CN101927008B - Diagnostic and therapeutic gastric cancer vascular specific binding peptide GEBP11 isotope probe - Google Patents

Abstract

The invention belongs to the field of biomedical technology, and in particular relates to a diagnostic and therapeutic gastric cancer vascular specific isotope-labeled short peptide probe <99>TC<m>-GEBP11 or <131>I-GEBP11 and a preparation method thereof. The probe is characterized in that the diagnostic isotope <99>Tc<m>O4<-> or therapeutic isotope <131>I is connected to GEBP11 short peptide, so that the short peptide probe is endowed with double functions of gastric cancer vascular targeting and diagnosis or therapy. In-vivo tracer experiments of the isotope show that the GEBP11 isotope probe has the characteristics of good in-vivo gastric cancer tissue targeting and specific distribution. SPECT imaging of <99>Tc<m>-GEBP11 in a gastric cancer bearing nude mice excellently shows a lesion site of transplanted tumor, and the ideal imaging time interval is within 18 to 24 hours after the injection. Results of tumor-suppressing experiments show that <131>I-GEBP11 has obvious effects of suppressing tumor growth and prolonging life time of the tumor bearing mice and weak toxic or side effect. The GEBP11 isotope probe has certain theoretical significance and great potential application value in gastric cancer diagnosis, vascular targeting therapy and evaluation of therapeutic effect and the like.

Description

Diagnosis and treatment gastric cancer vascular specific binding peptide GEBP11 isotope probe

Technical field

The invention belongs to field of biomedicine technology, be specifically related to diagnosis and treatment gastric cancer vascular specific property isotopic labeling short peptide probe ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11 and preparation method thereof also relates to this probe in this Application for Field.

Background technology

Gastric cancer is the modal malignant tumor of digestive tract, and its mortality rate occupies second in global cancer mortality.Traditional therapeutic modality such as operation, chemotherapy and outer radiotherapy can not obtain ideal therapeutic effect.Discover that angiogenesis is one of essential condition in the tumor development process, process, character and the density that the tumor tissues blood capillary generates is directly connected to the ability and the prognosis of tumor growth, invasion and attack, transfer.Based on the dependent theory of tumor vessel and the fact; The blood vessel suppression therapy becomes a focus of present oncotherapy research field; And with tumor tissues microvessel density (Microvascular density; MVD) and index of correlation be used for diagnosing tumor, therapeutic evaluation, relapse and metastasis early warning and prognosis evaluation etc., obtained certain clinical effectiveness, demonstrate good prospects for application.Yet; Present tumor vessel inhibition medicine and detection means are still not mature enough; Still unsatisfactory; Also have tumor vascular targeted undesirable, the special problems such as targeted molecular of shortage tumor vessel, the solution of these problems depends on tumor-blood-vessel growth characterization of molecules and the further investigation of mechanism and the continuous development of correlation technique means.

Research shows that tumor vessel is different from normal blood vessels, has unusual anatomical features and molecule heterogeneity, and tumor vessel is expressed the specific molecular that is different from normal blood vessels, and this is the molecular basis of tumor vascular targeted Clinics and Practices.Identify the heterogeneous molecule of tumor vascular endothelial cell; Study the effect of these key molecules in angiogenesis; Detect its Changing Pattern of its expression and dynamic monitoring; Will help the deep molecular mechanism of understanding tumor-blood-vessel growth, for diagnosing tumor, blood vessel suppression therapy, branch prediction and prognosis evaluation etc. provide important target spot.

The evaluation of tumor-blood-vessel growth at present still mainly relies on traditional pathology method immunohistochemical staining to carry out the MVD counting, and this method is time-consuming, require great effort, have wound, and is prone to cause personal error, can not estimate angiogenic activity from function again; And the molecular imaging that develops rapidly in recent years technology provides opportunity for addressing this problem; Utilize the specific molecular probe; Can be at body, in real time, cell, molecular events in the dynamic monitoring tumor development process, be expected to realize that the visual detection by quantitative of tumor-blood-vessel growth and dynamic real-time follow the trail of.Current tumor vessel suppression therapy is many as a kind of supplementary means, normal and other cytotoxic drug Combined application treatment tumor; And the treatment of tumor vessel radioreceptor combines receptor target property nucleic internal radiotherapy with the tumor vessel suppression therapy; Give tumor vessel treatment target killing vigor; Be expected to solve some problems in the tumor vessel suppression therapy, become a focus of research in recent years.Molecular imaging technology and radioreceptor treatment all depend on the selectively targeted molecule of high-affinity.In the previous work, we obtain gastric cancer vascular endothelial-cell specific binding peptide GEBP11 through phage random peptide library screening, for the property diagnosis of gastric cancer vascular targeting and treatment provide the candidate targeted molecular.

Summary of the invention

One of the object of the invention is, a kind of diagnosis and treatment gastric cancer vascular specific binding peptide GEBP11 isotope probe is provided ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11, this short peptide probe has the dual-use function of gastric cancer vascular targeting and diagnosis or treatment.

Another object of the present invention is, diagnosis and treatment gastric cancer vascular specific binding peptide GEBP11 is provided isotope probe ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11 preparation and authentication method.

A further object of the present invention is, with diagnosis and treatment gastric cancer vascular specific binding peptide GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11 is used for diagnosing gastric cancer, blood-vessels target treatment and therapeutic evaluation.

Technical scheme of the present invention is: provide to have people's gastric cancer vascular specific and combine active diagnosis and treatment GEBP11 isotope short peptide probe; It is characterized in that: this isotope probe has identical framing structure---people's gastric cancer vascular specific binding peptide GEBP11; It forms (CTKNSYLMC) by 9 aminoacid; Form disulfide bond and cyclisation through the two ends cysteine, with diagnosis type isotope ⁹⁹Tc ^mO ₄ ^-Or therapeutic type isotope ¹³¹I is connected in the GEBP11 small peptide, gives the dual-use function of short peptide probe gastric cancer vascular targeting and diagnosis or treatment.

Described people's gastric cancer vascular specific combines active diagnosis and treatment GEBP11 isotope short peptide probe, and its preparation or implementation are:

1) with radiosiotope ⁹⁹Tc ^mO ₄ ^-, ¹³¹I is marked on gastric cancer vascular specific binding peptide GEBP11 respectively, and its mark quality and BA are identified;

2) binding characteristic of GEBP11 small peptide and its receptor is analyzed;

3) GEBP11 or its isotope probe are detected in intravital gastric cancer vascular targeting property of mice with tumor and biodistribution characteristic;

4) right ⁹⁹Tc ^mThe diagnostic value of-GEBP11 in mice with tumor gastric cancer estimated;

5) right ¹³¹Therapeutic effect and the toxic and side effects of I-GEBP11 in mice with tumor gastric cancer estimated.

Described radiosiotope ⁹⁹T ^cMO ₄ ^-, ¹³¹I is marked on GEBP11 respectively, and its implementation is: the employing direct method will ⁹⁹Tc ^mO ₄ ^-Be marked on GEBP11, promptly utilize ascorbic acid reduction opened disulfide bond, expose sulfydryl, use the hydrosulfurous acid sodium reduction ⁹⁹Tc ^mO ₄ ^-, with technetium from+7 valencys be reduced to+3 ,+4 ,+5 valencys, thereby as the sulfydryl chelating lower valency of strong chelation group ⁹⁹Tc ^mGenerate ⁹⁹Tc ^m-GEBP11; Employing NBS method will ¹³¹I is marked on GEBP11, promptly based on the principle of electrophilic substitution on the aromatic rings, with NBS with Na ¹³¹I is oxidized to I ²Or I ⁺, I then ²Or I ⁺Substitute onto on the tyrosine residue of GEBP11 and generate ¹³¹I-GEBP11.

Said diagnosis type isotope ⁹⁹Tc ^mO ₄ ^-Be connected in the GEBP11 small peptide, adopt direct labelling method to realize; Its optimum mark or reaction condition are: fresh 1mol/L sodium ascorbate solution 20 μ L, add 50g/L hydrosulfurous acid sodium solution 20 μ L successively, and 100 μ L GEBP11 solution and 2-5mCi's is fresh ⁹⁹Tc ^mO ₄ ^-Leacheate 100 μ L, reaction system pH 4.8, and room temperature reaction 10min accomplishes labelling.

Said therapeutic type isotope ¹³¹I is connected in the GEBP11 small peptide, adopts the NBS labelling method to realize; Its optimum mark or reaction condition are: NBS is with deionized water dissolving, and GEBP11 adds GEBP11 solution 20 μ L earlier with the dissolving of PBS buffer in the reaction tube, add the Na of 7.4MBq then ¹³¹I solution respectively adds NBS solution 20 μ L at last, and mixing gently under the room temperature after 30 seconds, adds 2% human serum albumin, 10 μ L cessation reactions and promptly accomplishes labelling.

Described its mark quality and BA are identified that its implementation is: paper chromatography is measured mark rate, measures gained ⁹⁹Tc ^m-GEBP11, ¹³¹The radiochemicsl purity of I-GEBP11, specific activity, inside and outside stability, external microcosmic receptor autoradiography technology for detection ⁹⁹Tc ^m-GEBP11 reaches ¹³¹The bonded BA of I-GEBP11 and Co-HUVECs.

Described binding characteristic to GEBP11 small peptide and its receptor is analyzed; Its implementation is: analyze experimental identification receptor affinity and recipient cell density through the receptor radiative aglycone combination; Adopt immunocytochemistry or Subcellular Localization and the internalization of fluoroscopic examination GEBP11 in Co-HUVECs, the binding specificity of GEBP11 in people's stomach organization detects in immunohistofluorescence.

The detection of gastric cancer vascular targeting property and biodistribution characteristic in the described body; Its implementation is: through blue plaque form experiment and GEBP11, the IN11 phage suppresses experiment in the intravital competition of tumor bearing nude mice and detects the specificity of going back to the nest in the phage body; Immunofluorescence technique detects GEBP11 at the intravital binding specificity of tumor bearing nude mice, and isotope tracer technique detects ¹³¹I-GEBP11 is in the intravital biodistribution of tumor bearing nude mice.

