CN107184992A - PHRE Egr1 HSV TK/PEI PtFe NPs compounds and preparation method thereof - Google Patents

PHRE Egr1 HSV TK/PEI PtFe NPs compounds and preparation method thereof Download PDF

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CN107184992A
CN107184992A CN201710389528.6A CN201710389528A CN107184992A CN 107184992 A CN107184992 A CN 107184992A CN 201710389528 A CN201710389528 A CN 201710389528A CN 107184992 A CN107184992 A CN 107184992A
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林梅
肖艳红
姜兴茂
施裕娟
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Taizhou Peoples Hospital
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Abstract

The invention belongs to medical science and gene engineering technology field, and in particular to a kind of pHRE Egr1 HSV TK/PEI PtFe NPs compounds and preparation method thereof.The compound be the PtFe nanoparticles PEI PtFe NPs modified using Polyethylenimine as carrier, carry pHRE Egr1 HSV TK plasmids.The preparation of the compound is to dilute PEI FePt NPs and pHRE Egrl HSV TK plasmids with serum free medium respectively, after the dilution of the two is mixed evenly, and is incubated and obtains at room temperature.The present invention has also separated application of the compound in anti-tumor drugs targeting is prepared.Compound of the present invention is transfected to liver-cancer stem cell, adds I131Anti- CD133McAb, magnetic induction orientation heating, one side nucleic and thermotherapy can killing tumor cell, while inducing HSV TK expression of suicide gene under HRE Egr1 mediations, gene, nucleic, the multiple lethal effect of hyperpyrexia can be achieved.

Description

PHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds and preparation method thereof
Technical field
The invention belongs to medical science and gene engineering technology field, and in particular to a kind of pHRE-Egr1-HSV-TK/PEI- PtFe-NPs compounds and its preparation method and application.
Background technology
Liver cancer is clinically one of most common malignant tumour, is also the dead the third-largest main cause of cancer-related, With frequently-occurring and high lethal, the whole world there are about 600,000 people and dies from liver cancer every year.Operative treatment, through transcatheter arterial TAE (TACE), RF ablation (RFA), liver transfer operation, radiotherapy and chemotherapy are the common methods of current clinical treatment liver cancer.In mistake Go in many decades, although having been achieved for many progress in terms of the detection and treatment of liver cancer, onset of liver cancer concealment, patient's quilt Most when making a definite diagnosis to be in middle and advanced stage, its grade malignancy is high, and prognosis mala easily recurs and shifted, 5 years survival rate deficiencies 30%.Therefore seeking the new method of liver cancer treatment, the particularly liver cancer to middle and advanced stage, transfer or recurrence, there is important clinic to anticipate Justice.
In recent years, the theoretical proposition of tumor stem cell causes the extensive concern of domestic and foreign scholars.The theory thinks, tumour Stem cell (cancer stem cells, CSC) is the sub-fraction cell (about 5% or so) being present in tumor tissues, these Cell has the potential of self-renewing, unlimited multiplication capacity and Multidirectional Differentiation, is to cause one of root of Tumor Heterogeneity, they It is in mostly in G0 phases and micro-environmental hypoxia, and has certain repellence to radiotherapy, chemotherapy, in the generation of tumour, development, answers Key effect is played in hair and transfer process.At present in hematologic malignancies and breast cancer, colorectal cancer, liver cancer, lung Tumor stem cell is found that in the entity tumors such as cancer, cancer of pancreas and Huppert's disease.It is theoretical according to tumor stem cell, thoroughly disappear The liver-cancer stem cell (liver cancer stem cells, LCSCs) that goes out is to cure liver cancer and improve the key of prognosis, can be by it It is used as the development of monitoring liver cancer and the important indicator of assessment curative effect.Clinically being used to separate the method for liver-cancer stem cell at present mainly has Fluidic cell separating method and immunological magnetic bead sorting method, its principle are all by recognizing that various cell specific marker things sub-elect liver Cancer stem cell.Substantial amounts of LCSC surface markers have been found to and confirm, such as ALDH, CD133, CD13, CD90, CD44, CD24, OV6 and EpCAM etc..CD133 is a kind of five cross-film single chain glycoproteins, can be used as stomach cancer, lung cancer, liver cancer, colon cancer And the stem cell surface marker of many tumours such as cancer of pancreas.Found according to research, CD133+Liver cancer cells have propagation, divided Change the ability with self-renewing.Suetsugu etc. has found, from the human liver cell with stem cell-like properties or cancer progenitor cells characteristic CD133 can be isolated in cancerous cell line+Cell, CD133+Huh-7 cells compare CD133-Huh-7 cells have stronger external Multiplication capacity.Therefore, it is the important research direction in liver cancer treatment field to combine a variety of method targeted therapy of liver cancer stem cells.
