CN101818128B - Superparamagnetic iron oxide labeled human pancreas cancer cell strain as well as labeling method and application thereof - Google Patents
Superparamagnetic iron oxide labeled human pancreas cancer cell strain as well as labeling method and application thereof Download PDFInfo
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- CN101818128B CN101818128B CN201010138427XA CN201010138427A CN101818128B CN 101818128 B CN101818128 B CN 101818128B CN 201010138427X A CN201010138427X A CN 201010138427XA CN 201010138427 A CN201010138427 A CN 201010138427A CN 101818128 B CN101818128 B CN 101818128B
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Abstract
The invention relates to a superparamagnetic iron oxide labeled human pancreas cancer cell strain, wherein superparamagnetic iron oxide is contained in a cell. In a method for labeling the human pancreas cancer cell strain through the superparamagnetic iron oxide, polylysine is adopted as a transfection medium and is incubated together with the cell through a labeled liquid containing superparamagnetic iron oxide-polylysine, and the method comprises the following steps of: 1. preparation of the labeled liquid; 2. cultivation of the human pancreas cancer cell strain; and 3. labeling of the human pancreas cancer cell strain. The superparamagnetic iron oxide labeled human pancreas cancer cell strain can be applied to magnetic resonance imaging and applied to constructing a pancreas cancer animal model.
Description
Technical field
The invention belongs to the cell marking field, particularly the human pancreas cancer cell strain of marked by superparamagnetism iron oxide and marking method thereof and application.
Background technology
The cell iconography is rapid as branch's development of molecular imaging, and the cell marker that is used for nuclear magnetic resonance (being called for short MRI) research at present mainly contains: paramagnetism gadolinium chelate compound, SPIO nano particle and perfluorocarbon etc.Paramagnetism gadolinium ion contrast medium commonly used such as magnevist; This type of contrast medium itself can not permeates cell membranes get in the cell; It is cationic-liposome-mediated to use gadolinium ion contrast labeled cell to need, and need carry out very long common incubation time with cell, therefore the less mark that is used for cell.Perfluorocarbon be through
19The magnetic resonance signal of F changes and forms images, and can avoid
1The interference of H contrast medium water molecules signal in videograph process, thereby the video picture of the no background interference of realization, but owing to routine MRI is seldom used
19The nuclear magnetic resonance of F, this method are difficult to promote.SPIO (is called for short SPIO; In the specification sheets of present patent application, " SPIO " is SPIO) nano particle can produce instantaneous big magnetic field in magnetic field, and the magnetization of molecule is on every side produced disturb; That causes proton goes phase place fast; Externally-applied magnetic field is had hypersensitivity, and the simultaneous oxidation iron granules has excellent biological compatibility, thereby is widely used in the video picture of mr molecule with its special advantages.
Use the method for SPIO labeled cell to mainly contain mechanical process and cytophagy method.The cytophagy method is the most frequently used marking method, and mainly to be the endocytosis that utilizes cell be rolled into vesicles with macromole and particle swallows in the tenuigenin.The cytophagy of SPIO is mainly realized through three kinds of modes.A kind of mode is that SPIO is coated with by at virus coat or surface of liposome, gets in the cell through virus infected cell or liposome transfection.Another mode is to use monoclonal antibody, HIV virus reaction factor transcription factor class, peptide class, the polycation macromole transfection factor etc. that SPIO is carried out finishing, SPIO particle and cytolemma is pressed close to, thereby increased cytophagic probability.Another mode is through changing ferric oxide nanometer particle crystalline pan coating thing the avidity of ferric oxide particles and cell to be increased, thereby makes cell can engulf more ferric oxide particles.
At present, the modal SPIO of being is used for the mark of stem cell, but does not see the report that uses SPIO labelling human pancreatic cancer cell as yet.Yet carcinoma of the pancreas is a kind of malignant tumour of serious threat human health, the early diagnosis difficulty, and mortality ratio is high, presses for the effective ways of setting up early diagnosis, treatment and monitoring therapeuticing effect.Therefore with SPIO mark pancreas cancer cell strain; Set up the carcinoma of the pancreas animal model; And the early stage pancreatic cancer cell of observing of utilization MRI technology is in the intravital division growth situation of living Animal Models, to the incidence and development process of understanding carcinoma of the pancreas and diagnose in early days and open up new treatment approach, improve carcinoma of the pancreas result of treatment, change its prognosis, the survival time and the quality of life that improve patient be significant.
