CN109939247A - A kind of endothelial progenitor cells of radioisotope labeling and its preparation method and application - Google Patents
A kind of endothelial progenitor cells of radioisotope labeling and its preparation method and application Download PDFInfo
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Abstract
The technical problem to be solved in the present invention is that proposing a kind of endothelial progenitor cells and its preparation method and application of radioisotope labeling, the initiative discovery endothelial progenitor cells of the present invention can be specific be enriched in lung, the especially lung of pulmonary hypertension model, there is this characteristic of Lung targeting using endothelial progenitor cells, endothelial progenitor cells are prepared to the imaging agent or drug of Lung targeting, certain biological properties in real-time qualitative and quantitative detection active somatic cell on cell and molecular level, the anomalous variation of the cellular and molecular level of disease early stage and nontoxic can be observed, it is without side-effects, it is with good stability.
Description
Technical field
The present invention relates to cell biologies and in-vivo imaging tracer field, and in particular to a kind of radioisotope labeling it is interior
Skin progenitor cells and its preparation method and application.
Background technique
It is more than one kind of certain dividing value that pulmonary hypertension (pulmonary hypertension), which refers to that pulmonary artery pressure increases,
Haemodynamics and pathological and physiological condition, can lead to right heart failure, can be a kind of independent disease, are also possible to complication,
It can also be syndrome.Its haemodynamics diagnostic criteria are as follows: under the quiescent condition of sea level, it is average that right heart catheter detects pulmonary artery
Pressure >=25mmHg.Pulmonary hypertension is a kind of common disease, frequently-occurring disease, and disability rate and case fatality rate are very high, therefore, Ying Yinqi people
Great attention.And have for the tracer of the lung of patients with pulmonary hypertension observation for the treatment and prognosis of pulmonary hypertension
Greatly help.Pulmonary hypertension is mainly characterized by Pulmonary Vascular excess shrinkage, abnormal vascular reconstruct and lung parteriole obstruction.Blood
Pipe reconstruct, which is mainly largely increased by endothelial cell (endothelial cells, ECs) dysfunction and α-SMA positive cell, draws
It rises.Also become the direction of the research of numerous scholars using endothelial cell treatment pulmonary hypertension.
PET full name is Positron Emission Computed Tomography (positron emission tomography PET),
It is the picture reproducer of the gene for reflecting lesion, molecule, metabolism and functional status.It is to utilize positron radionuclide labelled glucose etc.
Human metabolite reflects its metabolic alterations to the intake of imaging agent as imaging agent, by lesion, to provide disease for clinic
The biological metabolism information of disease.It is the new milestone of current life science, Medical Imaging Technology development.CT full name is electronic computer
X-ray tomography technology (Computed Tomography), it is to carry out the inspection of body layer to human body using X-ray. PET/
CT imaging agent is that PET and CT combine, and shares an image workstation, PET/CT using the same examination couch
Have the function of PET, CT simultaneously and by PET image and CT image co-registration etc..
Endothelial progenitor cells have the ability of self-renewing and differentiation, extensive amplification are able to carry out in vitro, if can
It is proven to have pulmonary artery targeting, will be of great significance to applied to clinical treatment, however yet there are no endothelial progenitor cells
Relevant report with pulmonary artery targeting.Present invention discover that endothelial progenitor cells have pulmonary artery targeting, using this characteristic,
Initiative applies it in the imaging agent with Lung targeting, drug or other products, and demonstrates the feasible of its application
Property, the occurrence and development situation of pulmonary hypertension can be observed, and monitor distribution, proliferation and the body of carrier cell in vivo and repel
Process, can have the characteristics that noninvasive, real-time dynamic, quantitative observation, and can be right using the molecular images technology such as PET
Same animal is monitored in different time points, can assess the curative effect of targeted therapy well.
Summary of the invention
Therefore, the invention solves first technical problem be to propose that a kind of endothelial progenitor cells are preparing Lung targeting
Purposes in imaging agent, drug or product.
The invention solves second technical problem be to propose a kind of endothelium ancestral of image displaying function with Lung targeting
The endothelial progenitor cells of cell and its preparation method and application, the image displaying function with Lung targeting specific can be enriched in
The lung of lung, especially pulmonary hypertension model carries out tracer observation to lung, nontoxic, without side-effects, has good steady
It is qualitative.
For this purpose, the present invention provides the following technical scheme that
The purposes that the present invention provides endothelial progenitor cells in the imaging agent or drug for preparing Lung targeting.
Preferably, the Lung targeting is pulmonary artery targeting.
The present invention provides a kind of endothelial progenitor cells of image displaying function with Lung targeting, using radionuclide, fluorescence
Dyestuff, quantum dot or molecular probe handle endothelial progenitor cells by way of marking or transfecting.
