CN112251486B - In-vitro screening and biological effect evaluation method for three-dimensional cell model radiopharmaceuticals - Google Patents
In-vitro screening and biological effect evaluation method for three-dimensional cell model radiopharmaceuticals Download PDFInfo
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- 238000011156 evaluation Methods 0.000 title claims abstract description 15
- 238000012216 screening Methods 0.000 title claims abstract description 15
- 230000004071 biological effect Effects 0.000 title claims abstract description 12
- 229940121896 radiopharmaceutical Drugs 0.000 title claims description 12
- 239000012217 radiopharmaceutical Substances 0.000 title claims description 12
- 230000002799 radiopharmaceutical effect Effects 0.000 title claims description 12
- 201000008968 osteosarcoma Diseases 0.000 claims abstract description 19
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 20
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/20—Small organic molecules
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention provides a radioactive drug in-vitro screening and biological effect evaluation method based on a three-dimensional cell model, which comprises the following steps: (1) constructing an in vitro three-dimensional cell model culture substrate; (2) constructing an in-vitro three-dimensional cell model of human osteosarcoma; (3) To be of different radioactivity 177 Lu-Herceptin is added into a three-dimensional cell model of human osteosarcoma MG63, and incubated for 4 hours at 37 ℃; taking the supernatant culture solution of each hole, soaking the cell model for 2 minutes by using a 1M phosphate buffer solution precooled at 4 ℃, combining the soaking solution and the supernatant, and carrying out radioactive counting by using a Gamma Counter; cells in the model were digested with pancreatin, separated from the matrix, and cell fluid was collected and radiocounted using Gamma Counter. The method of the invention applies the three-dimensional cell model to the radioactive therapeutic drugs, carries out scientific evaluation on biological effects caused by radiation from multi-scale layers such as molecules, cells, sub-tissues and the like, and realizes the efficient screening and the evaluation of the drug effect of the radioactive therapeutic drugs in vitro.
Description
Technical Field
The invention belongs to the technical field of radiopharmaceutical screening and evaluation, and particularly relates to a radiopharmaceutical in-vitro screening and biological effect evaluation method based on a three-dimensional cell model.
Background
At present, in the research and development process of the medicine, the pre-clinical medicine effect of the medicine is generally researched and evaluated on a cell level by utilizing a two-dimensional cell model, but the two-dimensional cell model has great difference in solid tumors in vivo, because the two-dimensional cell model in an in-vitro environment is obviously subjected to phenotypic transformation, the culture environment is obviously different from the real in-vivo environment, and the effect of the medicine in the two-dimensional cell model is obviously different from the effect of the medicine in a patient.
In the process of screening and evaluating the radioactive therapeutic drugs, the limitations of the radioactive therapeutic drugs are that the experimental results are greatly different from the real conditions in the body because the two-dimensional cell model is used for carrying out preclinical in-vitro drug effect research. This is also one of the reasons why the systematic study at the cellular level after the radionuclide has been exposed to human tissue is still imperfect. In particular, the lack of systematic studies on the effects of targeting and in vivo residence time changes on the cellular level caused by organisms resulting from the loading of the prodrugs with nuclides has led to clinical radiopharmaceutical doses and dosing regimens that can only be tried by practice, with inadequate theoretical basis support, and a significant risk to the patient.
The growth environment provided in the three-dimensional cell model is closer to in-vivo tissues in the aspects of intercellular communication, extracellular matrix and the like, so that the cells are closer to in-vivo environment in the aspects of migration, differentiation, proliferation, apoptosis, gene expression and the like. The cell state in the three-dimensional cell model is between the two-dimensional cell model and the cells in the animal body, so that the in-vivo environment can be better simulated, and the difference between in-vitro cell culture and in-vivo research is made up. In particular, in preclinical studies of drugs, one important use of three-dimensional cell models is for the establishment of in vitro disease models to evaluate the response of cells to drugs. Studies have shown that the results obtained from testing drug toxicity using three-dimensional cell models are closer to those of in vivo toxicity tests.
