CN102010878B

CN102010878B - Super-paramagnetic nanometer vector carrying retinal pigment epithelium derived factor (PEDF)

Info

Publication number: CN102010878B
Application number: CN 200910195226
Authority: CN
Inventors: 张弢
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2009-09-07
Filing date: 2009-09-07
Publication date: 2013-06-19
Anticipated expiration: 2029-09-07
Also published as: CN102010878A

Abstract

The invention belongs to the technical field of biology and relates to a super-paramagnetic nanometer vector (USPIO) carrying retinal pigment epithelium derived factor (PEDF). In the invention, super-paramagnetic nanometer iron particles are prepared through a nanometer technology and the surfaces of the particles are functionally modified; the retinal pigment epithelium derived factor is constructed on an adenovirus related expression vector AAV-GFP containing green fluorescent protein report gene so as to crosslink USPIO with the AAV-GFP; and glioma cells are induced in vitro and devoured. By utilizing the magnetic marker function of the vector of the invention, the influences of the PEDF on the growth, the differentiation and the invasiveness of the brain glioma are noninvasively monitored in vivo, thereby breaking new incorporating and monitoring ways for the clinical application of the glioma gene treatment, and providing innovative operation means and gathering exploratory experience for the research of the clinical anti-tumor treatment.

Description

Carry the superparamagnetic nano-carrier of retinal pigment epithelium derivative factor

Technical field

The invention belongs to biological technical field, be specifically related to a kind of superparamagnetic nano-carrier (PEDF-USPIO) that carries retinal pigment epithelium derivative factor (PEDF).

Background technology

Studies show that, cerebral glioma is derived from neurogliocyte in brain, is modal brain tumor, accounts for 50% of intracranial tumors, and its malignant phenotype is take rapid propagation, local invasion and attack and vasculogenesis as principal character.Although the many research work to Treatment for Glioma are being carried out, its mechanism and related methods for the treatment of that occurs, develops and worsen has no obvious breakthrough always.Clinical practice shows, even experienced operation, postoperative irradiation and chemotherapy, the ripe methods such as biotherapy of using even that is that all right, Most patients recurrence and progress are still inevitable, so find and open up new methods for the treatment of to have become the focus that investigators pay close attention to.

Retinal pigment epithelium derivative factor (pigment epithelium derived factor, PEDF) be a kind of neurotrophic factor, at first at fetal retinal pigment epithelial cell (retinalpigment epithelial, RPE) find in nutrient solution, it can induce the Retinoblastoma cell Y79 differentiation of vitro culture, then, research finds that it is present in the many tissues of human body, has great expression especially in cerebral tissue.PEDF also has effect except in the Retinoblastoma Cells of cultivating, neurotrophic effect being arranged to normal neurocyte.Taniwaki etc. study discovery, and PEDF is to the nutritious effect of normal cerebellar granule cell (cerebellar granule cell, CGC); The discovery PEDF such as Bilak can also keep general form and the neurone sum of notochord; PEDF can promote the Growth and Differentiation of dynamoneure effectively, avoids neurodegeneration; Except the trophic nerve effect, PEDF can also be by activating NFkappaB and ERK1/2 approach, and effectively neuroprotective unit avoids the neurotoxicity infringement of glutaminate mediation.Sugita and colleague also observe another surprising characteristic of PEDF.Along with neuronic death, the spongiocyte that originally was mixed with in the CGC nutrient solution is propagation rapidly, can occupy whole culture dish at last.Yet exist in the situation of PEDF, the astroglia cell growth phase is when slow, and the microglia form changes, and accelerates old and feeble.PEDF stops G CFS (granulocyte-macrophage colony stimulation factor, GMCSF) to the splitting action of spongiocyte, to be referred to as " spongiocyte tranquillization " effect fully.PEDF can also effectively suppress new vessel and form, PEDF suppresses specific activity angiostatin (angiostatin), thrombospondin-1 (thrombospondin-1), endostatin (endostatin) that vascular endothelial cell divides a word with a hyphen at the end of a line and wants height, therefore PEDF is regarded as at present one of the most effective endogenic Angiogenesis Inhibitor.