The endothelial progenitor cells of the image displaying function with Lung targeting, the radionuclide are89Zr、45Ti、55Mn、59Fe、64Cu、94mTc、67Ga、71/72/74As、82Rb、89Y or124I。
The endothelial progenitor cells of the image displaying function with Lung targeting, the radionuclide are89Zr。
The present invention provides a kind of methods of endothelial progenitor cells for preparing the image displaying function with Lung targeting, will put
Penetrating property nucleic, fluorescent dye, quantum dot or molecular probe handle endothelial progenitor cells by way of marking or transfecting, and are had
The endothelial progenitor cells of image displaying function.
The preparation method, by being modified in progenitor endothelial cell surface, the modification of endothelial progenitor cells cell surface
Modifier can be by radioisotope labeling.Preferably, the modifier includes but is not limited to mark prothetic group, bifunctional chelating agent
Or complex compound.
Preferably, the complex compound is DFO complex compound.
Preferably, the preparation method, includes the following steps:
S1, the aqueous solution for taking DFO complex compound, are added in endothelial progenitor cells culture solution, adjust pH value, are incubated for, spare；
It is added in S2, the Incubating Solution into S1 step89Zr- oxalate solution, continue be incubated for get.
The present invention provides a kind of radioisotope labelings with Lung targeting being prepared by the preparation method
Endothelial progenitor cells.
What the endothelial progenitor cells of the image displaying function with Lung targeting or the preparation method were prepared has
Purposes of the endothelial progenitor cells of the radioisotope labeling of Lung targeting in the imaging agent or therapeutic agent for preparing pulmonary disease.It is excellent
Choosing, the lung disease includes pulmonary hypertension disease.
The present invention provides a kind of PET/CT imaging agent, the endothelium ancestral including the image displaying function with Lung targeting is thin
The endothelial progenitor cells for the radioisotope labeling with Lung targeting that born of the same parents or the preparation method are prepared.
Purposes of the PET/CT imaging agent in lung or pulmonary disease dynamic tracer.
Technical solution of the present invention has the advantages that
1. purposes of the endothelial progenitor cells of the present invention in the imaging agent or drug for preparing Lung targeting, the present invention is pioneering
Property discovery endothelial progenitor cells have the function of Lung targeting, especially pulmonary artery have high specific targeting, it is therefore, available
Endothelial progenitor cells have this characteristic of Lung targeting, by endothelial progenitor cells in the imaging agent or drug for preparing Lung targeting, by endothelium ancestral
The imaging agent of cell preparation can on cell and molecular level certain biology in real-time qualitative and quantitative detection active somatic cell
The anomalous variation of the cellular and molecular level of disease early stage can be observed in feature.
2. a kind of endothelial progenitor cells of image displaying function with Lung targeting of the present invention, the radionuclide are89Zr, with89Long half-lift positron radionuclide and labeling method based on Zr carry out Cellular tracking, overcome tradition18The tracers such as F
Half-life short and111The features such as In single photon tracer resolution ratio is poor, and have the characteristics that sensitivity and degrees of specificity are high.
3. the method that preparation provided by the invention has the endothelial progenitor cells of the image displaying function of Lung targeting, by endothelium ancestral
Cells cell surface is modified, and the modifier of endothelial progenitor cells cell surface modification can be by radioisotope labeling.It is preferred that
, the modifier includes but is not limited to mark prothetic group, bifunctional chelating agent or complex compound.Select modifier for DFO complex compound
Endothelial progenitor cells are modified, are then carried out89Zr label, does not produce bigger effect the survival of endothelial progenitor cell and state, PET
Carry out the migration course that Cellular tracking realizes noninvasive, the real-time same animal body internal labeling cell of dynamic observation.
4. PET/CT imaging agent of the present invention, the endothelial progenitor cells or described including the image displaying function with Lung targeting
The endothelial progenitor cells of the radioisotope labeling with Lung targeting that are prepared of preparation method, be a kind of extraordinary conversion
Medical tool can be realized in the intracorporal vivo tracking of people using identical labeling method, observe cell in the intracorporal dynamic of people
The therapeutic process of variation and pulmonary hypertension.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is in experimental example 1 of the present invention89Zr-DFO-NCS Quality Control result；
Fig. 2 is in experimental example 1 of the present invention89The stability result of Zr-DFO-NCS；
Fig. 3 is that the G1 group normal SD rats in experimental example 2 of the present invention respectively organize %ID/g-mean to change over time curve；
Fig. 4 is the G1-M-01 different time points PET/CT scan image in experimental example 2 of the present invention；
Fig. 5 is that the G2 group pulmonary hypertension SD rat in experimental example 2 of the present invention respectively organizes %ID/g-mean to change over time
Curve；
Fig. 6 is the G2-M-01 different time points PET/CT scan image in experimental example 2 of the present invention；
Fig. 7 is that the G3 group pulmonary hypertension SD rat in experimental example 2 of the present invention respectively organizes %ID/g-mean to change over time
Curve；
Fig. 8 is the G3-M-01 different time points PET/CT scan image in experimental example 2 of the present invention；
Fig. 9 is the reaction route of endothelial progenitor cells and DFO complex compound in the embodiment of the present invention 1；
Figure 10 is the endothelial progenitor cells and radionuclide that surface modification has DFO complex compound in the embodiment of the present invention 189Zr's
Reaction route.
