CN110404060A - Thrombin inhibitor is preparing the purposes in antineoplastic invasion diversion medicaments - Google Patents

Abstract

The invention belongs to field of biotechnology, are related to the new pharmaceutical usage of thrombin inhibitor, and in particular to thrombin inhibitor especially divalent direct thrombin inhibitor is preparing the purposes in antineoplastic invasion diversion medicaments.The present invention uses the direct thrombin inhibitor small peptide DTIP with anticoagulating active to carry out cell assay in vitro and interior animal experiment, and test result is shown, the divalent direct thrombin inhibitor DTIP can inhibit the invasion and transfer of tumour in vitro；Experiment in vivo show divalent direct thrombin inhibitor DTIP on tumor development have influence into；Further, the direct thrombin inhibitor small peptide DTIP can prepare antineoplastic invasion diversion medicaments.

Description

Thrombin inhibitor is preparing the purposes in antineoplastic invasion diversion medicaments

Technical field

The invention belongs to field of biotechnology, are related to the new pharmaceutical usage of thrombin inhibitor, and in particular to fibrin ferment inhibits Agent especially divalent direct thrombin inhibitor is preparing the purposes in antineoplastic invasion diversion medicaments.

Background technique

Data shows that Cancer Mortality is high, seriously threatens human health, has caused to people's lives huge It is big to influence.According to statistics, China's lung cancer, breast cancer are in high incidence and high mortality；By taking lung cancer as an example, it is diagnosed as TNM stage Be no more than 48 months for the mean survival time (MST) of II phase or II the patient more than phase, with the raising of grade malignancy, life cycle also with Reduction.The diagnosing and treating of the malignant tumours such as lung cancer is one of the hot spot studied at present, wherein being directed to the prevention of lung cancer metastasis It is especially important with the research for the treatment of.

Studies have shown that malignant tumour occurs, development is extremely closely related with blood coagulation system, shows as malignant tumour evening Phase, the blood of patient are in hypercoagulative state, blood coagulation system high level activation.The tumor patient of clinical studies show, 4%-20% is suffered from Phlebothrombosis, and show this data up to 50% according to Autopsy Report.The ratio of studies have shown that tumor patient phlebothrombosis is higher by it He is 4.1 times of crowd；After chemotherapy, this coefficient rises to 6.5 times.Studies have reported that occurrence and development and the high blood coagulation shape of tumour State has bidirectional relationship, i.e. tumor tissues can be generated coagulation factor, cause high blood coagulation state；And the formation of thrombus can be with Promote the deterioration of tumour, forms vicious circle；It is related to grind although this various relationship is related to diversified mechanism The person of studying carefully thinks that anticoagulation medicine is likely to become a kind of attractive anti-cancer therapies.

Since Armand Trousseau elaborates between tumour and blood coagulation system several researchs there is after certain relationship Scholar attempts to illustrate the pathogenic mechanism of correlation between cancer and blood coagulation；But wherein still has and do not know place.90 years 20th century Dai Chuyou research reports effect of the fibrin ferment in Nasopharyngeal neoplasms for the first time, it is related study in by aortic endothelial cell and W256 cell is incubated for altogether, and the adherency of fibrin ferment and aortic endothelial cell and fibrinogen increases 50%- as the result is shown 300%；It in animal experiment, gives breast cancer (3503), after the mouse model thrombin injection of the carcinoma of the rectum (C26), display can promote The lung of cancer cell shifts；In addition, Proliferation of Human Ovarian Cell is transplanted in Mice Body, thrombin injection, the volume of tumour can increase And improve mouse death rate.

It is the proteinase activated receptors found at first that research, which discloses PAR-1, after finding PAR-1 in health adult tissue Soon, confirm that its presence, subsequent research are found in breast cancer, nasopharyngeal carcinoma, gastric cancer, melanocyte in human tumor cells There is PAR-1 expression in the tumour cells such as tumor, glioblastoma and osteosarcoma；Protease-Activated Receptor (PARs) is a kind of Transmembrane G protein coupled receptor (GPCR), by seven cross-film α spirals, cytoplasmic domains and extracellular N-terminal composition；Fibrin ferment and PAR- The end 1N LDP R41-S42 sequence combines, and cuts R41-S42 peptide bond, to activate PAR-1, induction transmembrane signal conducts (such as Fig. 1 It is shown), play major function in the process is the exosite I site and C-terminal of fibrin ferment.