Summary of the invention
The human pancreas cancer cell strain that the purpose of this invention is to provide a kind of marked by superparamagnetism iron oxide; So that study human pancreatic cancer cell in external growing multiplication characteristic through nuclear magnetic resonance (MRI); And make up the carcinoma of the pancreas animal model through biological behaviours such as nuclear magnetic resonance (MRI) observation tumour cell growth in vivo, propagation and infiltrations, for the early diagnosis of carcinoma of the pancreas with open up new treatment approach and provide support; A purpose more of the present invention provides the method and optimization flag condition with the marked by superparamagnetism iron oxide human pancreas cancer cell strain.
The human pancreas cancer cell strain of marked by superparamagnetism iron oxide according to the invention, its cell contains SPIO.This kind human pancreas cancer cell strain still has the biological characteristics of the general population's pancreas cancer cell strain, and rate of propagation is fast, and its proterties, form remained unchanged after cell repeatedly went down to posterity, and can long-term frozen preserve.
Experiment shows, the human pancreatic cancer cell digestion of SPIO mark is centrifugal and resuspendedly be placed on centrifuge tube or EP manages expert magnetic resonance imaging, can realize nuclear magnetic resonance at cell levels, therefore can in nuclear magnetic resonance, use.The 3D-FIESTA sequence is the concentration change of responsive reaction iron ion more, can be used as the optimum scanning sequence that detects SPIO.
Experiment shows, the centrifugal back of human pancreatic cancer cell digestion of SPIO mark is resuspended with phosphate buffered saline buffer (PBS liquid), makes every ml cells suspension contain human pancreatic cancer cell number about 1 * 10
5The human pancreatic cancer cell suspension of said SPIO mark is pressed subcutaneous (injection once) of the dose ejection of every nude mice 0.3ml in the nude mice oxter; Continue under the SPF level condition to raise, injection back one week (seven days) promptly it is thus clear that subcutaneous have the knurl piece to form, therefore can be used in making up the carcinoma of the pancreas animal model.
Experiment shows that the MRI of constructed carcinoma of the pancreas animal model can know that the gathering of demonstration tumour central part iron ion is more, and a peripheral part tumor tissues contains iron ion but concentration obviously reduces.Point out intracellular iron ion along with the division of cell is distributed in the daughter cell, its concentration is reduced greatly, but the tumour cell division of tumour central authorities slowly or remain static.This provides the objective basis of a somatoscopy for pancreatic cancer growth Research on development and oncotherapy research.
Method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain according to the invention adopts poly-lysine (PLL) as transfection medium, hatches jointly through the marking fluid and the cell that contain SPIO, poly-lysine, and step is following:
(1) preparation of marking fluid
With SPIO nanoparticle, mass concentration 0.1% poly-lysine storing solution, the RPMI-1640 that contains NBCS is raw material; Said raw material mixed under normal pressure, room temperature promptly form marking fluid; In the said marking fluid; The volume ratio of SPIO nanoparticle and 0.1% poly-lysine storing solution is 3: 1, and the concentration of iron ion is at least 168 μ g/ml;
(2) cultivation of human pancreas cancer cell strain
With cell concn 0.5 * 10
6/ ml～1 * 10
6The human pancreatic cancer cell suspension of/ml is planted in culturing bottle, and adds the RPMI-1640 that contains NBCS, then said culturing bottle is put into CO
2In the incubator, cultivate in 37 ℃, behind 80% at the bottom of the human pancreatic cancer cell confluent culture bottle bottle, change liquid;
(3) mark of human pancreas cancer cell strain
The marking fluid of step (1) preparation is added step (2) cultivate and form the mark system in the nutrient solution that obtains, the add-on of said marking fluid reaches 4.2～84 μ g/ml with the concentration of iron ion in the mark system exceeds, and the mark system is put into CO
2In the incubator, cultivated 20～24 hours in 37 ℃, incubation time at the expiration after with the mark system from CO
2Take out in the incubator, abandon nutrient solution successively,, promptly obtain the human pancreas cancer cell strain of marked by superparamagnetism iron oxide with PBS liquid flushing,, the resuspended operation of RPMI-1640 that usefulness contains NBCS centrifugal with trysinization.
Nuclear magnetic resonance and cell bio-activity test experience show, the add-on of said marking fluid makes the biological activity of concentration human pancreatic cancer cell of formed SPIO mark when 21～42 μ g/ml of iron ion in the mark system good, and imaging effect is best.
