CN107050432B - Application of the gastrin in preparation diagnosing and treating breast cancer medicines - Google Patents

Abstract

The present invention discloses application of the gastrin in preparation diagnosing and treating breast cancer medicines.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 by up-regulation CCKBR protein abundance, and the CCKBR receptor of up-regulation combines starting with gastrin and amplifies the key link that the influence to downstream signaling molecule is the antitumor proliferation of gastrin.The present invention is to illustrate pathogenesis of breast carcinoma molecular mechanism, researches and develops new target therapeutic agent, explores the new therapeutic scheme of breast cancer and provides direction.

Description

Application of the gastrin in preparation diagnosing and treating breast cancer medicines

Technical field

The invention belongs to field of biological pharmacy, more particularly, are related to gastrin in preparation diagnosing and treating breast cancer drug Application in object.

Background technique

Breast cancer is one of the most common malignant tumors in women, seriously affects 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 It is not yet completely understood in 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 for illustrating pathogenesis of breast carcinoma, researches and develops new target therapeutic agent, explores new treatment side Case will become the Main way of breast cancer treatment from now on.

Gastrin is a kind of polypeptide hormone as one of the gastrointestinal hormone found earliest, by be located at antrum, duodenum and The G cell of upper section jejunum is secreted, in addition, the D cell in people's pancreas islet can also secrete gastrin [2], it is main adjust gastric acid secretion and Nutrition stomach lining.Gastrin includes two kinds of G-17, G-34, wherein G-17 be gastrin principal mode and diet after stomach secrete Element is present in the principal mode [3] in blood circulation.G-17 activity highest, about the 5 of G-34 times [4,5], and G-34 is then main It is present in blood, resolves into G-17 competence exertion physiological function.This experiment is using G-17 as reagent, to closer and reflect The biological action rule of gastrin.Gastrin-receptor, also known as cholecystokinin B receptor (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 and ECL cell of gastric mucosa [6,7].Gastrin mainly passes through [8] are played a role with the CCKBR specific binding starting downstream signaling pathway on cell membrane.Studies have shown that gastrin not only has Irritating gastric acid secretion and the effect of nutrition gastrointestinal mucosa, and the proliferation [9] of stomach and intestine tumor is had an effect on, but for gastrin There is also many disputes at present for relationship between malignant tumour.Part research thinks that gastrin is important rush cancer hormone, The cancer cell of the gastrin-receptor positive can be promoted to increase [10-12] by up-regulation Reg1 albumen etc..Also studies have found that stomach is secreted Element can effectively inhibit the proliferation [13-15] of colon tumor by inhibiting the activity of the intracellular NF- κ B of Colo320.This project The expression of gastrin is lower than cancer beside organism [16] in the early stage research discovery stomach organization of group, and the serum stomach of patients with gastric cancer is secreted Plain level is negatively correlated with its diameter of tumor, and confirms that gastrin can effectively inhibit stomach cancer cell by external and experiment in vivo Proliferation.In addition, the discovery gastrin of this seminar research in recent years, Herceptin is in the positive and negative stomach cancer cell of HER2 There are antitumor action [17] by raising CCKBR, this provides thinking for our research.

It sees " experimental study of gastrin releasing peptide DNA vaccination inhibition EMT6 growth of breast cancers " [18] of Ou Yangkedong et al. Examine the inhibiting effect that gastrin releasing peptide (GRP) DNA vaccination grows EMT6 mouse breast cancer, gastrin releasing peptide and gastrin Two substances are different, there is complete different protein sequence.

Gastrin releasing peptide, full name in English gastrin-releasing peptide (GRP) is most abundant in pancreas expression, Secondly there is expression in stomach, adrenal cortex and brain.Gastrin releasing peptide and GRP receptor (GRPR) are combined, and adjust nervous centralis Many functions of the internal organs such as system and stomach and intestine, the release (gastrin Gastrin belongs to one of which) including gastrointestinal hormone are 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 G cell secretion release gastrin in stimulation antrum portion and duodenum proximal end mucous membrane.Gastrin releasing peptide is in breast cancer Using mainly table of the receptor gastrin-releasing peptide Receptor (GRPR) of the peptide in tumor tissues It reaches, organism immune response is activated by gastrin releasing peptide vaccine, and our technology is research directly with 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 Jihong, 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] Ou Yangkedong, crossing is Wu Guojun, Zhang Shuya, Liu Jingjing.Gastrin releasing peptide DNA vaccination inhibits EMT6 cream The experimental study of gland cancer growth.Chinese Clinical pharmacology and acology, 2007,05:512-515.

