroblast (RIPA method):

Concrete steps are: handle cell on ice, behind the PBS washed cell of pre-cooling, every hole adds 200 μ lRIPA lysis (containing 1%PMSF) liquid, scrapes repeatedly on ice with the cell curet and gets cell, moves into a clean 1.5ml EP pipe, places 30min on ice; The ice-bath ultrasonic cracking, 4 ℃ of 14000rpm * 30min move into the supernatant after centrifugal in the new 1.5mlEP pipe; The BCA method is measured protein concentration (with reference to the U.S. BCA of Pierce company determination of protein concentration test kit description).Use ddH ₂O adjusts the concentration of sample to suitable concentration, adds 5 * SDS-PAGE sample-loading buffer; Albumen boils 5min; Last sample 20 μ l/ holes; SDS-PAGE electrophoresis, electrophoresis are through with and take off gel and put into electricity and change buffer and soak 20min, soak 6 filter paper simultaneously, and 1 pvdf membrane is installed " sandwich " structure, from the negative pole to the positive pole: three metafiltration paper-gel-pvdf membrane-three metafiltration paper, constant current 0.8Ma/cm ², electricity changeed 1.5 hours.Taking out PVDF room temperature in the TBST that contains 5% defatted milk powder (1 ‰ Tween20) confining liquid sealed 2 hours; Add one anti-(mouse anti human beta-actin monoclonal antibody 1: 400, mouse anti human TGF-β 1 monoclonal antibody 4 μ g/ml, mouse anti human α-SMA monoclonal antibody 1: 100) 4 ℃ of night incubation; Wash film 1 ‰ TBST * 10min * 3 time; Added two anti-(goat anti-mouse igg of peroxidase labelling, 1: 2000) incubated at room 1 hour; Wash film 1 ‰ TBST * 10min * 3 time; Develop, add each 500 μ l of chemical luminescence for liquid A liquid B liquid (be test kit carry) of U.S. Santa Cruz company after black out develop.

SDS-PAGE running gel system:

Separation gel (12%), 80V electrophoresis cumulative volume 5ml spacer gel (5%), 150V electrophoresis cumulative volume 2ml

30% acrylamide 2ml, 30% acrylamide 0.33ml

1.5mol/L?Tris-HCL 1.3ml 1.5mol/L?Tris-HCL 1.3ml

10％SDS 0.05ml 10％SDS 0.02ml

10% Ammonium persulfate. 0.05ml, 10% Ammonium persulfate. 0.02ml

TEMED 0.002ml TEMED 0.002ml

ddH ₂O 1.6ml ddH ₂O 1.4ml

Observe the influence of ITGB4BP 9.3 utilize the FPCL model to the fibroblast biological behaviour

The making of FPCL model: get and infect adenovirus Ad-EGFP and the hypertrophic cicatrix fibroblast of Ad-ITGB4BP-EGFP after 5 days, with the DMEM re-suspended cell of using serum-free behind 0.25% trypsinization, preparation cell suspension, and the adjustment cell density is 1 * 10 ⁶Individual cell/ml.By cell suspension: 5 * DMEM: the volume ratio of Mus tail collagen=1: 2: 7 mixes above-mentioned various composition, is specially and mixes Mus tail collagen and 5 * DMEM earlier on ice, and adjusting pH7.2 adds cell suspension again; Every hole added 3ml serum-free DMEM cultivation after incubator was placed 30min formation gel.Promptly obtain fibroblastic dimensional culture system (fibroblast-populated collagen lattice, FPCL).

Every group of each 3 FPCL.Observe, write down the vary in diameter of FPCL on the 8th day, calculate shrinkage index.FPCL shrinkage index computing formula is as follows:

CI＝[1-(D/D ₀) ²]×100％

Wherein, CI: shrinkage index; D: the diameter of collagen gel piece; D ₀: gel piece initial diameter (22mm).

In addition, should be noted that and in all embodiment of the present invention, carry out statistical analysis if desired that then The data SPSS 13.0 softwares are carried out the independent sample t check, P≤0.05 is considered to have statistical significance.

