CN102225209B - Preparation method of nano magnetic granule composite system - Google Patents

Abstract

The invention provides a preparation method of nano magnetic granule composite system. The method comprises steps of: preparing plasmid pHRE-Egr-1-EGFP and PEI-Mn0.5Zn0.5Fe2O4 nano magnetic granule respectively; diluting the plasmid pEgrl-HSV-TK and PEI-MZF-NPs respectively by a mass ratio of 1:40 with a serum-free medium, disposing at room temperature for 5min, mixing the two together and mixing well, and incubating at room temperature for 30min to obtain a pEgrl-HSV-TK / PEI-MZF-NPs compound. In transfection of liver cancer Bel-7402 cells, the compound prepared by the invention has high transfection efficiency, little cytotoxicity and obvious superiority to an electroporation transfection method and a liposome transfection method.

Description

A kind of method for preparing of nanometer magnetic grain hybrid system

Technical field

The invention belongs to the genetic engineering field, be specifically related to a kind of method for preparing of nanometer magnetic grain hybrid system.

Background technology

Tumour radiotherapy-gene therapy is the new approaches of the treatment malignant tumor that proposes to the problem of tumor radiotherapy and gene therapy existence in practice separately in the world in recent years; Its implication is with expressing through ray induction and the gene that tumor has a lethal effect being changed over to tumor cell; Inducible gene expression when then tumor being implemented local radiotherapy; Cause the dual lethal effect of ray and gene pairs tumor, make both advantages be able to complementation, collaborative performance GVT.This treatment pattern has become the new focus in tumor research field.The Egr-1 gene is the transcription factor that the early stage pair cell of radiation plays regulating and controlling effect, can under effects of ionizing radiation, induce downstream gene expression, realizes the space-time regulation and control to destination gene expression.The research of the radiation-gene therapy that utilizes the radiosensitivity of Egr-1 gene promoter regulating and controlling sequence and carry out has obtained challenging achievement; But this method still is faced with ubiquitous problem in the gene therapy---lack suitable gene transmission method and change over to genetic safety in the cell effectively and expression efficiently; And; Because the ubiquitous anoxia microenvironment of solid tumor also can make the induced activity of radiation promoter reduce, thereby the curative effect and the application of this therapy have been influenced.

Weary oxygen response element (HRE) is a kind of anoxia sensitivity enhancer, can combine with HIF-1 Hypoxia Inducible Factor-1 (HIF-1) specificity and induces downstream gene expression.The HRE/HIF regulating system is that mammalian cell and human tissue are total; HIF-1 α crosses in the tumor of 68-84% and expresses (Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer; 2003; 3:721-732), can use HRE sequence regulation and control destination gene expression in this prompting tumor hypoxia environment.(Shibata T such as Shibata; Giaccia AJ; Brown JM. Development of ahypoxia-responsive vecter for tumor-specific gene therapy.Gene Ther; 2000, the HRE that 7:403-498.) has confirmed 5 copies is connected with promoter can make 500 times of the gene expression increases that lack under the oxygen.

The selection of gene transfer vector is another critical problem of gene therapy.Gene transfer vector mainly is divided into viral vector and non-virus carrier two big classes at present.Virus carrier system is up to now efficient gene transfer method, but because the genes of interest capacity is little, targeting specific is poor, and the immunogenicity of self, especially biological safety problem, make its clinical application receive strict control; Although traditional non-virus carrier system has avoided great potential safety hazard, its transfection efficiency not as virus carrier system, is difficult to obtain significant gene expression always.Liposome transfection method and electroporation transfection method are non-virus carrier transfection methods the most commonly used at present, and liposome method has higher transfection efficiency, but can be removed by serum rapidly in vivo; And pair cell toxicity is bigger; Therefore to a great extent limit its use (Wu Haixia, Zhang Weiming, Li Xiaomian. the development and the present situation [J] of gene transfection technology. international biomedical engineering magazine; 2007,30 (6): 376.4-7 nano-carrier advantage).The electroporation transfection efficiency is very high, is fit to transient expression and stable conversion, but this method only is applicable to cell in vitro or organizes transfection, be unwell to transfection in the body, and the pair cell damage is big, has a large amount of cell deaths after the electric shock.

The appearance of nanotechnology and develop into and solve the gene transfer vector problem new thinking is provided is that the research of gene transfer vector has caused extensive concern with the nano-particle.Be that gene transfer vector is wrapped in gene therapy molecules such as DNA, RNA in the nano-particle exactly or is adsorbed on its surface with the nano-particle, under endocytosis, get into to discharge the gene therapy molecule in the cell.Compare with conventional carriers, nano-carrier has following advantage (Panyam, Jayanth at mediated gene transfer party mask; Labhasetwar; Vinod et al. Biodegradable nanoparticles for drug and gene delivery to cells and tissue [J] .Advanced Drug Delivery Reviews, 2003,55 (3): 329-347) (Bhakta G; Mitra S; Maitra A, et al.DNA encapsulated magnesium and manganous phosphate nanoparticles:potential non-viral vectors for gene delivery [J]. Biomaterials, 2005; 26 (14): 2157-2163): non-immunogenicity, can repeatedly inject repeatedly; Hereditary-less toxicity and cytotoxicity can not cause transformation and cell death; Allow gene slowly to discharge, prolong action time effectively, and keep effective production concentration, improve the bioavailability of transfection efficiency and transfection product; But the integration of mediate foreign gene in host cell chromosome DNA, thereby obtain genetically modified long-term, stably express.Therefore, nano-carrier had both combined the advantage of viral vector and traditional non-virus carrier, had avoided both defectives again; Become the novel carriers system that has application prospect; Especially magnetic Nano gene transfer vector, it also has superparamagnetism except the characteristic with general nano-particle; Can under the effect of externally-applied magnetic field, carry out efficient magnetic transfection and move, thereby carry out target gene therapy with directed.