It has now been found that fibrin ferment and PAR-1 have mediated intracellular signal transduction by different approaches, tumour is participated in The occurrence and development of cell；PAR-1 and gα protein (G α q, G α i and G α can be made by the PAR-1 conformation change of thrombin-mediated 12/13) it is coupled with G β γ；Mitogen-activated protein (MAP) kinases can be activated by the condensate that PAR-1 and G α q is formed, Improve Ca²⁺Concentration, and the complex of PAR-1 and G α 12/13 can be with activated G protein, Rho and Rac.PARs is also induced to be swashed with albumen Enzyme C and the relevant cascade reaction of tyrosine kinase；The condensate of PAR-1 and G β γ can activate 3 (PI3- of phosphatidyl inositol kinase K), adenyl cyclase (AC) can be inhibited with the compound of G α i；Studies have shown that activates PAR-1, and cell factor can be improved, The transcription of chemotactic factor (CF) and growth factor increases the expression of bioactivity lipid, adjusts cell Proliferation, apoptosis, adherency and migration, Promote tumour growth, invasion and transfer (as shown in Figure 2).

The transfer of studies have shown that tumor cell invasion is related to mesenchymal cell, extracellular matrix, blood and endothelial cell it Between complicated interaction；And fibrin ferment and its receptor, by being overexpressed inflammatory factor, adhere to and divide in tumor pathogenesis Son, angiogenesis factor and matrix degrading protease enzyme induce tumor cell proliferation, angiogenesis and transfer, research shows that fibrin ferment Can be expressed in tumor microenvironment independently of coagulation process, and can by activator protein enzyme activation receptor 1 (PAR-1) come Inducing cell signal transduction, PAR-1 are transmembrane G protein coupled receptor (GPCR), are swashed by unique proteolysis mechanism It is living.

It has also been found that fibrin ferment is the primary activation agent of PAR-1, fibrin ferment and PAR-1 have been mediated carefully by different approaches for research Signal transduction intracellular, participates in the occurrence and development of tumour cell, and fibrin ferment and PAR-1 interaction are mainly manifested in 3 sides Face (as shown in Figure 3)；Thrombin activation PAR-1, promotes the release of inflammatory factor, the increase of vasopermeability, platelet aggregation, Expression of Cell Adhesion Molecules up-regulation, extracellular matrix degradation promote the invasion of tumour cell；Fibrin ferment and PAR-1 are combined, and are promoted Angiogenesis.The growth and transfer of tumour are along with angiogenesis.And thrombin activation PAR-1, promote VEGF secretion, the latter lures New vessels in tumor microenvironment are led to be formed.Yamahata etc. proves that fibrin ferment is raised by the signal path that PAR-1 is mediated VEGF stimulates the proliferation and angiogenesis of people's glioma；Fibrin ferment and PAR-1 can activate NF- κ B signal access；In cancer of pancreas In, recombination thrombomodulin can be by blocking the activation of the PAR-1 and NF- κ B of thrombin induction to inhibit tumour growth； Tantivejkul etc. has found that in prostate gland cancer cell, thrombin activation PAR-1 promotes IL-6 by NF- κ B signal access, The expression of the inflammatory factors such as IL-8.

Thrombin inhibitor is current clinically most common anticoagulant anti-bolt drug.Research foundation based on the prior art, Present inventor is quasi- to provide thrombin inhibitor new pharmaceutical usage, and in particular to thrombin inhibitor especially divalent is straight It connects thrombin inhibitor and is preparing the purposes in antineoplastic invasion diversion medicaments.

