CN101792772A

CN101792772A - Gene transfer material and preparation method

Info

Publication number: CN101792772A
Application number: CN200910193766A
Authority: CN
Inventors: 王深明; 周鸿雁; 徐安武; 叶财盛; 张德元; 刘勇; 胡作军; 王冕; 常光其
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-11-09
Filing date: 2009-11-09
Publication date: 2010-08-04
Anticipated expiration: 2029-11-09
Also published as: CN101792772B

Abstract

The invention discloses a gene transfer material, which is a compound material consisting of calcium carbonate and calcium phytate and having the average grain diameter of about 200 nm, wherein the content of the calcium carbonate is 80%, and the content of the calcium phytate is 20%. The invention further discloses a preparation method of a gene transfer material. The gene transfer material has the advantages of higher transfection efficiency reaching about 40% in the human vascular smooth muscle cells, low toxicity and high cell survival because the calcium carbonate has good biocompatibility, good dispersity and suitability of the requirements of the transfection and low cost because the phytic acid is food additive and has low cost. In addition, the chemicals, i.e. calcium chloride and ammonia in the experiment are common cheap reagents; the preparation method of the material is simple and easy to operate and has good repeatability.

Description

Gene transfer material and preparation method

Technical field

The present invention relates to the gene leading-in technique.

Background technology

Gene import system or method can be divided into two classes: virus type gene import system is a carrier with retrovirus, adenovirus, adeno-associated virus; Non-virus type gene imports, as microinjection, particle gun, coprecipitation of calcium phosphate, cationic-liposome method and emerging nano gene transfer material.Because there are many serious deficiencies in virus type gene import system, might activate proto-oncogene etc. as virus transfection, so non-virus type rotaring transfecting mode is present research focus, in above-mentioned non-virus-type transfection, microinjection once can only be handled a cell; The particle gun penetration power is very limited; Coprecipitation of calcium phosphate method unstable result; Only the cationic-liposome method has showed good transfection efficiency, but too high toxicity has limited its application again.

Summary of the invention

At the shortcoming of prior art, the invention provides a kind of gene transfer material and preparation method, have good cell consistency and stable and higher transfection efficiency.

To achieve these goals, the technical scheme of gene transfer material of the present invention is: a kind of gene transfer material, and it is the matrix material that lime carbonate and phytic acid ca are formed, median size is about 200 nanometers, wherein calcium carbonate content is 80%, and phytic acid ca content is 20%.

Described gene transfer material and egfp grain gene pGFP in conjunction with forming the gene import system, are used for gene transfection in non-covalent mode.

The method for preparing gene transfer material is ultrasonic in ethanol for comprising the steps: that (1) gets analytically pure calcium chloride, volatile salt, cleans with distilled water, washing lotion, distilled water, acetone successively then, dries in air at last; (2) prepare the calcium chloride solution of 0.01M new system with deionized water dissolved chlorine calcium; (3) add the 80mg phytic acid in the 80mL0.01M calcium chloride solution, behind the stirring and evenly mixing it is divided in 4 beakers, in described each beaker the 0.25mL phytic acid is housed, initial concentration is 1g/L, initial PH=3.0; (4) described beaker is sealed with parafilm, and on parafilm, prick three pinpricks symmetrically.Be positioned over the moisture eliminator upper strata, the 10mL small beaker that 3 symmetries are placed is put by moisture eliminator lower floor, fills in each small beaker and grinds levigated volatile salt powder, and small beaker also seals with parafilm, and pricks three pinpricks on parafilm symmetrically; (5) cover dryer door and carry out the diffusion process reaction; Take out sample after (6) 6 hours, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, wash with ethanol again, at room temperature dry then.

Compared with prior art, gene transfer material of the present invention has the following advantages: (1) has higher transfection efficiency, can reach about 40% in human smooth muscle cell; (2) hypotoxicity, because of lime carbonate, calcium phosphate all have excellent biological compatibility, the cells survival rate is very high; (3) dispersion of materials is good, meets the requirement to transfection; (4) low-down cost, phytic acid is foodstuff additive, cost is very low, the pharmaceutical chemicals that uses in testing in addition, calcium chloride and ammoniacal liquor all are common cheap reagent; (5) the material preparation reaction is simple to operation, favorable repeatability.

