CN102242081B - Super paramagnetic iron oxide particle marked cell for magnetic-resonance tracing in vivo - Google Patents
Super paramagnetic iron oxide particle marked cell for magnetic-resonance tracing in vivo Download PDFInfo
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Abstract
The invention relates to a super paramagnetic iron oxide (SPIO) marked cell for magnetic-resonance tracing in vivo. The cell can produce a strong magnetic resonance signal and be traced for a long time. A cell for the preparation of the SPIO marked cell utilized for magnetic-resonance tracing in vivo is a cell with a high ferritin expression level. The SPIO marked cell is prepared from a SPIO and a cell with a high ferritin expression level by co-culture, wherein a concentration of the SPIO as a marker is in a range of 30 to 60 micrograms per milliliter. The cell with a high ferritin expression level is a cell transfected by ferritin gene plasmids. The preferred ferritin gene plasmids are ferritin heavy-chain whole gene plasmids. Research results confirm that through highly expressed ferritin, ferrum degraded by a SPIO is regarded as an additional ferrum source, is absorbed, and is stored in a cell; ferritin can produce an effect with the SPIO as a cell marker thus a tracing time of the SPIO in a living cell is prolonged and degraded SPIO can improve a signal intensity of a ferritin reporter gene; and through a combined use of ferritin and the SPIO, a tracing time of a living marked cell and an intensity of a magnetic-resonance signal are improved.
Description
Technical field
The present invention relates to a kind of cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking.
Background technology
The treatment of the various kinds of cell such as stem cell, progenitor cell is more and more extensive in clinical application.The treatment cell of non-invasive vivo tracking inoculation is monitored one of key that becomes the treatment success for a long time to the survival of inoculating cell, differentiation with in the histoorgan location of damage.Mr imaging technique (MRI) has height time and spatial resolution, and the information of body dissection and function aspects can be provided simultaneously, has become the important means of vivo tracking cell at present.Super-paramagnetism nano iron particle (hereinafter to be referred as SPIO) mark is the main method of mr vivo tracking cell, is widely used in basic scientific research and preclinical study.But the cell of SPIO mark is along with the metabolic degradation of cell fission and iron particle, and magnetic resonance signal weakens gradually to disappearance, for a long time spike treatment cell.Ferritin extensively is present in the biomass cells, participates in the body iron metabolism, has the function of storage iron.In recent years, relevant ferritin gene receives much attention as the research of mr reporter gene.Its principle is: manually the ferritin gene transfered cell is made cell high expression level ferritin, the ferritin of high expression level is caught the outer endogenous iron of body cell, forms the crystallization iron with superparamagnetism in born of the same parents, produces contrast under mr.But, the magnetic resonance signal that ferritin produces very a little less than, be difficult to be applied to clinical.
Summary of the invention
The invention provides a kind of cell of the SPIO mark for the mr vivo tracking, magnetic resonance signal is strong, and for a long time spike.
The cell of described SPIO mark for the mr vivo tracking, described cell is high expression level ferritin cell.
The cell of described SPIO mark is obtained by SPIO and high expression level ferritin cell co-culture, and the label concentration of SPIO is 30～60 μ g/mL.
Described high expression level ferritin cell is ferritin gene plasmid transfection cell.Described ferritin gene plasmid is preferably the full gene plasmid of ferritin heavy chain.