In the aforesaid method, said SPIO nanoparticle is with Fe
3O
4And Fe
2O
3Nanocrystal (monocrystalline) is the magnetic core, and the outside has encapsulated the nanoparticle of carbon oxygen VISOSE, and the particle diameter of said SPIO nanoparticle is 45nm～60nm (comprising its encrusting substance).
The present invention has following beneficial effect:
1, the human pancreas cancer cell strain of SPIO mark according to the invention; Can realize the MR imaging at cell levels; Can be sensitive and show the change of mr (MR) signal more clearly, thereby can study human pancreatic cancer cell in external growing multiplication characteristic through nuclear magnetic resonance (MRI).
2, the constructed carcinoma of the pancreas animal model of the human pancreas cancer cell strain of marked by superparamagnetism iron oxide according to the invention; Can observe biological behaviours such as tumour cell growth in vivo, propagation and infiltration through nuclear magnetic resonance (MRI); Inquire into the incidence and development of carcinoma of the pancreas and invade profit mechanism; For pancreatic cancer growth Research on development and oncotherapy research provides the objective basis of somatoscopy, for the early diagnosis of carcinoma of the pancreas with open up new treatment approach and provide support.
3, the human pancreas cancer cell strain of SPIO mark according to the invention, it is strong to have a multiplication capacity, and tumor formation rate is high, becomes characteristics such as the knurl time is short, thereby can make up the carcinoma of the pancreas animal model easy, apace through the human pancreas cancer cell strain of this SPIO mark.
4, the invention provides the concentration range of iron ion in the mark system that the human pancreatic cancer cell biological activity is good, imaging effect is best of SPIO mark, the optimum scanning sequence that detects SPIO is provided.
5, the method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain according to the invention; Owing to adopt poly-lysine (PLL) as transfection medium; Marking fluid and cell through containing SPIO, poly-lysine are hatched jointly; Thereby can be efficiently ferric oxide particles be shifted in the cell, thereby the cell marking rate is increased greatly.
6, the method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain according to the invention is because employed SPIO nanoparticle is with Fe
3O
4And Fe
2O
3Encapsulated the nanoparticle (ferric oxide particles after VISOSE encapsulates is electroneutral) of carbon oxygen VISOSE for magnetic core, outside; Thereby the combination rate of SPIO nanoparticle and cytolemma increases greatly; The phagolysis of cell increases greatly, and VISOSE provides good chemical platform to be beneficial to further biochemical regulation and control simultaneously.
Description of drawings
Fig. 1 is the human pancreas cancer cell strain of a SPIO mark according to the invention light micrograph (observation of 100x object lens) after with prussian blue staining, and visible a large amount of iron particles are arranged in tenuigenin (photochrome iron particle is blueness).
Fig. 2 is the external mr T1-SE sequence MR image of the human pancreas cancer cell strain of SPIO mark according to the invention;
Fig. 3 is the external mr T2-FRFSE sequence MR image of the human pancreas cancer cell strain of SPIO mark according to the invention;
Fig. 4 is the external mr T1-SPGR sequence MR image of the human pancreas cancer cell strain of SPIO mark according to the invention;
Fig. 5 is the external mr 3D-FIESTA sequence MR image of the human pancreas cancer cell strain of SPIO mark according to the invention;
Fig. 6 is the nude mice plantation property carcinoma of the pancreas model photo with the human pancreatic cancer cell structure of SPIO mark according to the invention, and the nude mice in the photo is the situation of human pancreatic cancer cell suspension after one week of injection SPIO mark;
Fig. 7 is the nude mice plantation property carcinoma of the pancreas model photo with the human pancreatic cancer cell structure of SPIO mark according to the invention, and the nude mice in the photo is the situation of human pancreatic cancer cell suspension after two weeks of injection SPIO mark;
Fig. 8 is the nude mice plantation property carcinoma of the pancreas model photo with the human pancreatic cancer cell structure of SPIO mark according to the invention, and the nude mice in the photo is the situation of human pancreatic cancer cell suspension after three weeks of injection SPIO mark;