The neurotrophy of PEDF, neuroprotective properties to new vessel restraining effect and unique " spongiocyte tranquillization " effect, allow the investigator associate PEDF to the tumour of many central nervous systems, and especially the treatment of glioma may be not without benefit,

Along with gene therapy enters the preclinical phase experiment, there is more dispute in its operability: how effectively therapeutic gene to be imported becomes the difficult point that the gene therapy clinical application need overcome in the patient tumors cell.Along with the arriving in " molecular image " epoch, the tumour-specific markers probe is combined with advanced imaging technique, revolutionaryly changed clinical means to tumor diagnosis and treatment research.Survey macromole with mr, must use the specific combination part, these parts are still needed with MR contrast medium phase conjugate to produce detectable signal difference; In like manner, to use contrast medium mark purpose cell to the demonstration of cell.Although the gadolinium sequestrant can be used for MR molecule or cell imaging, the relaxivity of this type of contrast medium is lower, can further lower after taking the photograph in cell, and the gadolinium agent does not possess biocompatibility, goes after chelating the understanding of genotoxic potential very few to it.USPIO possesses following characteristic: maximum that 1) can the metal M R of the unit of generation signal changes, and contains thousands of iron atoms, has greatly improved the susceptibility of MRI; 2) formed by biodegradable iron; 3) its pan coating thing (as dextran) can be directly and functional group and part carry out Chemical bond; 4) be easy to show with light microscopic and Electronic Speculum; 5) can have different magnetic attributes with size variation, be conducive to the research of its conformation.Make increasing research use USPIO as the live body non-invasive monitoring probe mark thing of MR molecule and cell imaging based on above reason.

Nano-carrier aspect the mediated gene transfer except the common advantage of non-virus carrier, due to its special structure and surface charge, have very high gene transfering efficiency; But the integration of mediate foreign gene in host cell chromosome DNA, thereby obtain genetically modified long-term, stably express; Can protect transducible gene not to be subjected to the destruction of various complements in body blood plasma or histocyte and various enzymes, be conducive to goal gene and can better, more play consistently its effect after transduction enters target cell; Nanometer itself has opposing or kills some virus (comprising HIV virus), so controlling and releasing system with nanotechnology has broad prospects in the application of gene transfection and field of gene.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of superparamagnetic nano-carrier that carries retinal pigment epithelium derivative factor PEDF is provided.

The invention provides the nano magnetic particle carrier of a kind of PEDF of carrying, and utilize its magnetic mark effect, live body non-invasive monitoring PEDF is on cerebral glioma growth, differentiation, invasive impact, for new importing and monitoring approach are opened up in the clinical application of glioma gene therapy, provide operational means and the exploratory test experience of accumulation of novelty for the research of clinical antineoplaston.

The present invention prepares superparamagnetic Nano iron Particles (USPIO) by nanotechnology, its surface of functional modification, retinal pigment epithelium derivative factor (PEDF) is structured on the adenovirus correlative expression vector AAV-GFP that contains Green fluorescent protein fusion vector, and then USPIO and AAV-GFP is crosslinked, external evoked glioma cell is engulfed it.

The present invention prepares superparamagnetic Nano iron Particles (USPIO) by following method and step

1) preparation surface-functionalized superparamagnetic Nano iron Particles (USPIO)

Utilize the coprecipitation synthesis nanometer Fe ₃O ₄Magnetic ion after surperficial dextran is coated, carries out carboxylated functional group and modifies, and utilizes transmission electron microscope to show nano magnetic particle (Fig. 1).

2) synthetic people AAV-PEDF and the crosslinked genophore (PEDF-USPIO) of making of USPIO

(1) preparation PEDF-USPIO carrier:

Pcr amplification PEDF cDNA according to a conventional method, the PEDF cDNA that pcr amplification is gone out with BamHI and NcoI double digestion after, be connected in carrier for expression of eukaryon, be built into the AAV-PEDF expression vector.

In the present invention, described carrier for expression of eukaryon is AAV-GFP.

(2) NMR (Nuclear Magnetic Resonance) imaging of PEDF-USPIO carrier:

Above-mentioned PEDF-USPIO adds in the glioma cell substratum, hatches with cell, determines concentration of iron and the concentration of PEDF-USPIO and the relation of incubation time in cell.

In the present invention, along with increasing of the concentration of USPIO, in cell, indigo plant is dyed the iron particle and is also increased.When USPIO concentration is 150～180 μ g/ml, show that activity on glioma cell is without obvious impact; But when the concentration of USPIO reaches 230 μ g/ml, show that USPIO affect the activity of glioma cell, show as mtt assay and detect that dead cell increases and a large amount of glioma cell apoptosis of flow cytometer detection.

The concentration of the preferred USPIO of the present invention is 180 μ g/ml, and incubation time is 24 hours.

Studies show that the restraining effect of the PEDF that the present invention makes to glioma growth.And for further PEDF is safe, special, effectively import glioma cell, observe its impact on growth of tumour cell, differentiation, invasion and attack the reference data that practice significance is arranged be provided in order to follow up.Can further develop live body non-invasive monitoring PEDF to cerebral glioma growth, differentiation, invasive preparation.

The present invention tests the reagent of employing, and cell, cell culture medium and carrier for expression of eukaryon etc. all can be by commercial or obtain by prior art.