The construction process of the full gene plasmid of described ferritin heavy chain is: mRNA is carried out reverse transcription, polymerase chain reaction, amplify the full gene segment of ferritin heavy chain, the full gene of ferritin heavy chain is inserted in the carrier, make up the full gene plasmid of ferritin heavy chain.As preferred version, the construction process of the full gene plasmid of described ferritin heavy chain is: mRNA is reversed into cDNA, according to the ferritin heavy chain gene order, application contains the polymerase chain reaction primer of restriction endonuclease sites, amplify the full gene fragment of ferritin heavy chain, the application limitations restriction endonuclease digests the full gene fragment of ferritin heavy chain and carrier pcDNA3.1, and digestion product is carried out behind the purifying the full gene insertion vector pcDNA3.1 of ferritin being formed the full gene plasmid of ferritin heavy chain.Preferred scheme is, described carrier is pcDNA3.1, the construction process of the full gene plasmid of described ferritin heavy chain is: mRNA is reversed into cDNA, according to the ferritin heavy chain gene order, application contains the polymerase chain reaction primer of NotI and BamHI restriction endonuclease sites, amplify the full gene fragment of ferritin heavy chain, use NotI and BamHI restriction enzyme the full gene fragment of ferritin heavy chain and carrier pcDNA3.1 are digested, digestion product is carried out purifying by the T4 ligase enzyme the full gene insertion vector pcDNA3.1 of ferritin is formed the full gene plasmid of ferritin heavy chain.For the structure of the full gene plasmid of people mouse ferritin heavy chain, preferred polymerase chain reaction primer sequence is as follows:
The upstream primer sequence: 5 '-ataagaatgcggccgctaaactataccatgaccaccgcgtctccctc-3 ';
The downstream primer sequence: 5 '-cgcggatccgcgttagctctcatcaccgtgtcccag-3 '.
Among the present invention, described mr vivo tracking cell is C 6 Cell of Glioma, and the method that obtains ferritin gene plasmid transfection mr vivo tracking cell is:
(1) C 6 Cell of Glioma is inoculated in the substratum, in the cell cultures hole, cultivates, make C 6 Cell of Glioma reach 90% degrees of fusion;
(2) transfection: the ferritin gene plasmid mixes with transfection agents, after the incubated at room, add described cell cultures hole, gently the mixing overnight incubation;
(3) peptic cell carries out going down to posterity at 1: 10 behind the transfection 24hr, at last screening.
Described transfection agents is the Lipofectamine 2000 that Invitrogen company produces.
Confirm that after deliberation the ferritin of high expression level can absorb the iron of SPIO degraded and be stored in the cell as extra source of iron.The magnetic resonance imaging strength of signal of different time points conforms to iron atom concentration results in the cell after the cell inoculation.Ferritin can with the SPIO effect of cell marking, prolong time of SPIO vivo tracking cell; The SPIO of degraded can strengthen the strength of signal of ferritin reporter gene simultaneously.Both unite use, can strengthen vivo tracking time and magnetic resonance signal intensity to labeled cell.This result of study has certain using value for carrying out clinical cell therapy and gene therapy to location, the in real time detection of survival and growth situation for the treatment of cell.
Description of drawings
Fig. 1. the blue iron dyeing of Immunohistochemical dyeing and general Lu Shi.Wherein, a, b are the immunohistochemical staining result, a: contrast C6 cell, b: full gene C 6 cells of transfection ferritin heavy chain; C, d are Prussian blue iron coloration result, c: contrast C6 cell marking SPIO.D: the C6 cell marking SPIO of transfection ferritin gene.The blue iron dyeing of general Lu Shi is presented at the iron particle that endocytosis is arranged in two groups of cells, and intracellular iron particle does not have notable difference at two groups of iuntercellulars.
Fig. 2. determining iron concentration in culturing cell and the tumor tissue cell.
Fig. 3. cell inoculation different time points is carried out magnetic resonance imaging.
Fig. 4. the tumor tissue pathology that forms after the inoculation checks, wherein, among a, the left side is the tumour of contrast C6 cell derived, the right side is the tumour that the full gene of transfection ferritin heavy chain does not have the C6 cell derived of mark SPIO, among the b, the left side is the tumour in contrast C6 mark SPIO source, and the right side is that the transfection ferritin gene is with the tumour of the C6 cell derived of tense marker SPIO.
Fig. 5 is the histological examination of the tumor tissues that forms after the inoculation, wherein a: the transfection ferritin gene is with the tumor tissues of the C6 cell derived of tense marker SPIO, b: the tumor tissues in contrast C6 cell marking SPIO source.