Specific embodiment
Reagent, material or the instrument being related in following embodiments are commercial product, using the reagent of different manufacturers, material
Material or instrument can't generate apparent difference.
If endothelial progenitor cells are according to " separation, culture and the identification of human endothelial progenitor cells from peripheral blood " (Qiao Wei, Ran Feng, Liu
Length build, Chinese Tissue Engineering Study, the 36th phase 2013-09-03 of volume 17) disclosed in method human peripheral endothelium is prepared
Progenitor cells.
Main test apparatus model, producer see the table below 1
1 key instrument of table
The model of main reagent, producer see the table below 2
2 main agents of table
Embodiment 1
Present embodiments provide a kind of radionuclide89The preparation method of the endothelial progenitor cells of Zr label, including walk as follows
It is rapid:
(1) acquisition of endothelial progenitor cells: acquisition 50ml peripheral blood is obtained outer using isometric PBS buffer solution dilution
Then the centrifuge tube of the separating liquid of human lymphocyte containing Ficoll-Paque (Amersham company, Switzerland) is added in all hemodilution liquid
In, it is located at Ficoll-Paque human lymphocyte separating liquid upper layer, peripheral blood dilution and Ficoll-Paque human lymphocyte
The height ratio of separating liquid is 4: 6, and high speed centrifugation is carried out under room temperature, is centrifuged radius 15cm, 2000r/min, is centrifuged 30min, carefully
After tunica albuginea confluent monolayer cells wash 2 times with PBS buffer solution among drawing, to be centrifuged radius 15cm, 1500r/min is centrifuged 8min and obtains
Monocyte；Monocyte is resuspended in containing the 10% full culture medium of fetal calf serum endothelial cell of volume fraction (U.S. Gibco public affairs
Department), with 1 × 106Concentration is inoculated in has been coated with fibronectin (Sigma Co., USA) (5 μ g/cm in advance2) 25cm2Carefully
In born of the same parents' culture bottle, 37 DEG C, volume fraction 5%CO are set2It is cultivated in incubator, liquid is changed after 3 days and removes not adherent suspension cell, it is adherent
Cell continues to cultivate, and changes within every 3 days liquid 1 time later.With 1: 3 ratio secondary culture after the fusion of cell 80% or so.Culture period
Between, the form sum number amount variation of attached cell is observed under inverted phase contrast microscope daily.
(2) take DFO complex compound (with the chemical formula 1 in comparative example 1) (DFO complex compound is purchased from macrocyclics company)
The concentration that 50 μ L are made is the aqueous solution of 6.9mM, and being added to cell concentration is 1 × 106That cultivates in the step of a/ml (1) is interior
Skin progenitor cells culture solution, is then added alkaline buffer, and the pH value of mixture is adjusted to 9, then in 5%CO2, at 37 DEG C
It is incubated for 30 minutes in incubator, states be added 1400KBq's in the cell culture fluid of incubation then up89Zr- oxalate solution, so
After continue in 5%CO2, be incubated for 30 minutes to get radionuclide in incubator at 37 DEG C89The endothelial progenitor cells of Zr label89Zr-EPC, the detection of cell marking retention rate are shown in experimental example 1.
The radionuclide of endothelial progenitor cells in above-mentioned steps (2)89The route of Zr label is as shown in attached drawing 8-9, Fig. 8
The substance of approximate Y type in middle reactant refers to endothelial progenitor cells, and spherical shape represents endothelial progenitor cells in Fig. 9.
Comparative example 1
Present embodiments provide one kind89The preparation method of Zr-DFO-NCS, include the following steps: to take DFO complex compound (under
State chemical formula 1) (the DFO complex compound can be synthesized by following reaction routes, and DFO complex compound is purchased from this embodiment
Macrocyclics company) aqueous solution that the concentration of 50 μ L is 6.9mM is made, then it is added 37MBq's89Zr- oxalate solution,
It is described89HEPES buffer solution tune pH value in Zr- oxalate solution containing 1M is 7.4, is incubated at room temperature 15min to get putting
The detection of penetrating property chemical purity is shown in experimental example 1.
Experimental example 1 investigates radionuclide89The quality control of the endothelial progenitor cells of Zr label and vitro stability
1. tested material: prepared by embodiment 189Zr-EPC, the lead can under the conditions of being stored in 2-8 DEG C, solvent are EGM-2 culture
Base；Prepared by comparative example 189Zr-DFO-NCS, the lead can under the conditions of being stored in 2-8 DEG C, solvent are slow for 0.15M acetic acid/sodium acetate
Rush solution (pH=7.2).