Summary of the invention

It is mesh of the invention the Research foundation based on the prior art, provides thrombin inhibitor new pharmaceutical usage, has Body is related to thrombin inhibitor especially divalent direct thrombin inhibitor and is preparing the purposes in antineoplastic invasion diversion medicaments.

Specifically, the application provides the new medicinal use of the direct thrombin inhibitor small peptide DTIP with anticoagulating active On the way, especially DTIP is preparing the purposes in antineoplastic invasion diversion medicaments.

The Research foundation of foundation of the present invention has: the thrombin inhibitor clinically used at present is divided into two kinds, wherein a kind of For indirect thrombin inhibitor, the work of its inhibition thrombin activity of the effect competence exertion of some confactors must be passed through With, if heparin must be combined first with Antithrombin III (Anti-thrombin III, AT III), AT structure is made to change, To inhibit thrombin activity, the indirect thrombin inhibitor can only inhibit free fibrin ferment；Another kind is direct blood coagulation Enzyme inhibitor (Direct thrombin inhibitors, DTIs), such as lepirudin 023 ludon；DTIs and indirect thrombin inhibitor It compares, the activity of fibrin ferment can be inhibited directly in conjunction with fibrin ferment, not need the participation of AT and other confactors；DTIs is not Only there is inhibition function to the activity of free fibrin ferment, also there is inhibition to make non-free fibrin ferment (in conjunction with fibrin) With so its anticoagulation is stronger；It includes recombination leech that DTIs, which is broadly divided into divalent DTIs and unit price DTIs, divalent DTIs mainly, It is special in conjunction with fibrin ferment to increase it in conjunction with the outer binding site I of fibrin ferment for element and hirudin analog, first c-terminus Property, while making amido end in conjunction with thrombin activity center, play inhibiting effect；Monovalent DTIs mainly includes some small molecules Object is closed, the activated centre of fibrin ferment is directly inserted into, to inhibit thrombin activity.

The thrombin inhibitor that the present invention uses is the direct fibrin ferment suppression of divalent direct thrombin inhibitor anticoagulating active Preparation small peptide DTIP, the present invention have carried out cell assay in vitro and interior animal experiment, and test result is shown, the divalent is straight Thrombin inhibitor DTIP is met, the invasion and transfer of tumour can be inhibited in vitro；Experiment in vivo shows the direct fibrin ferment suppression of divalent Preparation DTIP on tumor development have influence into.

The present invention has carried out following cell assay in vitro and interior animal experiment:

In mouse subcutaneous xenograft tumor model, after injecting DTIP, the mouse transfer quantity that Lung metastases occur has Conspicuousness declines (as illustrated in figure 10 c)；Tumor tissues HE dyeing discovery, DTIP can inhibit the peripherad muscle of tumour cell or Person's fat invades (as shown in Figure 10 D)；Make the quantity of immunohistochemical observation intratumoral vasculature new life of CD31, discovery DTIP can be with Significantly inhibit the quantity (as shown in figure 10e) of tumour cell medium vessels new life；

In the present invention, its effect to proliferation of lung cancer cells, migration is studied using DTIP, the results show that DTIP is in vitro To proliferation of lung cancer cells without apparent inhibiting effect；Lung carcinoma cell is cultivated in vitro, and it is solidifying that a certain concentration is added in the medium Hemase, DTIP can inhibit lung carcinoma cell invasion and transfer, and fibrin ferment, which is added, cannot restore its invasion and transfer ability, show DTIP is by inhibiting fibrin ferment to play a role；

In the present invention, normal pulmonary epithelial cells BEAS-2B and lung cell A549 and 95D are detected by Western blot The expression of PAR-1 in cell, the results show that the expression of PAR-1 is significantly higher than BEAS-2B, immunohistochemistry in A549 and 95D As the result is shown in lung neoplasm tissue, PAR-1 expression is higher than normal lung tissue, consistent with cell results, meanwhile, experimental result is aobvious Show, the mRNA level in-site of PAR-1 is higher by other 3-10 times of non-high-transfer cell strain in lung cancer height invasion cell strain, shows PAR-1 Expression quantity it is related to cell invasion and transfer ability；