But nano-particle (especially magnetic nano-particle) ubiquity soft-agglomerated phenomenon; And the more little easy more reunion of granule; Naked material can not be transferred to gene in the cell; Have only through its genophore function of competence exertion after the finishing, and the dispersibility, surface functional group etc. of modifying the back magnetic particle directly affect the further application of magnetic particle.PEI is a kind of powder body material surface modifier commonly used, for electric spatial stability mechanism type dispersant, can have Coulomb repulsion and space steric effect, monomer whose (CH-CH simultaneously at aqueous phase ₂-NH ₂-) in per three atoms contain an amino, be rich in cation, the stronger combination DNA and the ability of adherent cell are arranged.In the research in early stage; This laboratory adopts chemical coprecipitation successfully to prepare the temperature sensitive manganese-zinc ferrite nanometer of nanoscale magnetic grain; And carry out finishing with PEI; Improved agglomeration between magnetic particle effectively, COS-7 has obtained higher gene transfection efficient with its mediation pCMV-EGFP transfection MK cells.But can the manganese-zinc ferrite nanometer magnetic grain of modifying through PEI be used for the transfection of HCC, whether has good binding and releasability with other specific gene, and these are all unknown, remains further research.

Summary of the invention

In order to overcome the deficiency of liposome method and electroporation when the transfection HCC; The invention provides a kind of method for preparing of nanometer magnetic grain hybrid system; The nanometer magnetic grain hybrid system that obtains through the present invention is used for the transfection HCC, can efficiently solve the problem that exists in the prior art.

The said nanometer magnetic of the present invention grain hybrid system is by plasmid pHRE-Egr-1-EGFP and PEI-Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain is pressed the 1:40 mass ratio and is mixed the complex that constitutes.

Wherein said plasmid pHRE-Egr-1-EGFP, be with weary oxygen induce element HRE and promoter Egr-1 with radiosensitivity mutually Rhizoma Nelumbinis join, form the weary sense promoter HRE/Egr1 of oxygen radiation Lazer, be connected in the reporter gene EGFP upper reaches.PHRE-Egr-1-EGFP plasmid (being pCDNA3.1-5HRE-Egr1-EGFP) structural representation is seen Fig. 7.

The above-mentioned said composite Nano pHRE-Egr-1-EGFP/PEI-MZF – NPs of system is to obtain through following method:

(1) preparation of plasmid pHRE-Egr-1-EGFP: the Egr1 promoter is placed on the pIRES carrier, a cmv enhancer fragment is arranged before it, in order to strengthen Egr1 promoter expression efficient.With the CMVE-Egr1p sequence is template design primer, pcr amplification CMVE+Egr1p fragment.Reclaiming the CMVE-Egr1p fragment, is the sub-clone site with MluI+NheI, and its sub-clone to pCDNA3.1-EGFP, is made up the pCDNA3.1-Egr1-EGFP plasmid.After the conversion, several clones of picking extract plasmid, carry out PCR and identify.According to the PCR electrophoresis result, select the correct pCDNA3.1-Egr1-EGFP comparison of checking order.Synthesize 5HRE by list of references: for the ease of the carrying out of subsequent experimental; When synthetic 5HRE fragment; Both sides have respectively added several restriction enzyme sites; Be inserted on the pUC57 carrier (winning the biological company limited of profit available from Changsha), obtain pUC57-5HRE, it makes up collection of illustrative plates and sees Figure 13 (gene title: 5HRE; Mrna length: 185bp; Container name: pUC57; Cloning site: EcoRI, HindIII).With the evaluation of synthetic product enzyme action, gene sequencing.PUC57-5HRE is connected with the MluI enzyme action respectively with pCDNA3.1-Egr1p-EGFP, will connects product and be converted among the DH5 α, the picking clone carries out the BglII enzyme action and identifies.Select correct clone to check order, the 5HRE sequence of sequencing sequence and desire acquisition is compared.