Described right ⁹⁹Tc ^mThe diagnostic value of-GEBP11 in mice with tumor gastric cancer estimated, and its implementation is: adopt the SPECT imaging technique to carry out in the lotus people gastric cancer nude mouse ⁹⁹Tc ^m-GEBP11 video picture is developed to the bonded organizational structure of GEBP11 small peptide, and tracing study 24h observes continuously ⁹⁹Tc ^m-GEBP11 is the targeting property of tumor tissues in vivo, and its imaging law of desk study is inquired into the diagnostic value of GEBP11 isotope probe in the gastric cancer vascular molecular imaging.

Described right ¹³¹Therapeutic effect and the toxic and side effects of I-GEBP11 in mice with tumor gastric cancer estimated, and its implementation is: press down the tumor experimental evaluation through MTT cell proliferation experiment, mice with tumor ¹³¹I-GEBP11 is to the inhibition ability of endotheliocyte and mice with tumor tumor, and immunohistochemical staining is counted tumor tissue MVD, and pathology HE dyeing, blood blood cell analysis and biochemical indicator detect hepar damnification and the bone marrow depression situation of observing.

Described diagnosis and treatment gastric cancer vascular specific binding peptide GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11 has certain theoretical significance and great application value at aspects such as diagnosing gastric cancer, blood-vessels target treatment and therapeutic evaluatioies.

Characteristics of the present invention are:

1, the present invention is based on the gastric cancer vascular specific binding peptide GEBP11 that obtains through phage peptide library in-vitro screening technology in the previous work, have independent intellectual property right and sturdy working foundation, belong to original achievement, have novelty.The stomach cancer cell of previous work screening phage-display peptide library, HUVEC external co-culture model, based on " with the external altogether cultivation of tumor cell can inducing endothelial cell generation analog in the tumor environment endotheliocyte change (Microvascular Res.2002; Discovering 63:316) " has sufficient theoretical basis, and experimental result has also confirmed this point.In-vitro screening based on co-culture model; Avoided in the body that the screening influence factor is more, screening conditions are difficult to strict control and are difficult to confirm that the transplanted tumor tissue blood vessel is major issues such as animal derived or humanized; Thereby have certain advantage, lay a good foundation for obtaining desirable peptide section.The GEBP11 small peptide is the screening gained; Combine difference one of small peptide the most significantly with the normal blood vessels endotheliocyte at the gastric cancer vascular endotheliocyte; Its specificity is incorporated into the prompting of gastric cancer vascular endotheliocyte, and it might become the targeted molecular of gastric cancer vascular; In property diagnosis of gastric cancer vascular targeting and treatment, have potential using value, its bind receptor possibly play a significant role in gastric cancer vascular generates, and might become the novel targets of tumor vessel suppression therapy.

2, select classical radiosiotope for use ⁹⁹Tc ^mO ₄ ^-, ¹³¹I; Adopt direct method, NBS method that it is marked on gastric cancer vascular specific binding peptide GEBP11 respectively; Analyse the expansion system through groping definite its optimum mark condition and suitable ply of paper repeatedly; Qualification result shows that gained isotope probe GEBP11 has good radiochemicsl purity, specific activity, inside and outside stability and BA.Different isotope labeling principles are also inequality, and many aspects such as the composition structure of reactant concentration, reaction condition, institute's labelled molecule and character all might influence the labelling result, and therefore different molecules is carried out labelling need grope its optimum mark condition.And whether isotopic labelling influences the original BA of institute's labelled molecule, is the major issue of decision success or failure.The present invention has found out the direct method labelling ⁹⁹Tc ^mO ₄ ^-, NBS method labelling ¹³¹I is in the optimum mark condition of GEBP11 small peptide, and confirms that the short peptide probe behind the labelling still has original BA, and this just is that next step experiment is laid a good foundation.GEBP11 good biology combines activity be it as isotope probe at all, and classical radiosiotope ⁹⁹Tc ^mO ₄ ^-, ¹³¹The successful labelling of I is the range of application further changing other isotope of labelling, promote this targeted molecular, lays a good foundation and reference is provided.

3, based on good affinity of GEBP11 small peptide and its receptor and higher Rd; And with its receptors bind can be by the characteristic of internalization; It is had and the bonded activity of gastric cancer vascular endothelial-cell specific; Have more the targeting property of stomach organization in the good body and the distribution of specific characteristic of stomach organization, this makes the GEBP11 isotope probe carry out video picture to the gastric cancer focus in vivo preferably, gathers resident through radiosiotope dense in cancer; The performance GVT alleviates toxic and side effects.The correlational study achievement of this GEBP11 isotope probe; Lay a good foundation for developing the stomach cancer diagnosis reagent or the medicine that are fit to clinical practice; Might with treatment new means be provided for the molecular diagnosis of gastric cancer, have certain theoretical significance and great potential using value.

Description of drawings

Below in conjunction with the embodiment accompanying drawing the present invention is done further detailed description.

Fig. 1 ⁹⁹Tc ^m-GEBP11 stability test; Show among the figure: with labelling peptide room temperature held, and mix with normal saline, serum, EDTA, cysteine, its 0-24h mark rate does not have obvious reduction, shows ⁹⁹Tc ^m-GEBP11 has good stable property in vivo and in vitro.

Fig. 2 ⁹⁹Tc ^m-GEBP11 reaches ¹³¹The bioactive autoradiography of I-GEBP11 is identified; Shown in figure, ⁹⁹Tc ^m-GEBP11 combines (A) with Co-HUVECs, visible black color silver particle deposition, its intensity apparently higher than ⁹⁹Tc ^mThe combination (B) of-GEBP11 on HUVECs, ⁹⁹Tc ^m-URP (C) on the Co-HUVECs reaction (A-C is a cell smear, * 100) that is negative; ¹³¹I-GEBP11 combines (D) with Co-HUVECs, visible black color silver particle deposition, its intensity apparently higher than ¹³¹The combination (E) of I-GEBP11 on HUVECs, ¹³¹I-OXT (F) on the Co-HUVECs reaction (D-F is a cell climbing sheet, * 400) that is negative.

Fig. 3 ⁹⁹Tc ^mThe saturation curve of-GEBP11 and receptors bind and Scatchard mapping; ⁹⁹Tc ^mThe analysis of-GEBP11 receptor radiative aglycone combination shows, the affinity (KD=1.972nM) of it and Co-HUVECs is significantly higher than HUVECs (KD=2.489nM), the Rd (5.7 * 10 of Co-HUVECs ⁵/ cell) apparently higher than HUVECs (3.3 * 10 ⁵/ cell).

The binding specificity of Fig. 4 GEBP11 and the immunocytochemical stain of Subcellular Localization (* 400); A-E is Co-HUVECs among the figure, and F is HUVECs, and G is the GES cell, and H is the SGC7901 cell, and I is an ags cell.A GEBP11 is at after birth and to examine all endochylemas painted; The contrast of B VIII factor antibody, it is painted to examine all endochylemas; C CD31 antibody control, after birth is painted; D URP is negative; E PBS is negative; F-I GEBP11 is negative or weak positive reaction on wild type HUVECs, GES cell and gastric cancer SGC7901 and ags cell.

Fig. 5 laser co-focusing immunofluorescence technique detection GEBP11 small peptide combines and internalization experiment (* 100) diagram with its receptor; When 4 ℃ of A-C are hatched among the figure (endotheliocyte partly bounces back), green fluorescence mainly is present in the endotheliocyte after birth; After 37 ℃ of D-F were hatched, endochylema also sent fluorescence.Wherein B, E are that the DAPI lining dyes, and C, F are respectively the Merge image of A and B, D and E.Used cell is Co-HUVECs.

Fig. 6 GEBP11 small peptide combines active immunofluorescence dyeing (* 100) result with people's stomach organization blood vessel; A-E is a stomach organization among the figure, and F is chronic atrophic gastritis (CAG) tissue, and wherein A, B are serial section.A&F GEBP11, B CD31 antibody, C GEBP11 and CD31 antibody staining Merge image, D PBS, E URP.

Fig. 7 phage in the tumor bearing nude mice body, go back to the nest in each the tissue in titre; The titre that diagram IN11 phage reclaims at tumor tissues is significantly higher than contrast phage or its titre that in control tissue, reclaims, and the difference of contrast phage between each tissue is little.

Fig. 8 GEBP11 small peptide and IN11 phage suppress experiment in the intravital competition of tumor bearing nude mice; Show among the figure that the GEBP11 small peptide is inhibited to going back to the nest of mice with tumor tumor tissues to the IN11 phage, and be dose dependent, when small peptide consumption during at 50-100 μ g, suppression ratio increases gently, presents high inhibitory, and the control peptide that has nothing to do does not have this effect.

Fig. 9 GEBP11 small peptide combines active immunofluorescence dyeing (* 100) in the tumor bearing nude mice body; A-F is a tumor tissues among the figure, and G-I is a heart tissue, and A-C, G-I are that GEBP11 (green fluorescence) is total to positioning analysis with VIII factor antibody (red fluorescence), and D-F is irrelevant peptide contrast.Wherein A, D, G show green fluorescence, B, E, H exhibit red fluorescence, and C, F, I are respectively the Merge image of A and B, D and E, G and H.By visible among the figure, consistent at tumor tissues GEBP11 with VIII factor antibody location, be shown as yellow behind the Merge.

Figure 10 ⁹⁹Tc ^m-GEBP11 is in the intravital SPECT video picture of lotus people gastric cancer nude mice; A, B are respectively GEBP11 and URP and form as figure as a result among the figure, and C, D are respectively its T/NT ratio Analysis.

Figure 11 ¹³¹I-GEBP11 is to the external lethal effect of Co-HUVECs propagation; Among the figure ¹³¹(Cx=3.7 * 10ExKBq/ml) representes that the final concentration of this medicine effect is 3.7 * 10 to I-GEBP11 ^xKBq/ml (x=0,1,2,3).

