CN102344117A - Method for preparing composite nano microsphere for enriching lung cancer cells - Google Patents

Method for preparing composite nano microsphere for enriching lung cancer cells Download PDF

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CN102344117A
CN102344117A CN2010102463156A CN201010246315A CN102344117A CN 102344117 A CN102344117 A CN 102344117A CN 2010102463156 A CN2010102463156 A CN 2010102463156A CN 201010246315 A CN201010246315 A CN 201010246315A CN 102344117 A CN102344117 A CN 102344117A
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sputum
protein
solution
particle
cell
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CN102344117B (en
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陈岗
严彪
李成魁
李辉
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Nantong Deyi Technology Consulting Co.,Ltd.
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SHANGHAI PULMONARY HOSPITAL AFFILIATED TO TONGJI UNIVERSITY
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Abstract

The invention discloses a composite Fe3O4 magnetic nano particle for combining lung cancer cells and a preparation method thereof. Polyimide is modified on the surface of the Fe3O4 magnetic nano particle, and composite protein which has capacity of combining the lung cancer cells is marked on the Fe3O4 magnetic nano particle, wherein the composite protein which has capacity of combining the lung cancer cells consists of staphylococcus A (SPA) protein and one or two of anti-CD44 antibody and anti-CK7 antibody. The invention also discloses a method for enriching and detecting the lung cancer cells in sputum. The method comprises the following steps of: a) collecting sputum, and fully liquefying the sputum; b) adding a composite nano magnetic particle solution and reacting; c) centrifuging reaction liquid to enrich cells in the reaction liquid; and suspending the obtained cells again, tabletting the suspension, dying, sealing and performing microscopic examination by using a thinprep cytology test (TCT) system. Compared with the conventional method, the method has the advantages that the positive rate of the lung cancer cells which is detected in the sputum is improved by more than 30 percent, the detection efficiency is improved obviously and detection cost is reduced to certain extent.