Fig. 9 is the nuclear magnetic resonance image of nude mice plantation property carcinoma of the pancreas model, and in each image, the thin arrow indication of white is the knurl piece, and the thick arrow indication of white is the dense district of gathering of iron ion at knurl piece center; Wherein: image A is the spin echo t1 weighted image of unmarked transplanted tumor in nude mice model, and plantation knurl piece such as is shown as at signal; Image B is the gtadient echo t1 weighted image of unmarked transplanted tumor in nude mice model, and plantation knurl piece such as is shown as at signal; Image C is the spin echo t2 weighted image of unmarked transplanted tumor in nude mice model, and plantation knurl piece is shown as high signal; Image D is the 3D-FIESTA image of unmarked transplanted tumor in nude mice model, and plantation knurl piece is shown as high slightly signal; Image E plants the knurl piece and is shown as low signal for inject the spin echo t1 weighted image of the transplanted tumor in nude mice model after a week with the human pancreatic cancer cell suspension of SPIO mark according to the invention; Image F plants the knurl piece and is shown as low signal for inject the gtadient echo t1 weighted image of the transplanted tumor in nude mice model after a week with the human pancreatic cancer cell suspension of SPIO mark according to the invention; Image G plants the knurl piece and is shown as low signal for inject the spin echo t2 weighted image after a week with the human pancreatic cancer cell suspension of SPIO mark according to the invention; Image H plants the knurl piece and is shown as low signal for inject the 3D-FIESTA image after a week with the human pancreatic cancer cell suspension of SPIO mark according to the invention; Image I is for injecting the spin echo t1 weighted image after two weeks with the human pancreatic cancer cell suspension of SPIO mark according to the invention, plantation knurl piece is shown as and waits signal, and it is thus clear that it is interior the dense district of gathering of iron ion of low signal; Image J is for injecting the gtadient echo t1 weighted image after two weeks with the human pancreatic cancer cell suspension of SPIO mark according to the invention, plantation knurl piece is shown as and waits signal, and it is thus clear that it is interior the dense district of gathering of iron ion of low signal; Image K is for injecting the spin echo t2 weighted image after two weeks with the human pancreatic cancer cell suspension of SPIO mark according to the invention, plantation knurl piece is shown as high signal, in it it is thus clear that the dense district of gathering of iron ion of low signal; Image L is for injecting the 3D-FIESTA image after two weeks with the human pancreatic cancer cell suspension of SPIO mark according to the invention, plantation knurl piece is shown as high slightly signal, in it it is thus clear that the dense district of gathering of iron ion of low signal.
Embodiment
Through embodiment marked by superparamagnetism iron oxide human pancreas cancer cell strain according to the invention and preparation method thereof and application are described further below.
The external nuclear magnetic resonance of human pancreatic cancer cell of embodiment 1:SPIO mark
1, material and facility
Human pancreas cancer PANC-1 cell strain: purchase in Sichuan University;
SPIO: trade(brand)name Resovist (SHU555A) is with Fe
3O
4And Fe
2O
3Nanocrystal is the magnetic core, and the outside has encapsulated carbon oxygen VISOSE, and particle diameter (comprising its encrusting substance) 45nm-60nm purchases the company in German SCHERING (spirit earlier);
The poly-lysine storing solution of mass concentration 0.1%: purchase biological ltd in Bo Ruike;
NBCS: purchase the Heng Shengma of unit biotechnology research institute in Beijing;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase the Heng Shengma of unit biotechnology research institute in Beijing
Phosphate buffered saline buffer (PBS): pH value 7.4, preparation voluntarily;
Six orifice plates: purchase company in CORNING
CO
2Incubator: purchase model HF90 in Healforce company
Vortex oscillation device: purchase in sky, the west of a city, Jintan City and finish the model XH-C of laboratory apparatus factory
Superconduction MR imager: model 1.5T, U.S. GE company produces.