Continuing to develop and innovating with thermal biology and thermophysics, tumor thermotherapy has the development being exceedingly fast in recent years, The methods and techniques of tumor thermotherapy are updated and applied.Nanometer technology and magnetic induction thermotherapy are combined by Jordan etc., It has developed Magnetic Fluid Hyperthermia (Magnetic Fluid Hyperthermia, MFH) method to treat tumour, achieved aobvious The effect of work.Magnetic Fluid Hyperthermia is exactly that magnetic fluid is sent into tumor region by certain method, in alternating magnetic field, magnetic Grain can be heated up by reordering mechanism of the relaxation magnetic vector in magnetic field, and the temperature of tumour cell can be killed by reaching, and surrounding normal Magnetic fluid is not present in tissue, heating does not heat up substantially or, this kind of therapy is had the targeting and specificity of height.Spoke Penetrate-gene therapy is a kind of brand-new tumor therapeuticing method, it is therapeutic gene and the regulating and controlling sequence phase with radiation-induced property Lotus root connects, and is transferred to after forming recombinant plasmid in knurl body, the inducible gene expression when implementing partial radiation to tumour, realizes gene and penetrates Dual killing of the line to tumour.This treatment method with the equivalent exposure dose of relative reduction, can mitigate the damage of normal structure, also The localization and expression of therapeutic gene can be realized by the local irradiation of ray.But it is necessary that gene therapy will obtain preferable curative effect Two critical problems are solved, one is gene transfer vector problem, and another is the Modulatory character problem of gene expression.
In recent years, radioimmunotherapy (Radio Immunotherapy, RIT) achieves breakthrough in terms of tumour is treated Progress.This kind of therapy is the principle being combined using radiolabeled antigen with specific antibody, by for the spy of tumor associated antigen Heterogenetic antibody is marked as nucleic carrier with radionuclide, is injected in vivo and tumor cell specific antibodies phase With reference to making the dense poly- a large amount of radiation of absorption of target cell, realize the interior irradiation to tumour, be a kind of oncotherapy of CDCC New model.CD133 antigens are the specific mark molecules that kinds of tumors stem cell surface is independently expressed, self-renewing with tumour, Differentiation potential, chemicotherapy resistance, recurrence and prognosis etc. are closely related.Therefore, CD133 is likely to become treatment liver-cancer stem cell Novel targets.
Based on background above, it is contemplated that, tumor stem cell surface specific antigens c D133 is target, with PtFe magnetic Suicide gene recombinant plasmid is combined anti-CD133McAb targets by nanoparticle (PtFe-NPs) as thermotherapy magnetizing mediums and genophore Tumor locus is delivered to nucleic, under additional action of alternating magnetic field, magnetic nanosphere heat temperature raising is carried out thermotherapy, and in weary oxygen Destination gene expression efficiency is improved under radiation sensitive promoter regulation, so that by nucleic, gene, hyperpyrexia multiple treatments technology Organically combine, have complementary advantages, liver-cancer stem cell is killed in collaboration targeting, it is possible to obtain preferable therapeutic effect.