In the present invention, the nuclease based on CRISPR guiding has the advantages that simple, design is quick, and has been widely applied Gene editing etc. in organism, including mammality and primate, the present invention are knocked out in lung carcinoma cell by the technology PAR-1 gene, the results show that after knocking out lung carcinoma cell PAR-1 receptor, effect of the fibrin ferment to lung carcinoma cell invasion, transfer It is suppressed, shows that DTIP mainly plays a role to the inhibiting effect of invasion of lung cancer and transfer by PAR-1；

In mouse subcutaneous xenograft tumor model, successive administration one week, growth and increasing of the DTIP to tumour as the result is shown Length has no significant effect, consistent in vitro results；But the results show that DTIP can inhibit tumour cell to surrounding tissue Invasion；In mouse tumor metastasis models model, after tail vein injection lung carcinoma cell, successive administration one week, DTIP was given as the result is shown After medicine, the transfer that lung carcinoma cell is distally organized is suppressed, the results showed that, DTIP can inhibit the cancer of middle and advanced stage patient thin Dysuria with lower abdominal colic is moved；For infantile tumour patient, it can shift to an earlier date and kill tumour medicine or inhibit tumour growth drug combination, then not It is only capable of playing the effect of killing tumour, moreover it is possible to inhibit the invasion and transfer of tumour in advance；Further, the direct blood coagulation of the divalent Enzyme inhibitor can be used for preparing antineoplastic invasion diversion medicaments.

Advantages of the present invention has:

1. in vitro, DTIP can inhibit the invasion and transfer of lung carcinoma cell by the activity of inhibition fibrin ferment；

Expression of the 2.PAR-1 in lung carcinoma cell is significantly higher than normal pneumonocyte；After knocking out PAR-1, lung carcinoma cell Invasion and transfer ability are remarkably decreased, and after fibrin ferment is added, cannot significantly improve invasion and transfer ability again；

3. in mouse subcutaneous xenograft tumor model, DTIP can inhibit invasion of the lung carcinoma cell to surrounding tissue；It is small In mouse metastasis models, DTIP inhibits the transfer distally organized of lung carcinoma cell.

Detailed description of the invention

Fig. 1 .PAR-1 activates schematic diagram.

Fig. 2, effect of the fibrin ferment in tumor invasion, transfer process.

The signal transduction that Fig. 3, fibrin ferment and PAR-1 are mediated in tumour.

Fig. 4, DTIP do not have a significant impact to the proliferation of lung carcinoma cell.

Fig. 5, DTIP inhibit lung carcinoma cell invasion.

Fig. 6, DTIP inhibit lung carcinoma cell transfer.

The expression of Fig. 7, PAR-1 in lung carcinoma cell and tissue.

Fig. 8, electroresis appraisal LentiCRISPR-PXPR001 plasmid enzyme restriction result.

Fig. 9, after knocking out PAR-1, the invasion of lung carcinoma cell and transfer ability decline, fibrin ferment, which is added, cannot restore its invasion And transfer ability.

Figure 10, in mice lung cancer intravenous inoculation model, DTIP inhibits lung cancer metastasis.

Figure 11, in mouse subcutaneous xenograft tumor model, DTIP inhibits tumor cell invasion.

Specific embodiment

1 zoopery of embodiment

Include: using reagent

Key instrument includes:

Method includes:

Cell Growth Assays: I. takes 96 orifice plates, and the 1 × 10 of 200 μ L is added in every hole⁵/ mL cell suspension；II. proportionally Dosing, 50 50 μ g/mL of μ g/mL, DTIP of fibrin ferment 10U/mL, r-hirudin；III. 200 μ L serum-frees are used in blank well Culture medium compares；It after IV.24h, discards supernatant, 100 μ L MTT solution are added in every hole, and 37 DEG C of incubators place 4h；V. wavelength is used 490nm measurement.