2, experimental technique
(1) preparation of marking fluid
The poly-lysine storing solution 10 μ l of SPIO30 μ l (every microlitre contains iron ion 28 μ g), mass concentration 0.1%, contain the RPMI-1640 960 μ l of NBCS; In the said RPMI-1640 that contains NBCS, the volume ratio of NBCS and RPMI-1640 is 1: 4; The poly-lysine storing solution adding of SPIO and mass concentration 0.1% is contained in the RPMI-1640 of NBCS; Under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid through the sustained oscillation of vortex oscillation device; In the said marking fluid, iron particle concentration is 840ug/ml;
(2) preparation of external magnetic resonance imaging sample
With unlabelled human pancreas cancer PANC-1 cell strain with the trysinization of mass concentration 0.25% centrifugal and with the RPMI-1640 that contains NBCS (volume ratio of NBCS and RPMI-1640 is 1: 4) resuspended after, with about 1.0 * 10
6/ ml cell concn, every hole 2ml cell suspension inoculation are put into CO with said six orifice plates then in six orifice plates
2(humidity is saturated humidity to incubator, CO
2Volumetric concentration be 5%) in cultivate in 37 ℃, change liquid after 24 hours, when changing liquid the said RPMI-1640 that contains NBCS by 2000 μ l, 1990 μ l, 1980 μ l; 1950 μ l, 1900 μ l, 1800 μ l add respectively in six holes of six orifice plates, measure the marking fluid 0 μ l that in six holes of six orifice plates, adds step (1) preparation by said sequence respectively then; 10 μ l, 20 μ l, 50 μ l, 100 μ l; 200 μ l, (iron concentration is respectively 0 μ g/ml in each hole of six orifice plates, 4.2 μ g/ml, 8.4 μ g/ml to make total liquid measure in each hole of six orifice plates be 2000 μ l; 21 μ g/ml, 42 μ g/ml, 84 μ g/ml), after shaking up gently six orifice plates are put into CO
2(humidity is saturated humidity to incubator, CO
2Volumetric concentration be 5%) in hatched (cultivation) 24 hours in 37 ℃; Take out six orifice plates after (cultivation) time of hatching expires,, wash each hole repeatedly to remove free SPIO with PBS liquid with pipettor sucking-off nutrient solution; Then with the liquid sucking-off in each hole of six orifice plates; With adherent cell with the trysinization of mass concentration 0.25% centrifugal after, every hole 2ml RPMI-1640 re-suspended cell, the and respectively cell suspension in each hole being packed in six frozen pipes.6 frozen effective films that seal seal mixing on the vibrator.
(3) magnetic resonance imaging
6 frozen pipes in the step (2) are carried out T1-SE, T2-FRFSE, T1-SPGR, 3D-FIESTA sequence magnetic resonance imaging respectively.Image-forming condition: adopt eight passage head coils, the visual field (FOV) 12X12, bed thickness 2-3mm, interlamellar spacing 0-0.5mm, reconstruction matrix 256X192.
The image of T1-SE sequence magnetic resonance imaging is seen Fig. 2, and the image of T2-FRFSE sequence magnetic resonance imaging is seen Fig. 3, and the image of T1-SPGR sequence magnetic resonance imaging is seen Fig. 4; The image of 3D-FIESTA sequence magnetic resonance imaging is seen Fig. 5; Among each figure, iron concentration increases from left to right gradually in the PANC-1 cell, first figure among each figure; Iron concentration is 0 in the PANC-1 cell, is the blank image.
Can find out from Fig. 2, Fig. 3, Fig. 4, Fig. 5: with the human pancreatic cancer cell of SPIO mark; Increase along with iron concentration in the marking fluid; Magnetic resonance signal is reduction trend, and wherein iron concentration is 21ug/ml, 42ug/ml in the marking fluid, and the signal difference between the image of 84ug/ml and the blank image is obvious; Can be used as preferred label concentration, be beneficial to MRI and observe.In each MR sequence, the 3D-FIESTA sequence is the concentration change of responsive reaction iron ion more, can be used as the optimum scanning sequence that detects SPIO.
The growth activity of the human pancreatic cancer cell of embodiment 2:SPIO mark detects
1, material and facility
Human pancreas cancer PANC-1 cell strain: purchase in Sichuan University;
SPIO: trade(brand)name Resovist (SHU555A) is with Fe
3O
4And Fe
2O
3Nanocrystal is the magnetic core, and the outside has encapsulated carbon oxygen VISOSE, and particle diameter (comprising its encrusting substance) 45nm-60nm purchases the company in German SCHERING (spirit earlier);
The poly-lysine storing solution of mass concentration 0.1%: purchase biological ltd in Bo Ruike;
NBCS: purchase the Heng Shengma of unit biotechnology research institute in Beijing;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase the Heng Shengma of unit biotechnology research institute in Beijing;
Phosphate buffered saline buffer (PBS): pH value 7.4, preparation voluntarily;
96 orifice plates: purchase company in CORNING;
CO
2Incubator: purchase model HF90 in Healforce company;