The content of the invention
It is an object of the invention to provide a kind of pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds and its application.
Compound of the present invention is pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds, and it is with poly second The PtFe nanoparticles (PEI-PtFe-NPs) of alkene imines (PEI) modification are carrier, carry answering for pHRE-Egr1-HSV-TK plasmids Compound.The pHRE-Egr1-HSV-TK plasmids are the suicide gene HSV-TK of weary oxygen radiation sensitive promoter mediation.
Anti-tumor drugs targeting is being prepared the invention also discloses pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds In application.It is especially targeted the medicine for killing liver-cancer stem cell.
PHRE-Egr1-HSV-TK/PEI-PtFe-NPs of the present invention, which is prepared by the following method, to be obtained：
Diluted respectively with serum free medium by the PEI-FePt-NPs prepared and pHRE-Egrl-HSV-TK plasmids, room Temperature is placed more than 5 minutes, after the dilution of the two is mixed evenly, and is incubated more than 30 minutes at room temperature, that is, is obtained pHRE- Egr1-HSV-TK/PEI-PtFe-NPs compounds.Preferably, PEI-FePt-NPs and pHRE-Egrl-HSV-TK plasmid quality Than for 40：1.
, can be as gene transfer vector in order to inquire into PEI-FePt-NPs in the present invention, and study PEI-FePt-NPs Rear extracorporeal biology characteristic and cell transfecting situation are combined with DNA, using pHRE-Egrl-EGFP as experiment in DNA, carry out (1) EI-FePt-NPs and DNA combination Binding experiments, the digestion Protection of (2) PEI-FePt-NPs/DNA compounds, with PEI-FePt-NPs/DNA compounds are detected in transmitting procedure, whether DNA therein is by the DNase-I enzymic digestions in serum Fall, i.e. PEI-FePt-NPs is either with or without protecting digestions of the DNA from nuclease；(3) PEI-FePt-NPs-DNA compounds Middle DNA release experiments, to detect that PEI-FePt-NPs/DNA compounds are reached after target cell, can DNA discharge from compound Out.(4) cell transfection assays：DNA is transfected in cell with PEI-FePt-NPs, its transfection efficiency is detected.After above-mentioned confirmation, Again with being loaded with the suicide gene HSV-TK's that the i.e. weary oxygen radiation sensitive promoter of the pHRE-Egr1-HSV-TK plasmids is mediated PHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds are transfected to liver-cancer stem cell, add I131- anti-CD133McAb, magnetic strength Should orient heating, one side nucleic and thermotherapy can killing tumor cell, while induction HSV-TK commits suiside under HRE-Egr1 mediations Gene expression, can be achieved gene, nucleic, the multiple lethal effect of hyperpyrexia.
Brief description of the drawings
Fig. 1 is FePt-NPs transmission electron microscope pictures.
Fig. 2 is FePt-NPs x-ray diffraction pattern.
Fig. 3 is the FTIR collection of illustrative plates of FePt-NPs before and after PEI modifications.
