CN107050432A - Application of the gastrin in diagnosis and treatment breast cancer medicines is prepared - Google Patents

Abstract

The present invention discloses application of the gastrin in diagnosis and treatment breast cancer medicines is prepared.We have found that gastrin has antitumor action in breast cancer cell line MCF 7, T47D, experiment in vivo also further confirms the effect, its molecular mechanism is thought of as gastrin and is combined the key link that the influence for starting and amplifying to downstream signaling molecule is the antitumor propagation of gastrin with gastrin by raising CCKBR protein abundances, the CCKBR acceptors of up-regulation.The present invention researches and develops new target therapeutic agent to illustrate pathogenesis of breast carcinoma molecular mechanism, explores the new therapeutic scheme of breast cancer and provides direction.

Description

Application of the gastrin in diagnosis and treatment breast cancer medicines is prepared

Technical field

The invention belongs to field of biological pharmacy, more particularly, it is related to gastrin and is preparing diagnosis and treatment breast cancer drug Application in thing.

Background technology

Breast cancer is one of most common malignant tumour of women, has a strong impact on the quality of life [1] of women.Breast cancer according to Various criterion has different partings, such as can be divided into HER2+, HER2-, three feminine gender etc. according to different expression of receptor situations.It is current right Not yet it is fully apparent from the cause of disease of breast cancer, the method for clinical treatment breast cancer has operation, chemotherapy, endocrine etc., but has Certain limitation.Therefore the molecular mechanism of pathogenesis of breast carcinoma is illustrated, new target therapeutic agent is researched and developed, new treatment side is explored Case is by the Main way as breast cancer treatment from now on.

Gastrin, as one of gastrointestinal hormone found earliest, is a kind of polypeptide hormone, by positioned at antrum, duodenum and The G cells secretion of epimere jejunum, in addition, the D cells in people's pancreas islet can also secrete gastrin [2], main regulation gastric acid secretion and Nutrition stomach lining.Gastrin includes two kinds of G-17, G-34, and wherein G-17 is the principal mode of gastrin, be also after meals stomach secrete Element is present in the principal mode [3] in blood circulation.G-17 activity highests, about the 5 of G-34 times [4,5], and G-34 is then main It is present in blood, it resolves into G-17 competence exertion physiological functions.This experiment using G-17 as reagent, to closer to and reflect The biological action rule of gastrin.Gastrin-receptor, also known as cholecystokinin B acceptors (Cholecystokinin B Receptor, CCKBR), it belongs to g protein coupled receptor (G-protein coupled receptors, GPCRs) family, is One seven transmembrane protein.CCKBR is mainly distributed in the parietal cell of gastric mucosa and ECL cells [6,7].Gastrin mainly passes through Start downstream signaling pathway with the CCKBR specific bindings on cell membrane to play a role [8].Research shows that gastrin not only has There are gastric acid secretion and nutrition gastrointestinal mucosa, and have an effect on the propagation [9] of stomach and intestine tumor, but for gastrin Relation between malignant tumour also has many disputes at present.Part research thinks that gastrin is important rush cancer hormone, By raising Reg1 albumen etc. the positive cancer cell of gastrin-receptor can be promoted to increase [10-12].Also studies have found that stomach is secreted Element can effectively suppress the propagation [13-15] of colon tumor by suppressing the intracellular NF- κ B of Colo320 activity.This problem The early stage research of group finds that the expression of gastrin in stomach organization is less than cancer beside organism [16], and the serum stomach of patients with gastric cancer is secreted Plain level confirms that gastrin can effectively suppress stomach cancer cell with its diameter of tumor into negative correlation, and by external and experiment in vivo Propagation.In addition, this seminar research in recent years finds gastrin, Herceptin is in the positive and negative stomach cancer cells of HER2 There is antitumor action [17] by raising CCKBR, this provides thinking for our research.

Ouyang can et al. the experimental study of EMT6 growth of breast cancers " gastrin releasing peptide DNA vaccination suppress " [18] see Examine the inhibitory action that gastrin releasing peptide (GRP) DNA vaccination grows to EMT6 mouse breast cancers, gastrin releasing peptide and gastrin It is two different materials, with the protein sequence differed completely.