Vortex oscillation device: purchase in sky, the west of a city, Jintan City and finish the model XH-C of laboratory apparatus factory
MTT liquid: purchase biological ltd in Bo Ruike
DMSO 99.8MIN. (DMSO): purchase biological ltd in Bo Ruike
ELIASA: Bio-Rad 680 types
2, experimental technique
(1) preparation of marking fluid
The poly-lysine storing solution 10 μ l of SPIO30 μ l (every microlitre contains iron ion 28 μ g), mass concentration 0.1%, contain the RPMI-1640 960 μ l of NBCS; In the said RPMI-1640 that contains NBCS, the volume ratio of NBCS and RPMI-1640 is 1: 4; The poly-lysine storing solution adding of SPIO and mass concentration 0.1% is contained in the RPMI-1640 of NBCS; Under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid through the sustained oscillation of vortex oscillation device; In the said marking fluid, iron particle concentration is 840ug/ml;
(2) preparation of cytoactive test sample and active the detection
With unlabelled human pancreas cancer PANC-1 cell strain with the trysinization of mass concentration 0.25% centrifugal and with the RPMI-1640 that contains NBCS (volume ratio of NBCS and RPMI-1640 is 1: 4) resuspended after, with the every hole 200ul (about 2.0 * 10 of cell suspension
4Cell count) be inoculated in 96 orifice plates, totally 24 holes place CO
2(humidity is saturated humidity to incubator, CO
2Volumetric concentration be 5%) hatch (cultivation) in 37 ℃ and took out and changed liquid in 24 hours; The RPMI-1640 that every hole adds said marking fluid and contains NBCS when changing liquid is 200ul altogether; Make that per 4 hole iron concentrations are respectively 0,4.2,8.4,21,42,84ug/ml, after shaking up gently 96 orifice plates are put into CO
2(humidity is saturated humidity to incubator, CO
2Volumetric concentration be 5%) in hatched (cultivation) 24 hours in 37 ℃; Outwell marking fluid and nutrient solution after hatching completion; And add RPMI-1640 that 200ul contains NBCS with every hole after the PBS flushing 3 times and put into incubator with 20ulMTT liquid and hatch, outwell the nutrient solution that contains MTT behind the 4h, every then hole adding DMSO 99.8MIN. (DMSO) 200ul; 96 orifice plates that vibrate gently under the room temperature adopt 570nm wavelength measurement every hole absorbance [D (570)] on ELIASA behind the 20min.Take off data sees the following form:
| Fe ionic concn (ug/ml) | 0 | 4.2 | 8.4 | 21 | 42 | 84 |
| D (570) value | 0.79 | 0.80 | 0.80 | 0.82 | 0.76 | 0.63 |
Visible from last table, iron concentration is when 84ug/ml in the mark system, and the growth activity of the human pancreatic cancer cell PANC-1 of SPIO mark is suppressed.
Embodiment 3: with SPIO labelling human pancreas cancer cell strain
1, material and facility
Human pancreas cancer PANC-1 cell strain: purchase in Sichuan University;
SPIO: trade(brand)name Resovist (SHU555A) is with Fe
3O
4And Fe
2O
3Nanocrystal is the magnetic core, and the outside has encapsulated carbon oxygen VISOSE, and particle diameter (comprising its encrusting substance) 45nm-60nm purchases the company in German SCHERING (spirit earlier);
The poly-lysine storing solution of mass concentration 0.1%: purchase biological ltd in Bo Ruike;
NBCS: purchase the Heng Shengma of unit biotechnology research institute in Beijing;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase the Heng Shengma of unit biotechnology research institute in Beijing;
Phosphate buffered saline buffer (PBS): pH value 7.4, preparation voluntarily;
CO
2Incubator: purchase model HF90 in Healforce company;
Vortex oscillation device: purchase in sky, the west of a city, Jintan City and finish the model XH-C of laboratory apparatus factory.
2, experimental technique
(1) preparation of marking fluid
The poly-lysine storing solution 10 μ l of SPIO30 μ l (every microlitre contains iron ion 28 μ g), mass concentration 0.1%, contain the RPMI-1640 960 μ l of NBCS; In the said RPMI-1640 that contains NBCS, the volume ratio of NBCS and RPMI-1640 is 1: 4; The poly-lysine storing solution adding of SPIO and mass concentration 0.1% is contained in the RPMI-1640 of NBCS; Under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid through the sustained oscillation of vortex oscillation device; In the said marking fluid, iron particle concentration is 840ug/ml;
(2) cultivation of human pancreas cancer cell strain
With about 1 * 10
6/ ml human pancreas cancer PANC-1 cell suspension is planted in culturing bottle and is added the RPMI-1640 that contains NBCS, then said culturing bottle is put into CO
2In the incubator, cultivate in 37 ℃, behind 80% at the bottom of the human pancreatic cancer cell confluent culture bottle bottle, change liquid, in the said RPMI-1640 that contains NBCS, the volume ratio of NBCS and RPMI-1640 is 1: 4; Said CO
2Humidity in the incubator is saturated humidity, CO
2Volumetric concentration be 5%.