The variation of transplanted tumor volume in Figure 12 Drug therapy process; Result's demonstration, eADM, ¹³¹Two groups of tumor growths obviously slow (P＜0.05) after 2 weeks of I-GEBP11 show the effect that suppresses tumor growth; Na ¹³¹I, the effect of two groups of unrestraint tumor growths of GEBP11.

Each treatment group mice with tumor life span of Figure 13 relatively; ¹³¹I-GEBP11, eADM and GEBP11 group median survival interval obviously prolong Na than the NS group ¹³¹I organizes does not have prolongation life cycle.

The pathology that Figure 14 treats the back tumor tissues detect; A-C HE dyeing (* 100) among the figure, D-F the anti-FVIII-R antibody mediated immunity histochemical stain (* 200).The HE demonstration of dyeing, ¹³¹Visible slabbing degeneration necrosis district (B) in the I-GEBP11 treatment posterior tuberosity, and NS matched group oncocyte generates (A) in good condition, the downright bad special mess (C) of visible limitation in the tumor kitchen range after the GEBP11 treatment; Groupization dyeing demonstration, ¹³¹After I-GEBP11 (E) and GEBP11 (F) treatment, blood capillary is than the obvious minimizing of NS matched group (D) in the tumor.

Figure 15 treats back mice with tumor liver and bone marrow pathological examination; Show among the figure, eADM (A) or ¹³¹The visible hepatocellular degeneration necrotic reaction that is dispersed in of liver after I-GEBP11 (B) treatment is attached most importance to the eADM group, and the NS group does not see that hepatic injury sexually revises (C); The bone marrow section sees that eADM group bone marrow hematogenesis is suppressed (D), ¹³¹I-GEBP11 treatment group (E) and NS group (F) are not seen obviously unusual.

The specific embodiment

The treatment of tumor molecular imaging and radioreceptor is a big focus in emerging in recent years tumor research field, and its development depends on the selectively targeted molecule of high-affinity.The present invention is based on the gastric cancer vascular specific binding peptide GEBP11 that previous work obtains, use radiosiotope ⁹⁹Tc ^mO ₄ ^-, ¹³¹I carries out labelling respectively, successfully prepares diagnosis type and therapeutic type gastric cancer vascular specific property isotopic labeling short peptide probe, and through Quality Identification, the labelling peptide has good radiochemicsl purity, specific activity, inside and outside stability and BA.Experiments such as tracer experiment, immunofluorescence or chemical staining are further identified in the analysis of receptor radiative aglycone combination, the isotope body; The GEBP11 isotope probe combines to have good specificity and affinity with the gastric cancer vascular endotheliocyte; With can be after its receptors bind by internalization, and have the characteristic of stomach organization targeting and special distribution in the good body.The SPECT imaging results shows ⁹⁹Tc ^m-GEBP11 can show tumor kitchen range position preferably, presses down the tumor experimental result and shows ¹³¹I-GEBP11 has the obvious suppression tumor growth, prolongs the mice with tumor effect of life cycle.

Concrete grammar is following:

1.GEBP11 the preparation of isotope probe and evaluation

Adopt direct method, ⁹⁹Tc ^mO ₄ ^-Labelling GEBP11 and control peptide URP adopt the NBS method, ¹³¹I labelling GEBP11 and control peptide OXT, and labelled compound made Quality Identification (calculating mark rate, radiochemical purity, specific activity and stability) and BA is identified.

1.1 ⁹⁹Tc ^mThe preparation of-GEBP11

Adopt Reducing agent reduction disulfide bond, open it, will be reduced to by+7 valencys+5 ,+4 ,+3 valencys ⁹⁹Tc ^mBe connected on the sulfydryl that generates by disulfide bond reduction.The basic token method is following: fresh 1mol/L sodium ascorbate solution 20 μ L add 50g/L hydrosulfurous acid sodium solution 20 μ L, 100 μ L GEBP11 solution (contain GEBP1110 μ g, be dissolved in the SAS of 0.01mol/L pH4.2) and 3mCi's is fresh ⁹⁹Tc ^mO ₄ ^-Leacheate 100 μ L, pH value of reaction system 4.8 uses mass fraction to transfer to 500 μ L as 0.9%NaCl, and reaction mixture is put boiling water bath 15min and is accomplished labelling.Be the basis with above-mentioned reaction condition; Fixing other labelling factors are constant; Change the time labelling factors such as (1min, 5min, 10min, 20min, 30min) of small peptide consumption (10,20,30 μ g), ascorbic acid consumption (1mg/5 μ l, 2mg/10 μ l, 4mg/20 μ l, 8mg/40 μ l), sodium dithionite consumption (0.5mg/10 μ l, 1mg/20 μ l, 1.5mg/30 μ l), pH value of reaction system (2.0,3.0,4.0,4.8,5.0,6.0,7.0), reaction temperature (room temperature, 37 ℃, 60 ℃, 90 ℃, 100 ℃), labeled reactant; The observation mark rate changes, and confirms optimum reaction condition.

Through preliminary making repeatedly, find out the optimum mark condition and carry out ⁹⁹Tc ^mThe labelling of-GEBP11 and control peptide.The optimum mark condition is: fresh 1mol/L sodium ascorbate solution 20 μ L; Add 50g/L hydrosulfurous acid sodium solution 20 μ L successively; 100 μ LGEBP11 solution (contain GEBP11 20 μ g, be dissolved in the SAS of 0.01mol/L pH4.2) and 2-5mCi's is fresh ⁹⁹Tc ^mO ₄ ^-Leacheate 100 μ L, reaction system pH 4.8, and room temperature reaction 10min accomplishes labelling.

1.2 ⁹⁹Tc ^mThe Quality Identification of-GEBP 11

1.2.1 paper chromatography is measured mark rate

System I does developing solvent with acetone, labelling peptide and Rf ( ⁹⁹Tc ^m)=0.0-0.1, Rf ( ⁹⁹Tc ^mO ₄ ^-)=0.8-1.0, calculate the labelling peptide with ⁹⁹Tc ^mRadioactivity account for the percentage ratio of gross activity; System II uses ethanol: ammonia: water=2: 1: 5 is done developing solvent, Rf ( ⁹⁹Tc ^m)=0.0-0.1, labelling peptide and Rf ( ⁹⁹Tc ^mO ₄ ^-)=0.7-1.0 can calculate ⁹⁹Tc ^mRadioactivity account for the percentage ratio of gross activity.Wherein, certain peak radioactivity percentage rate=(this peak radiocounting/gross activity counting) * 100%.Concrete grammar is: with the mixture behind the labelling behind chromatographic paper one end points appearance, airing; Put into two kinds of developing solvents respectively; Take out airing when about 10min sample reaches another edge of chromatographic paper, chromatographic paper is cut by 10 equal portions one by one collected in the test tube, the radioactivity of each test tube of record on γ immunity enumerator; The radiocounting of collecting and writing down two peaks, calculate each peak radioactivity percentage rate with ⁹⁹Tc ^m-GEBP11 mark rate (among mark rate=system I the labelling peptide with ⁹⁹Tc ^mRadioactivity percentage rate-system II in ⁹⁹Tc ^mThe radioactivity percentage rate).

1.2.2 radiochemical purity is measured

Radiochemical purity refers to that the radiopharmaceutic radioactivity of particular chemical structure accounts for the percentage ratio of gross activity.If mark rate can carry out measuring radiochemical purity (detection of concrete operation method isolabeling rate) behind the purification when low.Among radiochemical purity=system I the labelling peptide with ⁹⁹Tc ^mRadioactivity percentage rate-system II in ⁹⁹Tc ^mThe radioactivity percentage rate.

1.2.3 specific activity

Specific activity refers to the radioactivity of certain radioactive substance of unit mass, requires to be at least 10Ci/mmol.The amount (mmol) of specific activity=label radioactivity/label.

1.2.4 stability

Measurement is in external room temperature and simulated in vivo environment ⁹⁹Tc ^mThe stability of-GEBP11 complex.Concrete grammar has: with the labelling peptide room temperature held 4h behind the purification, observe its radiochemicsl purity and change; With the labelling peptide of purification respectively with normal saline, excessive EDTA, Freshman serum, 37 ℃ of incubations of L-cysteine (CYS, mol ratio is 1: 100), the variation of paper chromatography certification mark peptide radiochemicsl purity.

Investigate as stated above ⁹⁹Tc ^mWith the bonded stability of GEBP11, thereby infer ⁹⁹Tc ^mThe body internal stability of-GEBP11.Because EDTA is a kind of metal-chelator, be prone to ⁹⁹Tc ^mChelating, and the main component HSA in cysteine and the serum all contain sulfydryl also be prone to ⁹⁹Tc ^mChelating equally maybe be to being sequestered on the peptide ⁹⁹Tc ^mExert an influence.Whether research EDTA, cysteine, HSA influence is attached to the peptide cystine linkage ⁹⁹Tc ^mStability, infer ⁹⁹Tc ^m-GEBP11 stability in vivo.If the gained result shows that mark rate does not have obvious reduction, explanation in each solution ⁹⁹Tc ^m-GEBP11 is very stable in vivo and in vitro.

Under above-mentioned flag condition, adopt direct method labelling GEBP11, paper chromatography is measured its mark rate and is reached 90-98%, need not purification.The gained specific activity has also satisfied the demand of video picture greater than 100Ci/mmol.With labelling peptide room temperature held, and mix with normal saline, serum, EDTA, cysteine, observe the variation of its 0-24h mark rate, the result is presented at that mark rate does not have obvious reduction in each mixed solution, shows ⁹⁹Tc ^m-GEBP11 has good stable property (Fig. 1) in vivo and in vitro.

1.3 ¹³¹The preparation of I-GEBP11 and Quality Identification

1.3.1 ¹³¹The preparation of I-GEBP11

With deionized water dissolving, GEBP11 is with the dissolving of PBS buffer as oxidant: NBS for employing NBS, and fixedly the GEBP11 consumption changes iodine consumption and NBS consumption, to confirm the optimum mark condition.Add GEBP11 solution 20 μ L (containing 10 μ g GEBP11) in each reaction tube earlier; The iodine solution that adds 7.4MBq or 14.8MBq then; Respectively add NBS solution 2 μ L, 10 μ L, 20 μ L, 40 μ L, 60 μ L, 80 μ L (containing NBS 1 μ g, 5 μ g, 10 μ g, 20 μ g, 30 μ g, 40 μ g) at last; Mixing gently under the room temperature after 30 seconds, adds 2% human serum albumin, 10 μ L cessation reactions and promptly accomplishes labelling.

