CN101143897B - Labeling technique for 99Tcm-His10-AnnexinV labeling compound and application of the same as developer in detecting cell apoptosis - Google Patents

Labeling technique for 99Tcm-His10-AnnexinV labeling compound and application of the same as developer in detecting cell apoptosis Download PDF

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CN101143897B
CN101143897B CN2007101520145A CN200710152014A CN101143897B CN 101143897 B CN101143897 B CN 101143897B CN 2007101520145 A CN2007101520145 A CN 2007101520145A CN 200710152014 A CN200710152014 A CN 200710152014A CN 101143897 B CN101143897 B CN 101143897B
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CN101143897A (en
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华子春
王峰
方纬
杨翔
王自正
季顺东
郑玉民
武婕
潘旭东
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JIANGSU TARGET BIOMEDICINE RESEARCH INSTITUTE Co Ltd
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Nanjing University
Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Fuwai Hospital of CAMS and PUMC
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Abstract

The invention relates to a labeling technique of <99>Tc<m>-His10-Annexin V and the applications of the <99>Tc<m>-His10-Annexin V as an imaging agent in aspects of apoptosis under the condition of detecting diseases. By applying a direct labeling method, the invention utilizes SnCl2. 2H2O reductant and citrate buffer to labeling <99>Tc<m> on His10-Annexin V. The method has the advantages of simple and convenient operation and low cost. Experiments on animals show that the injected <99>Tc<m>-His10-Annexin V can clearly display the apoptosis of different tissues, such as cardiac muscle, tumor, etc., which can be applied to the diagnosis of myocardial lesions, and the efficacy evaluation of tumor chemotherapy, etc.

Description

99Tc mThe labeling technique of-His10-AnnexinV tagged compound and as developer in the application that detects aspect the apoptosis
Technical field
The present invention relates to a kind of isotope labelling technique of compound, and the application of this radionuclide labelled compound, particularly 99Tc m-His10-AnnexinV tagged compound is as the application of developer.
Background technology
Apoptosis (apoptosis) is a kind of programmed cell death (PCD) of high conservative, plays an important role in many physiology and pathologic process.Excessively apoptosis can make function of organization's forfeiture, as acute myocardial infarction, chronic heart failure, graft-rejection, apoplexy, nervous system degeneration disease etc.On the contrary, systemic lupus erythematous and rheumatoid arthritis are insufficient with apoptosis to be feature, makes the immunocyte large number of viable and kills and destroy healthy tissues irrelevantly, makes and organizes organ function impaired.Apoptosis suppresses to cause cell hyperproliferation and tumour to form, and mostly many chemotherapeutics are by the apoptosis-induced therapeutic purpose that realize.
But conventional at present apoptosis detection method can't satisfy the needs of fundamental research and clinical practice, existing laboratory detection means can only be carried out external qualitative and quantitative analysis from morphocytology and biochemical change aspect, as electron microscopic observation, flow cytometry, dna degradation strip analysis and correlative protein expression level detection etc.Under condition of living body observation of cell apoptosis scope, the time mutually and tempo etc., be a great problem of research always.Particularly in the research of cardiovascular system diseases, the living specimen collection is difficulty particularly, so the live body video picture seems more necessary.
In recent years, carried out both at home and abroad the research of cells involved apoptosis radionuclide image, the key of research be synthetic can with the developer of apoptotic cell specific combination, and physics-chem characteristic is suitable for radioisotope labeling, and have blood remove fast, characteristics such as the target organ video picture is clear.Radioisotope labeling phospholipids incorporate albumen V (Annexin V) is one of apoptosis developer at present of greatest concern.
Annexin V is a kind of human body endogenous protein, belongs to Ca 2+Dependency phospholipids incorporate protein family is to separate from placenta the earliest, has some anti-freezings and reaches antiphlogistic effect admittedly, once is used as a kind of antithrombotics research.The chromosomal localization analysis shows that Annexin V is by the Annexin V genes encoding on the 4th the karyomit(e) 4q26-q28 that is positioned at the people, contains 319 amino acid, comprises a 70-80 amino acid repeating unit, and molecular weight is about 35kD.Studies confirm that, Annexin V can combine with phosphatidylserine (PS) specificity on apoptotic cell surface, and Normocellular PS is positioned at the cytolemma internal layer, is expressed in the cytolemma skin during apoptosis, be the features of apoptosis sign, the avidity of Annexin V and PS is up to 10 -9Mol/L.