Result and discussion

1.ITGB4BP low expression the in the hypertrophic cicatrix fibroblast

The testing result of embodiment 5 is presented among Fig. 1.Fig. 1 has shown that ITGB4BP can express in normal skin and hypertrophic cicatrix fibroblast, it represents original relative quantification (the raw relative quantitation of mRNA expression, hereinafter to be referred as original RQ) value is respectively 1 and 0.704, and ITGB4BP low expression in the hypertrophic cicatrix fibroblast is described.This shows that the low expression of ITGB4BP may be relevant with excessive fibrosis in the hypertrophic cicatrix, i.e. the low expression of ITGB4BP has increased the weight of the fibrosis of hypertrophic cicatrix.

2. in the hypertrophic cicatrix fibroblast, suppress ITGB4BP and express, can increase the weight of the fibroid of cicatrix

The result of the corresponding embodiment 6 of Fig. 2 A, show: slow virus-FIV-H1/U6-copGFP-ITGB4BPRNAi2# is best to the interference effect of ITGB4BP in the mRNA level.Particularly, negative control group is 1.00 original RQ on the mRNA level, and the 1# slow virus carrier is 2.068 original RQ to the ITGB4BP expression, and ITGB4BP is not had interference effect; The 2# carrier is 0.025 original RQ to the ITGB4BP expression, and suppression ratio reaches 99%, and interference effect is promptly arranged most; The 3# carrier is 0.253 original RQ to the ITGB4BP expression, and suppression ratio is about 75%.The result of the corresponding embodiment 7 of Fig. 2 B shows, in the hypertrophic cicatrix fibroblast, after suppressing the ITGB4BP expression on the mRNA level, TGF-β 1, MMP9 and type i collagen are expressed rising in the hypertrophic cicatrix fibroblast, and fibrosis increases the weight of.Compare with negative control, TGF-β 1 expression raises about 5 times, and MMP9 raises about 4.5 times, type i collagen raises about 6 times, show that on the mRNA level expression of can obviously raise after ITGB4BP suppresses fibrosis index of correlation such as TGF-β 1, MMP9 and type i collagen obviously increases the weight of fibrosis.

3. in normal skin fibroblast, increase ITGB4BP and express, can suppress fibroid

The result of embodiment 8 is presented among Fig. 3.The result shows that in normal skin fibroblast, after ITGB4BP mRNA expressed and raises, TGF-β 1, MMP9 and type i collagen were expressed reduction in normal skin fibroblast.Particularly, TGF-β 1 reduces about 2 times, and MMP9 reduces about 4.5 times, and type i collagen reduces about 6 times, promptly can obviously suppress the expression of fibrosis index of correlation such as TGF-β 1, MMP9 and type i collagen behind the ITGB4BP high expressed, suppresses fibrosis.

The concrete data of Fig. 2 and Fig. 3 correspondence are as follows:

Table 2.1 TGF-β 1 standard substance concentration and corresponding OD ₄₅₀Detected value

According to TGF-β 1 standard substance concentration (x) and its corresponding OD ₄₅₀Value (y) is carried out regression analysis (table 2.1), selecting for use the conic model match to get correlation coefficient (R) value is 1.000, F value=3464.810 in the variance analysis, significance probability SignifF=0.000＜0.05, the quadratic function relation that has highly significant between these two variablees is described, the quadratic curve equation formula of match is y=0.079+0.001x-0.00000066x ², and get TGF-β 1 concentration and OD in view of the above ₄₅₀The standard curve of value.

Table 2.2 fibroblast generates the OD of TGF-β 1 under the different stimulated condition ₄₅₀Detected value (detection of people TGF-β 1 ELISA detection kit)

The standard substance concentration of table 3.1 MMP-9 and corresponding OD ₄₅₀Detected value

Standard substance concentration (x) that obtains according to MMMP-9 and corresponding OD ₄₅₀Value (y) is carried out regression analysis (table 3), correlation coefficient (R) value of selecting for use the match of cubic curve model to obtain is 0.996, F value=154.414 in the variance analysis, significance probability SignifF=0.000＜0.05, the cubic function relation that has highly significant between these two variablees is described, its cubic curvilinear regression equation is y=0.106+0.003x-0.0000067x ²+ 0.00000000399x ³, get MMP-9 concentration and OD in view of the above ₄₅₀The standard curve of value.