Gastrin releasing peptide, English full name gastrin-releasing peptide (GRP) express most abundant in pancreas, Secondly there is expression in stomach, adrenal cortex and brain.Gastrin releasing peptide is combined with GRP acceptors (GRPR), adjusts nervous centralis Many functions of the internal organs such as system and stomach and intestine, include the release (gastrin Gastrin belongs to one of which) of gastrointestinal hormone, put down Sliding Muscle cell contract, epithelial cell proliferation and tumor tissues.That is gastrin releasing peptide effect is very wide, one of effect It is to stimulate G cells secretion release gastrin in antrum portion and duodenum near-end mucous membrane.Gastrin releasing peptide is in breast cancer Using, mainly the peptide acceptor gastrin-releasing peptide Receptor (GRPR) tumor tissues table Reach, organism immune response is activated by gastrin releasing peptide vaccine, and our technology is to study directly to use gastrin Therapy Breast cancer, is two different concepts.

[1].Siegel RL,Miller KD,Jemal A.

CA Cancer J Clin.2016Jan-Feb；66(1):7-30.doi:10.3322/caac.21332.PMID: 26742998

[2].Wu C.Y.,Wu M.S.,Kuo K.N.,et al.,Effective reduction of gastric cancer risk with regular use of nonsteroidal anti-inflammatory drugs in Helicobacter pylori-infected patients.J Clin Oncol,2010.28(18):p.2952-7.

[3].Grabowska,A.M.and Watson,S.A.Role of gastrin peptides in carcinogenesis.Cancer Lett 2007,257:1-15.

[4] Zou is after red, Li Jinrui, Zhang Zhongping.The new development (literature review) of gastrin research.Radioimmunology magazine, 1995,04：251-253.

[5].Grabowska,A.M.,Watson,S.A..Role of gastrin peptides in carcinogenesis.Cancer Lett,2007,257(1):1-15.

[6].Chen A.P.,Chang M.H.,and Romero M.F.,Functional analysis of nonsynonymous single nucleotide polymorphisms in human SLC26A9.Hum Mutat, 2012.33(8):p.1275-84.

[7].Sachs G.,Zeng N.,and Prinz C.,Physiology of isolated gastric endocrine cells.Annu Rev Physiol,1997.59:p.243-56.

[8].Schubert,M.L.,Peura,D.A.Control of gastric acid secretion in health and disease.Gastroenterology,2008,134(7):1842-60.

[9].Ashcroft F.J.,Varro A.,Dimaline R.,et al.,Control of expression of the lectin-like protein Reg-1 by gastrin:role of the Rho family GTPase RhoA and a C-rich promoter element.Biochem J,2004.381(Pt 2):p.397-403.

[10].Watson S.A.and Smith A.M.,Hypergastrinemia promotes adenoma progression in the APC(Min-/+)mouse model of familial adenomatous polyposis.Cancer Res,2001.61(2):p.625-31.

[11].Schubert M.L.and Peura D.A.,Control of gastric acid secretion in health and disease.Gastroenterology,2008.134(7):p.1842-60.

[12].Sebens Muerkoster S.,Rausch A.V.,Isberner A.,et al.,The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-kappa B inhibition.Oncogene,2008.27 (8):p.1122-34.

[13].Muerkoster S.,Isberner A.,Arlt A.,et al.,Gastrin suppresses growth of CCK2 receptor expressing colon cancer cells by inducing apoptosis in vitro and in vivo.Gastroenterology,2005.129(3):p.952-68.

[14].Obszynska J.A.,Atherfold P.A.,Nanji M.,et al.,Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo.Gut,2010.59(2): p.156-63.

[15].Tomita H.,Takaishi S.,Menheniott T.R.,et al.,Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.Gastroenterology,2011.140(3):p.879-91.

[16].Przemeck S.M.,Varro A.,Berry D.,et al.,Hypergastrinemia increases gastric epithelial susceptibility to apoptosis.Regul Pept,2008.146 (1-3):p.147-56.

[17].Tian H.,Zhang N.,Suo W.H.,et al.,Gastrin suppresses the interdependent expression of p16and anion exchanger 1favoring growth inhibition of gastric cancer cells.Int J Cancer,2010.127(6):p.1462-74.

[18] Ouyang can, cross be, Wu monarch, Zhang Shuya, Liu Jingjing.Gastrin releasing peptide DNA vaccination suppresses EMT6 breasts The experimental study of gland cancer growth.Chinese Clinical pharmacology and acology, 2007,05：512-515.

The content of the invention

It is an object of the invention to provide application of the gastrin in diagnosis and treatment breast cancer medicines is prepared.

To realize object above, the present invention discloses following technical scheme：Gastrin is preparing diagnosis and treatment breast cancer drug Application in thing.