Fig. 4 is the external magnetic induction heating curve (A of various concentrations nanometer Fe-Pt grain:FePt-NPs,B:PEI-FePt- NPs)。
Fig. 5 is the nanometer Fe-Pt grain magnetic induction heating empirical curve (C under different magnetic field intensity:FePt-NPs,D:PEI- FePt-NPs)。
Fig. 6 is PEI-FePt-NPs and DNA Binding experiment electrophoresis pattern.Lane 1：0:1 (i.e. 100ng DNAs, not Plus nano magnetic grain)；Lane 2：5:1；Lane 3：10:1；Lane 4：20:1；Lane 5：40:1；Lane 6：80:1；Lane M：DNA Marker (from top to bottom index zone be followed successively by 2000bp, 1500bp, 1000bp, 700bp, 500bp, 300bp, 100bp)。
Fig. 7 is that PEI-FePt-NPs digests Protection electrophoresis pattern to DNA DNase-I；
Lane 2：Compound 1min, Lane 3：Compound 10min, Lane 4：Compound 30min,
Lane 5：Compound 45min, Lane 6：Compound 1h,
Lane 7：Exposed plasmid 1min, Lane 8：Exposed plasmid 10min,
Lane 9：Exposed plasmid 30min, Lane 10：Exposed plasmid 45min
Lane M：DNA Marker (from top to bottom index zone be followed successively by 2000bp, 1500bp, 1000bp, 700bp, 500bp、300bp、100bp)。
Fig. 8 is DNA release experiment electrophoresis patterns in PEI-FePt-NPs-DNA compounds.Lane 1：100ng original plasmids DNA
Lane 1：100ng original plasmids DNA
Lane 2：1h、Lane 3：4h、Lane 4：8h、Lane 5：12h、
Lane 6：1d、Lane 7：2d、Lane 8：3d、Lane 8：4d、Lane 9：5d
Lane M：DNA Marker (from top to bottom index zone be followed successively by 2000bp, 1500bp, 1000bp, 700bp, 500bp、300bp、100bp)。
Embodiment
For a better understanding of the present invention, below in conjunction with the accompanying drawings, the present invention is further described for specific material, should not be understood For the specific restriction to the present invention.Material employed in the present invention is as follows：
Cell line and main agents
Huh-7 cells (human liver cancer cell) are purchased from Shanghai cell research institute of the Chinese Academy of Sciences
The monoclonal antibodies of CD 133 are purchased from Immunoway companies, the U.S.
Na131I Jiang Yuan hospitals provide
Embodiment one：The biological characteristics detection of PEI-PtFe-NPs/DNA compounds
1.FePt-NPs preparation and Polyethylenimine (PEI) surface modification, reference literature be (FePt magnetic nano particles The experimental study of preparation, magnetic resonance imaging and its magnetic induction heating external treatment oophoroma, Shi Yujuan master's thesis, 2016, Nantong, Nantong University) prepared.
FePt-NPs is prepared using reverse microemulsion method, basic skills is：0.087g Fe (NO3)3·9H20、0.1g NaCl and 5ml H2PtCl6·6H2O (concentration is 0.02g/ml) is put into three-necked flask, added after dissolving 0.73g CTAB, 90ml benzene, then magnetic agitation 6h at 70 DEG C, separates 2ml water, and magnetic stirring apparatus temperature rises to 90 DEG C, continues to stir 3h, separates Mixed liquor, is finally taken in flask by remaining water, is dried, air atmosphere calcining 5h, is taken out, in H210h is reduced under atmosphere, is washed Centrifugation removes NaCl, and FePt-NPs is can be obtained by after drying.
Take a certain amount of FePt-NPs dissolvings in deionized water, become the magnetic fluid that mass percent is 4%, surpass Sound disperses, and supernatant is removed after high speed centrifugation, and precipitation is suspended from PBS again, and PEI (PEI and FePt are slowly added into after ultrasonic disperse NPs mass ratioes are 1：5), fully mix, constant-temperature table 24h isolates nanoparticle, distilled water, methanol are repeatedly using magnetic separation method Washing, the vacuum dried rear FePt-NPs, i.e. PEI-FePt-NPs that can obtain PEI surface modifications.
Transmission electron microscope observing FePt-NPs average grain diameter about 3nm or so, size is more uniform, and dispersiveness is preferably (Fig. 1).Figure 2 be the X-ray diffracting spectrum of the iron platinum magnetic nano particle prepared, visible sharp diffraction maximum in figure, corresponding to each diffraction maximum Interplanar distance (d values) meet powder diffraction standard association can card (JCPDS：43-1359), it is the center of area four to show the material prepared The iron platinum magnetic nano particle of square.FTIR spectrum analysis (Fig. 3) display, the FePt-NPs after PEI modifications is in 1567cm-1、2851cm-1And 2920cm-1Place is visible new small characteristic peak, and this is PEI-NH2Produced with-CH2 stretching vibrations , point out PEI to be successfully coated on FePt-NPs.