(2) PEI-Mn _0.5Zn _0.5Fe ₂O ₄The preparation of nanometer magnetic grain: the mol ratio by Mn:Zn:Fe is that 0.5:0.5:2 takes by weighing raw material MnSO ₄H ₂O, ZnSO ₄7H ₂O, FeSO ₄7H ₂O pours into after the mixing and grinds the cup pulverize at a high speed, presses n (M): n (OH ^–)=1: 2.4 (M represents all metal ions) takes by weighing the amount of required NaOH, adds the suitable quantity of water dissolving, and powder is poured into wherein and mixing; Constantly stir into pasty state, after room temperature is placed 12h, 80 ℃ of dryings; Obtain Powdered presoma, put into 400 ℃ of roasting 1h of Muffle furnace, hot distilled water embathes behind the natural cooling; Filter, use BaCl ₂Detect in the filtrating behind the no white precipitate, dehydrated alcohol drip washing one time, 60 ℃ of oven dry promptly get Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain; Take by weighing a certain amount of Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain is dissolved in the deionized water, is mixed with the magnetic fluid of 4% (mass percent), and high speed centrifugation is abandoned supernatant behind the ultra-sonic dispersion; Deposition is resuspended in the PBS buffer, is that the amount of 1:5 slowly adds PEI according to PEI and MZF-NPs mass ratio behind the ultra-sonic dispersion, abundant mixing, and constant temperature shaking table 24h reacts fully.With magnetic separation method magnetic particle is separated from solution,, promptly obtain the Mn of finishing behind the vacuum drying through cyclic washings such as distilled water, methanol _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain (PEI-MZF-NPs).

(3) complex preparation: by PEI-MZF-NPs and pHRE-Egr1-EGFP mass ratio is 40:1; Plasmid pHRE-Egr1-EGFP and PEI-MZF-NPs are diluted with serum-free medium respectively; Room temperature was placed after 5 minutes; The two is blended into together and mixing, puts and hatched under the room temperature 30 minutes, promptly obtain the pHRE-Egr1-EGFP/PEI-MZF-NPs complex.

The present invention is a carrier with PEI-MZF-NPs; Made up pHRE-Egr-1-EGFP/PEI-MZF-NPs hybrid system; In DNA combination, protection and release experiment, all show; PEI-MZF-NPs not only can efficiently combine with pHRE-Egr-1-EGFP, and protection pHRE-Egr-1-EGFP avoids nuclease degradation, and can under appropriate condition, effectively discharge pHRE-Egr-1-EGFP.Why PEI-MZF-NPs can protect DNA to avoid DNase I digestion, possibly be because composite nano materials has been brought into play sterically hindered effect, promptly stops DNase I and Mg simultaneously ²⁺With contacting of DNA, the digestion that finally influences DNase I is active.In experiment, show the transfection of hepatocarcinoma Bel-7402 cells in vitro; Cytotoxicity is very little; And obtained higher transfection efficiency, shown than electroporation transfection method and liposome transfection method obvious superiority, and after the transfection of nanometer magnetic grain the gene expression efficiency under weary oxygen radiation is induced jointly apparently higher than independent radiation-induced group; Show that pHRE-Egr-1 lacks oxygen radiation Lazer sense promoter and can induce and improve the downstream gene expression level under the anaerobic environment, PEI-Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain can be used as a kind of novel gene vehicle; With pHRE-Egr-1-EGFP transfection Bel-7402 cell safely and effectively, the research of carrying out solid tumor radiation gene therapy for our follow-up mediation suicide gene provides strong theory and experimental basis.

Before among the present invention 5HRE being inserted the Egr1 promoter, make up weary oxygen radiation Lazer sense promoter, connect with reporter gene EGFP Rhizoma Nelumbinis again, made up the pHRE-Egr-1-EGFP plasmid, utilize weary oxygen, radiation is induced jointly and strengthen reporter gene in tumor cell, expresses.Wherein lonizing radiation promptly can be used as and start the switch that genes of interest is transcribed; Spatially go up the regulation and control expression of gene with the time; Because radiating dosage, scope, number of times can artificially be controlled; So might accomplish the horizontal expression of controlling gene at certain limit, reasonable time even needs, this has not only realized the Modulatory character target of gene expression to a certain extent, and can improve the cellular gene expression level in the solid tumor anoxia microenvironment.

Description of drawings

Fig. 1 is that the pCDNA3.1-Egr1-EGFP enzyme action identifies that ((each stripe size is swimming lane 1:Marker IV electrophoresis pattern from top to bottom: 7K, 5.5K, 3.5K, 2K, 1K, 500bp; Swimming lane 2:pCDNA3.1-Egr1-EGFP

; Swimming lane 3:pCDNA3.1-Egr1-EGFP

; Swimming lane 4:pCDNA3.1-Egr1-EGFP

).

Fig. 2 is. pCDNA3.1-Egr1-EGFP order-checking comparison (Sequence 0:Egr1 promoter sequence (the CMVE sequence excludes); Sequence 1:pCDNA3.1-Egr1-EGFP order-checking .ab1 reverse complementary sequence; Comparison software: Dnassit 2.0).

Fig. 3 is that the pUC57-5HRE enzyme action is identified (swimming lane 3:pUC57-5HRE; Swimming lane 2:pUC57-5HRE is through NdeI and HindIII enzyme action; Swimming lane 1:DL3000).