Fig. 6 is the blue iron dyeing of general Lu Shi of the tumor tissues that forms after the inoculation, wherein a: the transfection ferritin gene is with the tumor tissues of the C6 cell derived of tense marker SPIO, b: the tumor tissues in contrast C6 cell marking SPIO source.
Embodiment
1 materials and methods
1.1 cell cultures
Mouse C 6 Cell of Glioma cellar culture is (Gibico in F12Kaighn ' s substratum, Invitrogen, the U.S.), substratum adds the 1mM L-glutaminate, 15% horse serum (Hyclone, Thermo Scientific, the U.S.), 2.5% foetal calf serum (Gibico), 100U/mL penicillin and 100mg/mL Streptomycin sulphate.Cell culture condition is 37 ℃, 95% air, 5%CO
2
1.2 make up the full gene plasmid of mouse ferritin heavy chain
Use Trizol reagent (Invitrogen, the U.S.) and extract total RNA from Muscle Tissue.Under the effect of Oligo (dT) primer, utilize reverse transcription reagent (Promega, the U.S.) that mRNA is reversed into cDNA.According to mouse ferritin heavy chain gene order (GenBank, sequence number NM-010239) design contain NotI and BamHI restriction endonuclease sites polymerase chain reaction (PCR) primer (the upstream primer sequence: 5 '-ataagaatgcggccgctaaactataccatgaccaccgcgtctccctc-3 '; The downstream primer sequence: 5 '-cgcggatccgcgttagctctcatcaccgtgtcccag-3 '), pcr amplification goes out the full gene fragment of mouse ferritin heavy chain.Using NotI and BamHI restriction enzyme (Dalian is precious biological) digests the full gene fragment of mouse ferritin heavy chain and pcDNA3.1 (Invitrogen) carrier DNA, (QIAGEN, Germany) carries out purifying to digestion product by QIAquck PCR purification kit.Through T4 ligase enzyme (Dalian is precious biological) the full gene fragment insertion vector of ferritin heavy chain is formed the full gene plasmid of ferritin heavy chain (Fth-pcDNA3.1).
1.3Fth-pcDNA3.1 transfection C6 cell
Transfection the day before yesterday is with 10
5The C6 cell is inoculated in F12Kaighn ' the s substratum that 500 μ l do not contain antibiotic, is incubated at 24 hole plastic culture plates and makes cell reach 90% degrees of fusion.After mixing with Lipofectamine 2000 (Invitrogen), the full gene plasmid Fth-pcDNA3.1 of ferritin heavy chain behind incubated at room 20min, adds described 24 hole plastic culture plate, gently mixing overnight incubation.Peptic cell carries out going down to posterity at 1: 10 behind the transfection 24hr, adds G418 (Gibico) and screens, and filters out the cell of stably express ferritin, and the G418 final concentration is 500 μ g/ml.
1.4 immunocytochemistry dyeing
C6 cell (being called for short C6-Fth) and the contrast C6 cell cultures of transfection ferritin gene plasmid are attached on the coated slide of poly-lysine.With 10% neutral formalin fixed cell 30min, PBS buffer solution for cleaning.Be added on the cell after the polyclonal antibody of the anti-mouse ferritin heavy chain of specificity rabbit (Santa Cruz, the U.S.) dilution in 1: 100, incubate under 4 ℃ of conditions to bathe and spend the night.The PBS buffer solution for cleaning, add second antibody (goat-anti rabbit) (Dako, Denmark) after, incubate under the room temperature and bathe 30min.With benzidine colour developing, haematoxylin redyeing nucleus.
1.5 inductively coupled plasma spectrometer (ICP) is analyzed
Iron atom concentration is utilized inductively coupled plasma spectrometer (TJA IRISAdvantage/1000Radial ICAP, Thermo Scientific, the U.S.) analysis in culturing cell and the histocyte.Culturing cell adds the PBS buffer solution for cleaning three times after digesting with 0.05% trypsinase-EDTA (Gibico).Centrifugal rear collecting cell.Add 70% concentrated nitric acid in cultured cells and the tissue, 90 ℃ of digestion 1hr supply volume to 5ml with pure water.Measure simultaneously the pure water blank tube and contain iron atom 0.1,1,10 and the standard pipe of 100ppm, iron atom concentration calculates by typical curve.