As a preferred scheme, the gastrin refers to gastrin complete sequence, the peptide of activity form 17 and activity form 34 One or more in peptide.

As a preferred scheme, the breast cancer refers to the breast cancer with gastrin-receptor CCKBR.

The advantage of the invention is that：We have found that gastrin has antitumor work in breast cancer cell line MCF-7, T47D With, experiment in vivo also further confirms the effect, and its molecular mechanism is thought of as gastrin by raising CCKBR protein abundances, on It is the antitumor propagation of gastrin that the CCKBR acceptors of tune are combined the influence for starting and amplifying to downstream signaling molecule with gastrin Key link.The present invention researches and develops new target therapeutic agent to illustrate pathogenesis of breast carcinoma molecular mechanism, explores breast cancer controlling newly Treatment scheme provides direction.

Brief description of the drawings

The expression of CCKBR albumen in Fig. 1 difference breast carcinoma cell strains.

The expression of CCKBR albumen in Fig. 2 breast cancer tissues.

Inhibited proliferation of Fig. 3 gastrins to breast cancer cell.

Fig. 4 gastrins raise CCKBR protein abundances in MCF-7, T47D.

Fig. 5 gastrins raise erk, p65 protein abundances in MCF-7, T47D.

Fig. 6 gastrins suppress gastric cancer in nude mice lotus knurl propagation.

Fig. 7 gastrins promote the CCKBR expression of lotus knurl tissue.

Influence nude mice lotus knurl histone p-ERK, the p-P65 expression of Fig. 8 gastrins.

Gastrin Levels in Fig. 9 blood of patients with breast cancer.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.Experimental method used in following embodiments for example without Specified otherwise, is conventional method.Material, reagent used etc. in following embodiments, unless otherwise specified, can be from business way Footpath is obtained.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.

Embodiment 1.

1 materials and methods

1.1 experiment materials, reagent

(1) cell line：Tri- kinds of breast carcinoma cell strains of MCF-7, T47D, MDAMB231

(2) hyclone (Gibco BRL, Gaithersburg, MD, USA)

(3) DMEM culture mediums (Hyclone, Logan, UT, USA)

(4)Penicillin Streptomycin(Invitrogen,CA,USA)

(5) 0.25%Trypsin-EDTA (Gibco, Life technologies, USA)

(6) betulinic acid (Betulinic acid, Bio Vision, Mountain View, CA, USA)

(7)Parthenolide(Santa Cruz Biotechnology,Santa Cruz,CA,USA)

(8) rabbit-anti p65, rabbit-anti p-p65, rabbit-anti ERK and p-ERK antibody (Cell Signaling technology,

Everly,MA,USA)

(9) the anti-Vinculin antibody (Abcam, Cambridge, UK) of mouse

(10) HRP- rabbits secondary antibody, mouse secondary antibody (Sigma chemical Co., St.Louis, MO, USA)

(11) the super quick UltraSensitive TMSP kits of instant immunohistochemistry, DAB colour reagent box (good fortune State steps neoformation technological development Co., Ltd, China)

(12) protease inhibitors (Sigma chemical Co., St.Louis, MO, USA)

(13) gastrin (Gastrin) (strong credit is biological, Shanghai, China)

(14) lipopolysaccharides (LPS) (Sigma chemical Co., St.Louis, MO, USA)

(15)PD98059(Sigma chemical Co.,St.Louis,MO,USA)

(16) fast-type goat-anti rabbit/general secondary antibody of mouse (stepping neoformation technological development Co., Ltd, Fujian, China)

(17) RIPA lysates (green skies biotechnology research institute, Jiangsu, China)

(18)2×RNA loading dye(Fermentas Ins,Burlington,Canada)

1.2 key instrument equipment

(1) CO2 cell culture incubators (Thermo Fisher Scientific, USA)

(2) cell superclean bench (Thermo Fisher Scientific, USA)

(3) constant-temperature table (Forma, Milford, MA)

(4) low-temperature and high-speed centrifuge (Eppendorf, Germany)

(5) full-automatic embedding machine (Leica, Wetzlar, Germany)

(6) paraffin slicing machine (Leica, Wetzlar, Germany)

(7) ordinary optical microscope (Olympus, Tokyo, Japan)

(8) electrophoresis tank, transferring film groove (Bio-Rad, USA)

(9) water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory, China)

(10) electric-heated thermostatic water bath (the upper grand experimental facilities Co., Ltd of Nereid, China)

(11) the ultrapure water purification systems of MILLI-Q (Merck Millipore, USA)