Fig. 4 is that (Sequence 0:5HRE wishes to get sequence in pUC57-5HRE order-checking comparison; Sequence 1:5HRE.ab1 reverse complementary sequence; Comparison software: Dnassit 2.0).

Fig. 5 is that the pCDNA3.1-5HRE-Egr1-EGFP enzyme action is identified (swimming lane 3:pCDNA3.1-5HRE-Egr1p-EGFP; Swimming lane 2:pCDNA3.1-5HRE-Egr1p-EGFP is through the BglII enzyme action; Swimming lane 1:KB Ladder).

Fig. 6 is that (Sequence 0: the 5HRE sequence that expection is inserted in pCDNA3.1-5HRE-Egr1-EGFP order-checking comparison; Sequence 1:pCDNA3.1-5HRE-Egr1p-EGFP order-checking .ab1 sequence; Comparison software: Dnassit 2.0).

Fig. 7 is the pCDNA3.1-5HRE-Egr1-EGFP plasmid map.

To be different quality suppress experiment than the PEI-MZF-NPs-DNA complex gel electrophoresis swimming of (PEI-MZF-NPs:DNA) to Fig. 8 that (swimming lane 1 is Mark; Swimming lane 2 is 0:1; Swimming lane 3 is 5:1; Swimming lane 4 is 10:1; Swimming lane 5 is 20:1; Swimming lane 6 is 40:1; Swimming lane 7 is 80:1).

Fig. 9 is the DNase-I digestion protection experiment of PEI-MZF-NPs-DNA complex ( Swimming lane 1 is Mark; Swimming lane 2 is digestion 10min; Swimming lane 3 is digestion 20min; Swimming lane 4 is digestion 30min; Swimming lane 5 is 40min; Swimming lane 6 is 60min; Swimming lane 7 does not add DNase-I for the gymnoplasm grain; Swimming lane 8 adds DNase-I digestion 10min for the gymnoplasm grain; Swimming lane 9 adds DNase-I digestion 20min for the gymnoplasm grain; Swimming lane 10 adds DNase-I digestion 30min for the gymnoplasm grain; Swimming lane 10 digests 40 min for the gymnoplasm grain adds DNase-I; Swimming lane 11 digests 60 min for the gymnoplasm grain adds DNase-I).

The DNA release experiment of Figure 10 PEI-MZF-NPs-DNA complex ( Swimming lane 1 is Mark; Swimming lane 2 is for discharging 1h; Swimming lane 3 is digestion 4h; Swimming lane 4 is for discharging 8h; Swimming lane 5 is for discharging 12h; Swimming lane 6 is for discharging 24h; Swimming lane 7 is for discharging 48h; Swimming lane 8 is for discharging 3 days; Swimming lane 9 is for discharging 4 days; Swimming lane 10 is for discharging 5 days).

Figure 11 nanometer magnetic grain, electroporation, three kinds of method transfections of liposome Bel-7402 cell are induced or independent radiation-induced back fluorescence microscope photo (radiation-induced group of A, the weary oxygen of nanometer magnetic grain transfection in that weary oxygen radiation is dual; Radiation-induced group of B, the weary oxygen of electroporation transfection; Radiation-induced group of C, the weary oxygen of liposome transfection; Radiation-induced group of D, the transfection of nanometer magnetic grain).

Figure 12 nanometer magnetic grain, electroporation, three kinds of method transfections of liposome Bel-7402 cell are induced or independent radiation-induced back flow cytometer detects transfection efficiency (A, the transfection of nanometer magnetic grain lack radiation-induced group of oxygen with the fluorescence intensity result in that weary oxygen radiation is dual; Radiation-induced group of B, the weary oxygen of electroporation transfection; Radiation-induced group of C, the weary oxygen of liposome transfection; Radiation-induced group of D, the transfection of nanometer magnetic grain).

Figure 13 is that 5HRE inserts the sketch map that makes up pUC57-5HRE on the pUC57 carrier.

The specific embodiment

1 materials and methods

1.1 main agentsLipofectamine LipofectamineTM2000 (Invitrogen company); (polyethylenimine PEI) is Sigma to PEI; Agarose is MRI; DNaseI is Amercso; DMEM culture medium, hyclone are Gibco; Tetrazolium bromide (MTT, AMRESCO); Dimethyl sulfoxide (DMSO, Sigma company).PCDNA3.1-EGFP wins the profit bio tech ltd available from Changsha.

1.2 under the HRE-Egr1 promoter regulationEGFP The structure of eukaryon expression plasmid and evaluation

1.2.1 pcr amplification Egr1 promoter fragment

The Egr1 promoter is placed on the pIRES carrier (purchase in Changsha and win the profit bio tech ltd), a cmv enhancer fragment is arranged before it, in order to strengthen Egr1 promoter expression efficient.

The CMVE-Egr1p sequence is shown in SEQ ID NO:1, and (1-664bp is the CMVE sequence; 665-673bp is the SgfI restriction enzyme site; 678-1117bp is a mice Egr1 promoter sequence), be template design primer with the CMVE-Egr1p sequence.