1.6 the blue iron dyeing of general Lu Shi
The tissue slice dewaxing enters the distillation washing through ethanol at different levels.Section is immersed in the equal portions mixed solution of new 2% yellow prussiate of potash of preparing and 2% hydrochloric acid, 30-60min.Fully washing in the distilled water.Redye karyon with 1% toluylene red or 1% safranine, the distillation washing.Rapidly dehydration in the ethanol, dimethylbenzene is transparent, the neutral gum mounting.
1.7 live body magnetic resonance imaging
Two groups in the C6 cell of transfection ferritin heavy chain plasmid and contrast C6 cell be mark SPIO all.Adding SPIO (Ocean Nanotech, the U.S.) in the culturing cell, to make its final concentration be 60 μ g/ml, and the carbonic acid gas of rearmounted 37 ℃ of mixing is cultivated overnight incubation in the feather cockscomb gently.Culturing cell PBS buffer solution for cleaning three times, behind the tryptic digestion, the PBS buffer solution for cleaning, blood counting chamber calculates cell number.With 10
6Cell suspension is put for subsequent use on ice in 30 μ l PBS damping fluids.The nude mice in 6-10 week retreats subcutaneous respectively inoculating cell (the C6 cell of transfection ferritin gene and contrast C6 cell, mark SPIO or not mark SPIO, respectively two groups of nude mice back legs of subcutaneous vaccination both sides) in both sides behind the isoflurane inhalation anesthesia.
Different time points after the cell inoculation is carried out the GE7T magnetic resonance imaging.Scanning sequence is that FSE (fast spin echo, fast rotational echo) T2 adds entirely, and design parameter is: TR=4000ms, TE=40ms, FOV=40mm, Echo TrainLenghth=8ms.
1.8 statistical analysis
The result represents with mean ± s.d, the magnetic resonance signal intensity between on the same group not, and the difference between cell, the Iron In Tissue atomic percent is analyzed by t-test (SPSS10.5, the U.S.).The P value thinks that less than 0.05 statistical significance is arranged.
2 results
2.1 high expression level ferritin cell under condition of in vitro culture to the picked-up of iron
Mouse ferritin heavy chain gene plasmid transfection C6 cell screens by G418.The anti-mouse ferritin heavy chain of application specific antibody carries out the C6 cell high expression level ferritin heavy chain that immunocytochemistry dyeing confirms the transfection ferritin gene.The result utilizes specific murine ferritin heavy chain polyclonal antibody that cell is carried out immunohistochemical staining as shown in Figure 1, and the C6 cell cytosol of transfection ferritin gene presents brown color, is strong positive.It is faint yellow that cellular control unit is, and is the weak positive.
The C6 cell of transfection ferritin plasmid and contrast C6 cell use conventional F12kaighn ' s culture medium culturing, and the iron atom of cell derives from 15% horse serum and 2.5% foetal calf serum.The content of iron atom in these two groups of cells although it is slightly high to detect the cell iron atom concentration of finding the transfection ferritin gene by ICP, does not have notable difference (n=3, P=0.068 between two groups (C6 and C6-Fth); Fig. 2 a).
In cell culture medium, add 0.2mM Diammonium citrate iron (ferric ammonium citrate, during FAC) as the exogenous iron of cell, the interior iron atom concentration of C6 cell (C6-Fth+Fe) of transfection ferritin gene is apparently higher than control group C6 cell (C6+Fe) (n=3, P=0.001; Fig. 2 a).Shown in Fig. 2 b, when in substratum, adding the FAC of different concns (0.25,0.5,1.0 and 1.5mM), iron atom concentration is compared with contrast C6 cell in the C6 cell of transfection ferritin gene, (n=3, P=0.004,0.021 all obviously raise, 0.06 and 0.012), wherein 1,2,3,4 represent respectively 0.25,0.5,1.0 and the FAC concentration of 1.5mM, A represents control group C6 cell, and B represents the C6 cell of transfection ferritin gene.