Cell scratch (Wound Healing assay): 2 Well of Culture-Insert is placed into 24 orifice plates by I. In；II. the cell of 70 5 × 105/mL of μ L is added in Liang Ge culturing room, 37 DEG C of incubators place culture one day；III. will 2 Well element of Culture-Insert is gently taken out with tweezers；IV. serum free medium and different drug-treateds is added, It observes and takes pictures after 12h.

Transwell test: I. dilutes matrigel with serum free medium in 1: 3 ratio；II. cell is put into 24 In orifice plate, the matrigel dilution of 50 μ L of each small indoor addition is put into incubator and places 1h；III. cell is resuspended to 1-5 × 105/mL is inoculated with the cell suspension of 100 μ L in each cell；IV. in 10% serum free culture system of 500 μ L of lower indoor addition Drug-treated is added in upper and lower room in proportion in base；V.24h after, indoor culture medium is discarded；VI. cell is put solid in methyl alcohol Determine 30min；VII. violet staining 30min；After VIII.PBS is washed, observation and counting statistics after upper chamber are gently wiped with cotton swab.

PAR-1 is knocked out using CRISPR/Cas9 technology:

Design gRNA:I. finds target gene in Ensemble first, and point sequence, pink regions are exon, White area is introne；II. it replicates, is copied in ziFit part since first ATG of second exon；III. Purpose Target site is copied into retrieval (blast) in Ensemble, it is only available for 1hit as the result is shown；IV. plus viscosity End, as Oligos.

Plasmid enzyme restriction: I. is according to following system digestions

II. electroresis appraisal is run with 1%DNA glue；III. plasmid is recycled by plastic recovery kit, surveys concentration, -20 DEG C of preservations.

Annealing: I. designs PAR-1 gRNA, as follows:

II. according to following configuration schemes；

III.37℃30min；IV. after 95 DEG C of 5min of metal bath, metal bath is closed, room temperature is down to；

V. product 1: 200 is diluted.

Connection: linked system is configured as follows:

Conversion: I. is placed on ice after thawing from -80 DEG C of taking-up Stbl3 competence；II. 5 μ L connections is taken to produce in super-clean bench Object is mixed with Stbl3, is placed on 30min on ice；III. 42 DEG C of 90s of thermal shock；IV. it is placed on 5min on ice；V. 1mL is added, amp is not added LB culture medium, 37 DEG C in shaking table, 220rmp, 1h；VI.12000g is centrifuged 1min, discards supernatant, stays 100 μ L resuspended bacterium solutions, applies Cloth is on LB/Amp plate, constant temperature incubation 12-18h in 37 DEG C of incubators, observes bacterium colony growing state.

Slow virus packaging: I.293T cell density be 80-90% when, be changed to serum free medium DMEM (be not added FBS and PS)；II. plus LentiCRISPR-PXPR001/gRNA plasmid 8 μ g, PVSVG 6 μ g, PAX 24 μ g to 500 μ L free serum cultures In base, add 56 μ L of PEI (1mg/mL) (10cm dish, total plasmid: PEI=1: 4) into another 500 μ L serum free medium, 5min is stood in super-clean bench, then PEI is added in plasmid, 20min is stood in super-clean bench, is finally added to free serum culture In the 293T of base；Serum free medium is changed to the culture medium containing 10%FBS after III.6h；IV. become according to culture medium color Change, receive supernatant when can be used in 48h, be centrifuged, when 72h receives supernatant, is centrifuged, filtration sterilization after mixing, -80 DEG C of preservations.

Slow-virus transfection cell: I. when cell density be 80-90%, be added virus (viral liquid: complete medium=3: And polybrene (storing liquid concentration be 2mg/ml, 1000 ×) 1)；II. after cultivating for 24 hours, supernatant is abandoned, viral (virus liquid is added And polybrene body: complete medium=1: 1)；III. it is further cultured for for 24 hours, discarding supernatant, complete medium is added, makes cellular State is slowly restored；IV. puro (concentration concentration sieved before being all kills cell for that is, just 7 days) is added, screens 3-4 It, centre can change liquid；V. it discards supernatant, complete medium is added, restore cell state and passed on when growing to 80%, It after passage, can be screened according to cell state, then with puro, can suitably increase puro concentration.

