CN101818128A - 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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- CN101818128A CN101818128A CN201010138427A CN201010138427A CN101818128A CN 101818128 A CN101818128 A CN 101818128A CN 201010138427 A CN201010138427 A CN 201010138427A CN 201010138427 A CN201010138427 A CN 201010138427A CN 101818128 A CN101818128 A CN 101818128A
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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, Superparamagnetic Iron Oxide 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 enter 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 by
19The magnetic resonance signal of F changes and imaging can be avoided
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.Superparamagnetic Iron Oxide (is called for short SPIO, in the specification sheets of present patent application, " SPIO " is Superparamagnetic Iron Oxide) nano particle can produce instantaneous big magnetic field in magnetic field, the magnetization of molecule is on every side produced interference, 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 by three kinds of modes.A kind of mode is that SPIO is coated with by at virus coat or surface of liposome, enters in the cell by 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 by 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 the mortality ratio height 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 utilization MRI technology observes pancreatic cancer cell in early days in the intravital division growth situation of living Animal Models, to the generation evolution 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 by nuclear magnetic resonance (MRI), and make up the carcinoma of the pancreas animal model by 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 further object of the present invention provides with the method for marked by superparamagnetism iron oxide human pancreas cancer cell strain and optimizes flag condition.
The human pancreas cancer cell strain of marked by superparamagnetism iron oxide of the present invention, its cell contains Superparamagnetic Iron Oxide.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 use in nuclear magnetic resonance.The 3D-FIESTA sequence can be more responsive the change in concentration of reaction iron ion, can be used as the optimum scanning sequence that detects SPIO.
Experiment shows, uses phosphate buffered saline buffer (PBS liquid) resuspended the centrifugal back of human pancreatic cancer cell digestion of SPIO mark, makes every ml cells suspension contain human pancreatic cancer cell number about 1 * 10
5The human pancreatic cancer cell suspension of described SPIO mark is injected in subcutaneous (injection once) of nude mice oxter by the dosage of every nude mice 0.3ml, continue under the SPF level condition to raise, injection back one week (seven days) is promptly as seen subcutaneous to have the knurl piece to form, and therefore can use in making up the carcinoma of the pancreas animal model.
Experiment shows that the magnetic resonance image (MRI) of constructed carcinoma of the pancreas animal model can know that the gathering of demonstration tumour central part iron ion is more, and 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 of the present invention adopts poly-lysine (PLL) as transfection medium, hatches jointly by the marking fluid and the cell that contain Superparamagnetic Iron Oxide, poly-lysine, and step is as follows:
(1) preparation of marking fluid
With Superparamagnetic Iron Oxide nanoparticle, mass concentration 0.1% poly-lysine storing solution, the RPMI-1640 that contains new-born calf serum is raw material, described raw material mixed under normal pressure, room temperature promptly form marking fluid, in the described marking fluid, the volume ratio of Superparamagnetic Iron Oxide 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 new-born calf serum, then described 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 described 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, with PBS liquid flushing,, RPMI-1640 resuspended operation that usefulness contain new-born calf serum centrifugal, promptly obtain the human pancreas cancer cell strain of marked by superparamagnetism iron oxide with trysinization.
Nuclear magnetic resonance and cell bio-activity test experience show, the add-on of described 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 the best.
In the aforesaid method, described Superparamagnetic Iron Oxide nanoparticle is with Fe
3O
4And Fe
2O
3Nanocrystal (monocrystalline) is the magnetic core, and outer bread is by the nanoparticle of carbon oxygen dextran, and the particle diameter of described Superparamagnetic Iron Oxide 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 of the present 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 by 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 of the present invention, can observe biological behaviours such as tumour cell growth in vivo, propagation and infiltration by nuclear magnetic resonance (MRI), inquire into the generation 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 of the present invention, it is strong to have a multiplication capacity, and the tumor formation rate height becomes characteristics such as the knurl time is short, thereby can make up the carcinoma of the pancreas animal model easy, apace by 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 of the present invention, owing to adopt poly-lysine (PLL) as transfection medium, hatch jointly by the marking fluid and the cell that contain Superparamagnetic Iron Oxide, poly-lysine, thereby can 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 of the present invention is because employed SPIO nanoparticle is with Fe
3O
4And Fe
2O
3For magnetic core, outer bread by the nanoparticle of carbon oxygen dextran (the dextran bag by after ferric oxide particles be electric neutrality), thereby the combination rate of SPIO nanoparticle and cytolemma increases greatly, the phagolysis of cell increases greatly, and dextran 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 of the present 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 of the present invention;
Fig. 3 is the external mr T2-FRFSE sequence MR image of the human pancreas cancer cell strain of SPIO mark of the present invention;
Fig. 4 is the external mr T1-SPGR sequence MR image of the human pancreas cancer cell strain of SPIO mark of the present invention;
Fig. 5 is the external mr 3D-FIESTA sequence MR image of the human pancreas cancer cell strain of SPIO mark of the present 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 of the present 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 of the present 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 of the present 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 poly-district 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 of the present 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 of the present 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 of the present 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 of the present 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 of the present invention, plantation knurl piece is shown as and waits signal, its interior as seen dense poly-district 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 of the present invention, plantation knurl piece is shown as and waits signal, its interior as seen dense poly-district 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 of the present invention, plantation knurl piece is shown as high signal, visible dense poly-district of iron ion of low signal in it; Image L is for injecting the 3D-FIESTA image after two weeks with the human pancreatic cancer cell suspension of SPIO mark of the present invention, plantation knurl piece is shown as high slightly signal, visible dense poly-district of iron ion of low signal in it.