Through preliminary making discovery repeatedly, when the GEBP11 consumption is 10 μ g, ¹³¹The I consumption is that 7.4MBq tense marker rate is higher; The NBS consumption reaches 10 μ g tense marker rates and reaches the highest, increases not have obviously to raise or slightly decline again; And increase the GEBP11 consumption, when NBS10 μ g, still can obviously increase mark rate.Therefore, select for use the condition of reaction system to be: 20 μ g GEBP11,7.4MBq ¹³¹I, 10 μ g NBS.

1.3.2 ¹³¹The Quality Identification of I-GEBP11

Paper chromatography is measured its mark rate.Press the preliminary experiment result, development system is selected acetone for use, Rf ( ¹³¹I-GEBP11)=and 0.0-0.1, Rf (free-iodine)=0.4-0.6.Concrete grammar is: the 2cm place draw a starting line parallel with the base gently with pencil at chromatographic paper (width 1cm) apart from the base, with capillary glass tube point sample (being initial point) on starting line, free-iodine compares (point sample≤0.2cm); After drying chromatographic paper is suspended in vitro; Liquid level seals apart from initial point 1cm, when treating the solvent front apart from initial point 10cm; The taking-up chromatographic paper dries; Cut off piecemeal for every centimetre then, put respectively in vitro and directly measure, by formula calculate mark rate with γ line well type solid-state scintillation counter.Mark rate=(radioactivity/always the radiate activity at radiation peak) * 100%.

Select for use the optimum mark condition to carry out labelling, measure mark rate, can reach more than 90% with paper chromatography, average mark rate＞85%, good reproducibility generally need not purification, can directly be used for experiment.Its specific activity reaches requirement of experiment greater than 10Ci/mmol.Measure its radiochemicsl purity behind room temperature held 1h, 4h, the 8h and be respectively 90.3%, 89.1%, 88.7%, show the marked product that the method obtains ¹³¹I-GEBP11 has good stable property.

1.4GEBP11 the evaluation of isotope probe BA

Utilize micro autoradiography, whether the small peptide of observing behind the labelling still has BA, can combine with endothelial-cell specific, identifies that promptly whether the isotopic labeling process combines activity to impact to the biology of small peptide.Autoradiography concrete operations step is following.

1. conventional method prepares cell climbing sheet or smear, and 2.5% paraformaldehyde is 2min fixedly, and PBS cleans 5min * 3, drip respectively the GEBP11 isotope probe ( ⁹⁹Tc ^m-GEBP11 or ¹³¹I-GEBP11), behind the incubated at room 2h, all ice-cold buffer and mobile distilled water drip washing are used in section successively, and is gentle to liquid to remove unconjugated labelling aglucon, cold air drying.

2. in the darkroom under the HONGGUANG, 40 ℃ of water-baths fusing nuclear emulsion, dipping film method will immerse nuclear emulsion in microscope slide, put into Riker mount after the drying, add desiccant in the box, seal, and the black paper bag of external is tight, puts exposure 24h in 4 ℃ of refrigerators.

3. in the darkroom behind the equilibrium at room temperature; Microscope slide is put into the developer solution of constant water bath box; The temperature of developer solution is 19 ± 1 ℃, and developing time is 4min, and purpose is the light sensitive silver salt reduction that makes in the latex; Promptly from Silver monobromide, decomposite silver, and then make it to be reduced to visible ferrous metal silver granule under the light microscopic.

The microscope slide that 4. will reach developing time takes out, and washes, and puts into fixative solution then; The temperature of fixative solution is 19 ± 1 ℃, and the time is 8min, to remove not light sensitive as yet silver salt on the latex; Stop developing, prevent that simultaneously nuclear emulsion from continuing to absorb moisture and the generation illusion that expands.

5. microscope slide is washed 10-20min in clear water, fixative that flush away latex surface is left and dissolved silver salt.With the microscope slide gradient alcohol dehydration, xylene is transparent, the neutral gum mounting.

Whether 6. light microscopic is observed silver granuel distribution position and density down, observe the GEBP11 isotope probe and can be incorporated on the cell.

The biological activity of micro autoradiography technical appraisement isotopic labeling small peptide, result's demonstration, ⁹⁹Tc ^m-GEBP11 reaches ¹³¹I-GEBP11 and Co-HUVECs combine obviously to be better than HUVECs, and the contrasting marking peptide ⁹⁹Tc ^m-URP or ¹³¹I-OXT does not have obviously combination (Fig. 2) on Co-HUVECs.This explanation, labeling process does not have obvious influence, marked product to the ability of the specific bond gastric cancer vascular endotheliocyte that small peptide GEBP11 has ⁹⁹Tc ^m-GEBP11 reaches ¹³¹I-GEBP11 and Co-HUVECs still have specific binding capacity.

2. the binding characteristic of GEBP11 small peptide and its receptor is analyzed

Analyze experimental identification receptor affinity and recipient cell density through the receptor radiative aglycone combination; Adopt immunocytochemistry or Subcellular Localization and the internalization of fluoroscopic examination GEBP11 in Co-HUVECs, the binding specificity of GEBP11 in people's stomach organization detects in immunohistofluorescence.

2.1 the receptor radiative aglycone combination is analyzed

Analyze through the receptor radiative aglycone combination, can calculate average acceptor quantity of each cell surface and the bonded affinity of ligand receptor.The concrete operations step is following.

1. inoculate Co-HUVECs, HUVECs in 48 orifice plates, 2 * 10 ⁴/ every hole.Wherein every kind of cell divides 7 kinds of Concentraton gradient holes and corresponding non-specific binding hole; Totally 7 groups; Every group 5 hole comprises certain concentration 3 holes and non-specific bond 2 holes (non-specific bond hole is that concentration is higher than the unmark peptide with 100 times of group echo peptides), overnight incubation; Treat cell attachment, wash Co-HUVECs, HUVECs three times with PBS.

2. 400 μ L PBS-BSA (PBS, 10mg/mL BSA) seal 30min.

3. add Binding Buffer (PBS, 0.25%BSA, the variable concentrations (0.3125,0.625,1.25,2.5,5,10,20nM etc.) that pH7.4) dilutes ⁹⁹Tc ^mThe every hole 200 μ l of-GEBP11, every group of parallel three holes, 4 ℃ combine 2h, add ice-cold buffer 1ml cessation reaction again.Ice-cold PBST gives a baby a bath on the third day after its birth time, adds 600 μ L, 0.25% trypsin, and digestion 15min transfers to gamma and measures in the pipe, washes once with 600 μ L PBST, and washing liquid also joined measures in the pipe, and gamma is counted.

4. calculate specificity count value=total count value (every group of average cpm in certain concentration 3 holes)-non-specific count value (every group of non-specific bond 2 holes) according to formula.

5. utilize the bonded saturation curve of Prism 4.0 software development receptor-ligands, and carry out Scatchad and analyze, calculate the affinity of cell surface receptor density and part and receptors bind.

We carry out the analysis of extracorporeal receptor radiative aglycone combination on Co-HUVECs, HUVECs cell, by the multiple spot saturation analysis different markers peptide concentration is set, and analyze the affinity size of GEBP11 and endotheliocyte.Concentration with the labelling peptide is abscissa, is vertical coordinate with the concentration of the complex of labelling peptide and receptors bind, draws saturation curve.The receptor radiative aglycone combination is analyzed experimental result and shown, and is identical ⁹⁹Tc ^mDuring-GEBP11 labelling peptide concentration, the binding capacity of cultivating endotheliocyte and labelling peptide altogether will be higher than simple endothelial cells cultured.Article two, curve all is saturation curve, explains that GEBP11 belongs to specificity with two kinds of cells and combines.With the complex concentration is abscissa; With B/F is vertical coordinate; The Scatchard mapping shows (Fig. 3); The bonded Kd value of huve cell that GEBP11 cultivates together and cultivates merely is respectively 1.972nM, 2.489nM, and this explanation is compared with the huve cell of simple cultivation, and GEBP11 has and cultivates the higher affinity of endotheliocyte together.The binding site number (be recipient cell density) of GEBP11 on the huve cell of cultivating altogether and cultivating merely is respectively 5.7 * 10 ⁵/ cell, 3.3 * 10 ⁵/ cell shows that cultivating endotheliocyte altogether has higher GEBP11 Rd.

2.2 immunocytochemical stain detects the Subcellular Localization of GEBP11 in Co-HUVECs

2.2.1 the foundation of separation and Culture HUVECs and endotheliocyte, stomach cancer cell external co-culture model

Under aseptic condition, get the neonatal umbilical cord of normal pregnancy term labor, cut off pincers trace and sludged blood and block part, usefulness is added with 200,000 U/ and rises warm phosphate buffer (PBS) the lavation umbilical vein that penicillin, 200,000 U/ rise streptomycin; And the bloodstain on clean umbilical cord surface; With mosquito forceps umbilical cord one end folder is closed, 37 ℃ of 0.1%I Collagen Type VI enzyme 8-10ml of perfusion in umbilical vein make umbilical vein full; Again umbilical cord other end folder is closed, hatch 10-15min for 37 ℃.Decontrol mosquito forceps after taking out umbilical cord, collect the liquid in the umbilical vein, with 30-40ml PBS flushing tube chamber, Digestive system moves into centrifuge tube with flushing liquor, 800rpm, centrifugal 5min.Abandon supernatant, to contain the M200 culture medium 5ml re-suspended cell of 2%LSGS, with cell inoculation in 25cm ²In the plastics Tissue Culture Flask, put into 37 ℃, 5%CO ₂, cultivate in the saturated humidity incubator, avoid mobile in the 12h.Change liquid behind the 24h 1 time, after this, changed liquid 1 time in every 2-3 days, routine goes down to posterity or is frozen.

