CN113767879A - Cancer cell xenograft zebra fish model, and construction method and application thereof - Google Patents
Cancer cell xenograft zebra fish model, and construction method and application thereof Download PDFInfo
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Abstract
The scheme relates to a zebra fish model for cancer cell xenografting, a construction method thereof and application of quantitatively evaluating the proliferation of cancer cells in the zebra fish model body and screening effective anti-tumor drugs according to evaluation results; culturing and passaging cancer cells in a cell culture medium, then carrying out fluorescence labeling by using CFDA-SE fluorescent dye, injecting into a zebra fish embryo pericardial space through microinjection, and screening the zebra fish which develops normally and carries the fluorescence label, namely the cancer cell xenograft zebra fish model. The injection part can reduce the damage of allogenic cancer cell transplantation to the embryo in the pericardium space of the zebra fish embryo and improve the survival of the transplanted cancer cell, thereby ensuring the success of tumor allogenic transplantation; the method can quantitatively evaluate the growth and proliferation conditions of transplanted cancer cells in vivo; analyzing the effect of the target drug on inhibiting or eliminating cancer cells according to the relative expression quantity of the human genes, and further screening out effective anti-tumor drugs.
Description
Technical Field
The invention belongs to the field of biotechnology model construction, and particularly relates to a cancer cell xenograft zebra fish model, a construction method and application thereof.
Background
Cancer imposes a serious burden on China and even the world, and in order to provide clinical symptom detection and treatment decisions for cancer patients, a patient-derived tumor xenograft model is an important in vivo model for biological research, diagnostic marker search and drug screening of tumors.
At present, a mouse/rat is the most common patient-derived tumor allograft model animal, but because the mouse/rat patient-derived tumor xenograft model is a time-consuming and high-cost model, the time from tumor inoculation to drug treatment and pharmacodynamic analysis is usually 2-4 months, and the reason makes the mouse/rat patient-derived tumor xenograft model not suitable for clinical real-time guiding of individualized medication. More and more researches prove that the zebra fish model animal has irreplaceable advantages in the research of tumor-related gene functions and the screening research of anti-tumor drugs. Firstly, after the zebra fish is fertilized, no obvious immune system rejection reaction exists within 1 week, the embryo is transparent, and cells of a primary focus and a transfer focus can be clearly observed; the experimental period is short; the propagation is fast; the low feeding cost enables the zebra fish to become a platform for rapidly screening the in-vivo anti-tumor drugs in high flux. The zebra fish cancer cell allograft model marks cancer cells through fluorescence, then transplants the cancer cells onto zebra fish bodies, can be transplanted into different parts, and further observes and analyzes the proliferation and migration processes after the cancer cell transplantation.
In order to evaluate whether the targeted tumor drug has an anti-tumor effect, the most intuitive method is to observe the proliferation and migration conditions of injected fluorescence labeled cancer cells by utilizing the transparency of zebra fish embryos. The volume, number and migration distance of fluorescently labeled cancer cells are usually detected by imaging techniques such as confocal microscopy, which is time consuming and requires equipment and skilled laboratory personnel to perform, and the fluorescence labeling of cancer cells also has certain limitations. There are currently two common methods for marking cancer cells: one is to stably recombine a fluorescent reporter gene in cancer cells using a virus. The increase in proliferation of cancer cells only if the reporter gene is stably expressed in the cancer cells requires sufficient time to obtain human cancer cells stably transduced with the virus carrying the expressible fluorescent reporter gene; secondly, the cancer cells are directly marked by the fluorescent dye, but the fluorescent dye cannot increase along with the proliferation of the cells, and the proliferation condition of the cancer cells cannot be judged completely according to the fluorescence intensity.
Disclosure of Invention
Aiming at the defects of the imaging technology in the prior art and the difficult problems of quantitative detection of proliferation after cancer cells are transplanted into zebra fish embryos, the invention provides a zebra fish model for cancer cell xenografting from patients and a construction method thereof, and provides a method for quantitatively evaluating proliferation after cancer cells are transplanted into zebra fish embryos, so that the method is used for confirming the inhibition effect of drug treatment on human cancer cells in the embryos.
In order to achieve the purpose, the invention provides the following technical scheme:
a construction method of a cancer cell xenograft zebra fish model comprises the steps of culturing cancer cells in a cell culture medium for passage, then carrying out fluorescence labeling by CFDA-SE fluorescent dye, injecting the fluorescent dye into a zebra fish embryo pericardial space through microinjection, and screening normally-developed zebra fish carrying the fluorescence labeling, namely the cancer cell xenograft zebra fish model.