(12) PH counts (CyberScan, USA)

(13) FluorChem E sweep film instrument (Cell Biosciences, USA)

(14) low temperature refrigerator (SANY, Japan)

(15) ELIASA (Thermo, USA)

1.3 main agents are prepared

1.3.1 regular growth nutrient solution：

DMEM nutrient solutions：DMEM+10% hyclones

1.3.2 cells frozen storing liquid：

90% hyclone+10%DMSO

1.3.3 albumen transferring film buffer solution：

Tris base 3.04g, glycine 14.4g, plus distilled water 600ml dissolving are weighed, then adds methanol 200mL, finally Graduated cylinder is settled to 1L, and 4 DEG C stored refrigerated.

1.3.4SDS-PAGE electrophoretic buffer：

Tris base 3.03g, glycine 18.77g, SDS 1g, plus distilled water dissolving are weighed, graduated cylinder is settled to 1L.

1.3.5TBST(Tris-Buffered Saline and Tween-20)：

Nacl l8g are weighed, 1M Tris-Hcl (pH7.6) 20mL and Tween-20 1mL, plus distilled water graduated cylinder are settled to 1L。

1.3.6Western blot confining liquids：

10% (w/v) Western blot confining liquid (examples are made into TBST and bright skimmed milk power：50mL TBST+5.0g Skimmed milk power).

1.3.7Western blot cleaning fluids

Match somebody with somebody 2% (w/v) Western blot cleaning fluid (examples with TBST and bright skimmed milk power：100mL TBST+2.0g take off Fat milk powder).

1.3.8 the preparation of antigen retrieval buffer solution：

(1) preparation of citrate storing liquid：

0.1M citrate buffer solutions A：Every liter of 21g containing citric acid；

0.1M sodium citrate buffer solutions B：Every liter of 29.4g containing sodium citrate.

(2) preparation of citrate working solution：

0.01M citrate buffers (pH 6.0)：Every liter of 19mL of liquid containing A, B liquid 81mL, plus distilled water dissolving, adjust pH value To 6.0, graduated cylinder is titrated to 1L.

1.4 cell recovery

(1) cell cryopreservation tube frozen in -80 DEG C of refrigerators is circumferentially rocked rapidly in 42 DEG C of water-baths, as far as possible in 1 point Melt cell liquid in cryopreservation tube in clock；

(2) freeze after mouth of pipe alcolhol burner flame sterilization, cell liquid is moved into sterile 15ml centrifuge tubes, 800rpm, room Warm (room temperature, RT), centrifuges 5min, it is seen that cell mass is deposited on ttom of pipe；

(3) supernatant discarding, adds 1 × PBSs of 4ml (pH 7.4), after gently piping and druming is mixed, 800rpm, RT, from Heart 5min；

(4) supernatant discarding, single outstanding suspension cell is blown and beaten into 2ml DMEM culture mediums (containing 10% hyclone), will be thin Cytosol is transferred in Tissue Culture Dish, 10ml culture mediums is added, in 37 DEG C of incubators of the mixed gas containing 5%CO2 and 95%O2 Middle culture.

1.5 cell culture

(1) cell cellar culture in DMEM culture mediums (containing 10% hyclone), is incubated in containing 5%CO₂And 95%O₂It is mixed In 37 DEG C of incubators for closing gas；

(2) different processing are given according to the difference of cell doubling time, it is average to be passed on once per 2-3 days, to keep thin Born of the same parents are constantly in exponential phase；

(3) cell of culture carries out detection of mycoplasma during testing, and result is feminine gender；It is thin through Trypan Blue analysis Born of the same parents' vigor is maintained at more than 90%.

1.6 passage

(1) cell for intending passing on is taken, nutrient solution is discarded, 1 × PBS (pH 7.4) is added and jiggles rinsing cell Surface, totally 2 times, to remove influence of the serum composition in culture medium to trypsin digestion cell ability；

(2) discard 1 × PBS (pH 7.4), add appropriate pancreatin (0.25%Trypsin-EDTA), containing 5%CO₂And 95%O₂It is incubated several minutes in 37 DEG C of incubators of mixed gas；

(3) cellular morphology is observed under inverted phase contrast microscope, treats that cell loses original form, diminish and be rounded, in bright shape, When not fallen off in half adhered state, it is considered to eupepsia, pancreatin is discarded, appropriate culture medium is added and terminates digestion reaction；

(4) softly blow and beat cell with 1ml pipette tips to hang to single, it (is usually 1 that cell, which is divided into other culture dishes,:2 or 1：3 Passage), and be placed in incubator after adding enough culture mediums, complete passage.

