CN101980005A - Method for marking canine kidney cells through chitosan-quantum dot fluorescent probe - Google Patents
Method for marking canine kidney cells through chitosan-quantum dot fluorescent probe Download PDFInfo
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Abstract
The invention discloses a method for marking canine kidney cells through a chitosan-quantum dot fluorescent probe. The method comprises the following steps of: A, preparing solution of a carboxymethyl chitosan-CdTe quantum dot; transferring the solution of the carboxymethyl chitosan-CdTe quantum dot to a sterilized volumetric flask by using a pipette according to the concentration of the carboxymethyl chitosan-CdTe quantum dot, and adding high-purity water for constant volume; B, digesting the canine kidney cells overgrown in a T25 cell culture flask by using ethylene diamine tetraacetic acid (EDTA)-trypsin so as to form unicellular suspension, counting cells, inoculating the canine kidney cells to a cell culture dish, and adding nutrient solution for culturing; and C, after the canine kidney cells grow adherently, adding the chitosan-quantum dot fluorescent probe into the culture dish, and incubating the probe and the canine kidney cells together to obtain canine kidney cells marked with fluorescence. The method is easy and convenient to operate, has high fluorescent efficiency, good biocompatibility and long stable time, and can be used for observing the cells for a long time; and the fluorescent probe does not have cytotoxicity.
Description
Technical field
The invention belongs to nano material and be used for the technical field of analysis and detection of mark biology and living things system, more specifically relate to a kind of shitosan-quantum dot fluorescence probe and be used for the method for MDCK mark, mark by pair cell component and subcellular structure, the cells physiological active studies that can be used for also can be used for the transport pathway research of inside and outside each receptoroid of cell and the research of exogenous pollution thing and cell interaction.
Background technology
Shitosan is the product by the chitin deacetylate, and many hydroxyls and amino are distributing on its macromolecular chain.Shitosan removes to have outside the superiority such as multifunctionality, biocompatibility, biodegradability and low cytotoxicity, also has unique cross-cell membrane ability.But, limited its application greatly because shitosan only is dissolved in diluted acid and some specific solvent.CMC is a class chitin derivativ of the formation of shitosan behind carboxymethylation reaction, because contained-COO-has reduced-NH on the CMC
3+Electric density, make the cytotoxicity of CMC reduce, have than the better cell compatibility of shitosan; And, expanded its range of application because CMC is water-soluble.
Quantum dot (Quantum dots QDs) claims semiconductor nano microcrystal (Semiconductor nanocrystals) again, be radius less than or approach a based semiconductor nano particle of exciton Bohr radius.Quantum dot has fundamental property such as surface effect, bulk effect and the quantum size effect of general nanoparticle, has superior fluorescent characteristics such as wide excitation spectrum, narrow emission spectrum, emission wavelength that can be accurately tuning, insignificant photobleaching.The optical property that just is being based on the quantum dot uniqueness makes it in researchs such as biological chemistry, molecular biology, cell biology, genomics and proteomics great application prospect be arranged.But as a kind of nano material, because self physicochemical property and Effect of Environmental, quantum dot has also shown certain bio-toxicity effect in the process of using, the stability and the biocompatibility that how to improve the quantum dot fluorescence probe are the problems that presses for solution at present.
MDCK is generally used for separation and the evaluation of influenza virus, sets up this clone from the kidney of normal male Cocker in 1958.This clone is a kind of little Madin-Darby canine kidney(cell line), is characterized in that cell converges the epithelial cell characteristic that differentiation is expressed in the back, is easy to cultivate and go down to posterity.
Summary of the invention
The objective of the invention is at the defective of present all kinds of quantum dot fluorescence probes on using, the method of a kind of shitosan-quantum dot fluorescence probe mark MDCK is provided, CMC good cell compatibility and the superior fluorescence property of quantum dot are combined, utilize them to form fluorescence probe by self assembly, be used for cell marking then at water.With the mark situation of fluorescence microscope MDCK, detect the average fluorescent strength of MDCK in the double dish with flow cytometer.The present invention is easy and simple to handle, and fluorescence efficiency height, good biocompatibility, stabilization time are long, and the fluorescence probe no cytotoxicity can be used for the long observation of cell.