2. solvent:89Zr-EPC solvent is Endothelial cell culture base -2 (EGM-2) culture medium；89Zr-DFO-NCS solvent is
0.15M acetic acid/sodium acetate buffer (pH=7.2)
3. test method:
3.1 89The detection method of Zr-EPC
3.1.1 89The quality of Zr-EPC controls and vitro stability
Prepared by Example 189Zr-EPC (about 1 μ Ci, volume adjust to 1mL) by centrifugation respectively collect supernatant and
Cell precipitation detects its radioactivity using γ calculating instrument, and radioactivity total amount percentage is as its stabilization in vitro shared by cell precipitation
Property data.2 detection time points (first detection time point is 0h, and second detection time point is 16h) is set.Each detection
Radioactivity total amount percentage shared by the cell precipitation at time point should be not less than 80%.
3.1.2 89The detection of Zr-EPC Cell viability
89Zr-EPC carries out Cell viability detection using 0.4% trypan blue, when detection according to89Zr-EPC cell suspension:
0.4% trypan blue=1:1 (volume ratio), by what is prepared in embodiment 189Zr-EPC cell suspension is mixed with 0.4% trypan blue, so
Total cell number percentage (passing through micro- sem observation) shared by living cells is calculated afterwards.
3.2 89The detection method of Zr-DFO-NCS
3.2.1 89The quality of Zr-DFO-NCS controls
Using radio thin layer's chromatography (Radio-iTLC) method prepared by marked product comparative example 189Zr-DFO-NCS's
Radio-chemical purity is detected, and support is silica impregnated glass fiber item；Solvent is 0.5M citric acid/sodium citrate
Buffer solution (PH=5) is scanned with thin layer radioactive scanning instrument, will89In Zr- oxalic acid zirconium liquid dot to Radio-iTLC paper,
Ascending development as a control group, calculates R in 0.5M citric acid/sodium citrate buffer solution (pH=5) systemfValue, RfCalculation formula
It is as follows:
Wherein 0.15min is point sample origin, and 0.78min is solvent front.R of the retention time less than 0.15 minfValue
It is defined as 0, retention time is greater than the R of 0.78minfValue is defined as 1, it is desirable that the R of target productfValue 0-0.1,89Zr- oxalic acid zirconium liquid
The R of bodyfValue 0.8-1,89Zr-DFO-NCS radio-chemical purity is not less than 90%.
3.2.2 89The vitro stability of Zr-DFO-NCS
In comparative example 189Zr-DFO-NCS is detected as test sample (being stored in room temperature) by Radio-iTLC method
Its radio-chemical purity, as its vitro stability data, 2 detection time points are arranged, and (first detection time point is 0h
Purity detecting as in Quality Control, second detection time point are 11h).89Zr-DFO-NCS solvent stability radio chemistry is pure
Degree is not less than 90%.
4. test result
4.1 89Cell viability, quality control and the vitro stability of Zr-EPC
Table 389Zr-EPC Cell viability, Quality Control and stability result
By above-mentioned table 1 it is found that prepared by embodiment 189Zr-EPC is 76.52% in the Cell viability of 0h, and marker retains
Rate is 85.92%；It is 74.56% in the Cell viability of 16h, marker retention rate is 61.55%.
4.2 89Zr-DFO-NCS Quality Control and stability
89Zr-DFO-NCS Quality Control and stability are shown in attached drawing 1-2 and the following table 4-5, and test result shows89Zr-DFO-NCS
Radio-chemical purity be 100%, be greater than 90%；11h is saved in 0.15M acetic acid/hac buffer (pH=7.2)
Afterwards, radio-chemical purity 100% is greater than 90%.
Radio-chemical purity (RCP) result in 4 attached drawing 1 of table
RCP result in 5 attached drawing 2 of table
Experimental example 2 is investigated89The small animal position emission tomography (PET) of Zr-EPC/CT imaging
1. tested material: prepared by embodiment 189Zr-EPC, the lead can under the conditions of being stored in 2-8 DEG C, solvent are EGM-2 culture
Base；Prepared by comparative example 189Zr-DFO-NCS, the lead can under the conditions of being stored in 2-8 DEG C, solvent are slow for 0.15M acetic acid/sodium acetate
Rush solution (pH=7.2).
2. solvent:89Zr-EPC solvent is EGM-2 culture medium, and dilution is EGM-2 culture medium when administration；89Zr-DFO-
NCS solvent is 0.15M acetic acid/sodium acetate buffer (pH=7.2), and dilution is 0.15M acetic acid/sodium acetate buffer when administration
Solution (pH=7.2).