Sequencing detection PAR-1 is knocked out: I. extracts cellular genome；II. it from the preceding 100pb design primer of gRNA, carries out PCR, system are as follows:

Process is as follows:

III. sequencing is sent to after electroresis appraisal.

Q-PCR detects PAR-1 mRNA level in-site:

RNA extracting: I. cell seeding abandons supernatant to the greatest extent after handling the corresponding time, 1mL Trizol is added, stands in 6 orifice plates Cell is blown and beaten, 5min is stood, is put into the EP pipe of 1.5mL RNA enzyme Free, is placed on ice；II. 200 μ L chloroforms are added, cover 15sec fullys shake with oscillator after upper cover, is then stored at room temperature 5min, 4 DEG C, 12000rpm, 15min；III. on drawing In layer water phase to another new EP pipe, would rather inhale less also not draw liquid lower layer substance, and 500 μ L of isopropanol is then added, and use Power shakes 15sec, at room temperature static 10min, 4 DEG C, 12000rpm centrifugation 10min；IV. sediment is carefully left and taken, is added 75% 500 μ L of ethyl alcohol, vibrated in vortex oscillator, make precipitating mix, 4 DEG C, 8500rpm be centrifuged 5min；V. it is sucked with rifle point Clearly, it can be centrifuged 1min again, suck supernatant, place drying at room temperature 5-10min in draught cupboard；VI. RNA precipitate is dissolved in 10 μ L In DEPC water, 55 DEG C of -60 DEG C of placement 10min survey concentration after completely dissolution, and the preferably same day does reverse transcription after RNA has been extracted.

RNA reverse transcription is at cDNA:

I. reverse transcription system is configured according to following table

II. after mixing, room temperature 10min；III.42℃50min；Then IV.85 DEG C of 5min is placed on ice, -20 DEG C of preservations.

Q-PCR detects PAR-1 mRNA level in-site:

I. according toUniversal qPCR Master Mix kit specification configures reaction system；

II. Step one Plus Q-PCR instrument is used, parameter is as follows

III. it statisticallys analyze.

Western blot detects PAR-1 protein expression level:

I. total protein of cell extracts: the cell conditioned medium handled well in culture plate is sucked out, washes 2 times with sterile PBS, then to The mixed liquor of Ripe lysate, sample-loading buffer and protease inhibitors is added in hole, is stirred with cell scraper is repeatedly inverse along needle direction Even albumen, place 20-30min, be then transferred in 1.5ml EP pipe, in boiling water be denaturalized 5-10min 12000rpm, 4 DEG C from Heart 10min sets ice bath, sample loading or packing immediately, -20 DEG C of preservations immediately；

II. glue, 10% separation gel is concentrated by recipe configuration 5%；

III. the glass plate for preparing glue is filled it up with electrophoretic buffer and tests whether to seep water, then gently on electrophoresis frame It is stuck in electrophoresis tank；

IV. respective sample is added in every hole, and marks, and equivalent sample-loading buffer, former and later two holes are not added in well Add the Marker of 2 μ L pre-dyed；

V. 120v is set by electrophoresis apparatus voltage, until electrophoresis to bromophenol blue is swum out；

VI. transferring film, transfer box placement order are as follows: black flour, foam-rubber cushion, wet filter paper, separation gel, film, wet filter paper, sponge Pad, fine flour, pvdf membrane will be steeped with methanol in advance, and nitrocellulose membrane uses bubble in advance；

VII. in transfer box insertion transfer cell, while an ice cube box is aside placed, fills it up with transfering buffering liquid, fiber film surface (fine flour) is towards anode；

VIII. electric current constant current 250mA 90min, according to Marker judge destination protein and internal reference albumen locating for tunica fibrosa Position is marked with pencil, and redundance is cut off；

IX.TBST washes 5-10min, and three times, speed is slightly fast；

X.5%milk-TBST (be added 25%BSA at 1: 10) closing 30min-1h；TBST washes 5min, three times；