At present, the research on a three-dimensional cell model is less, and how to apply the three-dimensional cell model to the development process of the radioactive therapeutic drug is expected to scientifically evaluate the biological effect caused by radiation from a multi-scale layer such as molecules, cells, sub-tissues and the like, so that the efficient screening and the evaluation of the drug effect of the radioactive therapeutic drug in vitro are realized, and the technical problem to be solved urgently is achieved.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a radioactive drug in-vitro screening and biological effect evaluation method based on a three-dimensional cell model. The invention establishes a method for applying a three-dimensional cell model capable of well simulating in-vivo biological environment to a radioactive therapeutic drug, and the method can be used for better scientific evaluation of biological effects caused by radiation from multi-scale layers such as molecules, cells, sub-tissues and the like, so that efficient screening and drug effect evaluation of the radioactive therapeutic drug in vitro are realized.
The invention aims to provide a radiopharmaceutical in-vitro screening and biological effect evaluation method based on a three-dimensional cell model, which comprises the following steps of:
(1) Constructing a hydroxyapatite-phosphorylated hyaluronic acid in-vitro three-dimensional cell model culture medium;
(2) Constructing an in-vitro three-dimensional cell model of human osteosarcoma;
(3) To be of different radioactivity 177 Lu-Herceptin is added into a three-dimensional cell model of human osteosarcoma MG63, and incubated for 4 hours at 37 ℃; taking the supernatant culture solution of each hole, soaking the cell model for 2 minutes by using a 1M phosphate buffer solution precooled at 4 ℃, combining the soaking solution and the supernatant, and carrying out radioactive counting by using a Gamma Counter; cells in the model were digested with pancreatin, separated from the matrix, and cell fluid was collected and radiocounted using Gamma Counter.
Further, the method for constructing the culture medium of the in-vitro three-dimensional cell model in the step (1) comprises the following steps: the phosphorylated hyaluronic acid is dissolved in phosphate buffer solution to form 4% (w/v) solution, nano-hydroxyapatite nano particles are dispersed in pure water to obtain 12% (w/v) solution, the two solutions are evenly mixed in a syringe with equal volume, the mixture is rapidly injected into a fixed-shape die within 1 minute, and after the mixture is placed for 10 minutes, the concentration of the phosphorylated hyaluronic acid is 2% (w/v), and the concentration of the hydroxyapatite is 6% (w/v).
The percentage concentration refers to the mass volume percentage concentration of the corresponding substances, and means that phosphorylated hyaluronic acid is dissolved in phosphate buffer solution to form 40mg/ml solution, nano hydroxyapatite nano particles are dispersed in pure water to obtain 120mg/ml solution, the final phosphorylated hyaluronic acid concentration is 20mg/ml, and the hydroxyapatite concentration is 60mg/ml.
Further, the phosphate buffer was 20mM PBS.
Further, the method for constructing the in-vitro three-dimensional cell model of the human osteosarcoma in the step (2) comprises the following steps: human osteosarcoma MG63 cells were digested and counted, and the cells were resuspended in DMEM high-sugar medium containing 10% fetal bovine serum at 10 5 cell/well density was seeded in 24 well plates with hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture medium and placed at 37℃in 5% CO 2 In the incubator of (2), the culture was continued for 10 days, and fresh culture medium was changed every 2 days.
Further, the different activities in step (3) range from 3 to 0.0029 μCi.
Further, the step (3) is that 177 The specific activity of the Lu-Herceptin label was 2mci/mg.
Further, in step (3), the pH of the phosphate buffer solution is=7.4.
The beneficial effects of the invention are as follows:
the invention establishes a method for applying an in-vitro three-dimensional cell model to screening and evaluating the radioactive therapeutic drugs, and researches on the radiation biological effect generated by the radioactive therapeutic drugs based on the model, so that the in-vitro three-dimensional cell model can be proved to be used for screening and evaluating the drug effect of the radioactive drugs. The method of the invention realizes better scientific evaluation of biological effects caused by radiation from multi-scale layers such as molecules, cells, sub-tissues and the like, thereby realizing efficient screening of radioactive therapeutic drugs in vitro and evaluation of drug effects.