Description

The preparation of enrichment lung carcinoma cell composite nano-microsphere
Technical field
The invention belongs to polymer chemistry and cell biological medical domain, particularly a kind of compound Fe that combines lung carcinoma cell 3O 4Magnetic nanoparticle and preparation method thereof, and a kind of enrichment and the method that detects lung carcinoma cell in the sputum.
Background technology
Lung cancer occupies first of the ten big malignant tumours, has " three height " phenomenon: the incidence of disease is high, the death rate is high, ascensional range is high; Be the principal disease that influences human health, become one of main medical problem of 21 century side by side with AIDS.The means that rely on current diagnosis lung cancer in case find and make a definite diagnosis to be lung cancer, are in middle and advanced stage basically, still are difficult to obtain effective control.Therefore, the early diagnosis that realizes lung cancer be realize early treatment fail the international difficult medical problem that cracks for a long time.
Sputum cell pathology inspection is that the present utmost point is hopeful the effective method that develop and spread is an early diagnosis lung cancer.The main problem that exists is at present: 1, the sputum censorship is wasted time and energy; 2, false negative rate high (20～40%); Reason is: sputum film-making manual operations and to cause the quality of cell loss (sample can not all be taken), smear too poor (inhomogeneous, blocked up; Too much mucus, blood or inflammatory cell have hidden abnormal cell, cellular morphology is preserved not good); Cell component complicated (epithelial cell, inflammatory cell in a large number come off); The lung carcinoma cell that comes off is dispersed in, skewness.
So, must remove irrelevant cell, just can accomplish continuous detecting at low cost, increase substantially positive rate.Therefore, the lung carcinoma cell separation and concentration is the problem that realizes that this international difficult problem of lung cancer early diagnosis at first will solve.If can search out a kind of relatively simple and practical method lung carcinoma cell is separated enrichment from sputum, early treatment lung cancer just is hopeful.
Summary of the invention
Therefore, the technical problem that the present invention will solve is exactly complicated to cell component in the sputum, detects the high defective of lung cancer cast-off cells false negative rate in the sputum, and a kind of compound Fe that combines lung carcinoma cell is provided 3O 4Magnetic nanoparticle and preparation method thereof and a kind of method that detects lung carcinoma cell in the sputum.Lung carcinoma cell has the high positive rate in this method detection sputum.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: a kind of compound Fe that combines lung carcinoma cell 3O 4Magnetic nanoparticle, it is in finishing to gather acetimide, and is marked with the Fe that lung carcinoma cell is had the compound protein of binding ability 3O 4Magnetic nanoparticle, wherein, the described compound protein that lung carcinoma cell is had a binding ability by Protein S PA (staphylococcal protein A) and be selected from anti-CD44 antibody and anti-CK7 antibody in one or both compound proteins of forming.The preferred rabbit antihuman CD 44 of described anti-CD44 antibody, the anti-people CK7 of the preferred rabbit of anti-CK7 antibody.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: a kind of compound Fe of aforesaid combination lung carcinoma cell 3O 4The preparation method of magnetic nanoparticle may further comprise the steps:
1) preparation Fe 3O 4Magnetic nanoparticle;
2) at the Fe of step 1) gained 3O 4The magnetic nanoparticle finishing gathers acetimide;
3) in step 2) gained gather the Fe that acetimide is modified 3O 4The compound protein that the magnetic nanoparticle surface markers has binding ability to lung carcinoma cell, this compound protein is by Protein S PA and be selected from one or both compound proteins formed in anti-CD44 antibody and the anti-CK7 antibody.
Among the present invention, step 1) prepares Fe 3O 4The method of magnetic nanoparticle can this area conventional method, preferable employing coprecipitation.Better step 1) specifically comprises: is that 4: 1 ratio forms finely dispersed ethanolic solution with ferric nitrate and ferrous nitrate in ethanol with the molal weight ratio, and wherein iron concentration is 0.5～1.0mol/L; In the ethanolic solution that makes, add 10 times of epoxychloropropane of molal weight and form colloidal sol, leave standstill the back and form gel to iron ion; The gel that makes is also dry with washed with de-ionized water after ageing; Cleaning and dried gel carry out 400 ℃ of following annealing in process and promptly make Fe 3O 4Deposition; With the Fe that makes 3O 4Deposition is alternately cleaned with deionized water and ethanol successively, is neutral to the pH value, and vacuum drying promptly gets Fe 3O 4Magnetic nanoparticle.The described Fe of step 1) 3O 4The average particle size range of magnetic nanoparticle is 15～50nm preferably, and that better is 20nm.