Primer is right:

Egr1-f：5'-CCG?CTCGAG?CG?ACGCGT?GATCTTCAATATTGGCCATTAGC-3'（SEQ?ID?NO：2）

---MluI：CG?ACGCGT

---XhoI：CCG?CTCGAG

Egr1-r：5'-CCG?CTCGAG?CTA?GCTAGC?CCAAGTTCTGCGCGCTGGGAT-3'（SEQ?ID?NO：3）

---NheI：CTA?GCTAGC

---XhoI：CCG?CTCGAG

Product: sheet degree segment length=1117+17+18=1152bp, GC%=52.3, Ta=55.8

Pcr amplification CMVE+Egr1p fragment.

Fig. 1 is a pcr amplification rear electrophoresis collection of illustrative plates, can know that pCDNA3.1-Egr1-EGFP

, pCDNA3.1-Egr1-EGFP

are correct clones.

1.2.2 with Egr1p fragment sub-clone to pCDNA3.1-EGFP

Reclaiming the CMVE-Egr1p fragment in the first step, is the sub-clone site with MluI+NheI, and its sub-clone to pCDNA3.1-EGFP, is made up the pCDNA3.1-Egr1-EGFP plasmid.After the conversion, several clones of picking extract plasmid, carry out PCR and identify.

Primer is right:

Egr1-f：5'-CCG?CTCGAG?CG?ACGCGT?GATCTTCAATATTGGCCATTAGC-3'（SEQ?ID?NO：2）

Egr1-r：5'-CCG?CTCGAG?CTA?GCTAGC?CCAAGTTCTGCGCGCTGGGAT-3'（SEQ?ID?NO：3）

Product: sheet degree length=1152bp, GC%=52.3, Ta=55.8

According to the PCR electrophoresis result, select correct pCDNA3.1-Egr1-EGFP to send to order-checking.

Fig. 2 is pCDNA3.1-Egr1-EGFP order-checking collection of illustrative plates, with its sequence and the comparison of Egr1 template sequence, can know that the Egr1 sequence is entirely true, shows that pCDNA3.1-Egr1-EGFP makes up successfully.

1.2.3 synthetic 5HRE fragment

5HRE synthesized reference document: Zheng Aiqing, Song Xianrang, Yu Jinming is etc. the structure of mosaic promoter HRE. CArG and regulation and control that the weary oxygen of HCC and ray are replied. Chinese tumor biotherapy magazine, 2005,12 (1): 69-71.For the ease of the carrying out of subsequent experimental, when synthetic 5HRE fragment, both sides have respectively added several restriction enzyme sites (MluI, NheI, BglII, HindIII), and final synthetic 5HRE fragment sequence is shown in SEQ ID NO:4.The 5HRE fragment is inserted on the pUC57 carrier (winning the profit bio tech ltd available from Changsha), obtained pUC57-5HRE, it makes up collection of illustrative plates and sees Figure 13 (gene title: 5HRE; Mrna length: 185bp; Container name: pUC57; Cloning site: EcoRI, HindIII).With the evaluation of synthetic product enzyme action, gene sequencing.

Fig. 3 identifies collection of illustrative plates for the pUC57-5HRE enzyme action; On the empty pUC57 carrier; Fragment length between NdeI and the HindIII is about 290bp; If the 5HRE fragment is successfully inserted, then the fragment length between NdeI and the HindIII is about 480bp, can know that by Fig. 3 enzyme action result the pUC57-5HRE clone is correct.

1.2.4 pCDNA3.1-5HRE-Egr1p-EGFP makes up

PUC57-5HRE is connected with the MluI enzyme action respectively with pCDNA3.1-Egr1p-EGFP, will connects product and be converted among the DH5 α, the picking clone carries out the BglII enzyme action and identifies.Select correct clone to check order, the 5HRE sequence of sequencing sequence and desire acquisition is compared.

Fig. 4 is 5HRE order-checking collection of illustrative plates, but the major gene sequence is correct.

Fig. 5 identifies collection of illustrative plates for the pCDNA3.1-5HRE-Egr1-EGFP enzyme action; On the primary pCDNA3.1-Egr1p-EGFP; Have only a BglII restriction enzyme site, after the 5HRE fragment is successfully inserted, can bring a BglII restriction enzyme site into; Fragment length between two BglII restriction enzyme sites is about 380bp, can know that by Fig. 5 5HRE successfully inserts.Fig. 6 is pCDNA3.1-5HRE-Egr1-EGFP order-checking comparison collection of illustrative plates, but the major gene sequence is entirely true.

Can be known that by above result pCDNA3.1-5HRE-Egr1-EGFP makes up successfully, Fig. 7 is the pCDNA3.1-5HRE-Egr1-EGFP plasmid map.