To achieve these goals, the present invention adopts following technical measures:
Basic design of the present invention is: according to CMC and CdTe quantum dot self characteristics, form shitosan-quantum dot fluorescence probe at water by self assembly, with this fluorescence probe and MDCK in the DMEM nutrient solution, in 37 ℃, the CO of 5% volume fraction
2Hatch jointly in the cell culture incubator (down with), pinocytosis, endocytosis or the mode such as engulf of shitosan-quantum dot fluorescence probe by MDCK enters cell interior, with the protein bound in the cell, reaches MDCK is carried out fluorescently-labeled purpose.
The method of a kind of shitosan-quantum dot fluorescence probe mark MDCK comprises the steps:
1, (CMC-CdTe quantum dot self-control, the concise and to the point process of preparation is as follows: CdCl for CMC-CdTe quantum dot solution of preparation 0.0000025-0.001mol/L
2Adjusting its pH value with NaOH solution after solution and mercaptoacetic acid solution mix is alkalescence, adds freshly prepd KTeH again in this solution
4Solution.Change this mixed liquor over to teflon micro-wave digestion jar, the microwave heating system of putting into controllable temperature heats, and treats that solution is cooled to room temperature 20-25 ℃, can obtain the CdTe quantum dot solution after reaction is finished.According to the temperature difference of microwave heating, the fluorescence of CdTe quantum dot will be gradually varied to redness by green.CMC-CdTe quantum dot fluorescence probe that the CdTe quantum dot and the carboxymethyl chitosan sugar juice hybrid reaction of different colours can be generated corresponding fluorescence, those of ordinary skill in the art does not pay any creative work and all can prepare): according to the concentration of CMC-CdTe quantum dot, CMC-CdTe the quantum dot solution that pipettes 0.1 ~ 5ml with transfer pipet is in the 10ml volumetric flask of sterilization, add high purity water (resistivity is greater than 18 M Ω cm) constant volume, standby; Selected shitosan-quantum dot fluorescence probe is to be formed at aqueous phase reactions by CMC and CdTe quantum dot, has good water-solubility and biocompatibility;
2, will cover with in the T25 Tissue Culture Flask and MDCK (MDCK) that growth conditions is good is a single-cell suspension liquid with the EDTA-trypsinization of 0.05% volume ratio, got 1 * 10 after the cell count
4~ 1 * 10
7MDCK be inoculated in the Tissue Culture Dish, add the DMEM nutrient solution of the hyclone contain 10% volume ratio.At 37 ℃, the CO of 5% volume ratio
2Cultivate in the cell culture incubator, the normal adherent growth of cell is bred, obtain being used to implement the MDCK of mark.
3, treat that step 2 MDCK adherent growth after 12 ~ 36 hours, adds shitosan-quantum dot fluorescence probe in double dish, in 37 ℃, the CO of 5% volume ratio
2Hatched jointly 6 ~ 48 hours with cell in the cell culture incubator, can obtain having the MDCK of fluorescence.Discard nutrient solution, with phosphate buffered solution (pH=7.4) washing 2 times, promptly available fluorescence microscope mark situation.
Described shitosan-quantum dot fluorescence probe is to be formed by static complexing, covalent bond, the self assembly of chelating mode at water by CMC and CdTe quantum dot.
The cell of described mark is a MDCK.
The present invention compared with prior art has the following advantages and effect:
The method of shitosan of the present invention-quantum dot fluorescence probe mark MDCK is easy and simple to handle, cell is to the good biocompatibility of fluorescence probe, and fluorescence probe is showed cell toxicity not, the average fluorescent strength of cell and controllable color behind the mark, stabilization time is long, does not influence the normal growth of cell.The fluorescence intensity that cell can be labeled as different colors and cell as required is stable.This method can be used for long period (48 hours) observation and the cells physiological active studies of living cells, also can be used for the transport pathway research of inside and outside each receptoroid of cell and the research of exogenous pollution thing and cell interaction.
Description of drawings
Figure 1A is the fluorescent microscope photo synoptic diagram of green shitosan-quantum dot fluorescence probe mark MDCK.
Figure 1B is the fluorescent microscope photo synoptic diagram of red shitosan-quantum dot fluorescence probe mark MDCK.
The upgrowth situation light field photo synoptic diagram of MDCK when Fig. 2 A is unmarked.
Fig. 2 B is the upgrowth situation light field photo synoptic diagram of mark MDCK after 12 hours.