Adopt the Transwell culture dish or the culture plate in 0.4 μ m aperture, 3-5 is inoculated in last chamber and the following chamber of Transwell, co-cultivation respectively for HUVECs, people's adenocarcinoma of stomach cell SGC7901 cell.Two confluent monolayer cells are separated by semipermeable membrane, and culture fluid is traffic up and down then, and two kinds of cells are interacted through soluble factor, simulate intravital tumor microenvironment with this, and inducing endothelial cell changes.Operating procedure is following: recovery people adenocarcinoma of stomach cell line SGC7901, and with the RPMI1640 culture fluid that contains 10% hot deactivation NBCS, at 37 ℃, 5%CO ₂The conventional cultivation in the constant temperature incubator; The SGC7901 cell of results exponential phase is by 1 * 10 ⁵Individual cell/cm ²Density be inoculated in the following chamber or 24 orifice plates of the double-deck culture dish of Transwell single culture 24h; Simultaneously, gather in the crops the HUVECs of 3-5, according to 1 * 10 for exponential phase ⁵Individual/cm ²Density be inoculated into Transwell and go up chamber (Insert), place another culture dish single culture 24h; Behind the 24h; Treat that cell attachment stretches; With PBS SGC7901 cell and HUVECs are washed respectively 2 times; Move to the Transwell plug-in unit (insert) of having inoculated HUVECs in the culture dish or 24 orifice plate respective aperture of having inoculated the SGC7901 cell, add fresh M200 complete culture solution, making up and down, the chamber liquid level keeps balance; After this, every separated 24h changes liquid, continues to cultivate 3-4 days, forms the cell monolayer of subconfluence, is Co-HUVECs.

2.2.2 immunocytochemical stain

Conventional method prepares cell climbing sheet, adopts the method for immunocytochemical stain to identify the Subcellular Localization of GEBP11 small peptide on Co-HUVECs.After pressing routine immunization cytochemical staining step sealing completion, with 4 ℃ of incubated overnight of Bio-GEBP11 small peptide, hatch the DAB colour developing earlier then with the SP complex.Dye with anti-VIII factor antibody and anti-CD31 antibody respectively, as endochylema and the painted contrast of after birth.Optical microscope is observed coloration result down, judges the subcellular fraction position of brown conversion zone.

The immunocytochemical stain result shows; The GEBP11 small peptide dyes on Co-HUVECs and is positive; With after birth, all endochylemas of nuclear is outstanding, and VIII factor antibody and CD31 antibody staining are positive at all endochylemas of the nuclear of Co-HUVECs and after birth respectively, can be used as positive control; And URP and PBS contrast negative reaction, and the GEBP11 small peptide is negative or weak positive reaction on wild type HUVECs, GES cell, gastric cancer SGC7901 cell and ags cell. (see figure 4).

2.3 the immunocyte fluorescence staining detects the internalization of GEBP11 small peptide

The Co-HUVECs adherent growth is to exponential phase, and 1%BSA seals 20min, adds the FITC-GEBP11 of 100 μ g/ml, hatches after 2 hours fixedly 20min of cold acetone for 4 ℃, and laser confocal microscope is observed down.Or 4 ℃ hatch 2 hours after, hatched 2 hours in 37 ℃ again, cold acetone is 20min fixedly, laser confocal microscope is observed down.

Immunofluorescence dyeing laser confocal microscope observation GEBP11 small peptide combines and internalization with its receptor; The result shows; 4 ℃ of GEBP11 when hatching mainly are incorporated into the endotheliocyte after birth; After giving 37 ℃ of internalization conditions and hatching, the GEBP11 small peptide that is incorporated into the after birth receptor can be by (see figure 5) in the interior born of the same parents of dissolving.

2.4 the binding specificity of GEBP11 in people's stomach organization detects in immunohistofluorescence

Adopt immunofluorescence technique to identify FITC-GEBP11 small peptide and the bonded specificity of gastric cancer vascular, compare with non-cancer people's gastric tissue and irrelevant small peptide URP.The stomach organization frozen section is after the normal serum sealing; Add 4 ℃ of incubated overnight of anti-CD31 antibody earlier; Vibration washing 5min * 3 time, the FITC-GEBP11 that adds 100 μ g/ml simultaneously reaches the two anti-of Cy3 labelling approximately, hatches 1h for 37 ℃; Vibration washing 5min * 3 time are observed relatively down in fluorescence microscope.

The GEBP11 small peptide is the immunofluorescence dyeing result on people's stomach organization show; GEBP11 small peptide and the painted position consistency of CD31 antibody; Prompting GEBP11 small peptide can specific bond in people's stomach organization blood vessel; And control peptide URP does not have positive reaction, and the chronic gastritis tissue is not also seen obviously painted (Fig. 6).

3. GEBP11 is detected in the intravital gastric cancer vascular targeting property of mice with tumor

Through blue plaque form experiment and GEBP11, IN11 phage (the GEBP11 small peptide presents phage) suppresses experiment in the intravital competition of tumor bearing nude mice and detects the specificity of going back to the nest in the phage body; Immunofluorescence technique detects GEBP11 at the intravital binding specificity of tumor bearing nude mice, and isotope tracer technique detects ¹³¹I-GEBP11 is in the intravital biodistribution of tumor bearing nude mice.

3.1 blue plaque forms experiment and detects the specificity of going back to the nest in the phage body

3.1.1 the foundation of lotus people gastric cancer nude mice subcutaneous transplantation tumor model

Conventional method cultivating people's adenocarcinoma of stomach SGC7901 cell is to exponential phase, and also preparation single cell suspension is gathered in the crops in trypsinization, PBS centrifuge washing 2 times, and the adjustment cell density is 1 * 10 ⁷/ ml.Extracting tumor cell suspension with the 1ml syringe, to be inoculated in nude mice hind leg back subcutaneous, and each inoculation position injection 0.2ml contains viable count 2 * 10 ⁶Individual.Routine observation nude mice spirit, diet and body weight change situation.When treating that nude mice Subcutaneous tumor diameter grows to 0.8-1cm, be used for experiment.

3.1.2 the mensuration of phage titre

1. the recovery of recipient bacterium E.coli ER2738 and cultivation connect collarium with aseptic technique picking one from the frozen thing of the glycerol of E.coli ER2738; Coat on the LB-Tet flat board; 37 ℃ put upside down overnight incubation after, the single bacterium colony of picking places 3ml to contain the LB culture fluid of 1 μ g/ml Tet; 37 ℃ of shaken cultivation are spent the night, and the OD600 value is reached about 0.6.LB-Tet flat board and antibacterial amplification liquid place 4 ℃ of preservations, and is subsequent use;

2. phage carries out 10 with the LB culture fluid ⁿTimes serial dilution is (such as 10 ⁸-10 ¹¹If, for the elutriation eluate dilution range of not amplification is 10 ¹-10 ⁴, can adjust again according to the result);

3. the host bacterium 100 μ l that get phage diluent 10 μ l and incubated overnight respectively mix static 15-30min under the room temperature;

4. above-mentioned phage-host bacterium mixture is added respectively in the top agar of 45 ℃ of 5ml; And add 20 μ lX-gal/IPTG; Mixing, even shop are formed on the LB flat board rapidly, treat after top agar solidifies the LB flat board to be placed 37 ℃ of incubators, put upside down and cultivate 8-10h;

5. treat to grow on the flat board blue plaque, select density suitable (1 * 10 ²The pfu/ flat board) flat board carries out blue plaque counting, calculates the titre of phage.

Blue plaque forms experiment 3.1.3 go back to the nest in the phage body

1. get lotus people gastric cancer nude mice subcutaneous transplantation tumor model nude mice, pentobarbital sodium 150mg/kg intraperitoneal injection of anesthesia, 75% soak with ethanol 5min, with phage IN11 and URPh respectively with 1 * 10 ¹⁰Pfu 200 μ l RPMI1640 inject mouse tail vein;

2. after injecting phage 5min, fully expose heart, puncture into aorta through left ventricle with intravenous needle; Slowly push the RPMI1640 liquid 50ml of preparatory temperature to 37 ℃; Cut off an osculum at the place, right auricle simultaneously, visible blood flows out, and becomes pale red until effusive blood.

3. take out subcutaneous tumors piece and the control tissue (heart, liver, spleen, kidney, brain, flesh) of nude mice and weigh, add and deposit in 4 ℃ 1ml RPMI1640-PI (containing 1mM PMSF, 20 μ g/ml aprotinin, 1 μ g/mlleupeptin), carefully grind with Potter-Elvehjem Tissue Grinders.

4. move into the 50ml centrifuge tube to homogenate; Adding 5ml ice RPMI1640-PI (containing 1%BSA) washes 4 times; Vortex 10s is with abundant line and staff control and cleaning mixture, the centrifugal 4-5min of 3000-4000rpm, sucking-off supernatant; To vibrate with thorough washing homogenate deposition before noting adding cleaning mixture at every turn, reclaim the phage (operation on ice) of going back to the nest respectively in transplanted tumor and control tissue.

5. will organize with host bacterium 1ml and mix, static 15-30min under the room temperature adds temperature in advance to 37 ℃ LB culture fluid 10ml, incubated at room 30min.

6. get 2 μ l respectively, 20 μ l, 200 μ l tissue/host bacterium LB mixture add in 45 ℃ the 3ml top agar, add 20 μ l X-gal/IPTG, fully evenly are laid on the LB flat board behind the mixing.Treat after top agar solidifies the LB flat board to be placed 37 ℃ of incubators, put upside down and cultivate 8-10h.

7. treat to grow on the flat board blue plaque, the blue plaque of tumor tissue and control tissue is counted respectively, calculate the quantity of phage.Relatively the blue plaque quantity of phage IN11 and URPh is analyzed the specificity that phage IN11 goes back to the nest in the mice with tumor body.

