CN100501402C - Method of stably labelling liver cancer cell - Google Patents
Method of stably labelling liver cancer cell Download PDFInfo
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- CN100501402C CN100501402C CNB2005100200899A CN200510020089A CN100501402C CN 100501402 C CN100501402 C CN 100501402C CN B2005100200899 A CNB2005100200899 A CN B2005100200899A CN 200510020089 A CN200510020089 A CN 200510020089A CN 100501402 C CN100501402 C CN 100501402C
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Abstract
The present invention discloses a method for stably labeling liver cancer cells by fluorescence, which has the steps of liver cancer cell culture, liver cancer cell culture on tiny glass sheets, liverA method for stably labeling liver cancer cell includes combining liver cancer cell with specific AFP single cloning antibody used as the first resistance factor, washing and climbing plate of PBS, ca cancer cell fixing, washing and sealing of fixed liver cancer cell culturing tiny glass sheet, combination of liver cancer cell and special AFP monoclonal antibody (used as first resistance factors),rrying out fluorescent label by using QDs-IgG composite probe as the second resistance factor, washing and climbing plate of PBS, re-staining cell core with DAPI, washing and climbing plate of PBS and washing of the liver cancer cell culturing tiny glass sheet by PBS, fluorescence labeling (with QDs-IgG compound probes as second resistance factors), washing of the liver cancer cell culturing tiny preserving sample in light shielded condition. glass sheet by PBS, karyon counterstaining by 4', 6-diamidino-2-phenylindole (DAPI), washing of the liver cancer cell culturing tiny glass sheet by PBS and lucifugous preserving of samples. The method of the present invention has the advantages of easy implementation and convenient operation, and can stably labeling the liver cancer cells in high efficiency by the fluorescence. The present invention provides a proper experimental method for long-term observation and research on the liver cancer cells.
Description
Technical field
The invention belongs to quantum dot immune fluorescent cytochemistry technical field, be specifically related to a kind of method of stable labelling liver cancer cell.
Background technology
The immunofluorescence cell chemical technology is to adopt the known antibodies (or antigen) of fluorescent material mark as probe, detect the target antigen (or antibody) in tissue to be measured, the cell specimen, have fluorescent material on the antigen antibody complex that forms, under fluorescent microscope, owing to be subjected to the excitation source irradiation, fluorescent material sends bright fluorescence, so just can tell the position and the character thereof of antigen (or antibody).This class technology is widely used in modern biology and medical science, before more than 60 years, and by the immunofluorescence technique of establishments such as Coons, the existing so far very big progress of development.Continuous development along with high sensitivity and high specific probe technique, it is more and more obvious to be with FITC, rhodamine that the traditional organic fluorescent dye of representative is used the limitation that exists: the narrow spectrum width of launching of excitation spectrum, in imaging, be difficult for differentiating, vulnerable for biochemical reaction and metabolism, anti-photobleaching ability is low, and the imaging fluorescent lifetime is short etc.Since nineteen ninety-seven, along with quantum dot (quantum dots, QD
s) technology of preparing improve constantly QD
sApplication at biomedical aspect has also caused concern widely gradually.QD
sBe a class radius less than or approach the semiconductor nano crystal grain of exciton Bohr radius, as a kind of novel fluorescent material, QD
sCan overcome the deficiency of traditional organic fluorescent dye, have exciting light spectrum width and continuously, the detection sensitivity height, anti-photobleaching ability is strong, and fluorescence intensity is high and stablize characteristics such as good biocompatibility.The present external QD that takes
sThe method of labeled cell mainly is by chain (mould) Avidin-biotin system combination, this method operate comparatively complexity and cost higher, practicality is not strong.In the cancer research field, no-trump QD still both at home and abroad
sBe applied to the report of the immunofluorescence label research of hepatoma carcinoma cell.
Summary of the invention
The object of the present invention is to provide a kind of method of stable labelling liver cancer cell, adopted QD
s-IgG compound is as fluorescence probe, by specific AFP antibody indirect labelling liver cancer cell.Easy to implement the method, easy to operate, can carry out fluorescence labeling to hepatoma carcinoma cell efficiently and stably, provide suitable experimental technique to the long-time observational study of hepatoma carcinoma cell.