Among the present invention, step 2) at Fe 3O 4The method that the magnetic nanoparticle finishing gathers acetimide can adopt prior art, and is preferable, step 2) specifically comprise: with Fe 3O 4Magnetic nanoparticle places PBS cushioning liquid, and ultrasonic dispersion slowly adds and gathers acetimide (PEI), after stirring at a slow speed, moves to and continues in the water bath with thermostatic control to stir at a slow speed 24 hours, and bath temperature keeps 30 ℃; With magnetic separation method solid is separated from solution, use distilled water and methyl alcohol alternately to clean to the pH value successively solid and be neutrality, vacuum drying promptly gets the Fe that PEI modifies 3O 4Nano particle.
Among the present invention, preferable, step 3) specifically comprises: will gather the Fe that acetimide is modified 3O 4Nano particle is suspended among the 0.01M pH7.2TBS, gets nano-particle solution, wherein gathers the Fe that acetimide is modified 3O 4The concentration of nano particle is about 3 * 10 13Individual/ml; With 0.01M pH7.2TBS difference soluble protein A and protein B; Mixing; 4 ℃ were reacted 30 minutes; Promptly get compound protein solution, wherein, albumin A concentration is 10mg/ml; Protein B concentration is 1mg/ml; Albumin A is Protein S PA, and protein B is anti-CD44 antibody and/or anti-CK7 antibody, preferred rabbit antihuman CD 44 and/or the anti-people CK7 of rabbit; Above-mentioned compound protein solution is evenly mixed with above-mentioned nano-particle solution, and room temperature 60 minutes promptly gets composite Nano magnetic-particle solution.
The present invention solves the problems of the technologies described above three of the technical scheme that adopted: the method for lung carcinoma cell in a kind of enrichment sputum may further comprise the steps:
A) gather sputum, sputum fully liquefies;
B) in the liquefaction sputum of step a) gained, adding described composite Nano magnetic-particle solution reacts;
C) with the reactant liquor centrifugal enrichment cell wherein of step b).
The present invention solves the problems of the technologies described above four of the technical scheme that adopted: a kind of method that detects lung carcinoma cell in the sputum may further comprise the steps:
A) gather sputum, sputum fully liquefies;
B) in the liquefaction sputum of step a) gained, adding described composite Nano magnetic-particle solution reacts;
C) with the reactant liquor centrifugal enrichment cell wherein of step b), the gained cell is suspended again, adopt the liquid based thin-layer cell to learn detection system then this suspension film-making, dyeing, mounting, microscopy.
Among the present invention, the method for collection and liquefaction sputum is this area conventional method in the step a).Preferable, adding alkaline bleach liquor cleavage liquid in the sputum with abundant liquefaction sputum, described alkaline bleach liquor cleavage liquid is preferred: 1% Proteinase K, the aqueous solution of 10%NaOH.
Among the present invention, the preferred room temperature of the described reaction of step b) left standstill 15 minutes.
Among the present invention, the method for step c) centrifugal enrichment cell is a conventional method, described centrifugal preferred 500rpm, 5 minutes; The cell of centrifugal gained preferably is suspended in to be preserved in the liquid, and it is preferred to preserve liquid: 10% methanol aqueous solution.
Among the present invention, but above-mentioned optimum condition combination in any on the basis that meets this area general knowledge promptly gets each preferred embodiments of the present invention.
The present invention is except that specifying, used percentage all is mass percent.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
" room temperature " described in the present invention is meant the temperature between test operation, is generally 25 ℃.
Than prior art, beneficial effect of the present invention is following: the present invention adopts and gathers acetimide (PEI) to high magnetic characteristics Fe 3O 4Nano grain surface carries out amination to be handled, and makes the methylene and the iron coordination that gather on the acetimide, and through adjusting pH value and temperature, makes and gather the firm and Fe of acetimide 3O 4Nano particle is connected.This material can well be scattered in the aqueous solution, and has excellent biological compatibility and paramagnetism.In order to improve the ability that combines lung carcinoma cell, again mark can combine the compound protein of lung carcinoma cell, not only reduced cost effectively, and improved the efficient of enrichment lung carcinoma cell significantly.This compound Fe 3O 4The function that nano particle has enrichment, separates lung carcinoma cell in the sputum system, integrated liquid basal cell pathology method has improved lung carcinoma cell detects in the sputum reliability and accuracy.The positive rate that detects lung carcinoma cell in the sputum has improved more than 30% than conventional method.And increased the detection sample size, reduced coming and going of measured, detection efficiency obviously improves, and expense decreases, and is suitable for health check-up, generaI investigation.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 is the nano ferriferrous oxide nano particle that polymine coats.