The heating experiment of 2.FePt-NPs and PEI-FePt-NPs in AMF
The magnetic fluid magnetic induction heating experiment of 2.1 various concentrations
FePt-NPs, PEI-FePt-NPs be each made with physiological saline concentration be respectively 0.25,1.0,1.5,2.0g/ L magnetic fluid, numbers in order, and takes 5mL to be put into 25cm respectively2Flat based tubes, being individually placed to frequency in order at room temperature is 215KHz, output current make the bottom of test tube to heat 1h on 35A SPG-10A-II high-frequency magnetic induction firing equipment plate coils Portion and heating coil centre distance about 0.5cm, observer's temperature value of measurement record per 5min, using the time as abscissa, temperature Angle value is ordinate, FePt-NPs and PEI-FePt-NPs magnetic fluid heating curves are drawn respectively.
Magnetic fluid magnetic induction heating experiment under 2.2 different magnetic field intensity
FePt-NPs, PEI-FePt-NPs magnetic fluid for taking 5mL concentration to be 1.0g/L respectively are placed on 25cm2Flat based tubes In, it is placed at room temperature on SPG-10A-II high-frequency magnetic induction firing equipment plate coils and heats 1h, frequency-invariant is 215KHz, defeated Go out electric current and be adjusted to 25A, 30A, 35A, 40A respectively, the bottom of test tube is away from heating coil center about 0.5cm, and observer is every A 5min measurement temperature value of record, using the time as abscissa, temperature value is ordinate, and FePt-NPs and PEI- is drawn respectively FePt-NPs magnetic fluid heating curves.
Fig. 4 and Fig. 5 are respectively FePt-NPs and PEI-FePt-NPs external under various concentrations and different magnetic field intensity Heat up experimental result, and PEI-FePt-NPs and FePt-NPs are respectively provided with excellent heating performance under AC magnetic field effect, preceding Heating is very fast in 30min, then relatively slow, and tends towards stability, and can be used for Magnetic Fluid Hyperthermia as magnetizing mediums.
3.PEI-FePt-NPs is combined rear extracorporeal biology Characteristics Detection with DNA, is made by model of pHRE-Egr1-EGFP For DNA to be combined
3.1 PEI-FePt-NPs and DNA Binding experiment
PHRE-Egr1-EGFP can refer to document prepare (radiation of the weary oxygen of HRE-Egr1/radiation sensitive promoter mediation- The research of gene/nano magnetic heating treatment system joint anti-AFPMcAb targeted therapy of liver cancer of 131I- and its mechanism, doctor's Lin Mei opinion Text, 2013, Nanjing, Southeast China University)
PEI-FePt-NPs and pHRE-Egrl-EGFP is diluted with serum free medium respectively, room temperature places 5 points It is more than clock, in mass ratio 0：1、5：1、10：1、20：1、40：1、80：1 carries out mixing mixing, the final concentration of 20ng/ of DNA Ul, cumulative volume 20ul is supplied with ultra-pure water, at room temperature the quiet pHRE-Egrl-EGFP/ put more than 30min, form different ratio PEI-FePt-NPs compounds, take respectively 5ul compounds agarose gel electrophoresis (i.e. 100ng/ swimming lanes) detect magnetic nanosphere with DNA combination situations.Select the optimal proportions that PEI-FePt-NPs is combined with pHRE-Egr1-EGFP.
Fig. 6 is the agarose gel electrophoretogram that pHRE-Egr1-EGFP and PEI-FePt-NP binding abilities are detected, when receiving Rice magnetic grain and DNA mass ratio are 40:Can be substantially best combination ratio with plasmid all in articulated system when 1.