1.3 Mn _0.5 Zn _0.5 Fe ₂ O ₄ The preparation and the modification of nanometer magnetic grain (MZF-NPs)

1.3 .1 preparation

Adopt chemical coprecipitation to prepare MZF-NPs, its step: to take by weighing raw material MnSO by certain stoicheiometry (mol ratio of Mn:Zn:Fe is 0.5:0.5:2) ₄H ₂O, ZnSO ₄7H ₂O, FeSO ₄7H ₂O pours into after the mixing and grinds the cup pulverize at a high speed, presses n (M): n (OH ^–)=1: 2.4 (M represents all metal ions) takes by weighing the amount of required NaOH, adds the suitable quantity of water dissolving, and powder is poured into wherein and mixing; Constantly stir into pasty state, after room temperature is placed 12h, 80 ℃ of dryings; Obtain Powdered presoma, put into 400 ℃ of roasting 1h of Muffle furnace, hot distilled water embathes behind the natural cooling; Filter, use BaCl ₂Detect in the filtrating behind the no white precipitate, dehydrated alcohol drip washing one time, 60 ℃ of oven dry promptly obtain MZF-NPs

1.3 .2 finishing

Take by weighing a certain amount of MZF-NPs and be dissolved in the deionized water, be mixed with the magnetic fluid of 4% (mass percent), high speed centrifugation is abandoned supernatant behind the ultra-sonic dispersion; Deposition is resuspended in the PBS buffer, is that the amount of 1:5 slowly adds the abundant mixing of PEI according to PEI and MZF-NPs mass ratio behind the ultra-sonic dispersion, and constant temperature shaking table 24h reacts fully.With magnetic separation method magnetic particle is separated from solution,, promptly obtain the Mn of finishing behind the vacuum drying through cyclic washings such as distilled water, methanol _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain (PEI-MZF-NPs).

1.4 PEI-MZF-NPs combines experiment with plasmid pHRE-Egr-1-EGFP

PEI-MZF-NPs is mixed (final volume 500 μ l by mass ratio 0:1,5:1,10:1,20:1,40:1,80:1 respectively with plasmid pHRE-Egr-1-EGFP; Wherein the constant concentration of DNA is 0.01 μ g/ μ l); Room temperature left standstill 30 minutes; Form complex with sufficient reacting, get 10 μ l complex agarose gel electrophoresiies detection nanometer magnetic grain respectively and combine situation with DNA.Select PEI-MZF-NPs and combine optimal proportion with plasmid pHRE-Egr-1-EGFP.

Fig. 8 is the agarose gel electrophoretogram of PEI-MZF-NPs and DNA binding ability test, press 1:0,1:5,1:10 and add plasmid, on the swimming lane it is thus clear that DNA band clearly; 1:40 swimming lane and do not had obvious DNA band greater than the swimming lane of 1:40 shows PEI-MZF-NPs and plasmid pHRE-Egr-1-EGFP mass ratio since 1: 40, PEI-MZF-NPs all plasmids in can articulated system.

1.5 the DNase-I of PEI-MZF-NPs-DNA digestion protection experiment

Plasmid pHRE-Egr-1-EGFP and PEI-MZF-NPs are mixed in Tris Buffer by the 1:40 mass ratio (contain 60mM MgCl ₂) in, room temperature left standstill 30 minutes, added DNase I, 37 ℃ of water-baths, and respectively when digestion 10,20,40,60 min, stop buffer (400mmol/L NaCI, 100mmol/L EDTA) cessation reaction.The bonded plasmid pHRE-Egr-1-EGFP of PEI-MZF-NPs is eluted phenol, chloroform extracting, dehydrated alcohol deposition with SDS; 75% washing with alcohol; Be dissolved in an amount of distilled water, get the product agarose gel electrophoresis, observe the ability of complex opposing DNA enzymolysis.PHRE-Egr-1-EGFP handles with same procedure with the gymnoplasm grain, as contrast.

The nano Mn-Zn ferrite material DNA complex that PEI modifies was through DnaseI digestion 10-60 minute, and it is stable that plasmid still can keep, and electrophoretic band brightness does not obviously change; And after adding DNaseI digestion in the naked DNA; Along with the prolongation of digestion time, visible plasmid palliating degradation degree progressively increases, and is almost all digested after 40 minutes; Can't see band on the swimming lane, prompting PEI-MZF-NPs can protect DNA to avoid the digestion (see figure 9) of DNaseI effectively.

1.6 the DNA releasability detects in the PEI-MZF-NPs-DNA complex

Plasmid pHRE-Egr-1-EGFP is mixed by the 1:40 mass ratio with PEI-MZF-NPs, form complex.TE dissolving, the vibration of 37 constant temperature shaking tables, respectively at 1,4,8,12,24 h, 2 days, 3 days, 4 days, 15000r/min was centrifugal, gets the supernatant agarose gel electrophoresis, understanding complex DNA releasability, all supplied TE again after centrifugal each time and continued vibration.

The outer DNA release conditions of Figure 10 reaction dna-PEI-MZF-NPs composite body: in 1h, 4h, 8h, 12h, 1d, 2d, 3d; The burst size of DNA increases gradually; Showing as electrophoretic band brightness increases gradually, reaches maximum to the 3rd day burst size, does not have obvious difference in the 3rd day, the 4th day.Prompting PEI-MZF-NPs is except can efficiently combining with DNA, and protection DNA avoids degraded, can also be under appropriate condition effective released dna.