In the mice body, inject IN11 phage and URPh phage (1 * 10 respectively separately ¹⁰Pfu/ is only), the titre of transplanted tumor tissue and control tissue recovery phage is compared.The result is as shown in Figure 7, and the titre of the IN11 phage that from lotus people gastric cancer transplanted tumor in nude mice tissue, reclaims is 6.51 * 10 ⁶Pfu/g, the control tissue heart, liver,spleen,kidney, brain, muscular tissue are respectively 2.8 * 10 ⁵Pfu/g, 3.7 * 10 ⁵Pfu/g, 2.5 * 10 ⁵Pfu/g, 7.4 * 10 ⁵Pfu/g, 1.7 * 10 ⁵Pfu/g, 8.0 * 10 ⁴Pfu/g; The titre of the URPh contrast phage that tumor tissue reclaims is 3.1 * 10 ⁵Pfu/g, the control tissue heart, liver,spleen,kidney, brain, muscular tissue are respectively 4.5 * 10 ⁵Pfu/g, 5.3 * 10 ⁵Pfu/g, 5.1 * 10 ⁵Pfu/g, 6.8 * 10 ⁵Pfu/g, 3.6 * 10 ⁵Pfu/g, 1.0 * 10 ⁵Pfu/g.

The go back to the nest contrast of titre and contrast phage or control tissue of table 1 phage IN11 tumor tissue

The titre that the IN11 phage reclaims at tumor tissues is significantly higher than contrast phage or its titre that in control tissue, reclaims, and the difference of contrast phage between each tissue is little, and ratio Analysis is seen table 1.This shows, but the IN11 phage specificity in lotus people gastric cancer nude mouse that presents the GEBP11 small peptide is gone back to the nest in tumor tissues.

3.2GEBP11 small peptide and IN11 phage suppress experiment in the intravital competition of tumor bearing nude mice

Get lotus people gastric cancer nude mice subcutaneous transplantation tumor model nude mice, inject GEBP11 and URP small peptide 0.2ml respectively by mouse tail vein, concentration is respectively 31.25 μ g/ml; 62.5 μ g/ml, 125 μ g/ml, 250 μ g/ml and 500 μ g/ml; Be that the small peptide consumption is respectively 6.25 μ g; 12.5 μ g, 25 μ g, 50 μ g and 100 μ g.Inject corresponding phage 1 * 10 respectively from the tail vein again behind the 15min ¹⁰Pfu gets tumor tissue behind the 5min, reclaim phage, and the locus coeruleus number (concrete operation method is the same) in the transplanted tumor is calculated in titration.Calculate the competition suppression ratio of GEBP11 small peptide by following formula: locus coeruleus number * 100% in suppression ratio=(the locus coeruleus number in the locus coeruleus number-injection GEBP11 small peptide transplanted tumor in the injection PBS transplanted tumor)/injection PBS transplanted tumor to the IN11 phage.

GEBP11 small peptide and IN11 phage suppress experimental result in the intravital competition of tumor bearing nude mice and show that the GEBP11 small peptide is inhibited to going back to the nest of mice with tumor tumor tissues to the IN11 phage, and; Along with the rising of GEBP11 small peptide concentration, the phage quantity of going back to the nest in the transplanted tumor descends gradually, and suppression ratio increases gradually; When small peptide consumption during at 50-100 μ g; Suppression ratio increases gently, presents high inhibitory, and irrelevant control peptide does not have this effect (see figure 8).This explanation GEBP11 small peptide has competition and suppresses the IN11 phage to the special effect of going back to the nest of mice with tumor tumor tissues, and the GEBP11 small peptide that prompting IN11 phage is appeared has mediated its specificity in tumor and gone back to the nest.

3.3GEBP11 the immunofluorescence of small peptide binding specificity in the tumor bearing nude mice body detects

1. experimental group injects Bio-GEBP11 100 μ l/ only (1mg/ml) through nude mice tail vein, writes down inject time.Respectively at injection back 0.5h, 2h, 18h, 24h, 48h, pentobarbital sodium 150mg/kg intraperitoneal anesthesia.The tabula level is cut skin then, exposes whole thoracic wall, cuts off breastbone, notes avoiding damaging heart and trunk.

2. after fully exposing heart; Puncture into aorta through left ventricle with intravenous needle, the 50ml normal saline that slowly pushes 37 ℃ of temperature in advance behind the blood of resorption is arranged in the needle tubing, cut off an osculum at the place, right auricle simultaneously; It is thus clear that blood flows out, and becomes pale red until effusive blood.

3. get tumor, the heart, liver, kidney, lung, spleen, muscular tissue, respectively organize moisture mid-the swapping out of 25% sucrose solution, prepare frozen section after the OTC embedding, cold acetone is 20min fixedly.

4. detect with the routine immunization fluorescence detection method, add the anti-VIII factor antibody of rabbit, 4 ℃ of incubated overnight; PBS washing 5min * 3 time add anti-the rabbit igg two anti-and FITC-Avidins of FITC-, hatch 1h, and PBS washing 5min * 3 time are observed down in fluorescence microscope.

The immunofluorescence dyeing of GEBP11 small peptide binding specificity in tumor bearing nude mice body positioning analysis altogether shows; The GEBP11 small peptide distributes consistent with anti-VIII factor antibody in the tumor tissues; And control peptide or in control tissue such as heart, muscle; The VIII factor antibody red fluorescence clear demonstration blood vessel structure that dyes, but do not see the painted (see figure 9) of green fluorescence.This is prompting just, and the GEBP11 small peptide can be incorporated into gastric cancer vascular by specificity after injecting in the lotus people gastric cancer Mus body, and does not have obvious combination in control tissue.

3.4 isotope tracer technique detects ¹³¹I-GEBP11 is in the intravital biodistribution of tumor bearing nude mice

Totally 24 of lotus people gastric cancer transplanted tumor nude mices are divided into 8 groups, 3 every group. ¹³¹I labelling GEBP11, syringe extracts the 0.2ml label, approximately ¹³¹I-GEBP11200 μ Ci measures the radioactivity of every syringe, and writes down Measuring Time.In nude mice tail vein injects gastric cancer xenotransplantation tumor animal model, write down inject time, after injection finishes, measure the remaining radioactivity of every syringe, and the record Measuring Time.0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h put to death animal in batches in the injection back; Get blood, the heart, liver, kidney, lung, spleen, thyroid, stomach, small intestinal, tumor, a side lower limb muscles (15-20mg); Hollow organ cleans content, and filter paper blotted after normal saline was washed 3 times, and each internal organs is weighed; Measure each tissue radiation property counting; Correction for attenuation is done in the radioactive dosage unification that different time is measured,, calculated the increased radioactivity of each internal organs according to the ID after proofreading and correct; Calculate every gram tissue injection dose distribution [weight (the g)/ID of the radiocounting (cpm) of each tissue of %ID/g=, organ/each tissue, organ] respectively, verify whether it has the distribution of specific characteristic of tumor tissues.

As shown in table 2, adopt Radioactive tracer techniques to detect ¹³¹The distribution situation of I-GEBP11 each tissue in the tumor bearing nude mice body, result's demonstration, ¹³¹The most in vivo organs of I-GEBP11 are removed very fast, and radioactivity is the highest in the kidney, and checkout time is long, and the prompting kidney possibly be the main Excretory organ of GEBP11. ¹³¹It is interior after 2 hours that I-GEBP11 injects body, and radioactivity exceeds most of organs, delay in time in the tumor; Radioactive activity descends relatively slowly in the tumor, causes the relative ratio of it and other non-tumor organ to increase gradually, and ratios can reach (48h more than 15; Tumor/small intestinal); Show that GEBP11 has in vivo stomach organization targeting property, circulates through blood vessel ¹³¹The I-GEBP11 isotope probe can densely relatively gather in tumor.In addition, at organs such as stomach, lungs, arranged higher relatively dense gathering in early days, its meaning remains further to be studied.

Table 2 ¹³¹I-GEBP11 the intravital biodistribution of lotus people gastric cancer transplanted tumor nude mice (%ID/g, n=3)

?

0.5h

1h

2h

4h

8h

12h

24h

48h

Blood

1.032±0.07

0.489±0.10

0.282±0.05

0.075±0.008

0.056±0.005

0.031±0.007

0.022±0.004

0.011±0.002

Thyroid

0.586±0.03

0.345±0.05

0.338±0.05

0.232±0.04

0.048±0.006

0.067±0.009

0.028±0.003

0.022±0.003

Small intestinal

0.265±0.06

0.179±0.03

0.108±0.03

0.038±0.005

0.062±0.008

0.034±0.005

0.018±0.001

0.007±0.001

Stomach

0.979±0.08

0.576±0.08

0.346±0.03

0.115±0.03

0.048±0.004

0.042±0.007

0.034±0.004

0.018±0.004

The heart

0.356±0.04

0.168±0.03

0.099±0.01

0.031±0.02

0.025±0.004

0.017±0.003

0.014±0.002

0.014±.007

Spleen

0.471±0.04

0.277±0.04

0.155±0.02

0.033±0.001

0.017±0.003

0.015±0.002

0.011±0.004

0.010±0.002

Liver

0.341±0.04

0.177±0.02

0.107±0.02

0.037±0.03

0.022±0.006

0.016±0.003

0.014±0.002

0.011±0.001

Lung

0.767±0.05

0.333±0.04

0.189±0.03

0.045±0.007

0.042±0.001

0.038±0.004

0.024±0.004

0.012±0.001

Flesh

0.255±0.03

0.15±0.02

0.087±0.01

0.023±0.01

0.016±0.004

0.014±0.001

0.01±0.003

0.008±0.001

Tumor

0.625±0.07

0.531±0.04

0.381±0.04

0.182±0.02

0.165±0.03

0.149±0.03

0.13±0.02

0.11±0.01

Kidney

13.976±0.82

8.221±1.10

6.409±0.71

4.597±0.29

3.38±0.44

4.55±0.35

3.393±0.26

3.919±0.37

4. right ⁹⁹Tc ^mThe diagnostic value of-GEBP11 in mice with tumor gastric cancer estimated

Adopt the SPECT imaging technique to carry out in the lotus people gastric cancer nude mouse ⁹⁹Tc ^m-GEBP11 video picture is developed to the bonded organizational structure of GEBP11 small peptide, and tracing study 24h observes continuously ⁹⁹Tc ^m-GEBP11 is the targeting property of tumor tissues in vivo, and its imaging law of desk study is inquired into the diagnostic value of GEBP11 isotope probe in the gastric cancer vascular molecular imaging. ⁹⁹Tc ^m-GEBP11 at the concrete grammar of the intravital SPECT video picture of lotus people gastric cancer nude mice is:

Will ⁹⁹Tc ^m-GEBP11 500 μ Ci tail veins inject in the lotus people gastric cancer transplanted tumor in nude mice animal model, adopt SPECT, are equipped with mental retardation parallel hole high ersolution collimator; Gather ability peak 140Kev; Window width 20% is got reverse S-line of Golden, gathers counting 150K; Carry out ECT video picture, tracing study respectively at injection back 1h, 2h, 8h, 12h, 18h, 24h ⁹⁹Tc ^mThe variation that-GEBP11 distributes in animal model.The record tumor locus begins developing time, and concentrating reaches rush hour, and computer cropping tumor and heart region of interest are calculated T/NT ratio.With ⁹⁹Tc ^m-URP compares.