Fig. 2 is that composite nanometer particle is attached to lung cancer cast-off cells surface.
The specific embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1 Fe 3O 4The preparation of nano particle
1) be that 4: 1 ratio forms finely dispersed ethanolic solution with ferric nitrate and ferrous nitrate in ethanol with the molal weight ratio, wherein iron concentration is 0.5～1.0mol/L; In the ethanolic solution that makes, add 10 times of epoxychloropropane of molal weight and form colloidal sol, leave standstill the back and form gel to iron ion; The gel that makes cleans and drying with ethanol after ageing; Carry out 400 ℃ of following annealing in process subsequently and promptly make Fe 3O 4
2) after reaction finishes, with the Fe that makes 3O 4Alternately clean with deionized water and ethanol successively, to the pH value be 7.Under vacuum condition, carry out drying at last, promptly get Fe 3O 4Magnetic nanoparticle.
3) get the Fe of the above-mentioned preparation of 5g 3O 4Magnetic nanoparticle places the 20ml deionized water, and centrifugal (or magnetic) separated after ultrasonic (1000kHz) disperseed 30min, got nanometer Fe 3O 4Precipitate A.
4) precipitate A 5g is refitted in the 10ml PBS cushioning liquid, ultrasonic dispersion slowly adds 70mg and gathers acetimide (PEI), after 200r/min stirs at a slow speed, moves to and continues to stir at a slow speed 24hr in the water bath with thermostatic control, and bath temperature keeps 30 ℃.
5) with magnetic separation method solid is separated from solution, get deposit B and supernatant.
6) deposit B is used successively distilled water and methyl alcohol alternately clean to the pH value be 7, vacuum drying then promptly gets the Fe that PEI modifies 3O 4Nano particle is deposited in the drying basin subsequent use.
Transmission electron microscope and results of grain size analysis show, the Fe that this PEI modifies 3O 4Magnetic nanoparticle be basically single dispersion, spherical, size distribution is narrow, average grain diameter is about 20nm.Wherein Electronic Speculum figure sees Fig. 1.
The preparation of embodiment 2 composite Nano magnetic-particles
1, gets the Fe that PEI modifies 3O 4Nano particle 100mg is suspended among the 10ml 0.01M pH7.2TBS, gets nano-particle solution, wherein the Fe of PEI modification 3O 4The concentration of nano particle is about 3 * 10 13Individual/ml.
2, preparation compound protein: with 3ml 0.01M pH7.2TBS difference soluble protein A (SPA) and protein B (rabbit antihuman CD 44 and CK7) (all available from the gloomy bio tech ltd of treasure); Mixing; Put 4 ℃ of reactions 30 minutes, albumin A concentration is 10mg/ml, and protein B concentration is 1mg/ml.
3, get above-mentioned AB compound protein solution 0.5ml and evenly mix with above-mentioned nano-particle solution 10ml, room temperature 60 minutes promptly gets composite Nano magnetic-particle solution.
The pathology of lung carcinoma cell detect in embodiment 3 sputums
850 routine patients with lung cancer sputum samples are divided into 2 groups at random.The patient need stay phlegm in morning for three days on end, delivers to hospital in 2 hours, otherwise sputum is rotten, and aqtocytolysis can't be diagnosed.Gather patient's sputum 5ml in the phlegm box, add 0.5ml alkaline bleach liquor cleavage liquid (containing 1% Proteinase K, the aqueous solution of 10%NaOH) mixing, room temperature keeps 10min, and sputum fully liquefies.
Experimental group carries out the lung carcinoma cell enrichment and the liquid basal cell pathology detect.In above-mentioned liquefaction sputum, add the composite Nano magnetic-particle solution that 1/10 volume embodiment 2 makes, mixing left standstill 15 minutes.Centrifugal 5 minutes of 500rpm gets to be deposited in and preserves suspension in the liquid (10% methanol aqueous solution).(Thinprep cytology test is TCT) with above-mentioned suspension film-making, dyeing, mounting, microscopy (Fig. 2) to adopt new Bai Shi liquid based thin-layer cell to learn detection system then.
Control group carries out conventional cytology method and detects the lung cancer cast-off cells in the sputum.With the above-mentioned liquefaction sputum of bamboo let picking, smear dyes, mounting, microscopy then.
The result: it is 6.4% (54/850) that conventional cytology method detects positive rate, and it is 38.9% (331/850) that the liquid based cytology method of going again after composite nano-microsphere is handled detects positive rate.