In order to realize above-mentioned task, the present invention adopts following technical measures, and concrete steps are as follows:
1. cellular incubation human hepatoma cell strain HCCLM6 (Li Y, Tian B, Yang J, Zhao L, Wu X, Ye SL, Liu YK, Tang ZY.Stepwise metastatic human hepatocellular carcinoma cellmodel system with multiple metastatic potentials established through consecutivein vivo selection and studies on metastatic characteristics.J Cancer Res ClinOncol.2004; 130 (8): 460-468), nutrient solution for contain 10% hyclone (Hyclone, Utah, RPMI-1640 nutrient culture media USA) (Sigma, Saint Louis, Missouri, USA).Condition of culture: 37 ℃, 5%CO
2/ 95% air, saturated humidity.Change nutrient solution every other day, went down to posterity once in per 4～6 days.
2. the processing of cellular incubation slide and double dish, the glass culture dish of 20mm * 20mm cover glass and diameter 9cm is cleaned with clear water, 2% (vol/vol) hydrochloric acid solution soaked overnight, clear water soaks, and scrubs oven for drying gently with banister brush, temperature is controlled at 50～70 ℃, put in the sour cylinder and soaked 1～3 day through sulfuric acid-potassium dichromate cleaning solution (concentrated sulphuric acid 100ml, potassium dichromate 100g, distilled water 1000ml), tap water flushing 20 times, distilled water washing 10 times, soaked overnight is after the oven dry, do roasting sterilization, temperature is controlled at 150～170 ℃, and 2h is standby.
3. cell climbing sheet is tiled in cover glass on the double dish, each 4 cover glass in double dish shop.With 0.125% trypsase (AMRESCO, Solon, Ohio, USA) vitellophag is by 2 * 10
6Individual/each double dish inoculation, 37 ℃, 5%CO
2/ 95% air, saturated humidity is cultivated 3d, and cell covers with cover glass.
4. washing slide, cleaning fluid is 0.01mol/l, pH 7.2～7.4, PBS (KCl 0.2g, KH
2PO
40.2g, NaCl 8g, Na
2HPO
47H
2O 2.16g, distilled water 1000ml), wash each 3min 2 times;
5. fixing, immobile liquid is-20 ℃ of methyl alcohol, every slide 3～5ml, remove unnecessary PBS after, fixing 15min.
6. washing slide, cleaning fluid is 0.01mol/l, pH 7.2～7.4, PBS (above-mentioned steps 4), washs 3 times, each 3min.
7. sealing, (Ohio is USA) with 0.1% (vol/vol) triton x-100 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4) for AMRESCO, Solon in order to contain 1% (wt/vol) bovine serum albumin(BSA) for confining liquid.Sealing condition is: after removing unnecessary PBS, wet box is hatched, and 37 ℃, 20～30min.
8. add one anti-, remove unnecessary confining liquid after, drip mouse-anti people's AFP monoclonal antibody (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing), wet box is hatched, 37 ℃, 1～2h (or 4 ℃ of refrigerator overnight).
9. washing slide, cleaning fluid for contain 0.5% (vol/vol) tween #20 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4) washs 3 times, each 3min.
10. add two and resist, behind the remaining PBS, drip the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe and (synthesize with molecular science institute in the removal step 4 by Wuhan University's chemistry.Thank to petrel, Pang Daiwen .II-VI type quantum dot prepares and applied research progress in biological detection. the analytical chemistry research report, 2004,8 (32): 1099～1103), dilution is for containing 5% (wt/vol) bovine serum albumin(BSA) (AMRESCO, Solon, Ohio, USA) PBS (above-mentioned steps 4), dilution back IgG concentration is 10 μ g/ml.Wet box, room temperature 20-25 ℃ of lucifuge hatched 1h.