(3) mark of human pancreas cancer cell strain
The marking fluid of step (1) preparation is added step (2) cultivate and form the mark system in the nutrient solution that contains human pancreatic cancer cell that obtains, the add-on of said marking fluid reaches 42 μ g/ml with the concentration of iron ion in the marking fluid exceeds, and the mark system is put into CO
2In the incubator, cultivated 20 hours in 37 ℃, incubation time at the expiration after with the mark system from CO
2Take out in the incubator, abandon nutrient solution, wash 3 times repeatedly removing free SPIO with PBS liquid, centrifugal with the trysinization of mass concentration 0.25% then, use RPMI-1640 resuspended again, promptly obtain the human pancreas cancer cell strain of marked by superparamagnetism iron oxide.
With the human pancreas cancer cell strain of prepared SPIO mark with prussian blue staining after, its light micrograph (observation of 100x object lens) is seen Fig. 1, and is visible from Fig. 1, a large amount of iron particles are arranged in tenuigenin (photochrome iron particle is blueness).
Embodiment 4: make up nude mice plantation property tumor model
1, laboratory animal and material
Laboratory animal: nude mice, male, in age in 4-5 week, purchase animal center, feeding environment: SPF level Animal Lab. (Chuanbei Medical College is self-built) in Sichuan University;
The human pancreas cancer PANC-1 cell strain of the SPIO mark of embodiment 3 preparations;
Phosphate buffered saline buffer (PBS): pH value 7.4, preparation voluntarily.
2, experimental technique
30 of nude mices (are used the electronic scales weighing) about every original body weight 20g, and it is divided into experimental group and control group, and experimental group is 22 nude mices, and control group is 8 nude mices.
The human pancreas cancer PANC-1 cell strain of the SPIO mark of embodiment 3 preparation is resuspended with phosphate buffered saline buffer (PBS), make every ml cells suspension contain PANC-1 cell count about 1 * 10
5, the human pancreas cancer PANC-1 cell suspension of a SPIO mark of every nude mice injection of experimental group, ID is 0.3ml.
Unmarked human pancreas cancer PANC-1 cell strain is resuspended with phosphate buffered saline buffer (PBS), make every ml cells suspension contain PANC-1 cell count about 1 * 10
5, every once unmarked human pancreas cancer PANC-1 cell suspension of nude mice injection of control group, ID is 0.3ml.
With the alcohol disinfecting of the nude mice of experimental group left side underarm region skin with mass concentration 75%; Draw the human pancreas cancer PANC-1 cell suspension 0.3ml of SPIO mark with the 1ml asepsis injector; In nude mice left side oxter subcutaneous injection (patient wears sterile gloves), normally raise behind the injection operation.
With the alcohol disinfecting of the nude mice of control group left side underarm region skin with mass concentration 75%; Draw unmarked human pancreas cancer PANC-1 cell suspension 0.3ml with the 1ml asepsis injector; In nude mice left side oxter subcutaneous injection (patient wears sterile gloves), normally raise behind the injection operation.
Through observing, the promptly visible nude mice in human pancreas cancer PANC-1 cell suspension one all backs of injection SPIO mark is subcutaneous to have the knurl piece to form (see figure 6), and tumor growth rapid (seeing Fig. 7, Fig. 8).Also have the knurl piece to form after injecting unmarked one week of human pancreas cancer PANC-1 cell suspension, and tumor growth is rapid it is thus clear that nude mice is subcutaneous.
Embodiment 5: the nuclear magnetic resonance of tumor bearing nude mice
1, laboratory animal and material, equipment
The nude mice plantation property tumor model that laboratory animal: embodiment 4 makes up;
Chloral Hydrate: available from Cologne, Hunan medicine company limited-liability company;
Superconduction MR imager: model 1.5T, U.S. GE company produces;
Experimentation on animals coil: the special-purpose MRI coil of animalcule.
2, experimental technique
(1) patient is with sterile gloves, draws anesthesia medicament Chloral Hydrate 0.03ml (dosage of a model mouse) with the 1ml asepsis injector, to the capable abdominal injection of model mouse of experimental group and control group.