Behind the C6 cell of transfection ferritin gene and the contrast C6 cell mark SPIO, carry out continuous passage, and each passage cell is carried out ICP measure iron atom concentration in the cell.The result wherein 1,2,3,4,5,6 all represents passage number shown in Fig. 2 c, A represents control group C6 cell, and B represents the C6 cell of transfection ferritin gene.When the first-generation, iron atom concentration does not have notable difference (n=3, P=0.383) in two groups of cells.But from the s-generation, the C6 cell of transfection ferritin gene and mark SPIO, its iron atom concentration ratio do not have the transfection ferritin gene but the iron atom concentration of the C6 cell of mark SPIO obviously increases (n=3, P=0.03,0.02,0.004,0.002 and 0.002.Fig. 2 c).
2.2 in the high expression level ferritin cell living animal to the picked-up effect of iron
The C6 cell of transfection ferritin gene and contrast C6 cell, mark SPIO or not mark SPIO, respectively two groups of nude mice back legs of subcutaneous vaccination both sides.For the ferritin of exploring high expression level in vivo to endogenous iron with from degrade out the effect of catching of iron atom of SPIO, behind the animal inoculation pvaccination cell 13 days, put to death nude mice, collect tumor tissues, carry out ICP histocyte iron atom content analysis (Fig. 2 d) behind the weighing.Transfection ferritin gene but do not carry out the tumor tissues (C6-Fth) of the C6 cell derived of SPIO mark, iron atom content is than comparing with the tumor tissues (C6) of contrast C6 cell derived in its cell, although concentration slightly increases, but there is not notable difference (n=4, P=0.129).The transfection ferritin gene is with the tumor tissues (C6-Fth-SPIO) of the C6 cell derived of tense marker SPIO, the concentration of iron atom in its cell, compare with the tumor tissues (C6-SPIO) of the contrast C6 cell derived of mark SPIO, (n=5, P=0.034) obviously raises.Tumor tissues carries out pathological examination and the blue iron dyeing of general Lu Shi simultaneously.The histological examination confirmation, in the lump that the cell of nude mice both sides inoculation generates, dyskaryosis, karyon is out of proportion, does not have the structure of healthy tissues, is tumor tissues (Fig. 5 a is C6-SPIO, and Fig. 5 b is C6-Fth-SPIO).(a) obviously the tumour (Fig. 6 b) in contrast C6 cell marking SPIO source is many for Fig. 6 with iron particle in the tumor tissues of the C6 cell derived of tense marker SPIO for the transfection ferritin gene.
2.3 magnetic resonance imaging inoculation animal
Utilize GE 7T animalcule magnetic resonance device, adopt T2 to add behind tumor inoculation the 2nd, 6 and 13 day of complete sequence, nude mice is scanned.Analyze magnetic resonance signal intensity, the difference of comparative experiments group and control group.The result as shown in Figure 3, wherein, in the column diagram, 2,6,13 represent respectively postvaccinal the 2nd, 6,13 day, A, B, C, D represent respectively the full gene of transfection ferritin heavy chain and do not have the tumour of the C6 cell derived of mark SPIO, the tumour that contrasts the C6 cell derived, transfection ferritin gene with the tumour of the C6 cell derived of tense marker SPIO and the tumour in contrast C6 mark SPIO source.The result shows: transfection ferritin gene and do not have the tumour of the C6 cell derived of mark SPIO, compare different time points magnetic resonance signal intensity difference not obvious (Fig. 3 a.n=4, the 2nd day: P=0.379 with the tumour of contrast C6 cell derived; The 6th day: P=0.105; The 13rd day: P=0.056).And the transfection ferritin gene is compared with the tumour in contrast C6 mark SPIO source with the tumour of the C6 cell derived of tense marker SPIO, and the magnetic resonance signal intensity of different time points obviously exists notable difference (Fig. 3 b.n=5, the 2nd day: P=0.012; The 6th day: P=0.003; The 13rd day: P=0.021).