11. the washing slide, cleaning fluid is for containing 0.5% (vol/vol) tween #20 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4), lucifuge washing 3 times, each 3min.
12. nucleus is redyed, and behind the PBS in the removal step 4, drips 4 ', 6-diamidino-2-phenylindone (DAPI, Sigma, Saint Louis, Missouri, USA), dilution is for containing 5% (wt/vol) bovine serum albumin(BSA) (AMRESCO, Solon, Ohio, USA) PBS (above-mentioned steps 4), dilution back concentration is 2 μ g/ml.Wet box, room temperature 20-25 ℃ of lucifuge dyeing 15～20min.
13. washing slide, cleaning fluid are 0.01mol/l, pH 7.4, PBS (above-mentioned steps 4), and lucifuge washing 3 times, each 3min, buffering glycerine mounting, 4 ℃ keep in Dark Place.
14. fluorescent microscope or confocal laser scanning fluorescence microscope.
The present invention compares with existing traditional organic fluorescence labelling technique and has the following advantages and effect:
1, imaging is stable, and under identical continuous agitation condition, the fluorescence half life period of the fluorescently-labeled cell of the present invention is to be 180 times of traditional organic fluorescence mark of representative with fluorescein isothiocynate (FITC).
2, fluorescent brightness height, the fluorescence intensity of mark of the present invention can reach 5～20 times of traditional organic fluorescence mark (FITC).
3, the holding time long, the fluorescently-labeled cell of the present invention be saved under 4 ℃ of lucifuge conditions surpass a week after, fluorescence still exists, and the basic quencher of back fluorescence of spending the night of the sample of present traditional organic fluorescence mark.
4, mark high specificity can overcome the influence of sample autofluorescence.
The present invention compares with at present external Quantum Dot Labeling method, needn't adopt chain (mould) Avidin-biotin system to carry out combination, relevant Avidin covalent modification quantum dot, the steps such as biotinylation of antibody have been omitted, whole process can be finished in 3～4 hours, both simplified step, easy to operate, saved correlative charges again, practical.
Description of drawings
Fig. 1 is the immunofluorescence label image of the present invention to human liver cancer cell HCCLM6, wherein: figure A400 *, figure B1000 *.According to the invention described above and condition,, just obtained the external picture that is marked as of hepatoma cell strain HCCLM6AFP antigen clearly by confocal laser scanning fluorescent microscope.Bright Chinese red fluorescence partly is by QD
sExpress the zone of AFP antigen in the cytoplasm of mark, blue portion is the nuclear area of DAPI dye marker.
Fig. 2 for the present invention to the fluorescently-labeled specificity Test Drawing of human liver cancer cell HCCLM6,1000 *.In order further to prove the fluorescently-labeled high specific of present technique, adopt specificity mouse-anti people AFP antibody anti-as one, the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe is anti-as two, singly dye the AFP antigen of labelling human hepatoma carcinoma cell HCCLM6, replace mouse-anti people AFP antibody anti-with PBS simultaneously as one, and after the same method with condition flag as negative control, confocal laser scanning fluorescence microscope.Wherein scheme A for anti-as one with specificity mouse-anti people AFP antibody, the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe is as two anti-mark results.Figure B is for anti-as one with PBS, and the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe is as two anti-negative control mark results.The result shows: do not occur tangible QD in the visual field of negative control
sChinese red fluorescence, this forms a sharp contrast with the result who uses specificity one to resist, thereby proves the high specificity of this technology.
Fig. 3 is the fluorescent stability Test Drawing of labelling human hepatoma carcinoma cell HCCLM6 of the present invention, 1000 *.The present invention and traditional FITC fluorescence labeling are compared test.With fixing and place continuous agitation 1h under the Laser Scanning Confocal Microscope with the hepatoma carcinoma cell of CdSe/ZnS core/shell type water-soluble QDs-IgG compound probe and FITC mark respectively, every 3min obtains an image automatically, and compares the attenuation of both fluorescence intensities under laser continuous agitation.