3.2 PEI-FePt-NPs digest Protection to DNA DNase1
Tango Buffer (Thermo) and DNase-I enzymes are added and contain pHRE-Egr1-EGFP/PEI-FePt-NPs (PEI-FePt-NPs is 40 with pHRE-Egr1-EGFP mass ratioes：1) in serum free medium, 37 DEG C of water-baths digest respectively 1min, 10min, 30min, 45min, 1h, rapidly with isometric 0.5mol/L EDTA solution terminating reactions, and 55 DEG C of water-baths Further inactivate DNase1.The PEI-FePt-NPs pHRE-Egr1-EGFP combined are eluted with SDS, phenol, chloroform are taken out Carry, absolute ethyl alcohol precipitation, 75% ethanol washing is dissolved in appropriate distilled water, takes product agarose gel electrophoresis, observation is multiple Compound resists the ability of DNA enzymatic solution.Gymnoplasm grain pHRE-Egr1-EGFP is handled with same procedure, control is used as.
Fig. 7 digests Protection electrophoresis pattern for PEI-FePt-NPs and pHRE-Egr1-EGFP DNase-I.From figure It can be seen that the nanometer Fe-Pt grain DNA compounds of PEI modifications are after DNase-I digestion 10-60min, DNA remains to keep steady It is fixed, and exposed plasmid is added after DNase-I, almost all is digested after 45min, has not seen band, shows PEI-FePt- NPs can effectively protect plasmid from DNase1 digestion.
DNA releasabilities are detected in 3.3 pHRE-Egr1-EGFP/PEI-FePt-NPs
With TE solution by pHRE-Egr1-EGFP/PEI-FePt-NPs compounds (PEI-FePt-NPs and pHRE-Egr1- EGFP mass ratioes are 40：1) mend to 500 μ l (pHRE-Egr1-EGFP plasmids are 10 μ g), shaken to 37 DEG C of constant-temperature table 200rpm Swing, sample 8 μ l respectively at 1h, 4h, 8h, 12h, 1d, 2d, 3d, 4d, finally enter row agarose gel electrophoresis by 5 μ l/ holes.
Fig. 8 is DNA release experiment electrophoresis patterns in PEI-FePt-NPs-DNA compounds, and DNA burst sizes are gradually in 3d Increase, the 4th, almost indistinction after 5d, show that PEI-FePt-NPs under suitable condition can effectively released dna.
4.PEI-FePt-NPs efficiency gene transfection detection
4.1 CD133+The sorting and culture of Huh-7 cells
Cell is observed under inverted microscope, the good Huh-7 cells of growth conditions is chosen, is gone out with selected by flow cytometry apoptosis CD133+Huh-7 cells, by the CD133 sub-elected+Huh-7 cells are placed on EGF containing 40ng/mL, 20ng/mL bFGF, 1% B27,0.4% bovine serum albumin(BSA) and 5mg/mL insulin serum-free DMEM/F12 nutrient solution cultures.
4.2 flow cytomery PEI-FePt-NPs transfection efficiency
PHRE-Egr1-EGFP is transfected to CD133 with PEI-FePt-NPs infection protocols+Huh-7 cells：Take the logarithm growth Phase CD133+Huh-7 cells, by every hole 5 × 105Cell is inoculated in 6 orifice plates, 37 DEG C, 5%C02About 18h is cultivated in incubator Afterwards, original fluid is discarded, cell is washed with sterile PBS 3 times, then is washed 2 times with serum free medium, then adds and contains pHRE- The serum free medium (per the μ g pHRE-Egrl-EGFP of hole 3) of Egrl-EGFP/PEI-FePt-NPs compounds, is placed in incubator Middle incubation 5h, replaces the culture medium of serum-free with the culture medium of above-mentioned culture liver-cancer stem cell, continues cellar culture 2d.With routine Cultivate the CD133 of untransfected+As a control group, as a result flow cytomery transfection efficiency shows, PEI- Huh-7 cells FePt-NPs transfection efficiency is (53.65 ± 3.40) %, points out PEI-FePt-NPs to may be used as the carrier of gene transfer.