Further, the cancer cells are derived from primary cells of cancer patients and cancer cell lines, preferably liver cancer 468 cells; the cell culture medium is prepared from 84% of DMEM, 15% of fetal calf serum and 1% of double antibody.
Further, the cell density is controlled to be 5-10X 10 after the culture passage6/ml。
Further, the specific process of the fluorescence labeling is as follows: dissolving CFDA-SE in anhydrous dimethyl sulfoxide to obtain CFSE mother liquor with concentration of 10mM, diluting CFSE mother liquor with PBS to 10 μ M working concentrated solution to stain cancer cells after culture passage, removing dye after staining, washing with cell culture medium, and suspending with heavy suspension until cell density is 2-3 × 104/μL。
Further, the microinjection step includes: collecting fertilized eggs of zebra fish AB strains, anesthetizing the fertilized eggs with tricaine after the embryos grow to 48hpf, and fixing the fertilized eggs on an agar plate; injecting the fluorescence-labeled cells into the fluorescent labeling solution by using a microinjection capillary glass tube needle, wherein the injection volume is 9-10 mu L, and the number of the injected cells is 200-300 per zebra fish embryo.
A cancer cell xenograft zebra fish model constructed by the construction method.
The invention further provides application of the cancer cell xenograft zebra fish model in quantitative evaluation of cancer cell proliferation and screening of effective anti-tumor drugs.
Further, the application process comprises the following steps: screening embryos under a fluorescence microscope, grouping after screening and carrying out drug treatment; extracting genome DNA of each group of samples of the zebra fish, quantitatively detecting the relative expression quantity of human tumor genes in the zebra fish embryo by utilizing qPCR (quantitative polymerase chain reaction), and evaluating the growth and proliferation conditions of transplanted cancer cells in vivo; further, the inhibitory effect of the drug treatment on human cancer cells in the embryo was confirmed.
Further, the concentration of the drug is 0.675-1.350 nM, and the drug treatment time lasts for 2-4 days.
Compared with the prior art, the invention has the beneficial effects that: considering that the allogeneic cell transplantation has great damage to embryonic cells of the animal embryo, so that the normal survival of the embryo is ensured, and the survival rate of cancer cells after the allogeneic cell transplantation is also ensured, the optimal position of tumor injection is determined. The model construction method of the invention is to inject the cancer cells into the pericardium space of the zebra fish, compared with the injection into the yolk sac, the method of the invention can reduce the damage of the allogeneic cancer cell transplantation to the embryo, improve the survival of the transplanted cancer cells and ensure the success of the tumor allogeneic transplantation. The cancer cells injected by the invention can be primary cells of various cancer patients and cancer cell lines. The growth and proliferation conditions of transplanted cancer cells in vivo can be quantitatively evaluated by detecting the change of the amount of human genome DNA in the cancer cell xenograft zebra fish model; analyzing the effect of the target drug on inhibiting or eliminating cancer cells according to the relative expression quantity of the human genes, and further screening out effective anti-tumor drugs.
Drawings
Fig. 1 is a schematic diagram of injection of green fluorescence labeled tumor 468 cells into the pericardial space of zebrafish.
FIG. 2 is a graph showing the fluorescence distribution of tumor 468 cells in the pericardial space of zebra fish after 24h of drug treatment.
FIG. 3 is a graph showing the fluorescence distribution of tumor 468 cells in the pericardial space of zebra fish after 48h of drug treatment.
FIG. 4 is a statistical graph of fluorescence intensity expression of 468 cells injected in zebrafish 24h after treatment with different concentrations of drug.
FIG. 5 is a statistical graph showing the relative expression levels of human GADPH gene after 24h of drug treatment at different concentrations.
FIG. 6 is a statistical graph of fluorescence intensity expression of 468 cells injected in zebrafish after 48h treatment with different concentrations of drug.