Wherein: the image that figure A, B, C, D, E show obtains when being the 0th, 12,24,36,48 minute, adopt mark of the present invention;
The image that figure a, b, c, d, e show obtains when being the 0th, 12,24,36,48 minute adopts traditional FITC fluorescence labeling;
Fig. 4 shows for the fluorescence intensity standardized curve of continuous agitation 1h, result: in 1h, the fluorescence intensity of FITC obviously decays, and the QD of mark of the present invention
sThe fluorescence intensity kept stable is constant, has demonstrated fully the stability of mark fluorescent of the present invention.
Fig. 5 is for placing 4 ℃ to keep in Dark Place complete hepatoma carcinoma cell creep plate according to embodiment 1 step mark, and every 2d observes once, and figure A, B, C, D represent the 0th, 2,4,8 day image that obtains respectively.The result shows: the blue-fluorescence of the DAPI dyestuff of labeled cell nuclear just disappeared on the 2nd day, and the QD of specific marker cytoplasm AFP antigen
sChinese red fluorescence is still high-visible at the 8th day.This has further proved the characteristics that the mark fluorescent holding time of the present invention is long.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment only to be used to the present invention is described and be not used in restriction the scope of protection of present invention.
Embodiment 1:
The present invention is to the fluorescence labeling of human liver cancer cell HCCLM6, and step is as follows:
A. cellular incubation human hepatoma cell strain HCCLM6, nutrient solution for contain 10% hyclone (Hyclone, Utah, RPMI-1640 nutrient culture media USA) (Sigma, Saint Louis, Missouri, USA).Condition of culture: 37 ℃, 5%CO
2/ 95% air, saturated humidity.Change nutrient solution every other day, went down to posterity once in per 4～6 days.
B. the processing of cellular incubation slide and double dish, the glass culture dish of 20mm * 20mm cover glass and diameter 9cm is cleaned with clear water, 2% (vol/vol) hydrochloric acid solution soaked overnight, clear water soaks, scrub gently with banister brush, oven for drying, temperature are controlled at 50～70 ℃, put in the sour cylinder to soak 1～3 day through concentrated sulphuric acid 100ml, potassium dichromate cleaning solution, distilled water 1000ml, tap water flushing 20 times, distilled water washing 10 times, soaked overnight is after the oven dry, do roasting sterilization, temperature is controlled at 150～170 ℃, and 2h is standby.
C. cell climbing sheet is tiled in cover glass on the double dish, each 4 cover glass in double dish shop.With 0.125% trypsase (AMRESCO, Solon, Ohio, USA) vitellophag is by 2 * 10
6Individual/each double dish inoculation, 37 ℃, 5%CO
2/ 95% air, saturated humidity is cultivated 3d, and cell covers with cover glass.
D. wash slide, cleaning fluid is 0.01mol/l, and pH 7.2～7.4, PBS (KCl 0.2g, KH
2PO
40.2g, NaCl 8g, Na
2HPO
47H
2O 2.16g, distilled water 1000ml), wash each 3min 2 times;
E. fixing, immobile liquid is-20 ℃ of methyl alcohol, every slide 3～5ml, remove unnecessary PBS after, fixing 15min.
F. wash slide, cleaning fluid is 0.01mol/l, and pH 7.2～7.4, and PBS (above-mentioned steps 4) washs 3 times, each 3min.
G. sealing, (Ohio is USA) with 0.1% (vol/vol) triton x-100 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4) for AMRESCO, Solon in order to contain 1% (wt/vol) bovine serum albumin(BSA) for confining liquid.Sealing condition is: after removing unnecessary PBS, wet box is hatched, and 37 ℃, 20～30min.
H. add one anti-, remove unnecessary confining liquid after, drip mouse-anti people's AFP monoclonal antibody (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing), wet box is hatched, 37 ℃, 1～2h (or 4 ℃ of refrigerator overnight).
I. wash slide, cleaning fluid for contain 0.5% (vol/vol) tween #20 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4) washs 3 times, each 3min.