Summary of the invention

The purpose of the present invention is to provide application of the gastrin in preparation diagnosing and treating breast cancer medicines.

In order to achieve the above object, the present invention discloses following technical scheme: gastrin is in preparation diagnosing and treating breast cancer drug Application in object.

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

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

The present invention has the advantages 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 molecular mechanism is thought of as gastrin and passes through up-regulation CCKBR protein abundance, on The CCKBR receptor of tune combines starting with gastrin and the influence amplified to downstream signaling molecule is the antitumor proliferation of gastrin Key link.The present invention is to illustrate pathogenesis of breast carcinoma molecular mechanism, researches and develops new target therapeutic agent, and exploration breast cancer is new to be controlled Treatment scheme provides direction.

Detailed description of the invention

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

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

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

Fig. 4 gastrin raises CCKBR protein abundance in MCF-7, T47D.

Fig. 5 gastrin raises erk, p65 protein abundance in MCF-7, T47D.

Fig. 6 gastrin inhibits gastric cancer in nude mice lotus knurl proliferation.

Fig. 7 gastrin promotes the CCKBR expression of lotus knurl tissue.

Fig. 8 gastrin influences nude mice lotus knurl histone p-ERK, p-P65 expression.

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

Specific embodiment

Present invention will be further explained below with reference to specific examples.Experimental method used in following embodiments for example without Specified otherwise is conventional method.The materials, reagents and the like used in the following examples unless otherwise specified can be from business way Diameter obtains.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Embodiment 1.

1 materials and methods

1.1 experimental materials, reagent

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

(2) fetal calf serum (Gibco BRL, Gaithersburg, MD, USA)

(3) DMEM culture medium (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- rabbit secondary antibody, mouse secondary antibody (Sigma chemical Co., St.Louis, MO, USA)

(11) the super quick UltraSensitive TMSP kit 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 biology, 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 lysate (green skies biotechnology research institute, Jiangsu, China)

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

1.2 key instrument equipment

(1) CO2 cell incubator (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 slot (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 macro experimental facilities Co., Ltd of Nereid, China)

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

(12) PH counts (CyberScan, USA)

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

(14) low temperature refrigerator (SANY, Japan)

(15) microplate reader (Thermo, USA)

1.3 main agents are prepared

1.3.1 regular growth culture solution:

DMEM culture solution: DMEM+10% fetal calf serum

1.3.2 cells frozen storing liquid:

90% fetal calf serum+10%DMSO

1.3.3 albumen transferring film buffer:

Weigh Tris base 3.04g, glycine 14.4g, add distilled water 600ml dissolve, then plus 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 is weighed, glycine 18.77g, SDS 1g adds distilled water to dissolve, graduated cylinder is settled to 1L.

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

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

1.3.6Western blot confining liquid:

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

1.3.7Western blot cleaning solution

With TBST and bright skimmed milk power, with 2% (w/v) Western blot cleaning solution, (example: 100mL TBST+2.0g is de- Rouge milk powder).

1.3.8 the preparation of antigen retrieval buffer:

(1) preparation of citrate storing liquid:

A: every liter 21g containing citric acid of 0.1M citrate buffer solution；

B: every liter 29.4g containing sodium citrate of 0.1M sodium citrate buffer solution.

(2) preparation of citrate working solution:

0.01M citrate buffer (pH 6.0): every liter of 19mL of liquid containing A, B liquid 81mL add distilled water to dissolve, and 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) after freezing nozzle alcolhol burner flame sterilization, cell liquid is moved into sterile 15ml centrifuge tube, 800rpm, room Warm (room temperature, RT) is centrifuged 5min, it is seen that cell mass is deposited on tube bottom；

(3) it discards supernatant, is added 1 × PBS buffer solution of 4ml (pH 7.4), after gently piping and druming mixes, 800rpm, RT, from Heart 5min；

(4) it discards supernatant, it, will be thin at singly outstanding suspension cell with 2ml DMEM culture medium (containing 10% fetal calf serum) piping and druming Cytosol is transferred in Tissue Culture Dish, and 10ml culture medium, 37 DEG C of incubators of Yu Han 5%CO2 and 95%O2 mixed gas are added Middle culture.