FIG. 7 is a statistical graph showing the relative expression levels of human GADPH gene after 48h of drug treatment at different concentrations.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The pharmaceutical reagent apparatus and the like used in the following examples are described below:
1 XPBS (Wuhan Proxel), 0.25% pancreatin (Sigma), CFDA-SE (Invitrogen), Tricaine (Sigma, 400mg of Tricaine powder was weighed into about 97mL of ultrapure water, adjusted to pH 7.0 with 1M Tris (pH 9.0), 2 XPSYBR Green (Dow), Animal tissue Genomic DNA extraction Kit Animal tissue Genomic DNA Kit (Beijing Jianzhu), cell culture box: 37 ℃ and 5% CO2。
Example 1: obtaining fluorescently labeled cancer cells
1) Cell recovery: taking out a tube of frozen liver cancer 468 cells from a refrigerator at the temperature of minus 80 ℃, quickly placing the tube of frozen liver cancer 468 cells in a water bath kettle at the temperature of 37 ℃, placing the melted liver cancer 468 cells in a centrifuge tube, adding 1ml of cell culture medium, centrifuging, sucking out supernatant, continuously adding the cell culture medium, fully suspending, transferring the cell culture medium to a culture dish, supplementing 7ml of cell culture medium, and culturing in a cell culture box;
2) cell passage: when the cells grow to about 70-80% of the bottom of the culture dish for passage, firstly sucking off the cell culture medium, washing the cells for 2-3 times by using 2ml of 1 XPBS, covering the bottom by using 0.25% pancreatin, then putting the cells into a cell culture box for 3-4min, taking out the cells, adding the cell culture medium, then transferring the cells into a centrifuge tube, centrifuging, sucking out the supernatant, continuously adding the cell culture medium, fully suspending, and culturing in the cell culture box until the cells grow to about 80-90% of the bottom of the culture dish; repeating the above operation, resuspending the cells with 2ml of 1 × PBS, centrifuging, and removing the supernatant; adding 1 × PBS into the centrifuge tube to resuspend the cells and controlling the cell density at 5-10 × 106/ml；
3) Cell staining: the cell suspension is centrifuged, CFDA-SE is dissolved in anhydrous dimethylsulfone to prepare CFSE mother liquor with the concentration of 10mM, 2 mu of CFFSE mother liquor is added into 2mL of PBS to prepare staining solution, the cells are resuspended by the staining solution, and the cells are incubated for 25min at 37 ℃ in a dark place. The staining process was then quenched by adding 5 times the volume of the original stain to the mixture, the cells were washed twice with 10mL of cell culture medium after centrifugation, the unbound stain was removed sufficiently, and the cells were finally resuspended in cell culture medium.
Example 2: construction of cancer cell xenograft zebra fish model
1) Cell counting: counting a small amount of the suspension before cell injection, resuspending labeled liver cancer 468 cells by 2mL of 1 XPBS, taking out 10 uL of the suspension, adding 1 XPBS 90 uL of the suspension for 10 times dilution, mixing the suspension uniformly, sucking 10 uL of the diluted suspension, slightly blowing the suspension into a cell counting plate, and counting under a microscope: (33+40+36+ 25)/4X 104＝3.35×106The cell average density of the cell suspension was 3.35X 10/mL6/mL。
2) And (3) injection: collecting AB line fertilized eggs, after the embryos grow to 48hpf (hours after fertilization), anesthetizing with tricaine, placing on a gel plate made of agarose, if the embryos have not been broken, needing to be processedManual stripping was performed under a microscope. The cell culture medium for the cells labeled with fluorescence in example 1 was adjusted to 2 to 3X 104Density of/. mu.L. A microinjection capillary glass tube needle was prepared, and the pericardial space of the 48hpf zebrafish embryo was microinjected under a dissecting scope. As shown in fig. 1, green fluorescence labeled tumor 468 cells were injected into the pericardial space of zebrafish. The injection volume is about 10 mu L, and the number of injected cells is about 200-300 per zebra fish embryo. Culturing the injected embryo in a constant temperature incubator at 35 ℃.
Example 3:
and (3) treating the screened medicine: observing the green fluorescence carried by the injected embryos under a fluorescence microscope, selecting the embryos with normal development and good fluorescence, and randomly dividing into three groups. Two of the groups were treated with 0.675nM and 1.350nM drug, leaving one group as a control, and the drug treatments were sampled 24h and 48h, respectively. The drug is represented by X, and in the embodiment of the scheme, the zebra fish is subjected to drug treatment by apatinib.
Firstly, the tumor inhibition effect of the drug is directly observed through a fluorescence microscope, as shown in fig. 2 and fig. 3, the fluorescence expression of the liver cancer 468 cells in the zebra fish body after 24h and 48h of drug treatment is respectively shown, and the fluorescence expression of the liver cancer 468 cells with fluorescence labels is weakened after the drug treatment in the pericardial space of the zebra fish. The statistical graphs shown in fig. 4 and 6 can be respectively obtained according to the fluorescence expression intensity of the liver cancer 468 cells in the zebra fish body, compared with the blank control group, the fluorescence expression of the liver cancer 468 cells in the two groups of zebra fish treated by the medicine is reduced, and the inhibition effect of the medicine treatment for 24h and 48h on the growth of the liver cancer 468 cells is proved.