Description of drawings

Fig. 1 is the scanning electron microscope picture of gene transfer material of the present invention;

Fig. 2 is the projection Electronic Speculum picture of gene transfer material;

Fig. 3 is the fluorescence picture under the inverted fluorescence microscope after the transfection;

Fig. 4 is the cells survival rate picture after the transfection;

Fig. 5 is transfection efficiency figure;

Fig. 6 is the transfection test chart.

Fig. 7 is the figure of green fluorescent albumen (GFP) gene table.

Embodiment

One, the preparation of gene transfer material of the present invention (ACC modifies the nanometer amorphous calcium carbonate of back functionalization through phytic acid ca):

1, raw material is prepared

(molecular weight, MW660), aforementioned base materials all is Aldrich company products for analytically pure calcium chloride, volatile salt, phytic acid.All feedstock production glasswares, all in ethanol ultrasonic 5 minutes, distilled water cleaned then, and uses washing lotion H ₂O/HNO ₃(65%)/H ₂O ₂(35%) (1: 1: 1, v/v/v) clean, clean successively with distilled water, acetone more afterwards, in air, dry at last.

2, gene transfer material preparation method

Prepare the calcium chloride solution of 0.01M new system with deionized water dissolved chlorine calcium, at first, in the 80mL0.01M calcium chloride solution, add the 80mg phytic acid, behind the stirring and evenly mixing, it is divided in the beaker of 4 25mL 20mL in each beaker, initial phytic acid concentration is 1g/L, initial PH=3.0.The 25mL beaker seals with parafilm, and pricks three pinpricks on parafilm symmetrically.Be positioned over the moisture eliminator upper strata, the 10mL small beaker that 3 symmetries are placed is put by moisture eliminator lower floor, fills in each beaker and grinds levigated volatile salt powder, and beaker seals with parafilm, and pricks three pinpricks on parafilm symmetrically.Cover dryer door and carry out the diffusion process reaction.Take out sample after 6 hours, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, ethanol is washed, and final sample at room temperature dries.See also Fig. 1 and Fig. 2, be respectively scanning electron microscope sem and the transmission electron microscope TEM figure of ACC.

Two, the mensuration of bonding properties

1, main raw

Egfp grain (pEGFP-C1); HEPES balanced salt solution (autogamy); Electrophoretic buffer (0.5 * TBE, autogamy); The DNA sample-loading buffer; Bromination second pyridine (EB).

2, main method

1 μ LACC solution (5mgACC is dissolved in the 500 μ L distilled waters) mixes in the 1.5mL centrifuge tube with the 1 μ LpEGFP-C1 aqueous solution (0.1 μ g/ μ L) and 8 μ l distilled waters (pH=7.4water), places room temperature allow its abundant combination in following 30 minutes.The mass ratio of ACC and pEGFP-C1 is used 100: 1 respectively, and 50: 1,30: 1,10: 1,1: 1.Centrifugal 5min under the speed of 5000rpm.Throw out is loaded into 1% agarose (EB0.1 μ g/mL) and under the buffering of TAE, under 100V voltage, runs 40min, observe band then at the 320nm place.

3, result

The result observes, and has multiple band to occur, and this is because pEGFP-C1 has due to the multiple configuration.Mass ratio 30: 1,10: 1,1: 1 place's band is clear obviously illustrate under these mass ratioes, and DNA and nano particle be in conjunction with being not fine, by 100: 1, and band disappearance in 50: 1, description taken in conjunction is complete.

These results show: positively charged pEGFP-C1 and ACC have the binding ratio an of the best, exceed after 50: 1 too much nano particle and seem and recede into the background, and carry out transfection at 50: 1 so use.

