Plutonium-loaded hepatocyte model for plutonium biodynamics research

Abstract

The invention relates to a plutonium-loaded hepatocyte model for plutonium biodynamics research, and belongs to the technical field of nuclear biodynamics. The plutonium-loaded hepatocyte model for the plutonium biodynamics research is a plutonium-loaded hepatocyte model obtained by carrying out plutonium contamination on 4-5 days after hepatocyte inoculation, the inoculation number of hepatocytes is 5 * 10 < 5 >-10 * 10 < 5 >, and the plutonium contamination concentration is 800 pg / mL-1200 pg / mL. And the contamination time of the hepatocytes is 2-24 hours. The hepatocytes are L-02 human hepatocytes. According to the established trace plutonium cell model, species difference of animal models can be avoided, and meanwhile, the influence of an in-vivo neural immune system on a result in an in-vivo experiment is avoided. The established plutonium-loaded human hepatocyte model can be used for illuminating a plutonium transport mechanism and an effect mechanism, a basic support is provided for further estimation research of an internal irradiation dose, and meanwhile, a necessary experimental model can be provided for related research of development of plutonium excretion promoting drugs by utilizing the model to carry out a transfer mechanism of plutonium in cells.

Description

Technical Field

[0001] The invention discloses a plutonium-loaded liver cell model for plutonium biodynamics research, which belongs to the technical field of nuclear biodynamics. Background Art

[0002] Plutonium is an extremely toxic radionuclide and one of the main radiation hazards for radiation workers. It is also the focus of radiation protection. In order to assess the degree of harm to occupational workers, it is necessary to estimate the internal radiation dose. The basis for estimating the internal radiation dose of plutonium is to clarify the transfer and metabolism process of plutonium in the body, that is, the biokinetics of plutonium.

[0003] At present, the research on the biokinetics of plutonium in the liver mainly relies on personnel data and animal experiments. There is little personnel-related data on the biokinetics of plutonium in the liver and there are individual differences. Moreover, it is unrealistic to use personnel to conduct further research on plutonium biokinetics. There are obvious species differences between animal models and humans, especially the biokinetic data of the liver parts of different species vary greatly, and there is great uncertainty. In order to carry out relevant research on plutonium biokinetics, especially on the mechanism of plutonium transfer in cells, it is urgent to establish a cell model that can be used for experimental research. In addition, due to the particularity of plutonium, the purchase of large doses of plutonium is not allowed, which limits related research. In order to carry out relevant research on plutonium biokinetics, it is urgent to establish an in vitro cell model that can be used for experimental research. Therefore, this study can provide an experimental model for conducting relevant research on plutonium biokinetics by establishing a cell biokinetic experimental model for trace plutonium. Summary of the invention

[0004] Technical issues

[0005] At present, the research on the biodynamics of plutonium in the liver uses accident personnel who accidentally ingest plutonium, uses animal experiments to study the biodynamics of plutonium, and conducts research on the transfer coefficient of plutonium in the liver to provide data support for the estimation of internal irradiation dose of plutonium. The purpose of the present invention is to establish a plutonium-loaded hepatocyte model for the study of plutonium biodynamics, and to provide an in vitro experimental model for conducting relevant research on plutonium biodynamics.

[0006] Technical Solution

[0007] A plutonium-loaded hepatocyte model for plutonium biokinetics research, characterized in that the plutonium-loaded hepatocyte model is obtained by plutonium exposure on the 4th to 5th day after hepatocyte inoculation, wherein the number of hepatocytes inoculated in a 6-well plate is 5×10 5 pcs / hole~10×10 5 / well, the concentration of plutonium cell poisoning solution is 800pg / mL~1200pg / mL. The liver cell poisoning time is 2h-24h. The liver cells are L-02 human liver cells. The preparation method of plutonium cell poisoning solution is: use Na2CO3 solution to adjust the pH of 2mol / L plutonium nitrate solution to neutral, add plutonium nitrate solution to the cell culture medium inoculated with liver cells so that its final concentration is the plutonium poisoning concentration set by the poisoning solution, that is, the plutonium cell poisoning solution.