XI. primary antibody (primary antibody configures in TBST, and 25%BSA 1: 100 adds) is added to slowly shake, 4 DEG C overnight；

XII. same IX；

XIII. plus secondary antibody (1: 2000,25%BSA according to 1: 100 plus), room temperature shakes 1h slowly；TBST washes 10min, three times；

XIV. appropriate ECL developing solution A, B 1: 1 is mixed, and is made the entire film of liquid immersion, is drained liquid on film after 2min, put Enter in instrument darkroom；

XV. software is opened, white light scanning is used first, then exposes, takes pictures, analyzes, while detecting GAPDH as internal reference.

Mouse subcutaneous xenograft tumor model (subcutaneous vaccination):

I. 0.25% pancreatin of Lewis cell in logarithmic growth phase is digested, 1640 culture mediums containing serum is added It neutralizes, 1200rpm is centrifuged 5min；II. it is washed twice of cell with PBS, abandons supernatant, be resuspended with 1640 without FBS, adjustment is thin Born of the same parents' density is to 2 × 10⁷/mL；III. the cell suspension inoculation in 100 μ L steps (II) is extracted in small with the syringe of 1mL specification Dorsal sc on the right side of mouse is extracted and gently oppresses pin hole a moment with sterile cotton balls after syringe needle；IV. it is given at once after inoculated tumour Medicine, and successive administration 7 days, DTIP (1.0mg/kg, q.d)；R-hidudin (0.5mg/kg, q.d)；IV. Mice Body is weighed weekly Weight is secondary, observation treatment group and control group mice physiological status, diet, defecation and the state of mind, and small with vernier caliper measurement The length and width of mouse lump, tumor volume difference between statistics group after experiment, according to international standard formula V=0.5 × a × b²(a is length, and b is width)；V.28 tumor mass, lung tissue, subsequent pathological analysis tissue sample are collected behind day.

Mouse tumor metastasis models (intravenous inoculation):

I. it is same as above (I): II. and is same as above (II), cell density is adjusted to 1 × 10⁷/mL；III. iodine tincture disinfection tail vein is first used Then injection site is sterilized with 75% cotton ball soaked in alcohol, volatilize a moment to alcohol, extracts 100 μ L steps with the syringe of 1mL specification (II) cell suspension inoculation in is in mouse tail vein；IV. with 5.3.8 IV and V after, pulmonary nodule number is counted.

Experimental result is shown:

Influence of the DTIP to proliferation of lung cancer cells: mtt assay measures influence of the DTIP to proliferation of lung cancer cells.Proportionally plus Medicine, 50 50 μ g/mL of μ g/mL, DTIP of fibrin ferment 10U/mL, r-hirudin；Fibrin ferment+r-hirudin, fibrin ferment+DTIP, It after for 24 hours, discards supernatant, 100 μ L MTT solution are added in every hole, and 37 DEG C of incubators place 4h；It is measured with wavelength 490nm.Experimental result It was found that fibrin ferment does not have a significant impact (Fig. 4) to the growth of tumour cell, after DTIP is added, do not find that it is thin to tumour yet Significantly affecting on intracellular growth.

Influence of the DTIP to lung carcinoma cell invasion and transfer: being utilized respectively scratch and transwell experiment has detected DTIP Influence to lung carcinoma cell invasion and transfer.In scratch experiment, fibrin ferment is added can promote A549 (Fig. 5 A), 95D later The invasion of (Fig. 5 B) and Lewis (Fig. 5 C), but after DTIP is added, the facilitation of fibrin ferment can be significantly inhibited；In In transwell test, it is again seen that transfer of the cell to cell can be promoted after fibrin ferment is added with conspicuousness, but it is added After DTIP, the quantity of A549 (Fig. 6 A), 95D (Fig. 6 B) and Lewis (Fig. 6 C) room transfer downwards is substantially reduced.