3. experimental animal: male SD rat, 42, week old 6-9 weeks, weight 180-200g, wherein 32 are served only for pulmonary artery
High pressure modeling selects 13 pulmonary hypertension modeling animals for zoopery, remaining 10 normal SD rats, random selection 8
It is served only for zoopery.Quality certification number is 11400700259778, and supplier is that Beijing dimension tonneau China experimental animal technology has
Limit company, supplier's production licence number are SCXK (capital) 2016-0006.Every animal has unique animal identification number, adopts
It is marked with ear tag.Pulmonary hypertension model SD rat is modeled by meter Du (Nanjing) Bioisystech Co., Ltd, (the south meter Du
Capital) Bioisystech Co., Ltd assist, meter Du (Nanjing) Bioisystech Co., Ltd assisted in modeling process animal feeding and
Observation.
4. the reception of animal, quarantine, raising and management
SD rat can be into after quarantining 3 days qualifications to the Bioisystech Co., Ltd SPF grades of barrier animal room meter Du (Nanjing)
Enter test；
After SD rat completes quarantine, random selection 32 is served only for pulmonary hypertension model foundation.Pulmonary hypertension model is built
Cube method are as follows: (MCT is volumetric concentration or matter to every SD rat according to about 53mg/kg neck subcutaneous administration monocrotaline solution
Measure score 1-2%), it weighs weekly 3 times after giving MCT, continuous 3-4 weeks, the later period regarded animal state adjustment weighing number.According to
After animal weighing prediction models successfully, before administration and after the last one sweep time point, pulmonary arterial pressure is carried out
Measurement, the modeling animal before being administered after 13 pulmonary arterial pressure models of measurement selection are measured, and it is dynamic to obtain 6 pulmonary arterial pressure models
The pulmonary arterial pressure of object；After the last one sweep time point, before animal is put to death, to the normal rat (SD including non-administration
Rat), the remaining pulmonary arterial pressure animal pattern of non-administration, give89The pulmonary arterial pressure animal pattern of Zr-EPC carries out pulmonary arterial pressure
Measurement, obtain the pulmonary arterial pressure of 7 animals.
Illumination is 12 hours bright, dark alternatings during animal feeding, free water and is ingested, and drinking-water goes out through double door leafs pulsation high pressures
The sterilizing of bacterium cabinet, water standard are not less than urban life drinking water standard.Experimental animal uses licensing: SYXK (Soviet Union) 2015-0014.
5. the grouping and medication of experimental animal
The grouping and administration of 5.1 animals
The grouping and administration of 6 animal of table
A: increase by 1 animal as spare animal.NA: it is invalid, it is useless.IV: intravenous injection.
5.2 administration frequencies: single-dose
5.3 medications:
G1, G2, G4, G6 group:89Zr-EPC is prepared in panel sub-assembling platform through EGM-2 cell culture medium.Every animal is quiet through tail
Arteries and veins injection89Zr-EPC cell suspension, dosage are about 1.5 × 106-3.0×106Cells/ only, and records initial injection radiation
Property dosage, survey initial injection dose time, injection time, residual activity dosage, survey the Residual dose time.When in experimentation
Occur to be replaced when animal dead or other abnormal conditions using spare animal.
G3 group:89Zr-DFO-NCS is prepared in panel sub-assembling platform through 0.15M acetic acid/sodium-acetate buffer (pH=7.2) dilution.Often
Animal is through tail vein injection89Zr-DFO-NCS, dosage with89The injection radioactive dosage of Zr-EPC dosage is suitable, and records
Initial injection radioactive dosage surveys initial injection dose time, injection time, residual activity dosage, surveys the Residual dose time.
It is replaced when animal dead or other abnormal conditions occur in experimentation using spare animal.
Standard pipe is prepared: will with EGM-2 culture medium89Zr-EPC is diluted to suitable activity and concentration, and it is dilute to draw certain volume
After releasing89Zr-EPC is put in 6 to exempt from pipe, each puts and exempts from about 2 μ Ci of pipe radioactive dosage, when as in vitro tissue γ detection
Standard pipe.
5.4 Testing index
5.4.1 overview
During test, working day is observed 2 times (morning and afternoon are at least spaced 4 hours) daily, and festivals or holidays at weekend are seen daily
It examines 1 time, observation death, morbidity, breathing, secretion, excrement and diet, drinking-water situation etc..
5.4.2 weighing
Each the weight of animals is weighed before administration, for calculating dosage.
5.4.3 small animal position emission tomography (PET)/CT scanning method
Using toy Anesthesia machine by Animal Anesthesia, anesthetic: the isoflurane of about 1-2%, gas flow rate: about 400-
800mL/min；The good animal of induced anesthesia is put on small animal position emission tomography (PET)/CT bed, continues isoflurane and maintains anesthesia.Administration
After 1h, 1d, 3d, 7d and 14d carry out small animal position emission tomography (PET)/CT static state body scan, each bed scans 10~30min, note
It records sweep time, scanning energy window: 350-650 Kev.
Scan operation deviates the time no more than 5%.