J. add two anti-, remove unnecessary PBS after, it is (synthetic with molecular science institute by Wuhan University's chemistry to drip the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe.Thank to the preparation of petrel Pang Daiwen .II-VI type quantum dot and applied research progress in biological detection. the analytical chemistry research report, 2004,8 (32): 1099～1103), dilution is for containing 5% (wt/vol) bovine serum albumin(BSA) (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4), dilution back IgG concentration is 10 μ g/ml.Wet box, the room temperature lucifuge is hatched 1h.
K. wash slide, cleaning fluid is for containing 0.5% (vol/vol) tween #20 (AMRESCO, Solon, Ohio, PBS USA) (above-mentioned steps 4), lucifuge washing 3 times, each 3min.
L. nucleus is redyed, remove unnecessary PBS after, drip 4 ', 6-diamidino-2-phenylindone (DAPI, Sigma, Saint Louis, Missouri, USA), dilution is for containing 5% (wt/vol) bovine serum albumin(BSA) (AMRESCO, Solon, Ohio, USA) PBS (above-mentioned steps 4), dilution back concentration is 2 μ g/ml.Wet box, room temperature lucifuge dyeing 15～20min.
M. wash slide, cleaning fluid is 0.01mol/l, and pH 7.4, PBS (above-mentioned steps 4), lucifuge washing 3 times, each 3min, buffering glycerine mounting.
N. confocal laser scans fluorescence microscope.The results are shown in Figure 1.
Embodiment 2:
The specificity test of labelling liver cancer cell of the present invention, step is as follows:
A.～and G. is with embodiment 1, and the hepatoma carcinoma cell creep plate that sealing is good is divided into two groups, and one group is anti-as one with specificity mouse-anti people AFP antibody, and one group is anti-as one with PBS (with embodiment 1 step D), operates according to embodiment 1 step H..
I.～K. is with embodiment 1
L. cushion the glycerine mounting, confocal laser scanning fluorescence microscope.The results are shown in Figure 2.
Embodiment 3:
Labelling liver cancer cell fluorescent stability test of the present invention, step is as follows:
A.～and I. is with embodiment 1, and the hepatoma carcinoma cell creep plate that washing is good is divided into two groups, and one group is anti-as two with the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe, and one group is anti-as two with FITC-IgG, operates according to embodiment 1 step J..
K. with embodiment 1
L. cushion the glycerine mounting, confocal laser scanning fluorescence microscope.The results are shown in Figure 3, Fig. 4 and Fig. 5.
Claims (1)
1, a kind of method of stable labelling liver cancer cell, it comprise the following steps into:
A, cellular incubation human hepatoma cell strain HCCLM6, nutrient solution are the RPMI-1640 nutrient culture media that contains 10% hyclone, condition of culture: 37 ℃, the mixed gas volume ratio is 5%CO in the incubator
2/ 95% air, saturated humidity is changed nutrient solution every other day, goes down to posterity once in per 4～6 days;
The processing of B, cellular incubation slide and double dish, the glass culture dish of 20mm * 20mm cover glass and diameter 9cm is cleaned with clear water, 2%/vol/vol hydrochloric acid solution soaked overnight, clear water soaks, scrub with banister brush, oven for drying, temperature are controlled at 50～70 ℃, put in the sour cylinder to soak 1～3 day through sulfuric acid 100ml, potassium dichromate 100g, cleaning solution distilled water 1000ml, tap water flushing 20 times, distilled water washing 10 times, soaked overnight is after the oven dry, do roasting sterilization, temperature is controlled at 150～170 ℃, and 2h is standby;