The synthetic key of Annexin V class developer is the mark of radionuclide, should obtain higher radiochemical purity, and the combination that can not damage Annexin V simultaneously again is active.What research was the most successful at present is 99Tc mThe Annexin V of mark ( 99Tc m-AnnexinV).Radionuclide 99Tc mHave good physical properties, very suitable present video picture instrument, and obtain easily, cheap.
99Tc mThe method of mark can be divided into difunctional intercalating agent method and direct-reduction process.Difunctional intercalating agent method selects linking agent extremely important, should guarantee with 99Tc mVery high coordination ability is arranged, guarantee not change its original biological activity again.Commonly used have a disulfide dinitrogen (N 2S 2), quadrol halfcystine (EC), BTAP etc., wherein see so that hydrazino-nicotinamide (Hynic) more.At present, most preclinical studies employing Hynic are that sequestrant carries out 99Tc mMark Annexin V obtains 99Tc m-Hynic-Annexin V specific activity is higher, and radio chemistry purity is greater than 90%, and the good assessment of acquisition in the apoptosis animal model, is considered to monitor oncotherapy curative effect potential efficient emission medicine.But difunctional intercalating agent method process more complicated is strict to technical qualification, the preparation cost height.
Directly the reduction labelling method is more easy, ideal marking method, but research is in the past thought: with SnCl 2Under reductive agent, neutral PH condition, can obtain mark rate height (＞95%), good stability (＞10h) 99Tc m-AnnexinV marked product, and be easy to medicine boxization, but activity can be affected, and produces some metaproteins, causes the radioactive uptake of liver to increase.In order to overcome this shortcoming, scholars such as external Tait JF are by modifying the structure of Annexin V, prolong 7 amino acid (containing one or two cysteine residues) at the N of Annexin V end and formed metagon Annexin V116,117 and 118, three kinds of albumen can be expressed in Bacillus coli cells matter and but purifying becomes homoplasmon, with PS on the erythrocyte membrane higher avidity are arranged all.Be used in the method that adds peptide sequence on the Annexin V, make it to have formed with 99Tc mGive birth to the chelating site in the bonded.With SnCl 2Be reductive agent, gluceptate (GH) is a changing agent, but the combination of three kinds of albumen specificitys 99Tc mAt least 1850～3700MBq (50～100mCi), and stable in properties behind the mark, kept avidity with film PS.
Domestic scholars Hua Zichun, Zhang Lina etc. further in intestinal bacteria the clone, express and purifying a kind of new Annex in V derivative, 10 successive Histidines (His10-Annexin V) have been added at the N of Annexin V end, kept the AnnexinV primary characteristic, increased the ability of chelated metal ions again, will make in theory 99Tc mDirectly mark is handed over to such an extent that be more prone to.The invention achievement that this patent is related has at first realized His10-Annexin V's at home and abroad 99Tc mDirect mark, more domestic and international existing Annexin V class apoptosis developer technology of preparing is more simple and easy to do, and more practical, preparation cost is lower.
Summary of the invention
For overcoming the shortcoming of prior art, the object of the present invention is to provide the more simple and practical tagged compound of a kind of marking method, be used to carry out radionuclide image, detect apoptosis takes place in the various diseases degree and scope.
For achieving the above object, technical scheme of the present invention is to adopt direct labelling method to carry out the radionuclide of His10-AnnexinV compound 99Tc mMark.
Further, be 99Tc m-His10-AnnexinV tagged compound is used to detect the application of apoptosis of cardiac muscle aspect behind the ischemic injuries as developer.
Further, be 99Tc m-His10-AnnexinV tagged compound is used to detect the application of apoptosis of tumor cells aspect after the chemotherapy as developer.