Claims (9)

1. compound Fe who combines lung carcinoma cell 3O 4Magnetic nanoparticle is characterized in that, it is in finishing to gather acetimide, and is marked with the Fe that lung carcinoma cell is had the compound protein of binding ability 3O 4Magnetic nanoparticle, wherein, the described compound protein that lung carcinoma cell is had a binding ability by Protein S PA be selected from one or both compound proteins formed in anti-CD44 antibody and the anti-CK7 antibody.
2. the compound Fe of a combination lung carcinoma cell as claimed in claim 1 3O 4The preparation method of magnetic nanoparticle is characterized in that, may further comprise the steps:
1) preparation Fe 3O 4Magnetic nanoparticle;
2) at the Fe of step 1) gained 3O 4The magnetic nanoparticle finishing gathers acetimide;
3) in step 2) gained gather the Fe that acetimide is modified 3O 4The compound protein that the magnetic nanoparticle surface markers has binding ability to lung carcinoma cell, this compound protein is by Protein S PA and be selected from one or both compound proteins formed in anti-CD44 antibody and the anti-CK7 antibody.
3. preparation method as claimed in claim 2 is characterized in that, the described Fe of step 1) 3O 4The average particle size range of magnetic nanoparticle is 15～50nm.
4. preparation method as claimed in claim 2 is characterized in that step 1) comprises: is that 4: 1 ratio forms finely dispersed ethanolic solution with ferric nitrate and ferrous nitrate in ethanol with the molal weight ratio, and wherein iron concentration is 0.5～1.0mol/L; In the ethanolic solution that makes, add 10 times of epoxychloropropane of molal weight and form colloidal sol, leave standstill the back and form gel to iron ion; The gel that makes is also dry with washed with de-ionized water after ageing; Cleaning and dried gel carry out 400 ℃ of following annealing in process and promptly make Fe 3O 4Deposition; With the Fe that makes 3O 4Deposition is alternately cleaned with deionized water and ethanol successively, is neutral to the pH value, and vacuum drying promptly gets Fe 3O 4Magnetic nanoparticle;
Step 2) comprising:
With Fe 3O 4Magnetic nanoparticle places PBS cushioning liquid, and ultrasonic dispersion slowly adds and gathers acetimide (PEI), after stirring at a slow speed, moves to and continues in the water bath with thermostatic control to stir at a slow speed 24 hours, and bath temperature keeps 30 ℃;
With magnetic separation method solid is separated from solution, use distilled water and methyl alcohol alternately to clean to the pH value successively solid and be neutrality, vacuum drying promptly gets the Fe that PEI modifies 3O 4Nano particle;
Step 3) comprises:
To gather the Fe that acetimide is modified 3O 4Nano particle is suspended among the 0.01M pH7.2TBS, gets nano-particle solution, wherein gathers the Fe that acetimide is modified 3O 4The concentration of nano particle is about 3 * 10 13Individual/ml;
With 0.01M pH7.2TBS difference soluble protein A and protein B, mixing, 4 ℃ were reacted 30 minutes; Promptly get compound protein solution, wherein, albumin A concentration is 10mg/ml; Protein B concentration is 1mg/ml, and albumin A is Protein S PA, and protein B is anti-CD44 antibody and/or anti-CK7 antibody;
Above-mentioned compound protein solution is evenly mixed with above-mentioned nano-particle solution, and room temperature 60 minutes promptly gets composite Nano magnetic-particle solution.
5. the method for lung carcinoma cell in the enrichment sputum is characterized in that, may further comprise the steps:
A) gather sputum, sputum fully liquefies;
B) solution that in the liquefaction sputum of step a) gained, adds composite Nano magnetic-particle as claimed in claim 1 reacts;
C) with the reactant liquor centrifugal enrichment cell wherein of step b).
6. a method that detects lung carcinoma cell in the sputum is characterized in that, may further comprise the steps:
A) gather sputum, sputum fully liquefies;
B) solution that in the liquefaction sputum of step a) gained, adds composite Nano magnetic-particle as claimed in claim 1 reacts;
C) with the reactant liquor centrifugal enrichment cell wherein of step b), the gained cell is suspended again, adopt the liquid based thin-layer cell to learn detection system then this suspension film-making, dyeing, mounting, microscopy.
7. like claim 5 or 6 described methods, it is characterized in that, add alkaline bleach liquor cleavage liquid in the described sputum of step a) with abundant liquefaction sputum, described alkaline bleach liquor cleavage liquid: 1% Proteinase K, the aqueous solution of 10%NaOH.
8. like claim 5 or 6 described methods, it is characterized in that the described reaction of step b) is that room temperature left standstill 15 minutes.
9. like claim 5 or 6 described methods, it is characterized in that, step c) described centrifugal be 500rpm, 5 minutes; The cell of centrifugal gained is suspended in to be preserved in the liquid, preserves liquid and is: 10% methanol aqueous solution.
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CN105467112A (en) * 2015-11-27 2016-04-06 温州生物材料与工程研究所 Immunomagnetic beads applied to immunodetection and preparation method of immunomagnetic beads
CN105617406A (en) * 2014-11-07 2016-06-01 中国科学院苏州纳米技术与纳米仿生研究所 Targeting polypeptide-fluorescent magnetic nanometer complex, preparation method and applications thereof
CN109490526A (en) * 2017-09-13 2019-03-19 南京东纳生物科技有限公司 A kind of preparation method that antibody is orientated the fluorescent microsphere probe modified and the application in immunochromatography
CN109781702A (en) * 2019-01-18 2019-05-21 中国人民解放军军事科学院军事医学研究院 A kind of detection method of magnetic microsphere and preparation method thereof and microorganism

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CN1927400A (en) * 2006-09-25 2007-03-14 唐喜庆 Biomagnetism nano target anti-cancer drug and its preparation
CN101219218A (en) * 2007-01-11 2008-07-16 中国科学院过程工程研究所 Temperature sensing parents block polymer/iron oxide magnetic nano-carrier, preparation method and application thereof
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CN105617406A (en) * 2014-11-07 2016-06-01 中国科学院苏州纳米技术与纳米仿生研究所 Targeting polypeptide-fluorescent magnetic nanometer complex, preparation method and applications thereof
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CN109781702A (en) * 2019-01-18 2019-05-21 中国人民解放军军事科学院军事医学研究院 A kind of detection method of magnetic microsphere and preparation method thereof and microorganism

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