Embodiment
Below by embodiment marked by superparamagnetism iron oxide human pancreas cancer cell strain of the present invention and preparation method thereof and application are described further.
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 outer bread is by carbon oxygen dextran, 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 company limited in Bo Ruike;
New-born calf serum: purchase in Beijing Heng Shengma of unit biotechnology research institute;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase in Beijing Heng Shengma of unit biotechnology research institute
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 new-born calf serum, in the described RPMI-1640 that contains new-born calf serum, the volume ratio of new-born calf serum 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 new-born calf serum, under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid by the sustained oscillation of vortex oscillation device, in the described 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 new-born calf serum (volume ratio of new-born calf serum 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 described 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, the described RPMI-1640 of new-born calf serum that contains is by 2000 μ l when changing liquid, 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 to make total liquid measure in each hole of six orifice plates be 2000 μ l, 4.2 μ g/ml, 8.4 μ g/ml, 21 μ g/ml, 42 μ g/ml, 84 μ g/ml), six orifice plates are put into CO after shaking up gently
2(humidity is saturated humidity to incubator, CO
2Volumetric concentration be 5%) in hatched (cultivation) 24 hours in 37 ℃, after expiring, takes out (cultivation) time of hatching six orifice plates, with pipettor sucking-off nutrient solution, wash each hole repeatedly to remove free SPIO with PBS liquid, 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, and in six the frozen pipes of respectively cell suspension in each hole being packed into.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, the image of T2-FRFSE sequence magnetic resonance imaging is seen Fig. 3, 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.
From Fig. 2, Fig. 3, Fig. 4, Fig. 5 as can be seen: with the human pancreatic cancer cell of SPIO mark, increase along with iron concentration in the marking fluid, magnetic resonance signal is reduction trend, wherein iron concentration is 21ug/ml, 42ug/ml in the marking fluid, 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 change in concentration of the reaction iron ion that the 3D-FIESTA sequence can be more responsive 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 outer bread is by carbon oxygen dextran, 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 company limited in Bo Ruike;
New-born calf serum: purchase in Beijing Heng Shengma of unit biotechnology research institute;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase in Beijing Heng Shengma of unit biotechnology research institute;
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 company limited in Bo Ruike
Dimethyl sulfoxide (DMSO) (DMSO): purchase biological company limited in Bo Ruike
Microplate reader: 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 new-born calf serum, in the described RPMI-1640 that contains new-born calf serum, the volume ratio of new-born calf serum 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 new-born calf serum, under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid by the sustained oscillation of vortex oscillation device, in the described 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 new-born calf serum (volume ratio of new-born calf serum 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 described marking fluid and contains new-born calf serum 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 ℃, hatch and outwell marking fluid and nutrient solution after finishing, and add 200ul with every hole after the PBS flushing 3 times and contain the RPMI-1640 of new-born calf serum and 20ulMTT liquid and put into incubator and hatch, outwell the nutrient solution that contains MTT behind the 4h, every then hole adds dimethyl sulfoxide (DMSO) (DMSO) 200ul, 96 orifice plates that vibrate gently under the room temperature adopt 570nm wavelength measurement every hole absorbance [D (570)] on microplate reader 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 |
From last table as seen, 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 outer bread is by carbon oxygen dextran, 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 company limited in Bo Ruike;
New-born calf serum: purchase in Beijing Heng Shengma of unit biotechnology research institute;
RPMI-1640: purchase company in GIBICO;
Pancreatin: purchase in Beijing Heng Shengma of unit biotechnology research institute;
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 new-born calf serum, in the described RPMI-1640 that contains new-born calf serum, the volume ratio of new-born calf serum 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 new-born calf serum, under normal pressure, room temperature (25 ℃), they are mixed (about 60 minutes) and promptly form marking fluid by the sustained oscillation of vortex oscillation device, in the described 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 new-born calf serum, then described culturing bottle is put into CO
2In the incubator, cultivate in 37 ℃, change liquid behind 80% at the bottom of the human pancreatic cancer cell confluent culture bottle bottle, in the described RPMI-1640 that contains new-born calf serum, the volume ratio of new-born calf serum and RPMI-1640 is 1: 4; Described 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 described 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, as can be seen 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, injected dose 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, injected dose is 0.3ml.
The nude mice left side underarm region skin of experimental group is used the alcohol disinfecting of 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.
The nude mice left side underarm region skin of control group is used the alcohol disinfecting of 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, being that visible nude mice is subcutaneous after one week of human pancreas cancer PANC-1 cell suspension of injection SPIO mark has the knurl piece to form (see figure 6), and tumor growth rapid (seeing Fig. 7, Fig. 8).Inject that also visible nude mice is subcutaneous after unmarked one week of human pancreas cancer PANC-1 cell suspension has the knurl piece to form, and tumor growth is rapid.