Above-mentioned 99Tc m-His10-AnnexinV developer makes labeling process very simple and easy to do owing to adopted direct labelling method, has reduced the cost of developer preparation, and shows injection in the body by experimentation on animals 99Tc mBehind-the His10-AnnexinV, apoptosis such as cardiac muscle, tumour organize video picture clear, and picture quality is good.
Description of drawings
Fig. 1: ischemical reperfusion injury brings out the apoptosis of cardiac muscle animal model 99Tc m-His10-AnnexinV video picture figure.
Fig. 2: 99Tc mThe pathologic finding figure of the cardiac muscular tissue of the high picked-up of-His10-AnnexinV, result show that apoptosis takes place a large amount of myocardial cells.
Fig. 3: lotus H460 knurl nude mice model is through taxol chemotherapy induction apoptosis of tumor cells 99Tc m-His10-AnnexinV video picture figure.(A: control group; B: 24h group after the chemotherapy; C: 48h group after the chemotherapy; D: 72h group after the chemotherapy)
Fig. 4: 99Tc mThe pathologic finding figure of the nonsmall-cell lung cancer tumor tissues of the high picked-up of-His10-AnnexinV, the result shows massive tumor cell generation apoptosis.
Embodiment
One .His10-AnnexinV's 99Tc mLabeling technique
His10-Annexin V is provided by medical biotechnology National Key Laboratory of Nanjing University.Using gene engineering technique utilizes clone in the intestinal bacteria, expresses and purifying, adds 10 successive Histidines at the N of Annexin V end, uses the metal affinity chromatography purifying, reorganization preparation His10-Annexin V.
Adopt direct labelling method that His10-Annexin V is carried out 99Tc mMark.Get an amount of SnCl 22H 2O is the HCl dissolving of 0.05mol/L with concentration, and citrate buffer solution is diluted to 1mg/mL.Get the SnCl that contains that has prepared 2Citrate buffer solution 100 μ L, add His10-Annexin V 5 μ g successively, 99Tc mO 4 -0.8mL 10mg/mL vitamins C 100 μ L get final product behind the mixing.
Carry out marker detection with paper chromatography, measure radiochemical purity and the vitro stability of His10-Annexin V.Adopt two individual system: system 1 adopts No. 1 chromatographic paper of Xinhua as stationary phase, with acetone as developping agent, 99Tc m-His10-AnnexinV reaches 99Tc m-colloidal Rf value (Rf) is 0, 99Tc mO 4 -Rf=0.7-0.8; No. 1 chromatographic paper of Xinhua that system 2 was soaked with 2.5% bovine serum albumin is as stationary phase, and with ethanol: ammoniacal liquor: water (2: 1: 5 v/v) launches as developping agent, 99Tc m-colloidal Rf=0, and 99Tc m-His10-Annexin V reaches 99Tc mO 4 -Rf=0.7-0.8.
The radiochemical purity that paper chromatography is measured His10-Annexin V can reach (98.01 ± 1.67) %, colloidal content is (3.31 ± 1.37) %, radiochemical purity still remained on (95.45 ± 1.34) % after room temperature was placed 3h, reached the stability requirement of clinical application.
Two. 99Tc mThe biodistribution characteristics of-His10-AnnexinV tagged compound meet the requirement as developer, and distributing to test by the mouse intracorporeal organ describes.
Get 40 of Kunming kind small white mouses, be divided into 8 groups at random, 5 every group.Tail vein injection 99Tc m-His10-Annexin V, dosage are 11.1MBq, and volume is 0.2mL.Respectively at 5min, 30min, 1h, 2h, 4h, 6h, 12h, 24h after the administration totally 8 time points put to death a treated animal, take out 14 major organs such as blood, the heart, liver, spleen, lung, kidney, stomach, small intestine, pancreas, brain, muscle, bone, Tiroidina, sexual gland, weigh and measure radiocounting, draw the increased radioactivity-time curve of each internal organs with the γ calculating instrument; Calculate every gram and organize percentage injection dose rate (%ID/g, gross activity counting * 100% in every gram tissue or internal organs radiocounting/the be injected into mouse body).With time is X-coordinate, and radiocounting is that ordinate zou is drawn blood clearance curve, with the match of DAS2.0 software, analyzes at the intravital pharmacokinetic parameter of normal mouse.