Embodiment two：The preparation of pHRE-Egrl-HSV-TK/PEI-FePt-NPs compounds
1st, pHRE-Egr1-HSV-TK can refer to document and prepare (the weary oxygen of HRE-Egr1/radiation sensitive promoter mediation The research of radiation-gene/nano magnetic heating treatment system joint anti-AFPMcAb targeted therapy of liver cancer of 131I- and its mechanism, Lin Meibo Scholar's paper, 2013, Nanjing, Southeast China University)
2nd, the preparation of pHRE-Egrl-HSV-TK/PEI-FePt-NPs compounds
Plasmid pHRE-Egrl-HSV-TK and PEI-FePt-NPs are diluted with serum free medium respectively, room temperature places 5 More than minute, both are mixed into mixing, and (PEI-FePt-NPs and pHRE-Egrl-HSV-TK mass ratioes are 40：1), at room temperature More than 30min is incubated, that is, obtains pHRE-Egrl-HSV-TK/PEI-FePt-NPs compounds.
Embodiment three：The magnetic induction of pHRE-Egrl-HSV-TK/PEI-FePt-NPs compounds is heated to liver-cancer stem cell External treatment is studied
1、I131- anti-CD133McAb preparation, purifying
Used using improvement chloramine-t method131I marks CD133 antibody, and PD10 posts are isolated and purified.PD10 posts use preceding double steamings Washing 3 times, PB solution is washed 3 times, and PBS solution is washed 3 times.Take 1.5mL EP to manage, sequentially add CD133 antibody 50ul, 50ul Na131I (adds corresponding volume) according to activity, and 200ul PB solution, 10ul toluene-sodium-sulfonchloramides react 1min, added immediately after mixing 100ul inclined sol solution terminating reaction.Above-mentioned reaction solution is added post separation is crossed in PD10 posts, removed free131I, TLC are detected Its radio-chemical purity is 100%, and mark yield is 36%.
2nd, pHRE-Egrl-HSV-TK/PEI-FePt-NPs compounds magnetic induction heating is to CD133+Huh-7 liver cancer is dry thin The intervention of born of the same parents
By the CD133 after transfection+Huh-7 liver-cancer stem cells are diluted to 3 × 105/ ml single cell suspensions, connect according to 3ml/ bottles Plant in 5 culture dishes.Packet situation is as follows；(1) negative control group (untransfected)；(2)I131- anti-CD133McAb groups (do not turn Dye, nucleic group)；(3) Magnetic Fluid Hyperthermia group (untransfected group)；(4)pHRE-Egrl-HSV-TK/PEI-FePt-NPs/I131/ anti- CD133McAb groups (nucleic-genome)；(5)pHRE-Egrl-HSV-TK/PEI-FePt-NPs/I131/ anti-CD133McAb magnetic strengths Group (nucleic-hot group of gene-magnetic) should be heated.DMEM nutrient solutions, I are separately added into according to packet situation131- anti-CD133McAb is (whole The μ Ci of concentration 50), PEI-FePt-NPs.Magnetic Fluid Hyperthermia group cell 215kHz, 2.4kW, output current 35A SPG-10A- 1h is heated on II high-frequency magnetic induction heating instrument plate coils.Nucleic-genome and nucleic-hot group of gene-magnetic are placed in 37 DEG C of weary oxygen Cultivated under environment.Each group cell continues to cultivate 2d in incubator.
(1) proliferation inhibition rate of each group cell after MTT methods measure is handled
Part is taken out from each group cell after above-mentioned processing respectively, single cell suspension is made, 96 are inoculated in per hole 200ul Orifice plate, every group sets 5 multiple holes, continues to cultivate 24h, adds MTT 20ul/ holes, continues to cultivate 4h, discards liquid in hole, adds per hole Enter 150ul DMSO, vibration 10min mixes it, is placed on ELIASA and reads OD values at 493nm, calculates cell proliferation and suppresses Rate, cell proliferation inhibition rate=(1- experimental groups OD values/control group OD values) × 100%.