3 discuss
Because mr has higher time and spatial resolution, non-invasive, the advantage such as anatomical information can be provided, it has been used to the imaging of stem-cell research.The mr vivo tracking mainly is by cell endocytic super-paramagnetism nano iron particle (SPIO) at present, utilizes T
2Perhaps T
2Produce contrast gradient when * sequence is carried out magnetic resonance imaging.Behind the SPIO labeled cell, along with cell fission, intracellular concentration can reduce gradually.Its principle one be owing to SPIO particle itself along with cell fission is divided, physical property dilution; After the acidic substance such as the 2nd, SPIO particle and lysosome are combined, chemical degradation occuring, discharges the free iron atom.Dividing faster cell, after going down to posterity for 8 times, the SPIO particle has just disappeared in cell.Divide slower or nondividing cell, in cell, also can only keep about 2 months.So, the greatest drawback of carrying out the cell vivo tracking with nano iron particles be can't the long-term follow inoculation the treatment cell.Ferritin is as a kind of crude substance of cell itself, and Main Function is the storage iron atom in the body iron metabolism, is a kind of endogenous mr reporter gene.Ferritin picked-up endogenous iron forms super-paramagnetism nano iron and produces magnetic resonance signal, because body endogenous concentration of iron is lower, so a little less than relying on magnetic resonance signal that ferritin forms.In this research, we at first manually import the ferritin heavy chain gene, make C6 cell high expression level ferritin, use this cell of SPIO mark again, whether the ferritin of observing high expression level can absorb the SPIO iron atom of degrading in the cell as extra source of iron, thereby strengthens magnetic resonance signal.
The C6 cell of high expression level ferritin heavy chain gene is when vitro culture, if use conventional medium, the iron in foetal calf serum and the horse serum is as exogenous source of iron, cultivate certain hour after, intracellular concentration of iron and contrast C6 cell are not compared not obviously increase.If but in substratum, added Diammonium citrate iron as source of iron, the iron atom of the C6 cell contrast group cellular uptake of rotaring redyeing gene would obviously increase, and along with exogenous concentration of iron increases, intracellular concentration of iron also increases.This part in vitro tests result confirms that the ferritin heavy chain that we make up has the effect of picked-up and storage iron atom, if but the extracellular concentration of iron is low, and the effect of its picked-up storage iron can't fully show.Carry out continuous passage behind the C6 cell of transfection ferritin heavy chain gene and the contrast C6 cell marking SPIO, going down to posterity for the first time, concentration of iron does not have difference in both daughter cells, this may be because two groups of intracellular concentration of iron are very high, the iron of ferritin picked-up and the concentration of iron of cell endocytic differ too large, can't show.From s-generation daughter cell, concentration of iron begins to produce difference in two groups of cells, illustrates that along with cell fission intracellular SPIO is diluted rapidly and degrades, and confirms simultaneously under culture condition, and ferritin has the iron function that the SPIO degraded was received and utilized to resorption.
Experimental result shows in the body, and after the C6 of high expression level ferritin heavy chain cell is inoculated 13 days, the concentration of iron in its tumour cell was compared with the C6 that does not have the transfection ferritin gene, although slightly increase, not obvious.This phenomenon can be interpreted as, and ferritin heavy chain can absorb certain endogenous iron, but because this concentration of iron is very low, so ferritin can't be stored more iron by picked-up endogenous iron in cell.After transfection ferritin heavy chain gene was with tense marker SPIO, the C6 cell was inoculated the rear 13 days tumour cells that form, and the interior concentration of iron ratio of its cell only C6 cell of mark SPIO obviously increases.Our result confirms that the ferritin of high expression level can absorb the iron of SPIO degraded and be stored in the cell as extra source of iron.The magnetic resonance imaging strength of signal of different time points conforms to iron atom concentration results in the cell after the cell inoculation.The blue iron dyeing of the general Lu Shi of tumor cell tissue has further confirmed this result.