1.7 PEI-MZF-NPs transfection efficiency, weary oxygen-radiation-induced expression evaluation

1.7.1 cell culture

The Bel-7402 cell inoculation is in the RPMI RPMI-1640 that contains 10% calf serum, at 37 ℃, saturated humidity, 5%CO ₂Incubator in cultivate, went down to posterity once the trophophase cell of taking the logarithm during experiment in every 2-3 days.

1.7.2 transfection efficiency, weary oxygen-radiation-induced expression evaluation

With PEI-MZF-NPs the Bel-7402 cell is carried out transfection, and compare, its transfection efficiency is estimated with liposome transfection method, electroporation mediation infection protocol.

PEI-PEI-MZF-NPs transfection: before the transfection; In advance DNA and PEI-NPs are diluted with serum-free medium respectively; Hatched 30 minutes under the rearmounted room temperature of the two mixing, obtain DNA-PEI-PEI-MZF-NPs complex (PEI-MZF-NPs and DNA mass ratio are 40:1).With the Bel-7402 cell inoculation in the 6-orifice plate, every hole 5 * 10 ⁵Individual cell is hatched (cell has 80% fusion) after about 18 hours, discards original fluid; PBS washes cell 2 times, and reuse DMEM serum-free medium is washed 1 time, adds serum-free DMEM culture medium (the every hole 3 μ gDNA that contain DNA-PEI-MZF-NPs then; PEI-MZF-NPs and DNA mass ratio are 40:1); Place incubator to hatch 5h, the culture medium with the fresh full culture medium replacement serum-free that contains serum continues to hatch to 48 h.

Liposome transfection: before the transfection; In advance DNA and liposome (LipofectamineTM2000) are diluted with serum-free medium respectively; Hatched 30 minutes under the rearmounted room temperature of the two mixing, obtain DNA-liposome (LipofectamineTM2000) complex.Cell is handled and is organized with DNA-PEI-MZF-NPs before the transfection; The serum-free DMEM culture medium that will contain the DNA-liposome adds culture hole (every hole 3 μ gDNA; Liposome and DNA mass ratio are 5:3); Place incubator to hatch 5h, the culture medium with the fresh full culture medium replacement serum-free that contains serum continues to hatch to 48h.

Electroporation transfection: behind the trypsin digestion cell, stop pancreatin reaction, collecting cell centrifugal (revolution is 1000g 5 minutes) with full culture medium.Remove supernatant, wash cell twice with 1.25%E buffer (serum-free medium that contains 1.25%DMSO).With E buffer diluting cells, making cell concentration is 5 * 10 ⁶/ ml, obtained cell suspension 400ul (is that cell number is 2 * 10 ⁶/ ml) put into aseptic EP pipe, add the 3ugDNA plasmid then, change over to behind the mixing in the pole cup of 4mm.The selection electrode parameter is 250v/950uf, and pole cup thickness is 4mm, puts into the electrotransfection box to the pole cup that cell and DNA are housed, and electric shock back room temperature was placed 10 minutes, changed full culture medium culturing over to.Place incubator to hatch 24h, renew bright culture fluid liquid, continue to hatch to 48h to discard dead cell.

Behind the X ray irradiation (6 million electro-volt) of above-mentioned three kinds of method cells transfected, place (0.1% O under 37 ℃ of weary oxygen environment with linear accelerator 4Gy ₂, 5% CO ₂, N ₂Balance gas) 24h, the proteic expression of fluorescence microscope cell fluorescence, flow cytometer quantitative detecting analysis fluorescence intensity and transfection efficiency are cultivated in sealing.The PEI-PEI-MZF-NPs infection protocol establish simultaneously independent radiation-induced group as contrast, with without the negative contrast of the conventional cultured cells of transfection).

Figure 11 is an observed result under the fluorescence microscope; Each transfection group cell is all visible to have a green fluorescence, and it is more brighter that wherein the electroporation group is sent the cell of green fluorescence, and the PEI-MZF-NPs group is slightly taken second place; Liposome transfection group minimum (seeing Figure 11), untransfected group cell is not seen green fluorescence.

The transfection efficiency that flow cytometer detects (Figure 12) nanometer magnetic grain group, electroporation group, liposome group is respectively 64.65%, 67.60%, 45.77%; Average fluorescent strength is respectively 193.89,215.12,132.69 units; The transfection efficiency of nanometer magnetic grain infection protocol and average fluorescent strength are a little less than electroporation; But apparently higher than liposome method; And after the transfection of nanometer magnetic grain the transfection efficiency under weary oxygen radiation is induced jointly and fluorescence intensity all apparently higher than 48.94% and 169.15 independent radiation-induced group units and fluorescence microscope basically identical as a result.