Three, transfection

1, main raw

Human smooth muscle cell (HUSMC); ACC@pEGFP-C1 association (above-mentioned preparation); The DMEM substratum; Foetal calf serum FBS; 6 well culture plates.

2, main method

(1) cultivation of cell: HUSMC is digested with trypsinase-EDTA, join in 6 orifice plates (5 * 10 behind the counting ⁶/ hole), optimize reagent to be cultured to cell density be 60%～70% degrees of fusion with containing the DMEM solution of FBS10% and adding transfection.

(2) cell transfecting experiment: cultured cell conditioned medium is removed, the nutrient solution that renews also adds 50: 1 ACC@pEGFP-C1 association of mass ratio, cultivate after 48 hours and respectively its fluorescence, cell survival rate (iodate pyridine sign, flow cytometer detects), transfection efficiency (flow cytometer detection) are measured.

3, interpretation of result

See also Fig. 3, fluorogram has obvious green fluorescence as can be seen, and coverage rate is bigger; See also Fig. 4, be respectively the survival rate of no additive, ACC@pEGFP-C1 association, lipofectamine 2000@ pEGFP-C1 association, can see that the influence of ACC@pEGFP-C1 association pair cell survival rate is very little.Little more a lot of than the influence of lipofectamine 2000@ pEGFP-C1 association.See also Fig. 5, can see that ACC@pEGFP-C1 association can reach about 40% transfection efficiency, this is to be issued to so transfection efficiency in the prerequisite that has guaranteed the cells survival rate.Though not as good as lipofectamine 2000 height, its high cell compatibility is that lipofectamine 2000 is incomparable, has shown the great potential of ACC as transfection reagent; See also Fig. 6, in the transfection test, calcium carbonate granule combines with DNA and it is transferred among the human aortic smooth muscle cell, by fluorescent microscope, observes green fluorescence in cell.

To sum up, the matrix material of forming by the material of good biocompatibilities such as lime carbonate and low toxicity, show the good cell consistency, stablize and higher transfection efficiency, its principle is: phytic acid and calcium ion coordination, carbonate and calcium ion reaction then, self-assembly forms the mixture of amorphous calcium carbonate and phytic acid ca.Because the positive polarity of its surperficial calcium ion makes it possess the potentiality that become the transfection material.The amorphous calcium carbonate that phytic acid can be controlled generation is the spheric pattern, can control the particulate median size about 200nm by the control in reaction times, meets the transfection requirement.

Claims

1. a gene transfer material is characterized in that, it is the matrix material that lime carbonate and phytic acid ca are formed, and median size is about 200 nanometers, and wherein calcium carbonate content is 80%, and phytic acid ca content is 20%.

2. gene transfer material according to claim 1 is characterized in that, it and egfp grain gene pGFP in conjunction with forming the gene import system, are used for gene transfection in non-covalent mode.

3. the preparation method of a gene transfer material is characterized in that, it comprises the steps:

(1) it is ultrasonic in ethanol to get analytically pure calcium chloride, volatile salt, cleans with distilled water, washing lotion, distilled water, acetone successively then, dries in air at last;

(2) prepare the calcium chloride solution of 0.01M new system with deionized water dissolved chlorine calcium;

(3) add the 80mg phytic acid in the 80mL0.01M calcium chloride solution, behind the stirring and evenly mixing it is divided in 4 beakers, in described each beaker the 0.25mL phytic acid is housed, initial concentration is 1g/L, initial PH=3.0;

(4) described beaker is sealed with parafilm, and on parafilm, prick three pinpricks symmetrically.Be positioned over the moisture eliminator upper strata, the 10mL small beaker that 3 symmetries are placed is put by moisture eliminator lower floor, fills in each small beaker and grinds levigated volatile salt powder, and small beaker also seals with parafilm, and pricks three pinpricks on parafilm symmetrically;

(5) cover dryer door and carry out the diffusion process reaction;

Take out sample after (6) 6 hours, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, wash with ethanol again, at room temperature dry then.