1.7 cell cryopreservation

(1) cell frozen is needed, cell log growth period should be maintained it in, vigor is more than 90%；

(2) when cell culture is to when converging rate 90%-95%, nutrient solution is discarded, with 1 × PBS (pH of preheating 7.4) cleaning cell 2 times, adds appropriate trypsin digestion cell；

(3) postdigestive cell is moved into sterile 15ml centrifuge tubes, 800rpm, RT, centrifuges 5min, cell deposition exists Ttom of pipe；

(4) supernatant discarding, cell is cleaned with 1 × PBS (pH 7.4) of preheating, 800rpm, RT centrifuge 5min；

(5) supernatant discarding, is moved into cryopreservation tube after adding 1ml cells frozen storing liquids, soft piping and druming cell to single cell suspension, The title of freeze-stored cell is marked in detail, originates, freeze date etc., and -80 DEG C of refrigerators are transferred to after -20 DEG C of refrigerators are placed 30 minutes Stand overnight, finally move into and preserved for a long time in liquid nitrogen container.

1.8 cell counts are tested

Breast cancer cell MCF-7, T47D, 231 in increased logarithmic phase are arranged into control group, gastrin group to plant respectively 3000/hole of plate is in 96 orifice plates, and 3 multiple holes of every group of setting give various concentrations medicine as needed after 24 hour cells are adherent Thing processing, is administered once day, daily progress cell count in continuous 7 days

1.9 immunoblotting

Protein blot experiment (Western blot) continues to use existing experimental method, comprises the following steps that：

(1) albumen is received：Take the logarithm the cell in growth period, pre-cooled 1 × PBS (pH 7.4) cleaning twice, root Appropriate 2 × SDS protein lysates containing DTT are added according to cell density, are fully mixed with pipette tips, thorough crack protein, are drawn Cell protein is collected into 1.5ml EP pipes, is placed in 30min on ice, albumen is fully cracked；

(2) albuminous degeneration：The EP pipes that receipts have cell protein are placed in by Type17600Dri-Bath temperature adjustments to 95 DEG C 10min is boiled in metal bath, 10min is placed on ice, is repeated twice, albumen is thoroughly denatured；

(3) albumen supernatant is extracted：After albumen boils 10min through 95 DEG C again, 12,000rpm, RT centrifuge 5min, abandon precipitation, will Albumen supernatant is moved into another clean EP pipes；

(4) protein quantification：Albumen through BCA methods survey concentration quantify, be configured to SDS as volume, equally it is total Packing is stored in -80 DEG C after after amount；

(5) glue is matched somebody with somebody：According to destination protein molecular size range, the lauryl sodium sulfate for preparing corresponding 6%~12% is gathered Acrylamide gel (SDS-PAGE), Protein Separation is carried out by electrophoresis；

(6) albumen loading：By the albumen of packing from -80 DEG C of taking-ups, in boiling 10min in 95 DEG C of metal baths, by sample plus Enter in gel pore, while adding 1ul bromophenol blue spikes per hole；

(7) electrophoresis：Complete after loading, carry out electrophoresis with 80V voltages first to bromophenol blue spike band to separation gel time-varying Voltage is changed for 120V, until when bromophenol blue spike band reaches electrophoresis trench bottom, terminating electrophoresis；

(8) transferring film：Running gel is taken out, transferring film is carried out in the transferring film liquid of precooling after removing spacer gel, protein will be contained Separation gel lie in Western blot special sandwich style transferring films folder and clamp, placement order is followed successively by by negative pole to positive pole Blackboard, sponge, filter paper, separation gel, nitrocellulose filter (Nitrocellulose membrane, NC film), filter paper, sponge and Blank；Transferring film uses wet robin, selects constant current 300mA or constant pressure 100V according to requirement of experiment, the transferring film time is according to protein point Son amount is set as 60 to 100min, by protein delivery to NC films；

(9) Ponceaux is dyed：Transferring film takes out NC films after terminating, by the one side contacted with separation gel upward, cuts upper right corner work For mark, with TBST by NC films rinsing be once placed in antibody incubation box, Ponceaux dyeing substantially assess protein electrophoresis, transferring film and Protein quantification situation；

(10) rinse：With TBST short rinse NC films 3 times, Ponceaux is cleaned, antibody incubation box is then placed in 360 degree On shaking table, NC film about 10min are rinsed with TBST, fully washing to Ponceaux dyeing is taken off to the greatest extent；