Experimental result shows: kidney is right 99Tc mThe intake of-His10-Annexin V is the highest, and removes slowly, secondly is bone, liver, spleen, and the stomach and intestine picked-up is less, and brain does not absorb (seeing Table 1) substantially. 99Tc m-His10-Annexin V removes in blood rapidly, and DAS2.0 software fitting result shows that blood clearance curve meets the two-compartment model feature, distribution phase transformation period (T 1/2a) be 21.18 ± 5.95min, remove phase transformation period (T 1/2 β) be 69.32 ± 0.10min, average blood plasma clearance rate (CL) is 0.008 ± 0.002L/min/Kg, apparent volume of distribution (Vd) is 5.07 ± 0.86L/Kg, by the transfer rate constant (K of a Room to two Room 12) be 0.031 ± 0.010/min, by the transfer rate constant K of two Room to a Room 21Be 0.016 ± 0.002/min.
Table 1 99Tc m-His10-Annexin V the intravital bio distribution of Kunming kind small white mouse (
Figure S2007101520145D00041
N=5)
Organ The radioactive uptake mark (%ID) of different time points
?5min ?30min ?1h 2h 4h 6h 12h 24h
Blood conscience spleen lung kidney stomach-intestinal pancreas brain intermuscular bone Tiroidina ?3.53±0.63?1.30±0.28?1.64±0.68?0.94±0.31?2.29±0.52?4.90±3.74?1.58±0.89?2.71±0.65?0.82±0.35?0.11±0.06?1.01±0.26?3.37±0.75?0.94±0.31 ?2.34±0.63?0.66±0.21?0.70±0.33?0.29±0.20?0.70±0.54?4.33±3.64?0.76±0.45?0.43±0.35?0.33±0.28?0.09±0.05?0.54±0.29?2.36±0.64?0.29±0.20 ?1.27±0.21?0.30±0.09?0.82±0.12?0.23±0.10?0.74±0.12?5.20±0.93?0.53±0.19?0.40±0.15?0.34±0.26?0.07±0.01?0.24±0.09?2.20±0.35?0.23±0.10 1.43±0.580.36±0.120.96±0.330.45±0.300.69±0.205.31±0.840.57±0.210.35±0.100.34±0.110.08±0.010.43±0.092.51±0.290.45±0.30 0.87±0.120.25±0.120.62±0.210.18±0.090.46±0.234.00±2.350.34±0.100.29±0.190.16±0.080.05±0.010.14±0.091.07±0.550.18±0.09 0.75±0.100.20±0.090.50±0.320.11±0.050.28±0.172.59±1.960.29±0.260.10±0.070.11±0.040.04±0.010.22±0.102.23±0.700.11±0.05 0.92±0.180.34±0.050.73±0.110.38±0.090.60±0.162.74±0.630.67±0.190.38±0.090.30±0.140.07±0.010.33±0.193.21±0.620.38±0.09 0.58±0.090.15±0.080.62±0.210.16±0.090.30±0.231.71±1.130.36±0.170.20±0.050.13±0.030.02±0.000.12±0.051.44±0.820.16±0.09
The Tiroidina sexual gland 1.16±0.730.81±0.34 0.53±0.45 0.59±0.21 0.22±0.12 0.41±0.24 0.35±0.29 0.41±0.25 0.18±0.08 0.20±0.15 0.11±0.07 0.27±0.18 0.37±0.18 0.47±0.20 0.25±0.07 0.15±0.08
Three. 99Tc m-His10-AnnexinV tagged compound is used to detect the application of apoptosis aspect as developer.