5.4.4 the in vitro dissection of experimental animal
G4 and G6 group: in every animal CO of 3d after administration2Excessive inhalation is put to death and is dissected, take blood, the heart, liver, spleen,
Lung kidney, intestines, stomach, brain, pancreas, bone, muscle totally 12 tissue/body fluid carry out γ count detection.Wherein, when taking lung tissue, first
γ counting is carried out after handling according to common detection methods；After counting, after lung tissue is impregnated 0.5h in physiological saline, then
A γ is carried out to count.After γ is counted, after fixed lung tissue to 10 half-life period (i.e. 784h), it to be used for subsequent detection.
G5 and G7 group: after the last one time point end of scan of G1 and G2 group small animal position emission tomography (PET)/CT, every animal CO2It is excessive
Inhalation is put to death and is dissected, and blood, the heart, liver, spleen, lung kidney, intestines, stomach, brain, pancreas, bone, muscle totally 12 tissue/body fluid are taken, and carries out
γ count detection.Wherein, when taking lung tissue, γ counting is carried out after first handling according to common detection methods；It, will after counting
After lung tissue impregnates 0.5h in physiological saline, then carries out a γ and count.After γ is counted, fixed lung tissue declines to 10 half
After phase (i.e. 784h), it to be used for subsequent detection.
Sampling operation deviates the time no more than 5%.
6. data collection and analysis
6.1 small animal position emission tomography (PET)s/CT data acquisition
Data acquisition: image reconstruction, algorithm for reconstructing are as follows: 3D ordered subset expectation maximization value-based algorithm are carried out after the completion of acquisition
(OSEM 3D)+PSF；The number of iterations are as follows: 5 times.Image and data processing are carried out with image processing software PMOD after the completion of rebuilding,
Delineating the internal organs such as brain, the heart, liver, lung, kidney, intestines, knee joint, backbone, muscle is area-of-interest, obtains unit bodies after the completion of delineating
The radioactive activity value of product area-of-interest, and the %ID/g numerical value for obtaining each internal organs is calculated, formula is as follows:
The acquisition of 6.2 γ enumeration datas
Radioactive uptake value (%ID/g) of the test sample in experimental animal tissue/body fluid is calculated by γ count results,
And then test sample is obtained in the tissue distribution of experimental animal.
Tissue/body fluid89Zr-EPC radioactive uptake value (%ID/g)=sample count value/injection enters intracorporal tale
Value/tissue/body fluid quality * 100%.
Interpretation of result: it is analyzed using Graph pad software.
7. test result
The administration and observation of 7.1 animals
7 animal drug administration information of table
B: weight is the weight of animals on the administration same day.
C: dosage when radioactive dosage is administration.
During test, observation display daily, animal breath, secretion, excrement and diet, drinking-water situation are normal.Wherein
G2-M-02 and G2-M-04 is dead when 3d is scanned, and uses the experimental data of the spare M-01 replacement G2-M-04 of G2-, G2-M-04
The data of generation do not embody in this test.
7.2 small animal position emission tomography (PET)s/CT scan experimental result
G1 group normal SD rats are being given89After Zr-EPC, each time point animal tissue radioactive substance absorbs %ID/g-
Mean value, which is shown in Table 8, %ID/g-mean and changes over time curve, sees Fig. 3, and the detailed data of every animal sees attached list 9-13.PET/
CT scan representative image is shown in Fig. 4.By chart it is found that G1 group animal is being given89After Zr-EPC, radioactive substance intake mainly exists
Secondly lung is liver.Highest when the radioactive uptake 1h of lung, time point is basicly stable thereafter；When the radioactive uptake 1h of intestines
Highest, then decline rapidly；Liver, kidney, knee joint, backbone radioactive uptake first increase reduce afterwards at any time；The radioactivity of the heart
It absorbs relatively stable；The radioactive uptake of brain and muscle is close, very low.
8 G1 group normal SD rats of table respectively organize radioactive substance to absorb %ID/g-mean value (n=4)
9 G1 group normal SD rats of table are given89The %ID/g-mean value of each animal tissue of 1h after Zr-EPC
10 G1 group normal SD rats of table are given89The %ID/g-mean value of Zr-EPC Hou24hGe animal tissue
11 G1 group normal SD rats of table are given89The %ID/g-mean value of each animal tissue of 72h after Zr-EPC
12 G1 group normal SD rats of table are given89The %ID/g-mean value of each animal tissue of 168h after Zr-EPC
13 G1 group normal SD rats of table are given89The %ID/g-mean value of each animal tissue of 336h after Zr-EPC
G2 group pulmonary hypertension SD rat is being given89After Zr-EPC, each time point animal tissue radioactive substance absorbs %
ID/g-mean value, which is shown in Table 14, %ID/g-mean and changes over time curve, sees Fig. 5, and the detailed data of every animal sees attached list 15-
19.PET/CT scanning representative image is shown in Fig. 6.By chart it is found that G2 group animal is being given89After Zr-EPC, radioactive substance intake
It is secondly liver mainly in lung.Highest when the radioactive uptake 1h of lung, time point is basicly stable thereafter；The radioactivity of intestines is taken the photograph
Highest when taking 1h, then decline rapidly；Knee joint, backbone radioactive uptake first increase reduce afterwards at any time；Liver, kidney are put
Penetrating property fluctuates rising at any time；The radioactive uptake of the heart is relatively stable；The radioactive uptake of brain and muscle is close, very low.