1.5 cell culture

(1) cell routine culture in DMEM culture medium (containing 10% fetal calf serum), is incubated in containing 5%CO₂And 95%O₂It is mixed In 37 DEG C of incubators for closing gas；

(2) different processing is given according to the difference of cell doubling time, passage in average every 2-3 days is primary, to keep thin Born of the same parents are constantly in logarithmic growth phase；

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

The passage of 1.6 cells

(1) cell for taking quasi- passage, discards culture solution, and 1 × PBS buffer solution (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) 1 × PBS buffer solution (pH 7.4) is discarded, suitable pancreatin (0.25%Trypsin-EDTA) is added, is containing 5%CO₂And 95%O₂It is incubated for several minutes in 37 DEG C of incubators of mixed gas；

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

(4) cell is softly blown and beaten with 1ml pipette tips to hang to single, cell is divided into other culture dishes (usually 1:2 or 1:3 Passage), and enough culture mediums are added and are placed in incubator, complete cell passage.

1.7 cell cryopreservation

(1) cell for needing to freeze, should maintain it in cell log growth period, vigor is 90% or more；

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

(3) postdigestive cell is moved into sterile 15ml centrifuge tube, 800rpm, RT, is centrifuged 5min, cell deposition exists Tube bottom；

(4) it discards supernatant, cleans cell with 1 × PBS buffer solution (pH 7.4) of preheating, 800rpm, RT are centrifuged 5min；

(5) it discards supernatant, 1ml cells frozen storing liquid is added, is moved into cryopreservation tube after soft piping and druming cell to single cell suspension, In detail the label title of freeze-stored cell, source, freeze the date etc., be transferred to -80 DEG C of refrigerators after -20 DEG C of refrigerators are placed 30 minutes It stands overnight, finally moves into liquid nitrogen container and save for a long time.

1.8 cell counts experiment

By breast cancer cell MCF-7, T47D, 231 in increased logarithmic phase are arranged to control group, gastrin group is planted respectively In 96 orifice plates, 3 multiple holes of every group of setting give various concentration medicine as needed after 24 hour cells are adherent in plate 3000/hole Object 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, the specific steps are as follows:

(1) albumen: the cell of logarithmic growth phase is received, 1 pre-cooled × PBS buffer solution (pH 7.4) is cleaned twice, root Suitable 2 × SDS protein lysate containing DTT is added according to cell density, is mixed well with pipette tips, thorough crack protein, draws Cell protein is collected into 1.5ml EP pipe, is placed in 30min on ice, is cracked albumen sufficiently；

(2) albuminous degeneration: Type17600Dri-Bath temperature is adjusted to 95 DEG C, has the EP pipe of cell protein to be placed in receipts 10min is boiled in metal bath, places 10min on ice, is repeated twice, and is denaturalized albumen thoroughly；

(3) extract albumen supernatant: for albumen again after 95 DEG C are boiled 10min, 12,000rpm, RT are centrifuged 5min, abandon precipitating, will Albumen supernatant moves into another clean EP pipe；

(4) protein quantification: albumen is surveyed concentration through BCA method and quantify, is configured to the same volume, equally total with SDS Packing is stored in -80 DEG C after after amount；

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

(6) albumen loading: the albumen of packing is taken out from -80 DEG C, in boiling 10min in 95 DEG C of metal baths, by sample plus Enter in gel pore, while the tracer of 1ul bromophenol blue is added in every hole；

(7) electrophoresis: after completing loading, electrophoresis is carried out to bromophenol blue tracer band to separation gel time-varying with 80V voltage first Changing voltage is 120V, until terminating electrophoresis when bromophenol blue tracer band reaches electrophoresis trench bottom；

(8) transferring film: taking out running gel, carries out transferring film in the transferring film liquid of pre-cooling after removing spacer gel, will contain protein Separation gel lie in Western blot dedicated sandwich style transferring film folder and clamp, placement sequence is followed successively by by cathode to anode 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, will be on protein delivery to NC film；

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

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

(11) close: the NC film after rinsing is incubated in Western blot confining liquid (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 film；

(12) it is incubated for primary antibody: after terminating closing, NC film being incubated in prepared with Western blot cleaning solution and purpose In the corresponding primary antibody of albumen, horizontal shaker, RT is incubated for 2h or 4 DEG C of overnight incubation；

(13) it washs: after primary antibody is incubated for, TBST short rinse NC film 3 times, being subsequently placed on 360 degree of shaking tables and use again Western blot cleaning solution (2% skimmed milk power that TBST is prepared) washing 3 times, room temperature, each 10min；

(14) it is incubated for secondary antibody: sufficiently after the remaining primary antibody of washing, NC film being set in the secondary antibody that corresponding HRP is marked, RT is incubated Educate 1h；

(15) wash: after secondary antibody is incubated for, Western blot cleaning solution (2% skimmed milk power that TBST is prepared) is quickly Rinsing NC film 3 times, is subsequently placed on 360 degree of shaking tables and washs again 3 times, RT, each 10min；

(16) it images: by the Immobilon Western Chemilminescent HRP of the NC film after washing Substrate reagent develops, and antigen antibody complex can image in conjunction with developer in gel imager.