Table 3.2 fibroblast generates the OD of MMP-9 under the different stimulated condition ₄₅₀Detected value (detection of people MMP-9 ELISA detection kit)

Table 4.1 type i collagen standard substance concentration and corresponding OD ₄₅₀Detected value

According to type i collagen standard substance concentration (x) and corresponding OD ₄₅₀Value (y) is carried out regression analysis (table 4.1), correlation coefficient (R) value of selecting for use the match of cubic curve model to obtain is 0.999, F value=577.326 in the variance analysis, significance probability Signif F=0.000＜0.05, the cubic curvilinear regression equation of match is y=0.086+0.002x-0.0000028x ²+ 0.00000000139x ³, and get type i collagen concentration and OD in view of the above ₄₅₀The standard curve of value.

Table 4.2 fibroblast generates the OD of type i collagen under the different stimulated condition ₄₅₀Detected value (detection of people's type i collagen protein ELISA detection kit)

4. in the hypertrophic cicatrix fibroblast, the high expressed of ITGB4BP has tangible anti-fibrosis effect

The experimental result of embodiment 9 is presented among Fig. 4-7.The result of Fig. 4 shows, in the hypertrophic cicatrix fibroblast, has suppressed the expression of myofibroblast surface marker α-SMA after ITGB4BP expresses and raises, that is, suppressed fibroblast and transformed to the phenotype of myofibroblast.Behind the high expressed, expression is 0.329 original RQ to ITGB4BP on the mRNA level of α-SMA in the hypertrophic cicatrix fibroblast, is lower than 1.00 original RQ of matched group, and promptly the suppression ratio of α-SMA is 67% (Fig. 4 A).The Western blotting experimental result of Fig. 4 B clearly illustrates that also the high expressed of ITGB4BP suppresses the expression of α-SMA on protein level, quantize further to show that the α-SMA protein level relative expression quantity of ITGB4BP high expressed group is 0.016, be lower than matched group 0.023, its suppression ratio is 30% (Fig. 4 C).

The result of Fig. 5 shows that in hypertrophic cicatrix fibroblast (or model), ITGB4BP has suppressed the expression of TGF-β 1 in the hypertrophic cicatrix fibroblast after expressing and raising.Behind the high expressed, expression is 0.3979 original RQ to ITGB4BP on the mRNA level of TGF-β 1 in the hypertrophic cicatrix fibroblast, is lower than 1.00 original RQ of matched group, and promptly the suppression ratio of TGF-β 1 is 60%.The Western blotting experimental result of Fig. 5 B clearly illustrates that also the high expressed of ITGB4BP suppresses the expression of TGF-β 1 on protein level, quantize further to show that the TGF-β 1 protein level relative expression quantity of ITGB4BP high expressed group is 0.008, be lower than matched group 0.069, its suppression ratio is 88% (Fig. 5 C).

The result of Fig. 6 shows that in hypertrophic cicatrix fibroblast (or model), ITGB4BP has suppressed the expression of type i collagen in the hypertrophic cicatrix fibroblast after expressing and raising.Behind the high expressed, expression is 0.480 original RQ to ITGB4BP on the mRNA level of type i collagen in the hypertrophic cicatrix fibroblast, is lower than 1.00 original RQ of matched group, and promptly the suppression ratio to type i collagen is 52%.

The result of Fig. 7 shows that in hypertrophic cicatrix fibroblast (or model), ITGB4BP suppresses the contraction of myofibroblast.Behind the high expressed, the shrinkage index of three dimensional gel is 0.23 to ITGB4BP in the hypertrophic cicatrix fibroblast, and matched group is 0.90, and promptly the contractility of gel descends about 74%.

From The above results as can be seen, in fibrotic disease, the low expression of ITGB4BP may be the important initiating agent of a regulation and control fibroblast phenotype conversion and changing function, and ITGB4BP may be that a fibrosis with strong effect forms relevant negative tonal signal.Therefore the high expressed of ITGB4BP then is to have tangible anti-fibrosis effect.Therefore, ITGB4BP and therapeutic gene expression carrier thereof can be used to prevent and/or treat hypertrophic cicatrix disease or fibrosis lesion.

Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, can carry out the variation of various forms and details therein, and not deviate from by the defined the spirit and scope of the present invention of accompanying Claim.