14 G2 group pulmonary hypertension SD rat of table respectively organizes radioactive substance to absorb %ID/g-mean value (n=4)
D:n=3.
15 G2 group pulmonary hypertension model rat of table is given89The %ID/g-mean value of each animal tissue of 1h after Zr-EPC
16 G2 group pulmonary hypertension model rat of table is given89The %ID/g-mean value of Zr-EPC Hou24hGe animal tissue
17 G2 group pulmonary hypertension model rat of table is given89The %ID/g-mean value of each animal tissue of 72h after Zr-EPC
18 G2 group pulmonary hypertension model rat of table is given89The %ID/g-mean value of each animal tissue of 168h after Zr-EPC
NA: animal dead
19 G2 group pulmonary hypertension model rat of table is given89The %ID/g-mean value of each animal tissue of 336h after Zr-EPC
NA: animal dead
G3 group pulmonary hypertension SD rat is being given89After Zr-DFO-NCS, each time point animal tissue radioactive substance is taken the photograph
It takes %ID/g-mean value to be shown in Table 20, %ID/g-mean and change over time curve and sees Fig. 3, the detailed data of every animal is seen attached list
21-25.PET/CT scanning representative image is shown in attached drawing 4.By chart it is found that G3 group animal is being given89After Zr-DFO-NCS, radioactivity
Substance absorbs 1h and for 24 hours mainly in intestines, the organized radioactive uptake of institute is lower thereafter.When the radioactive uptake 1h of intestines most
Height reduces rapidly thereafter；The heart, lung, kidney radioactive uptake gradually decrease at any time；The radioactivity of liver, knee joint and backbone with
Time is in decreasing trend；The radioactive uptake of brain and muscle is close, very low.
20 G3 group pulmonary hypertension SD rat of table respectively organizes radioactive substance to absorb %ID/g-mean value (n=2)
21 G3 group pulmonary hypertension model rat of table is given89The %ID/g-mean of each animal tissue of 1h after Zr-DFO-NCS
Value
22 G3 group pulmonary hypertension model rat of table is given89The %ID/g- of Zr-DFO-NCS Hou24hGe animal tissue
Mean value
23 G3 group pulmonary hypertension model rat of table is given89The %ID/g- of each animal tissue of 72h after Zr-DFO-NCS
Mean value
24 G3 group pulmonary hypertension model rat of table is given89The %ID/g- of each animal tissue of 168h after Zr-DFO-NCS
Mean value
25 G3 group pulmonary hypertension model rat of table is given89The %ID/g- of each animal tissue of 336h after Zr-DFO-NCS
Mean value
7.3 in vitro tissue distribution results
Normal SD rats and pulmonary hypertension model SD rat are given893d and 14d, passes through CO after Zr-EPC2At inhalation
After dead animal, blood, the heart, liver, spleen, lung kidney, intestines, stomach, brain, pancreas, bone, muscle totally 12 tissue/body fluid are taken, γ is carried out and counts inspection
It surveys, calculates each tissue radioactive substance and absorb %ID/g, the results are shown in Table 26, table 27, table 28 and table 29.
By chart it is found that G4 group normal SD rats are given893d after Zr-EPC, radioactive substance mainly in lung, be secondly spleen,
Liver and blood, it is lower in remaining tissue.G5 group (i.e. G1 group) normal SD rat is given8914d after Zr-EPC, radioactive substance are main
It is secondly spleen and liver in lung, it is lower in remaining tissue.Compared with 3d, radioactive substance distribution is almost the same.
G6 group pulmonary hypertension SD rat is given893d after Zr-EPC, radioactive substance mainly in lung, be secondly spleen, blood and
Liver, it is lower in remaining tissue.G7 group (i.e. G2 group) Rats of Pulmonary Hypertension is given8914d after Zr-EPC, radioactive substance mainly exist
Secondly lung is spleen and liver, lower in remaining tissue.Compared with 3d, radioactive substance distribution is almost the same.
26 G4 group normal SD rats of table are given893d respectively organizes radioactive substance to absorb (%ID/g) after Zr-EPC
27 G5 group normal SD rats of table are given8914d respectively organizes radioactive substance to absorb (%ID/g) after Zr-EPC
28 G6 group pulmonary hypertension model SD rat of table is given893d respectively organizes radioactive substance to absorb (% after Zr-EPC
ID/g)
29 G7 group pulmonary hypertension model SD rat of table is given8914d respectively organizes radioactive substance to absorb (% after Zr-EPC
ID/g)
7.4PET/CT is scanned compared in vitro tissue γ count results
G4 group, G5 group, G6 group and each tissue γ of G7 group count the radioactive substance intake %ID/g obtained and same time point
The %ID/g that PET/CT scanning obtains is compared, and height intake organization type is almost the same.