Drawings
FIG. 1 is a graph of a rheological mechanical analysis of a hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture substrate;
FIG. 2 is a macroscopic morphology of a hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture substrate;
FIG. 3 is a microscopic morphology (SEM) map of a hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture substrate;
FIG. 4 is a two-dimensional cell model and three-dimensional cell model pair of MG63 177 Uptake results of Lu-Herceptin;
FIG. 5 is a schematic diagram of a preferred embodiment of the present invention 177 Effect of Lu-Herceptin on apoptosis in MG63 two-dimensional and three-dimensional cell models.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be specifically described with reference to the following examples, which are provided for explaining and illustrating the present invention only and are not intended to limit the present invention. Some non-essential modifications and adaptations of the invention according to the foregoing summary will still fall within the scope of the invention.
Example 1
Constructing a hydroxyapatite-phosphorylated hyaluronic acid in-vitro three-dimensional cell model culture matrix.
Experimental materials: hydroxyapatite nanoparticle (particle diameter about 200 nm), phosphorylated hyaluronic acid polymer, phosphate buffer solution, 5ml sterile syringe, self-made circular mold with diameter of 1.5 cm.
The experimental method comprises the following steps: the phosphorylated hyaluronic acid is dissolved in phosphate buffer (20 mM PBS) to form a 4% (w/v) solution, nano-hydroxyapatite nanoparticles are dispersed in pure water to obtain a 12% (w/v) solution, the two solutions are uniformly mixed in a syringe with a medium volume, the mixture is rapidly injected into a fixed-shape mold within 1 minute, and after the mixture is placed for 10 minutes, the hydroxyapatite-phosphorylated hyaluronic acid is cultured in vitro, the concentration of the final phosphorylated hyaluronic acid is 2% (w/v), and the concentration of the hydroxyapatite is 6% (w/v).
Example 2
Constructing an in-vitro three-dimensional cell model of human osteosarcoma:
human osteosarcoma MG63 cells were digested, counted and resuspended in DMEM high sugar medium containing 10% fetal bovine serum. At 10 5 cell/well density was seeded in 24 well plates with hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture medium and placed at 37℃in 5% CO 2 In the incubator of (2), the culture was continued for 10 days, and fresh culture medium was changed every 2 days.
Example 3
Constructing an in-vitro two-dimensional cell model of human osteosarcoma:
human osteosarcoma MG63 cells were digested, counted and resuspended in DMEM high sugar medium containing 10% fetal bovine serum. At 10 5 cell/well density was seeded in 24 well plates and placed at 37℃in 5% CO 2 The cells are cultured in the incubator until the cells form a compact monolayer, and fresh culture solution is changed every 2 days.
Example 4
Uptake and apoptosis experiments:
1. the method for constructing the three-dimensional cell model and the two-dimensional cell model of human osteosarcoma MG63 is the same as the experimental methods in example 2 and example 3
2. Uptake experiments of radiopharmaceuticals by cells
To different radioactivity (3-0.0029 muCi) 177 Lu-Herceptin (with a specific activity of 2 mci/MG) was added to the three-dimensional and two-dimensional cell models of human osteosarcoma MG63 and incubated at 37℃for 4 hours. The supernatant from each well was incubated with 1M phosphate buffer (ph=7.4) pre-chilled at 4 ℃ for 2 minutes, and the incubation was combined with the supernatant and counted radioactively using a Gamma Counter. Cells in the model were digested with pancreatin, separated from the matrix, and cell fluid was collected and radiocounted using Gamma Counter. Comparing the effect of different cell models on the uptake of radiopharmaceuticals, it was found that the uptake rate of MG63 cells on radiopharmaceuticals in the three-dimensional cell model was significantly higher than that of the two-dimensional cells.