Fig. 2 C is the upgrowth situation light field photo synoptic diagram of mark MDCK after 24 hours.
Fig. 2 D is the upgrowth situation light field photo synoptic diagram of mark MDCK after 48 hours.
Fig. 2 E is shitosan-quantum dot fluorescence probe different phase of entering MDCK (from 1 to 4 show be that fluorescence probe has just entered cell to the process that is full of whole cell) fluorescence photo synoptic diagram.
Fig. 2 F is shitosan-quantum dot fluorescence probe different phase of entering MDCK (from 1 to 4 show be that fluorescence probe has just entered cell to the process that is full of whole cell) light field photo synoptic diagram.
Fig. 3 A is the average fluorescent strength synoptic diagram (negative control) that flow cytometer detects MDCK when unmarked.
Fig. 3 B is that flow cytometer detects the average fluorescent strength synoptic diagram with cell behind the fluorescence probe mark MDCK of 0.00005mol/L.
Fig. 3 C is that flow cytometer detects the average fluorescent strength synoptic diagram with cell behind the fluorescence probe mark MDCK of 0.0001mol/L.
Fig. 3 D is that flow cytometer detects the average fluorescent strength synoptic diagram with cell behind the fluorescence probe mark MDCK of 0.00025mol/L.
Fig. 3 E is that flow cytometer detects the average fluorescent strength synoptic diagram with cell behind the fluorescence probe mark MDCK of 0.0005mol/L.
Fig. 3 F is that flow cytometer detects the average fluorescent strength synoptic diagram with cell behind the fluorescence probe mark MDCK of 0.001mol/L.
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, the following stated embodiment solvent for use is the high purity water of resistivity greater than 18 M Ω cm, and agents useful for same is analytical reagent.
Embodiment 1:
The method of a kind of shitosan-quantum dot fluorescence probe mark MDCK comprises the steps:
1. prepare green and the red CMC-CdTe quantum dot solution of 0.0005mol/L:
Pipette the green of 2.5mL0.002mol/L and red CMC-CdTe quantum dot solution in the 10mL volumetric flask of sterilization with transfer pipet respectively, add high purity water (resistivity is greater than 18 M Ω cm) constant volume, standby.Selected shitosan-quantum dot fluorescence probe is to be formed by mode self assemblies such as static complexing, covalent bond, chelations at water by CMC and CdTe quantum dot, has good water-solubility and biocompatibility; (CMC-CdTe quantum dot self-control, the concise and to the point process of preparation is as follows: CdCl
2Adjusting its pH value with NaOH solution after solution and mercaptoacetic acid solution mix is alkalescence, adds freshly prepd KTeH again in this solution
4Solution.Change this mixed liquor over to teflon micro-wave digestion jar, the microwave heating system of putting into controllable temperature heats, and treats that solution is cooled to room temperature 20-25 ℃, can obtain the CdTe quantum dot solution after reaction is finished.According to the temperature difference of microwave heating, the fluorescence of CdTe quantum dot will be gradually varied to redness by green.With CMC-CdTe quantum dot fluorescence probe that the CdTe quantum dot and the carboxymethyl chitosan sugar juice hybrid reaction of different colours can generate corresponding fluorescence, those of ordinary skill in the art does not pay any creative work and all can prepare):
2. the cultivation of MDCK:
Is single-cell suspension liquid with the MDCK (MDCK) that has covered with in the T25 Tissue Culture Flask and growth conditions is good with the 0.05%EDTA-trypsinization, gets 1 * 10 after the cell count
6MDCK be inoculated into respectively in 2 Tissue Culture Dishs, add the DMEM(U.S. Gbico company of the hyclone contain 10% volume ratio) nutrient solution.At 37 ℃, the CO of 5% volume ratio
2Cultivate in the cell culture incubator.Obtain being used to implement fluorescently-labeled MDCK.
3. CMC-CdTe quantum dot fluorescence probe is to the mark of MDCK:
Treat that the described MDCK adherent growth of step 2 after 18 hours, adds the described green of step 1 and red shitosan-quantum dot fluorescence probe respectively in double dish, in 37 ℃, the CO of 5% volume ratio
2Hatch jointly with cell in the cell culture incubator, discard nutrient solution after 24 hours, phosphate buffered solution (pH=7.4) washing 2 times, with fluorescence microscope mark situation, the result shows green and red fluorescence (seeing Figure 1A and Figure 1B) on the successful mark of MDCK.