7.5 pulmonary hypertension measurement results
Do not enter group preceding pulmonary hypertension information with after scanning of pulmonary hypertension SD rat administration and is shown in Table 30.As seen from table,
The pulmonary hypertension SD rat of non-administration pulmonary arterial pressure before formal experimental animal is administered is 41.38 ± 15.77mmHg, just
It is 33.69mmHg after the scanning of formula experimental animal.It gives89The pulmonary arterial pressure information of Zr-EPC treatment animal is shown in Table 31.It can by table
Know, pulmonary arterial pressure is 26.65 ± 7.65mmHg, lung after the pulmonary hypertension SD rat end of scan after the normal SD rats end of scan
Angiosthenia is 48.55 ± 11.87mmHg.
Table 30 does not enter the pulmonary hypertension information (mmHg) of group pulmonary hypertension SD rat
It is given after 31 end of scan of table89The pulmonary hypertension information (mmHg) of Zr-EPC animal
8. discussion of results
By the above test results show that prepared by the present invention89Zr-EPC has targeting to lung, while to pulmonary artery height
It presses the lung in animal pattern that there is higher targeting, further demonstrates that prepared by the present invention89Zr-EPC can be used for lung target
To research means.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. purposes of the endothelial progenitor cells in the imaging agent or drug for preparing Lung targeting.
2. a kind of endothelial progenitor cells of the image displaying function with Lung targeting, which is characterized in that contaminated using radionuclide, fluorescence
Material, quantum dot or molecular probe handle endothelial progenitor cells by way of marking or transfecting.
3. the endothelial progenitor cells of the image displaying function according to claim 2 with Lung targeting, which is characterized in that the radiation
Property nucleic is89Zr、45Ti、55Mn、59Fe、64Cu、94mTc、67Ga、71/72/74As、82Rb、89Y or124I。
4. the endothelial progenitor cells of the image displaying function according to claim 2 or 3 with Lung targeting, which is characterized in that described
Radionuclide is89Zr。
5. a kind of method for the endothelial progenitor cells for preparing the described in any item image displaying functions with Lung targeting of claim 2-4,
It is characterized in that, radionuclide, fluorescent dye, quantum dot or molecular probe are handled endothelium by way of marking or transfecting
Progenitor cells obtain the endothelial progenitor cells with image displaying function.
6. preparation method according to claim 5, which is characterized in that interior by being modified in progenitor endothelial cell surface
The modifier of skin progenitor cells surface modification can be by radioisotope labeling.Preferably, the modifier includes but unlimited
In label prothetic group, bifunctional chelating agent or complex compound.
7. a kind of radioisotope labeling with Lung targeting being prepared by preparation method described in claim 5 or 6
Endothelial progenitor cells.
8. the endothelial progenitor cells or claim 5 of the described in any item image displaying functions with Lung targeting of claim 2-4 or 6 institutes
The endothelial progenitor cells for the radioisotope labeling with Lung targeting that the preparation method stated is prepared are preparing pulmonary disease
Purposes in imaging agent or therapeutic agent.Preferably, the lung disease includes pulmonary hypertension disease.
9. a kind of PET/CT imaging agent, which is characterized in that including the described in any item imagings with Lung targeting of claim 2-4
The radionuclide with Lung targeting that the endothelial progenitor cells of function or preparation method described in claim 5 or 6 are prepared
The endothelial progenitor cells of label.
10. purposes of the PET/CT imaging agent as claimed in claim 9 in lung or pulmonary disease dynamic tracer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110917365A (en) * | 2019-12-20 | 2020-03-27 | 中国医学科学院北京协和医院 | CAR-T living body tracing method |
| CN113797360A (en) * | 2021-09-16 | 2021-12-17 | 南京艾尔普再生医学科技有限公司 | With radionuclides89Tracing method of Zr marked cardiac muscle cells in pharmacokinetics |
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2018
- 2018-09-27 CN CN201811131523.4A patent/CN109939247A/en active Pending
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| ADITYA BANSAL等: "Novel 89Zr cell labeling approach for PET-based cell trafficking studies", 《EJNMMI RESEARCH》 * |
| NORIKO SATO等: "89Zr-Oxine complex PET cell imaging in Monitoring cell-based Therapies", 《RADIOLOGY》 * |
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| CN110917365A (en) * | 2019-12-20 | 2020-03-27 | 中国医学科学院北京协和医院 | CAR-T living body tracing method |
| CN110917365B (en) * | 2019-12-20 | 2021-11-30 | 中国医学科学院北京协和医院 | CAR-T living body tracing method |
| CN113797360A (en) * | 2021-09-16 | 2021-12-17 | 南京艾尔普再生医学科技有限公司 | With radionuclides89Tracing method of Zr marked cardiac muscle cells in pharmacokinetics |
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