C, cell climbing sheet are tiled in cover glass on the double dish, and each 4 cover glass in double dish shop is used 0.125% trypsin digestion and cell, by 2 * 10
6Individual/each double dish inoculation, 37 ℃, the mixed gas volume ratio is 5%CO in the incubator
2/ 95% air, saturated humidity is cultivated 3d, and cell covers with cover glass;
D, washing slide are with KCl 0.2g, KH
2PO
40.2g, NaCl 8g, Na
2HPO
47H
2The PBS of O 2.16g, distilled water 1000ml preparation 0.01mol/l, pH7.2～7.4 washs 2 times as cleaning fluid, each 3min;
E, fixing, immobile liquid is-20 ℃ of methyl alcohol, every slide 3～5ml removes behind the above-mentioned residue PBS fixedly 15min;
F, washing slide, cleaning fluid is 0.01mol/l, pH7.2～7.4, PBS washs 3 times by the formulation of above-mentioned steps D, each 3min;
G, sealing, confining liquid are the PBS that contains 1%/wt/vol bovine serum albumin(BSA) and 0.1%/vol/vol triton x-100, and PBS is above-mentioned steps D, and sealing condition is: wet box is hatched, 37 ℃, and 20～30min;
H, add one anti-, removes above-mentioned residue confining liquid after, dropping mouse-anti people AFP monoclonal antibody, wet box is hatched, 37 ℃, 1～2h or 4 ℃ of refrigerator overnight;
I, washing slide, cleaning fluid is the PBS that contains 0.5%/vol/vol tween #20, PBS washs 3 times by the formulation of above-mentioned steps D, each 3min;
J, add two anti-, remove the remaining PBS of the rapid operation of previous step after, drip the water-soluble QDs-IgG compound of CdSe/ZnS core/shell type probe, dilution is the PBS that contains the 5%/wt/vol bovine serum albumin(BSA), dilution back IgG concentration is 10 μ g/ml, wet box, and 20～25 ℃ of lucifuges are hatched 1h;
K, washing slide, the used cleaning fluid of cleaning fluid and above-mentioned steps I is identical, and PBS is by the formulation of above-mentioned steps D, lucifuge washing 3 times, 3min at every turn;
L, nucleus are redyed, after removing the remaining PBS of the rapid operation of previous step, drip 4 ', 6-diamidino-2-phenylindone, dilution is the PBS that contains the 5%/wt/vol bovine serum albumin(BSA), and PBS is by the formulation of above-mentioned steps D, and dilution back concentration is 2 μ g/ml, wet box, 20～25 ℃ of lucifuge dyeing 15～20min;
M, washing slide, cleaning fluid is 0.01mol/l, pH7.2～7.4, PBS is by the formulation of above-mentioned steps D, and lucifuge is washed 3 times, each 3min, buffering glycerine mounting, 4 ℃ keep in Dark Place.
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| CN101531993B (en) * | 2008-03-12 | 2013-05-29 | 中国医学科学院肿瘤研究所 | Method and kit for stabilizing fluorescence labeling nucleus |
| CN102628869B (en) * | 2012-04-19 | 2014-04-02 | 上海蓝怡科技有限公司 | Preparation for improving freeze-drying stability of alpha fetal protein antibody |
| CN104458050A (en) * | 2014-11-27 | 2015-03-25 | 江南大学 | Fluorescent copper namo clusters for living cell temperature monitoring |
| CN109900895A (en) * | 2017-12-11 | 2019-06-18 | 北京和杰创新生物医学科技有限公司 | The adhesion process method on cell climbing sheet glass flake surface |
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Non-Patent Citations (4)
| Title |
|---|
| Ⅱ2 Ⅵ型量子点制备及其在生物检测中应用研究进展. 谢海燕，庞代文.分析化学研究报告，第32卷第8期. 2004 |
| Ⅱ2 Ⅵ型量子点制备及其在生物检测中应用研究进展. 谢海燕，庞代文.分析化学研究报告，第32卷第8期. 2004 * |
| Establishment of cell clones with different metastaticpotentialfrom the metastatic hepatocellular carcinoma celllineMHCC97. Yanl LI, Zhao-You Tang, Sheng-Long Ye etal.ORIGINAL RESEARCH,Vol.7 No.5. 2001 |
| Establishment of cell clones with different metastaticpotentialfrom the metastatic hepatocellular carcinoma celllineMHCC97. Yanl LI, Zhao-You Tang, Sheng-Long Ye etal.ORIGINAL RESEARCH,Vol.7 No.5. 2001 * |
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