Described shitosan-quantum dot fluorescence probe is to be formed by static complexing, covalent bond, the self assembly of chelating mode at water by CMC and CdTe quantum dot.
The cell of described mark is a MDCK.
Embodiment 2:
The method of a kind of shitosan-quantum dot fluorescence probe mark MDCK, its step is as follows:
The preparation 0.0001mol/L CMC-CdTe quantum dot solution:
Yellow green CMC-CdTe the quantum dot solution that pipettes 0.5mL0.002mol/L with transfer pipet adds high purity water (resistivity is greater than 18 M Ω cm) constant volume in the 10mL volumetric flask of sterilization, standby.Selected shitosan-quantum dot fluorescence probe is to be formed by mode self assemblies such as static complexing, covalent bond, chelations at water by CMC and CdTe quantum dot, has good water-solubility and biocompatibility;
2. the cultivation of MDCK:
Is single-cell suspension liquid with the MDCK (MDCK) that has covered with in the T25 Tissue Culture Flask and growth conditions is good with the 0.05%EDTA-trypsinization, gets 1 * 10 after the cell count
5MDCK be inoculated in the Tissue Culture Dish, add the DMEM nutrient solution contain 10% hyclone.At 37 ℃, the CO of 5% volume ratio
2Cultivate in the cell culture incubator, obtain being used to implement fluorescently-labeled MDCK.
CMC-CdTe quantum dot fluorescence probe to the MDCK mark after morphological observation
Treat the described MDCK adherent growth of step 2 after 28 hours, in double dish, add the described shitosan of step 1-quantum dot fluorescence probe, in 37 ℃, the CO of 5% volume ratio
2Hatch jointly with cell in the cell culture incubator.Used the microscope observing cell growing state in 0,12,24,48 hour, the result shows the MDCK equal normally adherent growth in 0 ~ 48 hour time behind the mark, swelling do not occur, come off and apoptosis, illustrate that cell is good to the biocompatibility of fluorescence probe, fluorescence probe is showed cell toxicity (seeing Fig. 2 A, 2B, 2C, 2D, 2E, 2F) not.
Described shitosan-quantum dot fluorescence probe is to be formed by static complexing, covalent bond, the self assembly of chelating mode at water by CMC and CdTe quantum dot.
The cell of described mark is a MDCK.
Other step is identical with embodiment 1.
Embodiment 3:
The method of a kind of shitosan-quantum dot fluorescence probe mark MDCK, its step is as follows:
1. prepare 0,0.00005,0.0001,0.00025,0.0005,0.001mol/L CMC-CdTe quantum dot solution:
With transfer pipet pipette 0,0.25,0.5,1.25,2.5, yellow green CMC-CdTe quantum dot solution of 5mL0.002mol/L in the 10mL volumetric flask of sterilization, add high purity water (resistivity is greater than 18 M Ω cm) constant volume, standby.Selected shitosan-quantum dot fluorescence probe is to be formed by mode self assemblies such as static complexing, covalent bond, chelations at water by CMC and CdTe quantum dot, has good water-solubility and biocompatibility;
2. the cultivation of MDCK:
Is single-cell suspension liquid with the MDCK (MDCK) that has covered with in the T25 Tissue Culture Flask and growth conditions is good with the 0.05%EDTA-trypsinization, gets 1 * 10 after the cell count
7MDCK be inoculated into respectively in 6 Tissue Culture Dishs, add the DMEM nutrient solution contain 10% hyclone.At 37 ℃, the CO of 5% volume ratio
2Cultivate in the cell culture incubator, obtain being used to implement fluorescently-labeled MDCK.