Will ⁹⁹Tc ^m-GEBP11 tail vein injects in the tumor-bearing mice body, carries out the SPECT video picture, at nude mice right back (TR) and left back (TL) limb dorsal part tumor locus; Can see the dense kitchen range that gathers of a radioactivity respectively; Continuous tracing study 24h, visible delay along with the time, the tumor locus radioactivity is dense, and to gather kitchen range clear gradually.Begin to be higher than painstaking effort pond background behind the 12h, the still visible dense shadow that gathers of radioactivity of 18h to 24h, and along with the quick reduction of other organ radioactive intensity, it is more clear that the tumor kitchen range shows.Calculate the radioactivity ratio (T/NT that respectively organizes tumor locus and non-tumor contrast position; H, M, B, K, L represent the heart, flesh, brain, kidney, liver respectively), visible along with change of time, T/NT increases gradually; Peak is T/M (muscle) in the 24h, reaches 14.4 (Figure 10-A, C) during 24h.Nude mice of control group tail vein injects ⁹⁹Tc ^m-URP carries out the SPECT video picture, and continuous tracing study 24h increased radioactivity is arranged in that right hind dorsal part tumor locus is visible, but its radioactivity is lower than painstaking effort pond background all the time, and its T/NT ratio is lower than experimental group, and postpones in time not have obviously to increase (Figure 10-B, D).

5. right ¹³¹Therapeutic effect and the toxic and side effects of I-GEBP11 in mice with tumor gastric cancer estimated

Press down the tumor experimental evaluation through MTT cell proliferation experiment, mice with tumor ¹³¹I-GEBP11 is to the inhibition ability of endotheliocyte and mice with tumor tumor, and immunohistochemical staining is counted tumor tissue MVD, and pathology HE dyeing, blood blood cell analysis and biochemical indicator detect hepar damnification and the bone marrow depression situation of observing.

5.1MTT cell proliferation experiment

Observe through the MTT cell proliferation experiment ¹³¹I-GEBP11 is to the external lethal effect of endotheliocyte, and operating procedure is following: exponential phase Co-HUVECs, using M200 culture fluid adjustment cell density is 5 * 10 ⁴/ ml, every hole 0.1ml is inoculated in 96 well culture plates, establishes 2 multiple holes.Divide ¹³¹I-GEBP11, Na ¹³¹Three groups of I, GEBP11, and establish normal saline (NS) contrast, blank.5%CO ₂After 37 ℃ of incubators are cultivated 24h, add final concentration respectively and be 3.7,37,370,3700KBq/ml ¹³¹I-GEBP11 or Na ¹³¹I, and final concentration is the GEBP11 of 0.01,0.1,1,10 μ g/ml, cultivate 2h after PBS wash one time; Be changed to the M200 culture fluid, continue to be cultured to 72h, every hole adds MTT 20 μ l (5mg/ml); Continue to cultivate 4h, abandon supernatant, add DMSO 150 μ l; Vibration 10min fully dissolves crystallization, wavelength 490nm place's photometry absorption value (A) on the spectrodensitometry appearance.Experimental result is represented with cell survival rate, and cell survival rate=(experimental group absorbance value/NS matched group absorbance value) * 100% is that the longitudinal axis, drug dose logarithm are the transverse axis mapping with the cell survival rate.Experiment repeats 3 times at least, and (expression of Mean ± SD) adopts SPSS software to carry out statistical analysis to data, and P＜0.05 o'clock thinks variant with mean ± standard deviation.

The MTT cell proliferation experiment is observed, ¹³¹I-GEBP11 concentration is the inhibitory action of killing and wounding that 37KBq/ml begins to show on cell proliferation, and is concentration dependent; Na ¹³¹The I pair cell does not have the specific killing effect; GEBP11 concentration begins to show the inhibitory action (seeing Figure 11) of on cell proliferation when being 10 μ g/ml.This prompting, ¹³¹The inhibitory action of I-GEBP11 on cell proliferation maybe be through the double route realization, promptly ¹³¹I and GEBP11 are during the medicine low concentration ¹³¹I plays a role, and when small peptide concentration reached 10 μ g/ml, small peptide also showed certain endotheliocyte inhibitory action.

5.2 mice with tumor presses down the tumor experiment

Set up lotus people gastric cancer nude mice subcutaneous transplantation tumor model by the preceding text method, when treating that nude mice Subcutaneous tumor diameter grows to 0.5-0.8cm., begin to press down the tumor experiment.Treat that to add final concentration in preceding 3 days in the beginning drinking water be 0.2% liquor kalii iodide (KI),, finish until treatment with the sealing thyroid.The mice with tumor that growth conditions is good is divided into two at random and organizes greatly; First group is carried out tumor killing effect and observes; Comprise that gross tumor volume changes the difference of (tumour inhibiting rate) and treatment back nude mice survival rate; Toxicity and tumor-blood-vessel growth situation are treated in second largest group of evaluation, and two big groups give identical grouping and treatment.Every big component (radiological dose 7.4MBq, GEBP1120 μ g), Na ¹³¹(eADM, 0.2mg/20g), the administration volume is 0.2ml, the negative matched group of NS wherein, the positive matched group of eADM for I (radiological dose 7.4MBq), GEBP11 (GEBP11 20 μ g), normal saline (NS) and epirubicin.In the 14th day (d14) repeat administration once, 28 days courses of treatment.Observe mice with tumor and tumor growth situation,, measure nude mice body weight and the nodular major diameter of transplanted tumor (L) and wide footpath (W), calculate transplanted tumor volume (V=0.5236 * L * W respectively at d0, d3.5, d7, d10.5, d14, d17.5, d21, d24.5, d28 ²), and calculate each smelting treatment group tumor control rate [tumor control rate=(the average tumor volume of average tumor volume recruitment/matched group recruitment is organized in the 1-treatment) * 100%].After treatment finished, first group was continued to observe nude mice and tumor growth situation, write down every nude mice life cycle, compared difference life cycle.Till 80 days, put to death the survival nude mice.Promptly put to death nude mice (pluck eyeball and get blood) after second largest group of treatment finishes, leave and take BIAO and BEN such as tumor tissues, liver organization, hind leg femur, blood sample respectively.

The growth change situation of gross tumor volume respectively organized in record in the therapeutic process, makes tumor growth curve, and the result shows that eADM is 64.9% to the most remarkable tumor control rate of inhibitory action of tumor growth, ¹³¹I-GEBP11 takes second place, and tumor control rate is 56.3%, two group of tumor growth obviously slow (P＜0.05) after 2 weeks; Na ¹³¹I, GEBP11 show the effect of certain inhibition tumor growth, and tumor control rate is respectively 18.3%, 21.6%, but not statistically significant (Figure 12).Observe the nude mice life cycle (seeing Figure 13) after treating, NS group median survival interval is 42 days, ¹³¹I-GEBP11, eADM obviously prolong two groups of life cycles, and median survival interval was respectively 61 days, 55 days, and in addition, GEBP11 group median survival interval is 53 days, also prolongs (P＜0.05), Na than the NS group ¹³¹I organizes does not have the prolongation (not shown) life cycle.The above results shows, ¹³¹I-GEBP11, eADM have obvious tumor-inhibiting action, can obviously prolong mice with tumor life cycle, and GEBP11 is not obvious to tumor inhibition effect, but can prolong life span.

5.3 immunohistochemical staining counting tumor tissue MVD

The frozen section of preparation tumor tissues, 4 ℃ of acetone fixed 20min, tumor tissues is observed in HE dyeing, carries out the anti-FVIII-R antibody staining (SP method) by the method for routine immunization histochemical stain, counting tumor tissue MVD.The concrete operations step is following: PBS washes section 5min * 3 time, 3%H ₂O ₂Room temperature effect 30min, PBS wash 3min * 3 time; 37 ℃ of sealings of normal goats serum 30min inclines and does not wash; Press the operation instructions recommended density, preparation also drips the anti-FVIII-R antibody, 4 ℃ of incubated overnight; 37 ℃ of rewarming 1h, PBS wash 5min * 3 time, and it is anti-to add biotinylation two, hatches 30-40min for 37 ℃; PBS washes 5min * 4 time, adds the SP complex, hatches 20min for 37 ℃; Redye, break up and dewater transparently through DAB colour developing, haematoxylin, the resinene mounting is observed coloration result down in optical microscope, row MVD counting.The detection method of MVD: sweep whole section with 40 times of light microscopics earlier; After seeking high vessel density zone; Counting is dyed brown blood capillary under 200 times of visuals field again, gets the MVD of the meansigma methods in 5 zones as this tumor biopsy, every group of MVD that amounts to several 10 sections; Calculating mean value is as the average MVD of this group transplanted tumor.Adopt SPSS software to carry out statistical procedures, relatively the difference between each group.