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8.Taylor?GA，Hudson?E，Resau?JH?et?al.Regulation?of?P311?expression?byMet-hepatocyte?growth?factor/scatter?factor?and?the?ubiquitin/proteasomesystem.J?Biol?Chem.2000；275(6)：4215-9.

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19.Sanvito?F，Vivoli?F，Gambini?S，et?al.Expression?of?a?highly?conservedprotein，p27BBP，during?the?progression?of?human?colorectal?cancer.CancerRes?2000；60(3)：510.

20.Si?K，Maitra?U.The?Saccharomyces?cerevisiae?homologue?of?mammaliantranslation?initiation?factor?6?does?not?function?as?a?translation?initiation?factor.Mol?Cell?Biol?1999；19(2)：1416.

21.Wood?LC，Ashby?MN，Grunfeld?C，Feingold?KR.Cloning?of?murinetranslation?initiation?factor?6?and?functional?analysis?of?the?homologoussequence?YPR016c?in?Saccharomyces?cerevisiae.J?Biol?Chem?1999；274(17)：11653.

22.Basu?U，Si?K，Warner?JR，Maitra?U.The?Saccharomyces?cerevisiae?TIF6gene?encoding?translation?initiation?factor?6?is?required?for?60S?ribosomalsubunit?biogenesis.Mol?Cell?Biol?2001；21(5)：1453.

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Sequence table

＜110〉Xinan Hospital, Chongqing

＜120〉ITGB4BP and derivant thereof are used to prevent and/or treat hypertrophic cicatrix and fibrosis lesion