(11) close：NC films after rinsing are incubated in Western blot confining liquids (TBST prepares 10% skimmed milk power) In, it is incubated at room temperature 1 hour on horizontal shaker, to close the nonspecific binding site on NC films；

(12) it is incubated primary antibody：Terminate after closing, NC films are incubated in prepared with Western blot cleaning solutions and purpose In the corresponding primary antibody of albumen, horizontal shaker, RT is incubated 2h, or 4 DEG C of overnight incubations；

(13) wash：After primary antibody incubation terminates, TBST short rinse NC films 3 times are subsequently placed on 360 degree of shaking tables and used again Western blot cleaning solutions (2% skimmed milk power that TBST is prepared) washing 3 times, room temperature, each 10min；

(14) it is incubated secondary antibody：Fully after the remaining primary antibody of washing, in the secondary antibody that NC films are put to corresponding HRP marks, RT is incubated Educate 1h；

(15) wash：After secondary antibody incubation terminates, Western blot cleaning solutions (2% skimmed milk power that TBST is prepared) are quick Rinse NC films 3 times, be subsequently placed on 360 degree of shaking tables and wash again 3 times, RT, each 10min；

(16) image：By the Immobilon Western Chemilminescent HRP of the NC films after washing Substrate reagents are developed, and antigen antibody complex is combined with developer, can be imaged in gel imager.

1.10 immunohistochemical staining is analyzed

Whole samples are routinely fixed through 10% formalin, FFPE processing, are cut into 4 μm of histotomies, specific immune Histochemical stain is tested with reference to existing experimental method, is comprised the following steps that：

(1) piece is baked：Histotomy is inserted baking 30 in 60 DEG C of constant temperature roasters and stayed overnight；

(2) dewax：Section, which is placed in 45 DEG C of dimethylbenzene I in 20min, dimethylbenzene II, places 30min；

(3) aquation：Section is sequentially placed into 100% ethanol I, 100% ethanol II, 95% ethanol I and 95% ethanol II, Each 2min；

(4) section is taken out, residual ethanol is rinsed with running water after standing 1min in running water, single water that steams is changed and stands 1min；

(5) antigen retrieval：Section is put in reparation box, antigen retrieval buffers (citrate buffer) is added, covers lid, 90 DEG C of 3min of microwave stove heat maintain 12min to buffer solution boiling, then with 60 DEG C；

(6) antigen repairing box is taken out, opens the lid, antigen retrieval buffers is stood at room temperature, up to temperature drop to room Untill temperature；

(7) antigen retrieval buffers are discarded, section is moved into staining jar, 1 × PBS (pH 7.4), gentle punching is added Wash section 3 times, each 5min；

(8) PBS is discarded, 3% hydrogen peroxide of Fresh is added, 10min is incubated at room temperature, to remove endogenous peroxidating The influence of thing enzyme；

(9) 3% hydrogen peroxide is discarded, section 3 times, each 5min are gently rinsed with 1 × PBS (pH 7.4)；

(10) section is moved into wet box, 5%BSA (distilled water preparation) is added dropwise in section, be incubated at room temperature 30min, closing Nonspecific binding site；

(11) 5%BSA is discarded, the primary antibody diluted by 5%BSA is directly added dropwise on biopsy tissues, 37 DEG C are incubated 1h, or 4 DEG C be incubated overnight；

(12) after the completion of primary antibody is incubated, section 3 times, each 10min are gently rinsed with 1 × PBS (pH 7.4)；

(13) 1 × PBS (pH 7.4) is discarded, the secondary antibody of HRP marks, incubation at room temperature are added dropwise on biopsy tissues 15min；

(14) after the completion of secondary antibody is incubated, section 3 times, each 10min are gently rinsed with 1 × PBS (pH 7.4)；

(15) it is incubated at room temperature and is developed the color with the diaminobenzidine solution (DAB) of fresh configuration, micro- Microscopic observation colour developing feelings Condition；

(16) distilled water rinses section, removes the DAB of residual；

(17) section is moved into staining jar, carries out redying for nucleus with haematoxylin dye liquor, 1min is placed at room temperature；

(18) haematoxylin dye liquor is reclaimed, section is rinsed for several times with running water, the haematoxylin of residual is removed, at 50 DEG C to 60 DEG C Running water in stand 5-10min, carry out anti-blue；

(19) after the completion of anti-basket, cut into slices with originally washing, then sequentially pass through 95% ethanol I, 95% ethanol II, 100% second Alcohol I, 100% ethanol II carry out section dehydration, and each 1min thoroughly dries at room temperature；

(20) section dried is each in dimethylbenzene I, dimethylbenzene II to place 5min, and progress section is transparent, dries；

(21) it is last that neutral gum mounting, rearmounted optical microphotograph Microscopic observation is added dropwise on section sample；

(22) result judges：Brownish black dyed particles are positive expression.