Originally studies confirm that, ferritin can with the SPIO effect of cell marking, prolong time of SPIO vivo tracking cell; The SPIO of degraded can strengthen the strength of signal of ferritin reporter gene simultaneously.Both unite use, can strengthen vivo tracking time and magnetic resonance signal intensity to labeled cell.This result of study has certain using value for carrying out clinical cell therapy and gene therapy to location, the in real time detection of survival and growth situation for the treatment of cell.
Claims (8)
1. cell that is used for the super-paramagnetism nano iron particle marker of mr vivo tracking, it is characterized in that described cell is ferritin gene plasmid transfection cell, obtained by super-paramagnetism nano iron particle and high expression level ferritin cell co-culture, the label concentration of super-paramagnetism nano iron particle is 30～60 μ g/mL.
2. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 1 is characterized in that described ferritin gene plasmid is the full gene plasmid of ferritin heavy chain.
3. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 2, the construction process that it is characterized in that the full gene plasmid of described ferritin heavy chain is: mRNA is carried out reverse transcription, polymerase chain reaction, amplify the full gene fragment of ferritin heavy chain, the full gene of ferritin heavy chain is inserted in the carrier, makes up the full gene plasmid of ferritin heavy chain.
4. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 3, the construction process that it is characterized in that the full gene plasmid of described ferritin heavy chain is: mRNA is reversed into cDNA, according to the ferritin heavy chain gene order, application contains the polymerase chain reaction primer of restriction endonuclease sites, amplify the full gene fragment of ferritin heavy chain, the application limitations restriction endonuclease digests the full gene fragment of ferritin heavy chain and carrier, and digestion product is carried out behind the purifying the full gene insertion vector of ferritin being formed the full gene plasmid of ferritin heavy chain.
5. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 4, it is characterized in that described carrier is pcDNA3.1, the construction process of the full gene plasmid of described ferritin heavy chain is: mRNA is reversed into cDNA, according to the ferritin heavy chain gene order, application contains the polymerase chain reaction primer of NotI and BamHI restriction endonuclease sites, amplify the full gene fragment of ferritin heavy chain, use NotI and BamHI restriction enzyme the full gene fragment of ferritin heavy chain and carrier pcDNA3.1 are digested, digestion product is carried out purifying by the T4 ligase enzyme the full gene insertion vector pcDNA3.1 of ferritin is formed the full gene plasmid of ferritin heavy chain.
6. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 5 is characterized in that described polymerase chain reaction primer sequence is as follows:
The upstream primer sequence: 5 '-ataagaatgcggccgctaaactataccatgaccaccgcgtctccctc-3 ';
The downstream primer sequence: 5 '-cgcggatccgcgttagctctcatcaccgtgtcccag-3 '.
7. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 1 is characterized in that described cell is C 6 Cell of Glioma, and the method that obtains ferritin gene plasmid transfection cell is:
(1) C 6 Cell of Glioma is inoculated in the substratum, in the cell cultures hole, cultivates, make C 6 Cell of Glioma reach 80%～90% degrees of fusion;
(2) transfection: the ferritin gene plasmid mixes with transfection agents, after the incubated at room, add described cell cultures hole, gently the mixing overnight incubation;
(3) peptic cell carries out 1:10 and goes down to posterity behind the transfection 24hr, at last screening.
8. the cell of the super-paramagnetism nano iron particle marker for the mr vivo tracking as claimed in claim 7 is characterized in that described transfection agents is the Lipofectamine 2000 that Invitrogen company produces.
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| CN1299249A (en) * | 1997-11-20 | 2001-06-13 | D·普拉特 | Reagent for tumor therapy and/or imaging |
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