1.10 immunohistochemical staining is analyzed

Whole samples are routinely fixed through 10% formalin, and 4 μm of histotomies are cut into paraffin embedding processing, specific immune Histochemical stain is tested referring to existing experimental method, the specific steps are as follows:

(1) bake piece: histotomy is placed in baking 30 in 60 DEG C of constant temperature roasters and stays overnight；

(2) dewax: slice places 20min in 45 DEG C of dimethylbenzene I, places 30min in dimethylbenzene II；

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

(4) slice is taken out, stands 1min in tap water after rinsing residual ethanol with tap water, single water that steams is changed and stands 1min；

(5) antigen retrieval: slice being set and is repaired in box, is added antigen retrieval buffers (citrate buffer), is covered box cover, 90 DEG C of 3min of microwave stove heating boil to buffer, then with 60 DEG C of maintenance 12min；

(6) antigen repairing box is taken out, opens the lid, stands antigen retrieval buffers at room temperature, until temperature drops to room Until temperature；

(7) antigen retrieval buffers are discarded, slice is moved into staining jar, are added 1 × PBS buffer solution (pH 7.4), it is mild to rush Wash slice 3 times, each 5min；

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

(9) 3% hydrogen peroxide is discarded, is mildly rinsed with 1 × PBS buffer solution (pH 7.4) slice 3 times, each 5min；

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

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

(12) it after the completion of primary antibody is incubated for, is mildly rinsed with 1 × PBS buffer solution (pH 7.4) slice 3 times, each 10min；

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

(14) it after the completion of secondary antibody is incubated for, is mildly rinsed with 1 × PBS buffer solution (pH 7.4) slice 3 times, each 10min；

(15) colour developing, microscopically observation colour developing feelings are incubated at room temperature with the diaminobenzidine solution (DAB) of fresh configuration Condition；

(16) distilled water rinses slice, removes remaining DAB；

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

(18) haematoxylin dye liquor is recycled, slice is rinsed for several times with tap water, remaining haematoxylin is removed, at 50 DEG C to 60 DEG C Tap water in stand 5-10min, carry out anti-blue；

(19) it after the completion of anti-basket, is sliced with originally washing, then successively pass through 95% ethyl alcohol I, 95% ethyl alcohol II, 100% second Alcohol I, 100% ethyl alcohol II carry out slice dehydration, and each 1min thoroughly dries at room temperature；

(20) slice dried respectively places 5min in dimethylbenzene I, dimethylbenzene II, be sliced transparent, dries；

(21) neutral gum mounting is finally added dropwise on slice sample, postposition optical microphotograph is under the microscope；

(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 mice (about 4~6 weeks) with mouse, and 16~18g of weight is purchased from Chinese science Institute's Shanghai Experimental Animal Center, in the laboratory of Experimental Animal Center SPF grades of this school, experimental design and operation are abided by for animal feeding 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 cell about 5 × 10⁶It is a, 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, it is above be administered 2 weeks.The administration same day is set as D0；Transplantable tumor is measured 2 times a week Major diameter and minor axis calculate gross tumor volume: gross tumor volume (mm according to following formula³)=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 the thin slice with a thickness of 4mm, partially with Formalin is fixed, and partially with liquid nitrogen cryopreservation, is used for subsequent experimental.

2 results

Expression of the 2.1CCKBR receptor in breast cancer cell

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

Expression of the 2.2CCKBR receptor in breast cancer tissue

This research carries out the expression of immunohistochemistry detection 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 cell is administered once day, continuous 7 days, daily into Row cell count.Tumor cell number significantly reduces (* P compared with control group after gastrin is handled in MCF-7 and T47D as the result is shown < 0.05, compared with the control group, n=3), but there is no inhibiting effect to 231.Fig. 3 is proliferation of the gastrin to breast cancer cell Inhibiting effect.