Technical scheme of the present invention is 99Tc m-His10-AnnexinV tagged compound is used to detect the application of apoptosis aspect as developer.Above-mentioned apoptosis can be any one apoptotic process normal or the pathological tissues cell in the organism in theory, as myocardial cell, tumour cell etc.Apoptosis of cardiac muscle and the apoptosis of tumor cells that causes respectively with ischemia-reperfusion-acute myocardial infarction and chemotherapy of tumors is that example describes below.
Example 1. 99Tc m-His10-AnnexinV tagged compound detects apoptosis of cardiac muscle as developer
Select experiment 6 of miniature pigs (Nanjing Military Command's hospital general's Experimental Animal Center provides), body weight 15～20Kg, after the vetatar intramuscular injection 10mg/kg anesthesia, intravenous drip Jing'an 6mg/kg/h keeps anesthesia.Under the X line monitored, femoral artery puncture was sent sacculus into the main leg distal end of coronary artery by the 6F conduit, inflated, blocking-up coronary flow.Bloodstream blocking 20 is to 30min, and decompression removes conduit, causes myocardial ischemia-reperfusion-acute myocardial infarction damage.Omnidistance cardioelectric monitor.
Animal model prepares successfully back 1h, auricular vein injection 99Tc m-His10-AnnexinV, dosage are 370MBq (10mCi), and 3h carries out emission type single photon tomography (SPECT video picture) and CT location scanning behind the injection developer.Adopt the two probe of the GE Millenium VG Hawkeye of company imager, at first carry out the CT location scanning, every layer of scanning 15s scans 40 layers altogether.Carry out the SPECT video picture subsequently, energy peak 140KeV, energy window 20%, image array 128 * 128, gather 1 two field picture for per 6 °, gather 60 two field pictures altogether, every frame is gathered 45s, behind the OSEM software iterative approximation, obtain SPECT and CT image respectively, carry out the localized fusion of SPECT image and CT again.Calculate (T/NT) ratio of target organ (apoptosis cardiac muscle)/non-target organ (normal myocardium) with the method for delineating " interested " district (ROI).
After video picture finishes, sacrifice of animal, take out heart immediately, according to framing and visual inspection, take out the apoptosis cardiac muscle that ischemical reperfusion injury causes, and get normal myocardium and organize in contrast, measure radiocounting respectively with the γ calculating instrument respectively, and weigh, calculate both unit weight radioactive uptake ratio.
Get ischemical reperfusion injury cardiac muscle and normal myocardium sample, carry out the dna content analysis respectively, and electron microscopic examination.The Bec Ton Dic kinson FACS Calibur of company type flow cytometer is adopted in the dna content analysis.Electronic Speculum adopts the Japanese Electronic Speculum JEM-1010 of company type, and 5% glutaraldehyde is fixed, and 1% osmic acid is fixed, and acetone dewaters step by step, embedding, and thin section, after acetic acid oil, the lead citrate dyeing, last machine is observed.
Experimental result shows: developer injection back 3h, and (Fig. 1), blood pond shadow develops lighter the visible tangible local radioactivity anomaly of myocardial sites dense poly-(picked-up of apoptosis cardiac muscle), and T/NT ratio is 3.89 ± 0.84.External test, the apoptosis cardiac muscle is 10.35 ± 4.33 with the unit mass radioactive uptake ratio of normal myocardium.
The dna content analytical results: the cardiac muscular tissue of the high radioactivity of video picture and external test picked-up, dna content is analyzed visible obvious apoptosis cell peak on the histogram, and normal myocardium is organized and is not seen that obvious apoptosis cell peak forms.Electron microscopic examination also confirms: cardiac muscular tissue's sample of high radioactivity picked-up, visible significantly a large amount of apoptosis myocardial cells form (Fig. 2).
Experimental result shows: 99Tc m-His10-AnnexinV tagged compound can show cardiac cellular apoptosis as developer, and apoptosis of cardiac muscle is the cardiomyopathy common pathological characters such as myocardium rejection after acute myocardial infarction, myocardosis, heart failure, the heart transplantation, and imaging technique of the present invention can be used for the diagnosis and the therapeutic evaluation of above-mentioned disease.