CMC-CdTe quantum dot fluorescence probe to the MDCK mark after cell show different average fluorescent strengths;
Treat that the described MDCK adherent growth of step 2 after 24 hours, adds the described shitosan of step 1-quantum dot fluorescence probe respectively in double dish, in 37 ℃, the CO of 5% volume ratio
2Hatch jointly with cell in the cell culture incubator, do negative control simultaneously.Discard nutrient solution after 24 hours, phosphate buffered solution (pH=7.4) washing 2 times, is single-cell suspension liquid with 0.05%EDTA-trypsase with the cell dissociation on the double dish, place and use fluorescence microscope mark situation on the microslide, detect the average fluorescent strength of MDCK in the different double dish simultaneously with flow cytometer.The result shows fluorescence on the successful mark of MDCK, and demonstrate different fluorescence intensities, increase with fluorescence probe concentration, cell average fluorescent strength changes to 5000 a.u.(by 50 a.u. and sees Fig. 3 A, 3B, 3C, 3D, 3E, 3F), explanation can come the fluorescence intensity of cell behind the control mark by the concentration that changes fluorescence probe.
Described shitosan-quantum dot fluorescence probe is to be formed by static complexing, covalent bond, the self assembly of chelating mode at water by CMC and CdTe quantum dot.
The cell of described mark is a MDCK.
Other step is identical with embodiment 1.
Claims (4)
1. the method for shitosan-quantum dot fluorescence probe mark MDCK the steps include:
CMC-CdTe quantum dot solution of A, preparation 0.0000025-0.001mol/L: according to the concentration of CMC-CdTe quantum dot, CMC-CdTe the quantum dot solution that pipettes 0.1 ~ 5ml with transfer pipet is in the 10ml volumetric flask of sterilization, add high purity water, resistivity is greater than 18 M Ω cm, constant volume, standby;
B, be single-cell suspension liquid with the EDTA-trypsinization of 0.05% volume ratio, get 1 * 10 after the cell count the MDCK that covered with in the T25 Tissue Culture Flask
4~ 1 * 10
7MDCK be inoculated in the Tissue Culture Dish, add the DMEM nutrient solution of the hyclone contain 10% volume ratio, at 37 ℃, the CO of 5% volume ratio
2Cultivate in the cell culture incubator, the normal adherent growth of cell is bred, obtain being used to implement the MDCK of mark;
C, treat that step (B) MDCK adherent growth after 12 ~ 36 hours, adds shitosan-quantum dot fluorescence probe in double dish, in 37 ℃, the CO of 5% volume ratio
2Hatched jointly 6 ~ 48 hours with cell in the cell culture incubator, obtain having the MDCK of fluorescence, discard nutrient solution, with pH=7.4 phosphate buffered solution washing 2 times, with fluorescence microscope mark situation.
2. the method for a kind of shitosan according to claim 1-quantum dot fluorescence probe mark MDCK is characterized in that: described shitosan-quantum dot fluorescence probe is to be formed by static complexing, covalent bond, the self assembly of chelating mode at water by CMC and CdTe quantum dot.
3. the method for a kind of shitosan according to claim 1-quantum dot fluorescence probe mark MDCK is characterized in that: the cell of described mark is a MDCK.
4. the method for a kind of shitosan according to claim 1-quantum dot fluorescence probe mark MDCK is characterized in that: described CMC-CdTe quantum dot preparation process is as follows, CdCl
2Adjusting its pH value with NaOH solution after solution and mercaptoacetic acid solution mix is alkalescence, adds freshly prepd KTeH again in this solution
4Solution, change this mixed liquor over to teflon micro-wave digestion jar, the microwave heating system of putting into controllable temperature heats, and treats that solution is cooled to room temperature after reaction is finished, obtain the CdTe quantum dot solution, itself and carboxymethyl chitosan sugar juice hybrid reaction are generated CMC-CdTe quantum dot.
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Cited By (3)
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| CN102998291A (en) * | 2012-11-28 | 2013-03-27 | 武汉市疾病预防控制中心 | Quantum-dot-based method for carrying out in-situ and real-time detection on heavy metal ions in cells |
| CN103076312A (en) * | 2012-12-17 | 2013-05-01 | 深圳先进技术研究院 | Cell fluorescent labeling method |
| CN109298180A (en) * | 2018-11-19 | 2019-02-01 | 中南大学 | A method of detection salmonella typhimurium |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103076312A (en) * | 2012-12-17 | 2013-05-01 | 深圳先进技术研究院 | Cell fluorescent labeling method |
| CN103076312B (en) * | 2012-12-17 | 2015-04-01 | 深圳先进技术研究院 | Cell fluorescent labeling method |
| CN109298180A (en) * | 2018-11-19 | 2019-02-01 | 中南大学 | A method of detection salmonella typhimurium |
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