1.11 pharmaceutical intervention nude mice lotus knurl proliferation experiments

This research experiment is female BAl BIc/c nude mices (about 4~6 weeks) with mouse, and 16~18g of body weight is purchased from Chinese science Institute's Shanghai Experimental Animal Center, animal feeding is in the laboratory of Experimental Animal Center SPF grades of this school, and experimental design and operation are observed The relevant regulations and requirement of Shanghai Communications University's Experimental Animal Center and the animal welfare committee.

Left and right fat pad with disposable sterilized injector in nude mice is inoculated with MCF-7 cells about 5 × 10⁶It is individual, and by nude mice It is randomly divided into 2 groups, respectively control group, gastrin group.Every group each 5；After cell seeding 15 days, gross tumor volume reaches about 100-200mm³, be administered from this day, control group 0.9%NaCl 100ul/ times, day 1 tail vein injection；Gastrin group gives Gastrin 2mg/kg, day 1 tail vein injection, the above is administered 2 weeks.The administration same day is set to D0；Transplantable tumor is measured 2 times a week Major diameter and minor axis, gross tumor volume is calculated according to following formula：Gross tumor volume (mm³)=long (mm) × wide by 2 (mm)/2；In D28 Giving nude mice, excessively anesthesia is put to death, and is taken out lotus knurl and is weighed respectively record, and tumour is cut into thin slice of the thickness for 4mm, partly with Formalin is fixed, partly with liquid nitrogen cryopreservation, for subsequent experimental.

2 results

Expression of the 2.1CCKBR acceptors in breast cancer cell

The Western blot detection results that albumen has carried out CCKBR albumen are extracted in this research to 3 kinds of breast carcinoma cell strains There is CCKBR expression in MCF-7 and T47D, and CCKBR expression is extremely low in 231, Fig. 1 is in different breast carcinoma cell strain The expression of CCKBR albumen.

Expression of the 2.2CCKBR acceptors in breast cancer tissue

This research carries out the expression that SABC detects CCKBR albumen to the tissue of 20 patient with breast cancers.As a result CCKBR albumen in the tissue of these patients is shown in generally to express.Fig. 2 is the expression of CCKBR albumen in breast cancer tissue.

Influence of 2.3 gastrins to Cells Proliferation of Human Breast Cancer

Gastrin is respectively applied to MCF-7, T47D and MDAMB231 cells are administered once day, continuous 7 days, enter daily Row cell count.As a result it is shown in MCF-7 and T47D tumor cell number after gastrin processing and significantly reduces (* P compared with control group <0.05, compared with control group, n=3), but to 231 without inhibitory action.Fig. 3 is propagation of the gastrin to breast cancer cell Inhibitory action.

Influence of 2.4 gastrins to CCKBR protein levels

By gastrin 10^-7M is respectively acting on MCF-7, T47D cell, day single administration, totally 3 days, then respectively at administration Afterwards 1d, 3d, 5d collect cell extraction albumen be Western-blot detection CCKBR albumen, as a result show MCF-7 gastrin to 1d can substantially raise CCKBR protein abundances after medicine, and with administration number of times increase and the extension of time still keeps high abundance And in obvious chronic up-regulation trend；And T47D cells 1d CCKBR protein abundances after gastrin administration start to increase, and with The extension of administration number of times and time are in gradually rise trend.And the speed that CCKBR rises in MCF-7 cells is significantly faster than that T47D Cell.Fig. 4 is that gastrin raises CCKBR protein abundances in MCF-7, T47D.

2.5 gastrins raise erk, p65 protein expressions

By gastrin 10^-7M is respectively acting on MCF-7, T47D cell, day single administration, totally 3 days, then respectively at administration 0d, 1d, 2d, 3d collect cell extraction albumen and are Western-blot detection erk afterwards, and as a result p65 albumen show MCF-7 in stomach 1d can substantially raise CCKBR protein abundances after secretin administration, and with administration number of times increase and the extension of time is still kept High abundance and in obvious chronic up-regulation trend；And T47D cells 1d CCKBR protein abundances after gastrin administration start increase, And as the extension of administration number of times and time are in gradually rise trend.And the speed that CCKBR rises in MCF-7 cells is substantially fast In T47D cells.Fig. 5 is that gastrin raises erk, p65 protein abundances in MCF-7, T47D.