Second, quantitative PCR detection
Extracting genomic DNA of each group of samples: the procedures were performed according to the instructions provided in the kit for extracting genomic DNA from animal tissues.
Preparing a quantitative PCR reaction system according to the following requirements: 5 μ L of 2 XSSYBR Green; 0.4 μ L of forward primer: zebrafish primer beta-actin (primer sequence: CCCCATTGAGCACGGTATTG; 10. mu.M), human primer GADPH (primer sequence: CCCCATTGAGCACGGTATTG; 10. mu.M); 0.4 μ L reverse primer: zebrafish primer beta-actin (primer sequence: GTTGGCTTTGGGATTCAGGG; 10. mu.M), 0.4. mu.L human primer GADPH (primer sequence: GTTGGCTTTGGGATTCAGGG; 10. mu.M); 1 μ L of genomic DNA template (24 h or 48h embryonic genomic DNA template sample after drug treatment) and 3.2 μ L of ultrapure water.
After the sample is added, the machine is arranged, the instrument is a fluorescent quantitative PCR instrument, and the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 2 min; 40 cycles: (95 ℃, 10 s; 60 ℃, 10 s; 72 ℃, 20 s).
The quantitative PCR results are shown in FIG. 5 and FIG. 7, compared with the blank control group, the relative expression level of the human GADPH gene of the drug-added group is significantly reduced, which indicates that the drug treatment has an inhibitory effect on the growth of the liver cancer 468 cells injected into the zebra fish body.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor with which the invention may be practiced, and further modifications may readily be effected therein by those skilled in the art, without departing from the general concept as defined by the claims and their equivalents, which are not limited to the details given herein and the examples shown and described herein.
Claims (9)
1. A construction method of a cancer cell xenograft zebra fish model is characterized in that cancer cells are cultured in a cell culture medium for passage, then CFDA-SE fluorescent dye is used for fluorescence labeling, and the fluorescence labeled zebra fish is injected into a pericardial space of a zebra fish embryo through microinjection, so that the zebra fish which develops normally and carries the fluorescence label is screened out to be the cancer cell xenograft zebra fish model.
2. The method for constructing the cancer cell xenograft zebra fish model according to claim 1, wherein the cancer cells are derived from primary cells of cancer patients and cancer cell lines; the cancer cells are liver cancer 468 cells; the cell culture medium is prepared from 84% of DMEM, 15% of fetal calf serum and 1% of double antibody.
3. The method of constructing the cancer cell xenografted zebrafish model of claim 1, wherein the cell density is controlled to 5 to 10 x 10 after the culture passage6/ml。
4. The method for constructing the cancer cell xenograft zebra fish model according to claim 1, wherein the fluorescence labeling comprises the following specific steps: dissolving CFDA-SE in anhydrous dimethyl sulfoxide to obtain CFSE mother liquor with the concentration of 10mM, diluting the CFSE mother liquor with PBS to 10 mu M working concentrated liquor to stain cancer cells after culture passage, removing dye after staining, washing with a cell culture medium, and resuspending until the cell density is 2-3 multiplied by 104/μL。
5. The method of constructing a cancer cell xenograft zebrafish model according to claim 1, wherein the step of operating microinjection comprises: collecting fertilized eggs of zebra fish AB strains, anesthetizing the fertilized eggs with tricaine after the embryos grow to 48hpf, and fixing the fertilized eggs on an agar plate; injecting the fluorescence-labeled cells into the fluorescent labeling solution by using a microinjection capillary glass tube needle, wherein the injection volume is 9-10 mu L, and the number of the injected cells is 200-300 per zebra fish embryo.
6. A cancer cell xenograft zebrafish model constructed using the construction method according to any one of claims 1 to 5.
7. Use of the cancer cell xenograft zebra fish model of claim 6 for quantitatively evaluating cancer cell proliferation in vivo and for screening effective antitumor drugs.
8. The use of the cancer cell xenograft zebrafish model of claim 7, wherein the use comprises the steps of: screening embryos under a fluorescence microscope, grouping after screening and carrying out drug treatment; extracting genome DNA of each group of samples, and quantitatively detecting the relative expression quantity of human tumor genes in zebra fish embryos by utilizing qPCR (quantitative polymerase chain reaction) to evaluate the growth and proliferation conditions of transplanted cancer cells in vivo; further, the inhibitory effect of the drug treatment on human cancer cells in the embryo was confirmed.
9. The use of the cancer cell xenograft zebrafish model of claim 8, wherein the drug concentration is 0.675 to 1.350nM and the drug treatment time is 2 to 4 days.
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