Example 2. 99Tc m-His10-AnnexinV tagged compound detects through the apoptosis of tumor cells (is example with the nonsmall-cell lung cancer) after the chemotherapy as developer
Set up lotus H460 knurl nude mice model.Get people's nonsmall-cell lung cancer (NSCLC) cell strain H460, with the RPMI-1640 perfect medium that contains massfraction 15% calf serum, in 37 ℃, volume fraction 5%CO 2Cultivate under the condition, when the long 80%-9 of arriving 0% of cell merges, use trysinization, guarantee that living cell rate is greater than 99%.Get the BALB/C nude mice in 4 to 5 ages in week, the cell suspension 0.2mL that will contain 1 * 106 tumour cell is injected in right side of mice forelimb oxter.Treat that knurl body diameter grows to about 1cm, through tail vein injection taxol induced chemotherapy, dosage is according to 100mg/m 2, 24h, 48h, 72h are used for the video picture experiment after the chemotherapy.
Untreated mice with tumor control group, after the taxol chemotherapy is induced 24h, 48h, 72h lotus H460 nude mice each 5, tail vein injection 99Tc m-His10-Annexin V 18.5MBq/0.2mL carries out the SPECT video picture respectively at injection back 2h, 4h and 6h, adopts pinhole collimator, and matrix is 256 * 256, gathers counting and is 500Kcounts.Delineate region of interest (ROI), calculate tumour and offside normal muscle tissue radiation count ratio (T/NT).After the video picture, the disconnected neck of nude mice is put to death, dissect and take out tumour, weigh and measure radiocounting, calculate every gram and organize percentage injection dose rate (%ID/g).
Subsequently, tumour being carried out the cell in vitro apoptosis detects.Comprise:
(1) flow cytometry (FCM) quantitative analysis activation caspase-3: flow cytometry quantitative analysis activation caspase-3 concrete steps are as follows: 1. fresh tumor tissues shreds, add 5mL physiological saline, draw tissue suspension with suction pipe, with 300 order nylon net filters, the centrifugal 5min of 1000r/min, the 0.01M PBS 2mL that adds pH7.4 again, 500 to 800r/min centrifugal 5min wash 2 times to remove cell debris, and 4 ℃ of preservations are standby.2. add cell suspension 300 μ L (1 * 10 in the centrifuge tube 6Individual cell), adds 1 μ LFITC-DEVD-FMK, 37 ℃, 5%CO 2Hatch 30min under the condition.3. add 1 μ L/mL caspase-3 inhibitor Z-VAD-FMK and suppress the caspase-3 activation as negative control group.4. with damping fluid 0.5mL, the centrifugal 5min of 3000r/min abandons supernatant.Repeat once again.5. add damping fluid 0.5mL, the vortex mixing.6. the upflowing cell instrument carries out quantitative analysis.
(2) light microscopic and situ end labeling (TUNEL) detect apoptosis: the TUNEL method is used for unicellular horizontal apoptosis immunohistochemical methods and detects and quantitative analysis.Its principle is marker DNA fracture position, and detailed step is as follows: 1. dimethylbenzene dewaxing, the PBS flushing is 2 times after the gradient alcohol aquation, each 3min.2. gastric pepsin digestion, PBS flushing 2 times, each 3min.3. dry tissue moisture content on every side, every 0.3%H that adds 50 μ L 2O 2Methanol solution, blocking-up endogenous peroxidase, PBS flushing 2 times, each 3min.4. every adds the 50ulTUNEL reaction solution, hatches in the box 37 ℃, 60min, PBS flushing 2 times, each 3min.5. every adds 50 μ L horseradish peroxidase antibody, hatches in the box 37 ℃, PBS flushing 3 times, each 3min.6. one of the DAB that adds fresh configuration, incubated at room, PBS flushing 3 times, each 3min.Hematorylin is redyed, dehydration of alcohol, drying.7. neutral gum mounting.Om observation, nucleus is brown positive.