[0008] Beneficial Effects

[0009] The present invention obtains the minimum plutonium poisoning dose range for modeling through experimental exploration, which satisfies the minimum dose required for measurement. Under this dose, the experimental conditions selected by the present invention are used for poisoning, which can meet the subsequent ICP-MS measurement requirements for accuracy.

[0010] The trace plutonium cell model established by the present invention can avoid the species differences of animal models and avoid the influence of the in vivo neuroimmune system on the results in in vivo experiments. The established plutonium-loaded human liver cell model can be used to clarify the plutonium transport mechanism and effect mechanism, providing basic support for further internal irradiation dose estimation research. At the same time, the model can be used to study the transfer mechanism of plutonium in cells, and can also provide a necessary experimental model for related research on the development of plutonium excretion-promoting drugs. It opens up a new research path for the study of plutonium biokinetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Flow chart of the construction of the plutonium-loaded hepatocyte model;

[0012] Figure 2 Hepatocyte growth curve. DETAILED DESCRIPTION

[0013] 1. In vitro culture of normal human hepatocytes and control of cell state stability

[0014] By measuring the temporal changes in the growth state of hepatocytes, the factors affecting the growth state of hepatocytes during the poisoning process are regulated, the cell state during the poisoning process is stabilized, and the uncertainty caused by changes in the cell state in the subsequent inductively coupled plasma mass spectrometry (ICP-MS) measurement process and the in vitro experimental model of plutonium hepatocyte biokinetics is reduced.

[0015] 1) Cell culture

[0016] Normal human liver cell line (L-02) (purchased from Kunming Cell Bank, Chinese Academy of Sciences, human fetal liver cells) was placed in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in an incubator at 37°C and 5% CO2. The medium was replaced every 2-3 days, and the cells were passaged when the cell density reached 80%-90%.

[0017] 2) Hepatocyte growth curve

[0018] CCK-8 reagent (Severn Innovation Biotechnology Co., Ltd., 100 times / box) was used to detect the growth curve of hepatocytes. Hepatocytes were inoculated in 96-well plates, with 1×10 4 Cells, 8 parallel wells per group, a total of 7 groups. Place the culture plate in a 37°C, 5% CO2 incubator for culture. Measure one group every day for 7 consecutive days. During this period, the culture medium was replaced every 2 days for the group not measured. Detection method: Before the measurement, mix the culture medium with the CCK-8 reagent at a volume ratio of 10:1. Aspirate the culture medium in the culture plate and add 200 μL of the mixed reagent in the step to each well. Place the 96-well plate in the incubator and incubate for 1-4 hours. Use an enzyme reader to measure the OD value at 450nm and draw a cell growth curve.

[0019] The results of CCK-8 assay showed that the first 3 days after inoculation of hepatocytes were the retention period, the 3rd to 4th days were the exponential proliferation period, and the hepatocytes reached the growth plateau period on the 4th to 5th day ( Figure 2 ). The growth and proliferation of liver cells in the plateau phase are relatively stable. Under normal circumstances, the liver is also in a stable state in the body. In order to be closer to the state of the liver under normal conditions and to avoid the uncertainty caused by cell proliferation, plutonium poisoning was selected on the 4th to 5th day after liver cell inoculation according to the test results.

[0020] 2. Selection of cell number and plutonium concentration

[0021] Plutonium poisoning: Hepatocytes were seeded in 24-well plates and 6-well plates, with 1×10 5 / well (24-well plate, 5 groups) and 5×10 5 / well (6-well plate, 2 groups), each 6 wells as a group. After 4 days of culture, the 24-well plate was given plutonium cell poisoning solution with plutonium poisoning concentrations of 0, 5, 10, 20, and 40 pg / mL, and the 6-well plate was given plutonium cell poisoning solution with plutonium poisoning concentrations of 0 and 1000 pg / mL. The treated cell culture plate was placed in an incubator for 24 hours and the cells were collected. Preparation of plutonium cell poisoning solution: Use Na2CO3 solution to adjust the pH of 2 mol / L plutonium nitrate solution to neutral, add plutonium nitrate solution to the cell culture medium inoculated with hepatocytes so that its final concentration is the plutonium poisoning concentration set by the poisoning solution, which is the plutonium cell poisoning solution. Plutonium (2 mol / L plutonium nitrate solution) was purchased from China Nuclear Power Technology Co., Ltd., 120 Bq / bottle.