Radiopharmaceutical uptake% = cell radioactivity count/(cell radioactivity count + supernatant radioactivity count + soak radioactivity count) ×100%
3. Influence of radiopharmaceuticals on apoptosis
50. Mu. Ci 177 Lu-Herceptin (with a specific activity of 2 mci/MG) was added to a three-dimensional and two-dimensional cell model of human osteosarcoma MG63 (10) 5 cells/well), 37 ℃,5% CO 2 Incubate under conditions for 4 days. The supernatant culture was discarded, the cell model was soaked in 1M phosphate buffer (ph=7.4) for 2 minutes, cells in the model were digested with pancreatin, separated from the matrix, and the cell fluid was collected. Cells were stained using the annexin v-PI standard procedure and apoptosis was determined using a flow cytometer. From the apoptosis results, it can be found that the tolerance of MG63 cells in the three-dimensional cell model to radiation damage caused by radionuclides is significantly better than that of the two-dimensional cell model.
Claims (6)
1. A radiopharmaceutical in vitro screening and biological effect evaluation method based on a three-dimensional cell model, which is characterized by comprising the following steps:
(1) Constructing a hydroxyapatite-phosphorylated hyaluronic acid in-vitro three-dimensional cell model culture medium;
(2) Constructing an in-vitro three-dimensional cell model of human osteosarcoma; the method for constructing the human osteosarcoma in-vitro three-dimensional cell model comprises the steps of inoculating human osteosarcoma MG63 cells on a hydroxyapatite-phosphorylated hyaluronic acid in-vitro three-dimensional cell model culture medium for culture;
(3) To be of different radioactivity 177 Lu-Herceptin is added into a three-dimensional cell model of human osteosarcoma MG63, and incubated for 4 hours at 37 ℃; taking the supernatant culture solution of each hole, soaking the cell model for 2 minutes by using a 1M phosphate buffer solution precooled at 4 ℃, combining the soaking solution and the supernatant, and carrying out radioactive counting by using a Gamma Counter; digesting cells in the model by using pancreatin to separate the cells from a matrix, collecting cell sap, and performing radioactive counting by using Gamma Counter;
the construction method of the in-vitro three-dimensional cell model culture medium comprises the following steps: the phosphorylated hyaluronic acid is dissolved in phosphate buffer solution to form 40mg/ml solution, nano-hydroxyapatite nano-particles are dispersed in pure water to obtain 120mg/ml solution, the two solutions are uniformly mixed in a syringe with equal volume, the mixture is rapidly injected into a fixed-shape die within 1 minute, and after the mixture is placed for 10 minutes, the concentration of the phosphorylated hyaluronic acid is 20mg/ml, and the concentration of the hydroxyapatite is 60mg/ml.
2. The method of claim 1, wherein the phosphate buffer is 20mM PBS.
3. The method according to claim 1, wherein the specific construction method of the human osteosarcoma in vitro three-dimensional cell model in step (2) is as follows: human osteosarcoma MG63 cells were digested and counted, and the cells were resuspended in DMEM high-sugar medium containing 10% fetal bovine serum at 10 5 Density of individual cells/well was seeded in 24 well plates with hydroxyapatite-phosphorylated hyaluronic acid in vitro cell culture medium and placed at 37 ℃,5% co 2 In the incubator of (2), the culture was continued for 10 days, and fresh culture medium was changed every 2 days.
4. The method of claim 1, wherein the different activities in step (3) range from 3 to 0.0029 μci.
5. The method according to claim 1, wherein in step (3) the 177 The specific activity of the Lu-Herceptin label was 2mci/mg.
6. The method of claim 1, wherein the phosphate buffer in step (3) has a ph=7.4.
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| 抗骨质疏松药物细胞筛选模型的研究进展;刘璐;张西正;邓桂茹;;中国药学杂志(06);第406页左栏倒数第1段-右栏第1段、第407页左栏倒数第1段-右栏倒数第2段 * |
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