The expression of PAR-1: normal pulmonary epithelial cells BEAS-2B cell and lung carcinoma cell are had detected by Western blot The expression of PAR-1 in A549 and 95D cell.Experimental result discovery expression of PAR-1 in A549 and 95D is significantly higher than BEAS- 2B (such as Fig. 7 A, shown in B) takes normal mouse lung tissue and mouse tumor tissue to do immunohistochemistry, it was also found that PAR-1 is in tumour Expression in tissue will be significantly higher than normal lung tissue (as seen in figure 7 c).

It knocks out influence of the PAR-1 later to lung carcinoma cell: utilizing CRISPR/cas9 technology and slow-virus transfection, it will PAR-1 in A549 and Lewis cell is knocked out.First by LentiCRISPR-PXPR001 plasmid enzyme restriction, electroresis appraisal digestion is just Really, there is the band (Fig. 8) of treaty a 10000bp and 2000pb, 10000bp band is recycled, connect gRNA, pass through sequencing Identify that connection product is correct.By packaging virus, slow-virus transfection, the gRNA of sequencing result display design is in A549 (such as Fig. 9 A It is shown) and Lewis (as shown in fig. 9d) cytogene in played function；Western blot detects A549 (as shown in Figure 9 B) 80% is reduced with the expression of PAR-1 in Lewis (as shown in fig. 9e)；

After knocking out PAR-1, the invasion of A549 (as shown in Figure 9 C) and lewis (as shown in fig. 9f) cell and transfer ability are all Conspicuousness reduces, and can not restore its transfer ability after fibrin ferment is added.

Experimental studies results show that DTIP is shown in vitro, and DTIP inhibits the invasion and transfer of tumour；DTIP experiment in vivo In, the Anti-tumor metastasis Potential Evaluation of DTIP is carried out by intravenous inoculation Lewis cell, after inoculating cell, successive administration one week, is tied After fruit shows medication, mouse lung tubercle number substantially reduces (such as Figure 10 A, shown in B) compared with the control group, and pathological examination is shown DTIP can significantly inhibit lung's transfer (as illustrated in figure 10 c) of tumour；The mouse quantity statistics of liver metastasis shows physiology salt There are 5 (5/9) that hepatic metastases has occurred in water group, hepatic metastases (such as Figure 10 D, shown in E) is had no in DTIP medication group mouse；

In mouse subcutaneous xenograft tumor model, r-hirudin has slight suppression to the size of tumour as the result is shown Production is used, and DTIP acts on unobvious (shown in such as Figure 11 A, B) to the size of tumour；After cell inoculation four weeks, mouse lung Transfer case statistics shows have 6 (7/9) that Lung metastases have occurred in physiological saline group, has 2 respectively in r-hirudin and DTIP Only Lung metastases only have occurred in (2/8) and 3 (3/9), and after injecting DTIP, the mouse transfer quantity that Lung metastases occur has conspicuousness decline (as shown in Figure 11 C)；Tumor tissues are taken to do HE dyeing discovery, DTIP can inhibit the peripherad muscle of tumour cell or fat It invades (as shown in Figure 11 D)；The quantity of immunohistochemical observation intratumoral vasculature new life is made of CD31, display DTIP significantly inhibits swollen The quantity (as depicted in fig. 11E) of oncocyte medium vessels new life.

Claims (5)

1. thrombin inhibitor is directly solidifying preparing the purposes in antineoplastic invasion diversion medicaments, the thrombin inhibitor Thrombin inhibitor small peptide DTIP.

2. purposes according to claim 1, which is characterized in that the direct thrombin inhibitor is lepirudin 023 ludon.

3. purposes according to claim 1, which is characterized in that the direct thrombin inhibitor small peptide DTIP presses down in vitro The invasion and transfer of tumour processed.

4. purposes according to claim 1, which is characterized in that the direct thrombin inhibitor small peptide DTIP inhibits tumour The peripherad muscle of cell or fat invasion.

5. purposes according to claim 1, which is characterized in that the direct thrombin inhibitor small peptide DTIP inhibits tumour The quantity of cell medium vessels new life.