1.7.3 mtt assay detects the DNA-PEI-MZF-NPs cytotoxicity

The Bel-7402 cell after above-mentioned three kinds of method transfections, changes and plants to 96 orifice plates (2 * 10 respectively ³/ hole), establishes the negative contrast of Bel-7402 cell simultaneously, establish 6 multiple holes for every group without transfection.Place 37 ℃, 5% CO ₂Cultivate 72h in the incubator, every hole adds MTT liquid 20 μ l (5 ㎎/ml, PBS preparation), exhausts supernatant after continuing to cultivate 4 h, adds DMSO (the every hole of 150 μ l/), and vibration dissolving 10 minutes is surveyed the OD value with ELIASA under 493nm.Calculate cell survival rate according to formula " survival rate of cell (%)=each experimental group OD value/negative control group OD value * 100% ".

Table 1 is the MTT experimental result, can find out: the cell relative survival rate of PEI-MZF-NPs transfection group is apparently higher than liposome transfection group and electroporation transfection group.Liposome transfection group survival rate is minimum, and the electroporation transfection group show that electroporation transfection method and liposome transfection method all have certain cytotoxicity to the Bel-7402 cell, and the cytotoxicity of PEI-MZF-NPs infection protocol is less a little more than liposome group (seeing table).

Table 1. PEI-MZF-NPS, electroporation, three kinds of transfection method cytotoxicities of liposome (MTT) experimental result (

± S, n=5)

Group optical density (OD) viability (%)

negative control group 0.599 ± 0.012

PEI-MZF-NPS group 0.581 ± 0.009 97.1

Electroporation group 0.394 ± 0.009 65.8

Liposome group 0.249 ± 0.073 41.5

SEQUENCE?LISTING

 

< 110>Southeast China University

 

< 120>a kind of method for preparing of nanometer magnetic grain hybrid system

 

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<170> PatentIn?version?3.3

 

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Claims (1)

1. the method for preparing of a nanometer magnetic grain hybrid system comprises the steps:

(1) preparation of plasmid pHRE-Egr-1-EGFP: the Egr1 promoter is placed on the pIRES carrier, a cmv enhancer fragment is arranged before it, in order to strengthen Egr1 promoter expression efficient; With the CMVE-Egr1p sequence is template design primer; Pcr amplification CMVE+Egr1p fragment reclaims the CMVE-Egr1p fragment, is the sub-clone site with MluI+NheI; Its sub-clone to pCDNA3.1-EGFP, is made up the pCDNA3.1-Egr1-EGFP plasmid;

After the conversion, several clones of picking extract plasmid, carry out PCR and identify; According to the PCR electrophoresis result, select the correct pCDNA3.1-Egr1-EGFP comparison of checking order; Synthetic 5HRE, and when synthetic 5HRE fragment, both sides respectively add several restriction enzyme sites, are inserted on the pUC57 carrier, obtain pUC57-5HRE, with the evaluation of synthetic product enzyme action, gene sequencing; PUC57-5HRE is connected with the MluI enzyme action respectively with pCDNA3.1-Egr1p-EGFP; To connect product is converted among the DH5 α; The picking clone carries out the BglII enzyme action and identifies, selects correct clone to check order, and the 5HRE sequence of sequencing sequence and desire acquisition is compared;

(2) PEI-Mn _0.5Zn _0.5Fe ₂O ₄The preparation of nanometer magnetic grain: the mol ratio by Mn:Zn:Fe is that 0.5:0.5:2 takes by weighing raw material MnSO ₄H ₂O, ZnSO ₄7H ₂O, FeSO ₄7H ₂O pours into after the mixing and grinds the cup pulverize at a high speed, presses n (M): n (OH ^–)=1: 2.4, wherein M represents all metal ions, takes by weighing the amount of required NaOH, adds the suitable quantity of water dissolving; Powder is poured into wherein and mixing, constantly stirred into pasty state, after room temperature is placed 12h; 80 ℃ of dryings obtain Powdered presoma, put into 400 ℃ of roasting 1h of Muffle furnace; Hot distilled water embathes behind the natural cooling, filters, and uses BaCl ₂Detect in the filtrating behind the no white precipitate, dehydrated alcohol drip washing one time, 60 ℃ of oven dry promptly get Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain; Take by weighing a certain amount of Mn _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain is dissolved in the deionized water, is mixed with mass percent and is 4% magnetic fluid, and high speed centrifugation is abandoned supernatant behind the ultra-sonic dispersion; Deposition is resuspended in the PBS buffer, is that the amount of 1:5 slowly adds PEI according to PEI and MZF-NPs mass ratio behind the ultra-sonic dispersion, abundant mixing, and constant temperature shaking table 24h reacts fully;

With magnetic separation method magnetic particle is separated from solution,, promptly obtain the Mn of finishing behind the vacuum drying through distilled water, methanol cyclic washing _0.5Zn _0.5Fe ₂O ₄Nanometer magnetic grain;

(3) complex preparation: by PEI-MZF-NPs and pHRE-Egr-1-EGFP mass ratio is 40:1; Plasmid pHRE-Egr-1-EGFP and PEI-MZF-NPs are diluted with serum-free medium respectively; Room temperature was placed after 5 minutes; The two is blended into together and mixing, puts and hatched under the room temperature 30 minutes, promptly obtain pHRE-Egr-1-EGFP/PEI-MZF-NPs complex.

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