For the ease of understanding, below will describe in detail of the present invention by concrete drawings and Examples.It needs to be noted, specific examples and accompanying drawing are only in order to illustrate, obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in scope of the present invention.

Description of drawings

The surface-functionalized superparamagnetic Nano iron Particles that Fig. 1 has successfully synthesized

Fig. 2 recombinant plasmid AAV-PEDF double digestion collection of illustrative plates

Fig. 3 PEDF-USPIO engulfs iron granules during 24h in containing the Nano iron Particles substratum.

Embodiment

Embodiment 1

One, the preparation of surface-functionalized superparamagnetic Nano iron Particles (USPIO)

Utilize the coprecipitation synthesis nanometer Fe ₃O ₄Magnetic ion after surperficial dextran is coated, carries out carboxylated functional group and modifies, and utilizes transmission electron microscope to show nano magnetic particle, sees Fig. 1.

Two, synthetic people AAV-PEDF and the crosslinked genophore (PEDF-USPIO) of making of USPIO

1) preparation of PEDF-USPIO carrier:

The PEDF cDNA that pcr amplification is gone out with BamHI and NcoI double digestion after, be connected in carrier for expression of eukaryon AAV-GFP, be built into the AAV-PEDF expression vector.After the double digestion order-checking is correct (accompanying drawing 2), get 1mg magnetic microsphere and 20 μ g AAV-PEDF, be dispersed in the crosslinked damping fluid of 0.6ml, after the oscillatory reaction 3h of 50 ℃, under the citrate buffer solution room temperature, the washing magnetic microsphere is 3 times, washs 2 times at 65 ℃ without ribonuclease T. water.

2) NMR (Nuclear Magnetic Resonance) imaging of PEDF-USPIO carrier:

The PEDF-USPIO of 50 μ g/mL adds in the glioma cell substratum, hatches 3 to 96 hours with cell, determines concentration of iron and the concentration of PEDF-USPIO and the relation of incubation time in cell.

Fig. 3 shows that clearly PEDF-USPIO engulfs iron granules during 24h in containing the Nano iron Particles substratum, prove the successful structure of PEDF-USPIO carrier.

The content that comes from iron due to the main side effects of USPIO is if iron too high levels in vivo can cause the harm such as liver cirrhosis, therefore, the present invention has determined the concentration of USPIO, studies show that, along with increasing of the concentration of USPIO, in cell, indigo plant is dyed the iron particle and is also increased.When USPIO concentration is 150～180 μ g/ml, on the activity of glioma cell without obvious impact; When the concentration of USPIO reaches 230 μ g/ml, USPIO affects the activity of glioma cell, shows as mtt assay and detects that dead cell increases and flow cytometer detects a large amount of glioma cell apoptosis.Therefore the USPIO of 180 μ g/ml is used for later experiments as optimum concn.

Claims

1. carry the superparamagnetic nano-carrier USPIO of retinal pigment epithelium derivative factor PEDF, it is characterized in that preparing the superparamagnetic Nano iron Particles by nanotechnology, its surface of functional modification, the retinal pigment epithelium derivative factor is structured on the adenovirus correlative expression vector that contains Green fluorescent protein fusion vector, and then crosslinked, make described carrier, it prepares by following step:

1) the surface-functionalized superparamagnetic Nano iron Particles of preparation

Utilize the coprecipitation synthesis nanometer Fe ₃O ₄Magnetic ion after surperficial dextran is coated, carries out carboxylated functional group and modifies, and utilizes transmission electron microscope to show nano magnetic particle;

2) with people AAV-PEDF and the crosslinked genophore of making of USPIO

(1) preparation PEDF-USPIO carrier:

Pcr amplification PEDF cDNA according to a conventional method, the PEDF cDNA that pcr amplification is gone out with BamHI and NcoI double digestion after, be connected in carrier for expression of eukaryon AAV-GFP, be built into the AAV-PEDF expression vector;

(2) NMR (Nuclear Magnetic Resonance) imaging of PEDF-USPIO carrier:

The PEDF-USPIO that makes adds in the glioma cell substratum, hatches with cell, determines concentration of iron and the concentration of PEDF-USPIO and the relation of incubation time in cell.

2. by the superparamagnetic nano-carrier that carries the retinal pigment epithelium derivative factor claimed in claim 1, it is characterized in that, the concentration of described USPIO is 180 μ g/ml.

3. by the superparamagnetic nano-carrier that carries the retinal pigment epithelium derivative factor claimed in claim 1, it is characterized in that, described incubation time is 24 hours.

4. the purposes of the superparamagnetic nano-carrier that carries the retinal pigment epithelium derivative factor of claim 1 in preparation monitor cerebral glioma growth, differentiation or aggressive preparation.