(2) measure of flow cytometer
Above-mentioned each group remaining cell is collected respectively, and cold PBS washs cell 3 times, and abandoning supernatant after centrifugation suspends cell In 100ul × annexin-binding buffer, add after each 5ul of Annexin V and PI, gently shake up, lucifuge room temperature 15min is reacted, takes 400ul × annexin-binding buffer to be added in sample, 1h in-flows cell instrument is examined after mixing Survey analysis each group Apoptosis situation.
MTT experiment the results are shown in Table 1, nucleic group, Magnetic Fluid Hyperthermia group, nucleic-genome and nucleic-hot group of gene-magnetic Cell proliferation inhibition rate is obviously higher than negative control group (p<, but nucleic-gene-thermotherapy group is respectively controlled apparently higher than other 0.05) Treatment group (p<0.05).Table 2 is flow cytomery result, and nucleic-gene-magnetic heat group cell mortality is up to 49.22%, bright It is aobvious to be higher than other each treatment groups.Show that there is the hot triple combination of nucleic, gene, magnetic preferable Proliferation Ability to make to liver-cancer stem cell With, and its apoptosis is induced, effect is substantially better than any of which single therapy, it is shown that the clear superiority of therapeutic alliance.
Table 1.pHRE-Egrl-HSV-TK/PEI-FePt-NPs/I131/ anti-CD133McAb magnetic induction heating is to CD133+ The inhibited proliferation of Huh-7 cells
aCompared with control group, p<0.05；bCompared with nucleic group, p<0.05；cCompared with Magnetic Fluid Hyperthermia group, p<0.05；d With nucleic-genome comparison, p<0.05；eCompared with nucleic-hot group of gene-magnetic, p<0.05.
The pHRE-Egrl-HSV-TK/PEI-FePt-NPs/I of table 2131/ anti-CD133McAb magnetic induction heating is to CD133+ The apoptosis-induced effect of Huh-7 cells

Claims (5)

1. a kind of pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds, it is characterised in that：It is to be modified with Polyethylenimine PtFe nanoparticles PEI-PtFe-NPs be carrier, carry pHRE-Egr1-HSV-TK plasmids.
2. the pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds described in claim 1, it is characterised in that PEI-FePt- NPs is 40 with pHRE-Egrl-HSV-TK plasmids mass ratio：1.
3. the pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds described in claim 1 are preparing anti-tumor drugs targeting In application.
4. the preparation method of pHRE-Egr1-HSV-TK/PEI-PtFe-NPs compounds described in claim 1, it is characterised in that Diluted respectively with serum free medium by the PEI-FePt-NPs prepared and pHRE-Egrl-HSV-TK plasmids, room temperature places 5 More than minute, after the dilution of the two is mixed evenly, it is incubated more than 30 minutes at room temperature, that is, obtains pHRE-Egr1-HSV- TK/PEI-PtFe-NPs compounds.
5. claim 4 methods described, it is characterised in that PEI-FePt-NPs and pHRE-Egrl-HSV-TK plasmid mass ratioes are 40：1.
CN201710389528.6A 2017-05-27 2017-05-27 PHRE Egr1 HSV TK/PEI PtFe NPs compounds and preparation method thereof Pending CN107184992A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102225209A (en) * 2011-06-26 2011-10-26 东南大学 Preparation method of nano magnetic granule composite system
CN103667345A (en) * 2013-12-02 2014-03-26 东南大学 Anti-oncogene magnetic composite nanoparticle and preparation method thereof

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Publication number Priority date Publication date Assignee Title
CN102225209A (en) * 2011-06-26 2011-10-26 东南大学 Preparation method of nano magnetic granule composite system
CN103667345A (en) * 2013-12-02 2014-03-26 东南大学 Anti-oncogene magnetic composite nanoparticle and preparation method thereof

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