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 with medicine Chloral Hydrate 0.03ml (dosage of a model mouse), to the capable abdominal injection of model mouse of experimental group and control group with the 1ml asepsis injector.
(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, magnetic resonance image (MRI) is reached workstation.
The magnetic resonance image (MRI) that obtains is seen Fig. 9.As can be seen from Figure 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, 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 tumour cell division herein slowly or remain static.Therefore can be pancreatic cancer growth Research on development and oncotherapy research provides the objective basis of somatoscopy.
Claims (10)
1. the human pancreas cancer cell strain of a marked by superparamagnetism iron oxide is characterized in that described human pancreatic cancer cell contains Superparamagnetic Iron Oxide.
2. method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain is characterized in that step is as follows:
(1) preparation of marking fluid
With Superparamagnetic Iron Oxide nanoparticle, mass concentration 0.1% poly-lysine storing solution, the RPMI-1640 that contains new-born calf serum is raw material, described raw material mixed under normal pressure, room temperature promptly form marking fluid, in the described marking fluid, the volume ratio of Superparamagnetic Iron Oxide 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 new-born calf serum, then described 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 described marking fluid reaches 4.2 μ g/ml～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 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, with PBS liquid flushing,, RPMI-1640 resuspended operation that usefulness contain new-born calf serum centrifugal, promptly obtain the human pancreas cancer cell strain of marked by superparamagnetism iron oxide with trysinization.
3. the method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain according to claim 2, when it is characterized in that the mark of human pancreas cancer cell strain, the add-on of described marking fluid reaches 21 μ g/ml～42 μ g/ml with the concentration of iron ion in the mark system and exceeds.
4. according to claim 2 or 3 described methods, it is characterized in that described Superparamagnetic Iron Oxide nanoparticle is with Fe with the marked by superparamagnetism iron oxide human pancreas cancer cell strain
3O
4And Fe
2O
3Nanocrystal is the magnetic core, and outer bread is by the nanoparticle of carbon oxygen dextran.
5. according to claim 2 or 3 described methods with the marked by superparamagnetism iron oxide human pancreas cancer cell strain, the particle diameter that it is characterized in that described Superparamagnetic Iron Oxide nanoparticle is 45nm～60nm.
6. the method with the marked by superparamagnetism iron oxide human pancreas cancer cell strain according to claim 4, the particle diameter that it is characterized in that described Superparamagnetic Iron Oxide nanoparticle is 45nm～60nm.
7. the application of the human pancreas cancer cell strain of the described marked by superparamagnetism iron oxide of claim 1 in making up the carcinoma of the pancreas animal model.
8. application according to claim 7 is characterized in that the human pancreatic cancer cell suspension of marked by superparamagnetism iron oxide is injected in the subcutaneous of nude mice oxter, continues under the SPF level condition to raise, and the injection back promptly had the knurl piece to form in one week.
9. the application of the human pancreas cancer cell strain of the described marked by superparamagnetism iron oxide of claim 1 in nuclear magnetic resonance.
10. application according to claim 9 is characterized in that comprising the nuclear magnetic resonance of cell levels and the nuclear magnetic resonance of animal model.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (6)
| Title |
|---|
| 《Cancer Biomarks》 20090504 Tina Islam et al. Current state and future applications of active targeting in malignancies using superparamagnetic iron oxide nanoparticles 99-107 1-10 第5卷, 第2期 2 * |
| 《Nanomedicine: Nanotechnology, Biology, and Medicine》 20081231 Chengli Yang et al. Nanoimmunoliposome delivery of superparamagnetic iron oxide markedly enhances targeting and uptake in human cancer cells in vitro and in vivo 318-329 1-10 第4卷, 第4期 2 * |
| 《NMR In Biomedicine》 20041130 Jeff W. M. Bulte et al. Iron oxide MR contrast agents for molecular and cellular imaging 484-489 1-10 第17卷, 第7期 2 * |
| 《国外医学临床放射学分册》 20070731 杨华等 超顺磁性氧化铁微粒在分子影像学中的研究现状 221-223，239 1-10 第30卷, 第4期 2 * |
| 《实用医学杂志》 20091231 陈国东等 超顺磁性氧化铁标记对大鼠骨髓间充质干细胞生物学特性的影响 3566-3568 1-10 第25卷, 第21期 2 * |
| 《第三军医大学学报》 20080930 杨华等 SPIO标记大鼠骨髓基质干细胞及体外磁共振成像研究 1626-1629 1-10 第30卷, 第17期 2 * |
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| CN105055447A (en) * | 2015-06-23 | 2015-11-18 | 中南大学湘雅二医院 | Applications of iron oxide nanoparticles in preparation of drug for improving erlotinib resistance |
| CN105055447B (en) * | 2015-06-23 | 2018-03-13 | 中南大学湘雅二医院 | Ferric oxide nanometer particle is used to prepare the application for improving Tarceva drug resistance medicine |
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