<130>IB091679

<160>2

<170>Patent?In?version?3.1

<210>1

<211>245

<212>PRT

＜213〉people

<400>1

Met?Ala?Val?Arg?Ala?Ser?Phe?Glu?Asn?Asn?Cys?Glu?Ile?Gly?Cys?Phe

1 5 10 15

Ala?Lys?Leu?Thr?Asn?Thr?Tyr?Cys?Leu?Val?Ala?Ile?Gly?Gly?Ser?Glu

20 25 30

Asn?Phe?Tyr?Ser?Val?Phe?Glu?Gly?Glu?Leu?Ser?Asp?Thr?Ile?Pro?Val

35 40 45

Val?His?Ala?Ser?Ile?Ala?Gly?Cys?Arg?Ile?Ile?Gly?Arg?Met?Cys?Val

50 55 60

Gly?Asn?Arg?His?Gly?Leu?Leu?Val?Pro?Asn?Asn?Thr?Thr?Asp?Gln?Glu

65 70 75 80

Leu?Gln?His?Ile?Arg?Asn?Ser?Leu?Pro?Asp?Thr?Val?Gln?Ile?Arg?Arg

85 90 95

Val?Glu?Glu?Arg?Leu?Ser?Ala?Leu?Gly?Asn?Val?Thr?Thr?Cys?Asn?Asp

100 105 110

Tyr?Val?Ala?Leu?Val?His?Pro?Asp?Leu?Asp?Arg?Glu?Thr?Glu?Glu?Ile

115 120 125

Leu?Ala?Asp?Val?Leu?Lys?Val?Glu?Val?Phe?Arg?Gln?Thr?Val?Ala?Asp

130 135 140

Gln?Val?Leu?Val?Gly?Ser?Tyr?Cys?Val?Phe?Ser?Asn?Gln?Gly?Gly?Leu

145 150 155 160

Val?His?Pro?Lys?Thr?Ser?Ile?Glu?Asp?Gln?Asp?Glu?Leu?Ser?Ser?Leu

165 170 175

Leu?Gln?Val?Pro?Leu?Val?Ala?Gly?Thr?Val?Asn?Arg?Gly?Ser?Glu?Val

180 185 190

Ile?Ala?Ala?Gly?Met?Val?Val?Asn?Asp?Trp?Cys?Ala?Phe?Cys?Gly?Leu

195 200 205

Asp?Thr?Thr?Ser?Thr?Glu?Leu?Ser?Val?Val?Glu?Ser?Val?Phe?Lys?Leu

210 215 220

Asn?Glu?Ala?Gln?Pro?Ser?Thr?Ile?Ala?Thr?Ser?Met?Arg?Asp?Ser?Leu

225 230 235 240

Ile?Asp?Ser?Leu?Thr

245

<210>2

<211>738

<212>DNA

＜213〉people

<400>2

atggcggtcc?gagcttcgtt?cgagaacaac?tgtgagatcg?gctgctttgc?caagctcacc 60

aacacctact?gtctggtagc?gatcggaggc?tcagagaact?tctacagtgt?gttcgagggc 120

gagctctccg?ataccatccc?cgtggtgcac?gcgtctatcg?ccggctgccg?catcatcggg 180

cgcatgtgtg?tggggaacag?gcacggtctc?ctggtaccca?acaataccac?cgaccaggag 240

ctgcaacaca?ttcgcaacag?cctcccagac?acagtgcaga?ttaggcgggt?ggaggagcgg 300

ctctcagcct?tgggcaatgt?caccacctgc?aatgactacg?tggccttggt?ccacccagac 360

ttggacaggg?agacagaaga?aattctggca?gatgtgctca?aggtggaagt?cttcagacag 420

acagtggccg?accaggtgct?agtaggaagc?tactgtgtct?tcagcaatca?gggagggctg 480

gtgcatccca?agacttcaat?tgaagaccag?gatgagctgt?cctctcttct?tcaagtcccc 540

cttgtggcgg?ggactgtgaa?ccgaggcagt?gaggtgattg?ctgctgggat?ggtggtgaat 600

gactggtgtg?ccttctgtgg?cctggacaca?accagcacag?agctgtcagt?ggtggagagt 660

gtcttcaagc?tgaatgaagc?ccagcctagc?accattgcca?ccagcatgcg?ggattccctc 720

attgacagcc?tcacctga 738

Claims (10)

1. the recombinant gene expression vector of β 4 integration plain conjugated protein (ITGB4BP) and derivant thereof or expression ITGB4BP and derivant thereof is used to prevent and/or treat the application of hypertrophic cicatrix or fibrosis lesion.

2. the application of claim 1, wherein said ITGB4BP derivant is to keep the binding site of ITGB4BP and the protein derivatives of functional part, comprises the expression that keeps fibrosis indexs of correlation such as suppressing TGF-β 1, MMP9 or type i collagen or is suppressed to the protein derivatives of ability of the expression of fibrocyte surface marker α-SMA.

3. claim 1 or 2 application, wherein said hypertrophic cicatrix comprises skin injurys such as burn, scald, chemical damage, frostbite, incision.

4. claim 1 or 2 application, wherein said fibrosis lesion comprises the pathologic fibrosis of various histoorgans, comprises skin histology fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis etc.

5. the application of claim 4, wherein said fibrosis lesion comprise the various pathologic contractions that caused by fibrosis, comprise the pathologic contraction of body surface and internal organs of the body, tissue etc.

6. the application of claim 1, wherein in normal skin fibroblast behind the described ITGB4BP of high expressed, index TGF-β 1, the MMP9 that fibrosis is relevant and the expression of type i collagen significantly reduce, and suppress fibrosis.

7. the application of claim 6, wherein in the hypertrophic cicatrix fibroblast behind the described ITGB4BP of high expressed, myofibroblast surface marker α-SMA expression reduces, suppress of the conversion of hypertrophic cicatrix fibroblast, and suppress the contractility of myofibroblast to myofibroblast.

8. be used to prevent and/or treat the pharmaceutical composition or the test kit of hypertrophic cicatrix disease or fibrosis lesion, described pharmaceutical composition or test kit comprise ITGB4BP or derivatives thereof for the treatment of effective dose or recombinant gene expression vector and the pharmaceutical excipient or the carrier etc. of expressing the ITGB4BP or derivatives thereof.

9. the pharmaceutical composition of claim 8 or test kit, described pharmaceutical composition or test kit also comprise other cicatrix or the fibrosis lesion inhibitor for the treatment of effective dose.

10.ITGB4BP the or derivatives thereof or the recombinant gene expression vector of expressing the ITGB4BP or derivatives thereof are used for preventing and/or treating the application of the pharmaceutical composition or the test kit of hypertrophic cicatrix disease or fibrosis lesion in preparation.

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