(2) anaesthetic effect is good model mouse is put into the special-purpose experimentation on animals coil of mouse, gets dorsal position, and head is advanced.
(3) with superconduction MR imager model mouse is carried out magnetic resonance imaging, the visual field (FOV) 6 * 6cm, bed thickness 2-3mm, interlamellar spacing 0-0.5mm, reconstruction matrix 256 * 192 or 256 * 160, imaging sequence are T1-SE, T1-SPGR, T2-FRFSE, 3D-FIESTA.
(4) behind the end of scan, MRI is reached workstation.
The MRI that obtains is seen Fig. 9.As can beappreciated from fig. 9: the knurl piece in human pancreatic cancer cell plantation 1 week of growth behind the SPIO mark all is low signal in each sequence image.Continued growth along with tumour; The tumour central area still is a low signal in each sequence image; And the lump peripheral portion signal such as is in T1WI and 3D-FIESTA sequence, be high slightly signal in the T2WI sequence, but its signal is still low than the control group signal; Show that the gathering of tumour central area iron ion is more, a peripheral part tumor tissues contains iron ion but concentration obviously reduces.Comprehensive each sequence image prompting; Along with growth of tumor, intracellular iron ion also can be distributed in the daughter cell along with the division of cell, and its concentration is reduced greatly; But the still visible more iron ion of tumour central area is assembled, and prompting the tumour cell division here slowly or remain static.Therefore can be pancreatic cancer growth Research on development and oncotherapy research provides the objective basis of somatoscopy.
Claims (3)
1. the human pancreas cancer cell strain of a marked by superparamagnetism iron oxide, said human pancreatic cancer cell contains SPIO, it is characterized in that marking method is following:
(1) preparation of marking fluid
With SPIO nanoparticle, mass concentration 0.1% poly-lysine storing solution, the RPMI-1640 that contains NBCS is raw material; Said raw material mixed under normal pressure, room temperature promptly form marking fluid; In the said marking fluid; The volume ratio of SPIO nanoparticle and 0.1% poly-lysine storing solution is 3: 1, and the concentration of iron ion is at least 168 μ g/ml;
Said SPIO nanoparticle is with Fe
3O
4And Fe
2O
3Nanocrystal is the magnetic core, and the outside has encapsulated the nanoparticle of carbon oxygen VISOSE, and particle diameter is 45nm～60nm;
(2) cultivation of human pancreas cancer cell strain
With cell concn 0.5 * 10
6/ ml～1 * 10
6The human pancreatic cancer cell suspension of/ml is planted in culturing bottle, and adds the RPMI-1640 that contains NBCS, then said culturing bottle is put into CO
2In the incubator, cultivate in 37 ℃, behind 80% at the bottom of the human pancreatic cancer cell confluent culture bottle bottle, change liquid;
(3) mark of human pancreas cancer cell strain
The marking fluid of step (1) preparation is added step (2) cultivate and form the mark system in the nutrient solution that obtains, the add-on of said marking fluid reaches 21 μ g/ml～42 μ g/ml with the concentration of iron ion in the mark system exceeds, and the mark system is put into CO
2In the incubator, cultivated 20 hours～24 hours in 37 ℃, incubation time at the expiration after with the mark system from CO
2Take out in the incubator, abandon nutrient solution successively,, promptly obtain the human pancreas cancer cell strain of marked by superparamagnetism iron oxide with PBS liquid flushing,, the resuspended operation of RPMI-1640 that usefulness contains NBCS centrifugal with trysinization.
2. the application of the human pancreas cancer cell strain of the said marked by superparamagnetism iron oxide of claim 1 in making up the carcinoma of the pancreas animal model.
3. the application of the human pancreas cancer cell strain of the said marked by superparamagnetism iron oxide of claim 1 in nuclear magnetic resonance comprises the nuclear magnetic resonance of cell levels and the nuclear magnetic resonance of animal model.
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| CN101130093A (en) * | 2007-08-01 | 2008-02-27 | 重庆医科大学 | Antisense oligonucleotide probe contrast agent marked by superparamagnetism iron oxide and production of the same |
| CN101444630A (en) * | 2008-12-31 | 2009-06-03 | 中山大学 | Method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function |
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| CN101130093A (en) * | 2007-08-01 | 2008-02-27 | 重庆医科大学 | Antisense oligonucleotide probe contrast agent marked by superparamagnetism iron oxide and production of the same |
| CN101444630A (en) * | 2008-12-31 | 2009-06-03 | 中山大学 | Method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function |
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