2.6 gastrins test in vivo in antitumor action

2.6.1 gastrin suppresses tumor proliferation in nude mice lotus knurl model

The BALB/c nude mices of average 6 week old are taken to inject 5 × 10 respectively in both sides fat pad⁶Individual MCF-7 cells, build nude mice Lotus knurl model.It is random after nude mice injection tumour cell that nude mice is divided into 2 groups, i.e. control group, gastrin group, inoculated tumour cell After about 15 days, nude mice oxter tumour grows to 100-200mm³After start to give drug-treated, control group 0.9%NaCl 100ul/ times, day 1 tail vein injection, gastrin 2mg/kg, day 1 tail vein injection, altogether be administered 2 weeks.By measuring lotus knurl Size calculates tumor growth rate, gross tumor volume=length × wide²/2.As a result the growth of each group lotus knurl is started after being shown in administration 2 weeks Curve is separated, and control group (P is significantly lower than to gastrin group lotus knurl mean size when being administered 2 weeks<0.05), it was demonstrated that the two Also there is antitumor action really in vivo.And take tumour to take pictures to weigh.Fig. 6 is that gastrin suppresses gastric cancer in nude mice lotus knurl propagation.

2.6.2 gastrin promotes the expression of the CCKBR albumen of nude mice lotus knurl tissue

Fig. 7 is the CCKBR expression that gastrin promotes lotus knurl tissue.

2.6.3 gastrin is by influenceing CCKBR downstream signaling pathway to suppress tumor proliferation

Take lotus knurl tissue extraction albumen to carry out GAP-associated protein GAP such as ERK, P65 etc. Western blot detections, as a result show Show in lotus knurl tissue gastrin can upregulated protein abundance, this is consistent with cell experiment result, further supports ours Hypothesis.Fig. 8 is gastrin influence nude mice lotus knurl histone p-ERK, p-P65 expression.

In summary test, it has been found that gastrin has antitumor action in MCF-7, T47D, experiment in vivo also enters One step confirms the effect.Its molecular mechanism is thought of as gastrin by raising CCKBR protein abundances, the CCKBR acceptors of up-regulation with Gastrin is combined the key link that the influence for starting and amplifying to downstream signaling molecule is the antitumor propagation of gastrin.

2.9 clinical patients Gastrin Levels are detected

Internal experiment in vitro confirms that gastrin can suppress two kinds of breast cancer cell lines of MCF-7, T47D.Analyze its molecule special Levy is HER2 negative, ER and/or PR positive.We have collected 38 this type patient with breast cancers serum and 15 it is normal The serum of people, two groups of Gastrin Levels are detected by ELISA.As a result show that Gastrin Levels are general in the blood of this kind of patient with breast cancer All over less than normal person.Fig. 9 is Gastrin Levels in blood of patients with breast cancer.

We handle breast cancer cell MCF-7 and T47D with gastrin, it is found that cell line MCF-7 and T47D propagation are obtained Suppress, detect that CCKBR, the ERK of phosphorylation and the P65 of phosphorylation have been raised in discovery by Westernblot.This result is dynamic Preliminary identification has also been obtained on thing preliminary experiment.Its precise mechanism also needs to ensuing further checking and probed into.MCF-7 and T47D is that HER2 negative, ER and/or PR are positive, and according to this characterization of molecules, we have collected the blood of 38 clinical breast cancer patients The serum of clear and 15 normal persons, its level of serum gastrin is detected by ELISA, finds this type patients serum's gastrin water It is flat to be less than normal person's level.Furthermore it has been found that in the low patient of these Gastrin Levels 25 main suits have stomach medical history or Gastrocopy has stomach trouble.Accordingly, it is presumed that Gastrin Levels it is low be pathogenesis of breast carcinoma a risk factors.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (3)

1. application of the gastrin in diagnosis and treatment breast cancer medicines is prepared.

2. application of the gastrin according to claim 1 in diagnosis and treatment breast cancer medicines is prepared, it is characterised in that The gastrin refers to the one or more in gastrin complete sequence, the peptide of activity form 17 and the peptide of activity form 34.

3. application of the gastrin according to claim 1 in diagnosis and treatment breast cancer medicines is prepared, it is characterised in that The breast cancer refers to the breast cancer with gastrin-receptor CCKBR.