Experimental result shows: injection 99Tc mThe promptly visible significantly high radioactivity of 2h behind the-His10-AnnexinV, the tumor locus of chemotherapy group is absorbed, and thickens gradually along with time lengthening; And the tumor locus of control group is not seen obvious radioactive uptake (Fig. 3).
The T/NT ratio of 24h, 48h, 72h group is respectively 2.63 ± 0.76,3.41 ± 0.90 and 3.85 ± 0.62 after the chemotherapy, and the T/NT of control group only is 1.42 ± 0.19, is starkly lower than chemotherapy group (P＜0.01).24h, 48h and 72h after the chemotherapy, the radioactive uptake value (%ID/g) of tumor tissues is respectively 2.55 ± 0.73,3.60 ± 1.09 and 3.73 ± 0.97, and the control group tumour only has a spot of radioactive uptake, is 1.09 ± 0.18, is starkly lower than chemotherapy group (P＜0.01).
Flow cytometry apoptosis result: FCM quantitative analysis activation caspase-3, control group is 3.70% ± 0.74%; After the taxol induced chemotherapy, a large amount of caspase-3 are activated, and 24h, 48h and 72h chemotherapy group are respectively 23.46% ± 2.23%, 62.85% ± 6.13% and 70.44 ± 6.09%, and chemotherapy group is compared with control group, and significant difference (P＜0.01) is all arranged.
Light microscopic and TUNEL method detect the apoptosis result: pathology detection the results are shown in Figure 4.The small amounts of cells apoptosis only appears in the tumor tissues of control group, and Hematorylin Yihong (HE) dyeing and TUNEL method only visible several are dispersed in apoptotic body, and the cell outline complete packet is around nuclear fragment.And chemotherapy is induced back 24h, and sheet or polynesic apoptosis have then taken place, and HE dyeing sees that a large amount of nuclear fragments, pyknosis, TUNEL show the apoptotic cell of a large amount of xanthochromias; Chemotherapy is induced back 48h, large stretch of cell generation apoptosis; Chemotherapy is induced back 72h, and apoptosis has taken place most cells, only around the lumen of vessels part cell survival is arranged, and the nuclear fission phase appears in small amounts of cells.Apoptosis cell accounts for the per-cent of total cell count in 10 high power fields, and averaging is apoptotic index (apoptoticindex).The apoptotic index of control group is 3.31% ± 0.61%, and 24h, 48h and 72h chemotherapy induce the apoptotic index of group to be respectively 32.90% ± 6.64%, 70.42% ± 7.54% and 8 3.23% ± 9.71%, and chemotherapy induces each group to compare with control group all has significant difference (P＜0.01).
Interpretation further shows: T/NT value and FCM activation caspase-3 have good dependency (r=0.847, P＜0.01), and the apoptotic index of measuring with the TUNEL method also has fine dependency (r=0.833, P＜0.01).The radioactive uptake of tumor tissues (%ID/g) has good dependency (r=0.774, P＜0.01) with FCM activation caspase-3, with TUNEL method apoptotic index good dependency (r=0.850, P＜0.01) is also arranged.
Experimental result shows: 99Tc m-His10-AnnexinV tagged compound can show the apoptosis situation of tumour cell after the chemotherapy as developer, because the induced tumor apoptosis is the therapeutic purpose of many chemotherapeutics such as taxol, therefore, imaging technique of the present invention can be used for the evaluation of malignant tumor chemotherapy curative effect.

Claims (1)

1. one kind 99Tc mTagged compound 99Tc mThe preparation method of-His10-AnnexinV, its marking method are characterised in that and adopt direct labelling method that His10-Annexin V is carried out 99Tc mMark, step is: get an amount of SnCl 22H 2O is the HCl dissolving of 0.05mol/L with concentration, and citrate buffer solution is diluted to 1mg/mL, gets the above-mentioned SnCl of containing that has prepared 2Citrate buffer solution 100 μ L, add His10-Annexin V 5 μ g successively, 99Tc mO 4 -0.8mL 10mg/mL vitamins C 100 μ L place behind the mixing, promptly form 99Tc m-His10-Annexin V.
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