Influence of 2.4 gastrins to CCKBR protein level

By gastrin 10^-7M is respectively acting on MCF-7, T47D cell, day single administration, totally 3 days, then respectively at administration 1d, 3d, 5d collect cell extraction albumen and do Western-blot detection CCKBR albumen afterwards, and MCF-7 gives in gastrin as the result is shown 1d can obviously raise CCKBR protein abundance after medicine, and with administration number of times increase and the extension of time still keeps high abundance It and is in obvious chronic up-regulation trend；And T47D cell 1d CCKBR protein abundance after gastrin administration starts 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 cell is significantly faster than that T47D Cell.Fig. 4 is that gastrin raises CCKBR protein abundance in MCF-7, T47D.

2.5 gastrins raise erk, p65 protein expression

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 p65 albumen, MCF-7 is in stomach as the result is shown 1d can obviously raise CCKBR protein abundance after secretin administration, and with administration number of times increase and the extension of time is still kept High abundance and be in obvious chronic up-regulation trend；And T47D cell 1d CCKBR protein abundance after gastrin administration starts to 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 cell is obviously fast In T47D cell.Fig. 5 is that gastrin raises erk, p65 protein abundance in MCF-7, T47D.

The antitumor action of 2.6 gastrins in vivo experiment

2.6.1 gastrin inhibits tumor proliferation in nude mice lotus knurl model

The BALB/c nude mice of average 6 week old is taken to inject 5 × 10 respectively in two sides fat pad⁶A MCF-7 cell builds nude mice Lotus knurl model.Nude mice is divided into 2 groups, i.e. control group, gastrin group, inoculated tumour cell at random after nude mice injects 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.Start the growth of each group lotus knurl after administration 2 weeks as the result is shown Curve separates, and is significantly lower than control group (P < 0.05) to gastrin group lotus knurl mean size when being administered 2 weeks, it was demonstrated that the two exists Also there is antitumor action really in vivo.And tumour is taken to take pictures weighing.Fig. 6 is that gastrin inhibits gastric cancer in nude mice lotus knurl proliferation.

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 inhibits tumor proliferation by influencing the downstream signaling pathway of CCKBR

Lotus knurl tissue extraction albumen is taken to carry out GAP-associated protein GAP such as ERK, as a result the Western blot detection of P65 etc. is shown Show in lotus knurl tissue gastrin can upregulated protein abundance, this is consistent with cell experiment result, further supports ours Hypothesis.Fig. 8 is that gastrin influences nude mice lotus knurl histone p-ERK, p-P65 expression.

In summary test, it has been found that gastrin has an antitumor action in MCF-7, T47D, experiment in vivo also into One step confirms the effect.Its molecular mechanism be thought of as gastrin by up-regulation CCKBR protein abundance, the CCKBR receptor of up-regulation with Gastrin combines starting and amplifies the key link that the influence to downstream signaling molecule is the antitumor proliferation of gastrin.

The detection of 2.9 clinical patients Gastrin Levels

Internal experiment in vitro confirms that gastrin is able to suppress two kinds of breast cancer cell lines of MCF-7, T47D.Analyze its molecule spy Sign is HER2 feminine gender, and ER and/or PR are positive.We have collected 38 this type patient with breast cancers serum and 15 it is normal The serum of people detects two groups of Gastrin Levels by ELISA.Gastrin Levels are general in the blood of this kind of patient with breast cancer as the result is shown All over lower 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, and the proliferation of discovery cell strain MCF-7 and T47D obtain Inhibit, CCKBR, the ERK of phosphorylation and the P65 of phosphorylation have been raised by Westernblot detection discovery.This result is dynamic Preliminary identification has also been obtained on object preliminary experiment.Its precise mechanism also needs next further verifying and probes into.MCF-7 and T47D is HER2 feminine gender, and ER and/or PR are positive, and according to this characterization of molecules, we have collected the blood of 38 clinical breast cancer patients Clear and 15 normal persons serum detects its level of serum gastrin by ELISA, finds this type patients serum gastrin water It is flat horizontal lower than normal person.Furthermore it has been found that in the low patient of these Gastrin Levels 25 main suits have stomach trouble history or Gastrocopy has stomach trouble.Accordingly, it is presumed that it is a risk factors of pathogenesis of breast carcinoma that Gastrin Levels are low.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (2)

1. application of the gastrin in preparation diagnosing and treating breast cancer medicines, which is characterized in that cause the thin of the breast cancer Born of the same parents refer to breast cancer cell MCF-7 and T47D.

2. application of the gastrin according to claim 1 in preparation diagnosing and treating breast cancer medicines, which is characterized in that The gastrin refers to one or more of 34 peptide of gastrin complete sequence, 17 peptide of active form and active form.