The tumor tissues of treating the back mice with tumor is carried out pathology detect, result's demonstration, ¹³¹Visible slabbing degeneration necrosis district in the I-GEBP11 treatment posterior tuberosity, the downright bad special mess of also visible limitation in the tumor kitchen range after the GEBP11 treatment, and NS group oncocyte generates in good condition; ¹³¹After I-GEBP11 and the GEBP11 treatment, the blood vessel of mice with tumor tumor tissues reduces (seeing Figure 14) than the NS matched group.It is right to have listed in the following table ¹³¹I-GEBP11, GEBP11 and NS group are carried out result's (table 3) of MVD counting statistics.

Table 3 ¹³¹The comparison of I-GEBP11, GEBP11 and NS group treatment back tumor MVD

Group	Microvessel density (MVD)
		NS	12.2±3.2
131I-GEBP11	3.5±0.9 **
		GEBP11	7.1±1.6 *

Annotate: ¹³¹I-GEBP11 and GEBP11 group MVD counting be starkly lower than the NS group ( ^*P＜0.05, ^*P＜0.01)

5.4 observe hepar damnification and bone marrow depression situation

5.4.1 pathology HE dyeing

Liver is prepared frozen section, cut into slices after the capable plastic embedding of femur, conventional H E dyeing.Examine under a microscope liver and bone marrow form, relatively each treatment group liver damage and bone marrow depression situation.

5.4.2 blood blood cell analysis and biochemical indicator detect

By conventional method, each group blood sample is carried out the detection of blood cell analysis and biochemical hepatic and renal function, relatively each treatment group hepatic and renal function is undermined the PBC situation.

According to differences such as each treatment group nude mice body weight situation of change, liver damage and bone marrow depression, overall merit is respectively organized the toxic and side effects of medicine in therapeutic process.

Nude mice body weight situation of change respectively organized in record during the treatment, and the result shows, gives 1 week behind the medicine, and except that two groups of NS, GEBP11, all the other respectively organize the average body weight average has in various degree and descend, and increases gradually again subsequently, and weight loss is the most obvious with the eADM group, ¹³¹I-GEBP11, Na ¹³¹Two groups of weight increase of I are extremely near the NS group, and the eADM group still can not be recovered initial body weight level.This shows that in the therapeutic process, eADM rings bigger to the nude mouse ghost image, ¹³¹I-GEBP11, Na ¹³¹I has a mistake gonosome heavily to alleviate, but can recover.Adopt nude mice blood and carry out hemocyte and biochemical analysis, as reference, the blood cell analysis result shows that eADM group leucocyte level reduces with the NS group, ¹³¹I-GEBP11 group platelet levels reduces all the other no significant changes; The blood biochemical analysis shows that eADM reaches ¹³¹I-GEBP11 group transaminase ALT, AST all increase, wherein the eADM group increase more remarkable, kidney merit no abnormality seen (seeing table 4).By finding out that toxic and side effects mainly is reflected in the variation of liver function and hemocyte quantity in the table, the medicine that causes apparent side effect mainly contain eADM and ¹³¹I-GEBP11, and ¹³¹The I-GEBP11 group obviously is lighter than eADM treatment group.For further understanding the side reaction situation, get treatment back nude mice liver and myeloid tissue, carry out pathology and detect.The result finds, eADM or ¹³¹The visible hepatocellular degeneration necrotic reaction that is dispersed in of liver after the I-GEBP11 treatment, and ¹³¹The I-GEBP11 group obviously is lighter than the eADM group; The bone marrow section sees that eADM group bone marrow hematogenesis is suppressed, ¹³¹I-GEBP11 treatment group is not seen obviously unusual (seeing Figure 15).

Table 4 each treatment group blood cell analysis and biochemical analysis

^*P＜0.05 is compared with the NS group

This shows that cytotoxic drug eADM but brings big side effect in effective antitumor growth, cause that weight increase receives to press down, bone marrow hematogenesis and liver function injury, and ¹³¹I-GEBP11 has under the prerequisite of obvious antitumous effect, and toxic and side effects is light than eADM.

The gastric cancer vascular specific binding peptide GEBP11 that the present invention selected for use is to obtain through the in-vitro screening phage peptide library in applicant's previous work, has independent intellectual property right and sturdy working foundation, belongs to original achievement, has novelty.With radiosiotope ⁹⁹Tc ^mO ₄ ^-, ¹³¹I is marked on the GEBP11 small peptide, resulting GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11, ¹³¹I-GEBP11 is the further transformation to the GEBP11 small peptide, makes it have new function, do not have same molecular both at home and abroad, so the present invention has uniqueness.

The GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11, ¹³¹The successful preparation of I-GEBP11 is in the aspects such as diagnosis, blood-vessels target treatment and therapeutic evaluation of gastric cancer, the potential using value and the theoretical significance that have.Can be used for the following aspects at present:

1. the development of stomach cancer diagnosis reagent box: the probe for preparing among the present invention ⁹⁹Tc ^m-GEBP11 has confirmed to have the ability that shows the gastric cancer focus in zoopery, through to its research and optimization further, be expected to become a kind of new tool of gastric cancer early diagnosis, MET or recurrent foci diagnosis and anti-angiogenic treatment therapeutic evaluation.

2. the exploitation of blood-vessels target medicine: the probe for preparing among the present invention ¹³¹I-GEBP11; In zoopery, confirmed to have the effect that suppresses the gastric cancer growth, prolongs the mice with tumor life span; And toxic and side effects is lighter, through to its transformation and optimization further, is expected to develop the blood-vessels target new drug that can be used in clinical curing gastric cancer.

3.GEBP11 the transformation and optimization of isotope probe and expansion: the radiosiotope that the present invention adopts ⁹⁹Tc ^mO ₄ ^-, ¹³¹I constantly have new radionuclide to be applied to experimentation or clinical diagnosis and treatment in recent years, and its labeling method has similarity for to be widely used in clinical classical isotope.Labeling method so that the present invention was set up is the basis, can change other consanguinity radionuclide of labelling, thereby make it have better diagnosis or therapeutic effect.In addition, also can set about carrying out structure of modification and optimization, obtain better diagnosis or therapeutic type gastric cancer vascular specific property isotopic labeling short peptide probe from small peptide.Be 5 groups, 5 every group, through the administration of tail vein injection mode, each is organized dosage and is: ¹³¹I-GEBP11.

Claims (5)

1. diagnosis type gastric cancer vascular specific binding peptide GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11 is characterized in that: with diagnosis type isotope ⁹⁹Tc ^mO ₄ ^-Be connected in the GEBP11 small peptide, give the dual-use function of short peptide probe gastric cancer vascular targeting and diagnosis; Said diagnosis type isotope ⁹⁹Tc _mO ₄ ^-Be connected in the GEBP11 small peptide, adopt direct labelling method to realize; Its labelling or reaction condition are: fresh 1mol/L sodium ascorbate solution 20 μ L, add 50g/L hydrosulfurous acid sodium solution 20 μ L successively, and 100 μ LGEBP11 solution and 2-5mCi's is fresh ⁹⁹Tc ^mO ₄ ^-Leacheate 100 μ L, reaction system pH 4.8, and room temperature reaction 10min accomplishes labelling.

2. therapeutic type gastric cancer vascular specific binding peptide GEBP11 isotope probe ¹³¹I-GEBP11 is characterized in that: with the therapeutic type isotope ¹³¹I is connected in the GEBP11 small peptide, gives the dual-use function of short peptide probe gastric cancer vascular targeting and treatment; Said therapeutic type isotope ¹³¹I is connected in the GEBP11 small peptide, adopts the NBS labelling method to realize; Its labelling or reaction condition are: NBS is with deionized water dissolving, and GEBP11 adds GEBP11 solution 20 μ L earlier with the dissolving of PBS buffer in the reaction tube, add the Na of 7.4MBq then ¹³¹I solution respectively adds NBS solution 20 μ L at last, and mixing gently under the room temperature after 30 seconds, adds 2% human serum albumin, 10 μ L cessation reactions and promptly accomplishes labelling.

3. according to claim 1 or 2 said diagnosis type gastric cancer vascular specific binding peptide GEBP11 isotope probes ⁹⁹Tc ^m-GEBP11 or therapeutic type gastric cancer vascular specific binding peptide GEBP11 isotope probe ¹³¹I-GEBP11 is characterized in that: with radiosiotope ⁹⁹Tc ^mO ₄ ^-, ¹³¹I is marked on GEBP11 respectively, and its mark quality and BA authentication method are that paper chromatography is measured mark rate, measure gained ⁹⁹Tc ^m-GEBP11, ¹³¹The radiochemicsl purity of I-GEBP11, specific activity, inside and outside stability, external microcosmic receptor autoradiography technology for detection ⁹⁹Tc ^m-GEBP11 reaches ¹³¹The bonded BA of I-GEBP11 and Co-HUVECs.

4. according to claim 1 or 2 said diagnosis type gastric cancer vascular specific binding peptide GEBP11 isotope probes ⁹⁹Tc ^m-GEBP11 or therapeutic type gastric cancer vascular specific binding peptide GEBP11 isotope probe ¹³¹I-GEBP11; It is characterized in that: the analytical method to GEBP11 small peptide and its receptor-binding characteristic is to analyze experimental identification receptor affinity and recipient cell density through the receptor radiative aglycone combination; Adopt immunocytochemistry or Subcellular Localization and the internalization of fluoroscopic examination GEBP11 in Co-HUVECs, the binding specificity of GEBP11 in people's stomach organization detects in immunohistofluorescence.

5. according to claim 1 or 2 said diagnosis type gastric cancer vascular specific binding peptide GEBP11 isotope probes ⁹⁹Tc ^m-GEBP11 or therapeutic type gastric cancer vascular specific binding peptide GEBP11 isotope probe ¹³¹I-GEBP11 is characterized in that: described diagnosis type gastric cancer vascular specific binding peptide GEBP11 isotope probe ⁹⁹Tc ^m-GEBP11 or therapeutic type gastric cancer vascular specific binding peptide GEBP11 isotope probe ¹³¹The application of I-GEBP11 in diagnosing gastric cancer, blood-vessels target treatment and therapeutic evaluation.

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