[0022] ICP-MS detection: Inductively coupled plasma mass spectrometry (ICP-MS) is used to measure the plutonium content in cells. The plutonium nitrate solution is diluted with ultrafiltered water to a concentration of 0.1, 0.25, 0.5, 1, and 2 pg / mL for the preparation of the standard curve for ICP-MS detection. The solution must be weighed at each step of the dilution process, and the plutonium concentration in the solution is calculated based on this. The nuclide concentration in the sample is calculated based on the standard curve.

[0023] The results showed that as the number of hepatocytes and the concentration of plutonium increased, the uptake of plutonium by hepatocytes also increased significantly. 5 When the plutonium concentration was 5-40 pg / mL, the plutonium content in liver cells was less than 1 pg, and the ICP-MS measurement error exceeded 10%. 5 When the plutonium concentration was 1000 pg / mL, the plutonium uptake by hepatocytes was 95.502±13.037 pg / 10 6 The maximum measurement error of ICP-MS is 1.8%. According to the above experimental results, the number of cells to be inoculated is 5×10 5 Plutonium cell poisoning solution with a plutonium poisoning concentration of 1000pg / mL was used for each well.

[0024] 3. Establishment of the biokinetic model of plutonium-loaded hepatocytes

[0025] T75 cell culture flasks were inoculated with hepatocytes, and 5×10 6 cells. Fresh culture medium was replaced every 2 days. After 4 days of culture, 10 mL of plutonium-poisoned culture medium (1000 pg / mL) was added and cultured for 24 h. After cell poisoning culture, cells in T75 flask were digested with trypsin and counted. Hepatocytes after poisoning culture were inoculated in 6-well plates, with 1.5×10 6Each well was a group of 6 cells, with a total of 6 groups. In 5 of the groups, the plutonium content in the cells was measured at 2, 4, 8, 24 and 48 hours of culture, and in the other group, the plutonium content in the culture medium was measured at 1, 3, 5 and 7 days. When collecting the culture medium samples, fresh culture medium should be replaced and cultured continuously. On the 7th day, the cells in the culture plate were collected and the plutonium content in the cells was measured using ICP-MS.

[0026] The experimental results showed that the plutonium uptake by liver cells increased with the extension of exposure time. The plutonium content in liver cells was 8.52±0.55pg / 10 at 2h of exposure. 6 cells, and the plutonium uptake by hepatocytes increased to 104.53±1.54pg / 10 at 24h. 6 Cells (Table 1). By continuing to culture the plutonium-loaded hepatocytes, the cells were collected at different time points for measurement, and the natural excretion of plutonium by the hepatocytes was obtained. It was found that as the culture time increased, the plutonium content in the cells gradually decreased (Table 2).

[0027] Table 1 Effects of different exposure time on plutonium uptake by hepatocytes (unit: pg / 10 6 cells, n=6)

[0028]

[0029] Table 2 Plutonium content in the culture medium of the culture medium group at different time periods (unit: pg, n = 6)

[0030]

[0031] 4 Feasibility verification of plutonium-loaded hepatocyte model

[0032] The established in vitro experimental model was used to study the effect of DTPA (diethylenetriaminepentaacetic acid) on the transfer process of plutonium in hepatocytes, and the feasibility of the established in vitro experimental model was verified. 2 mL of culture medium containing 0.5 mmol / LDTPA was added to the plutonium-loaded cells, and 2 mL of ordinary culture medium was added to the control group. The cells were cultured for 2, 4, 8, 24 and 48 hours, and the cells were collected and the plutonium content in the cells was measured by ICP-MS.

[0033] The results showed that the plutonium content in hepatocytes increased with time, regardless of the presence or absence of DTPA. The ratio of the DTPA group to the control group was lower than that of the non-DTPA group, and the ratio of the non-DTPA group to the DTPA group was 1.12±0.11 to 1.37±0.03. The data obtained by the model experiment had a good degree of dispersion, and the intra-group systematic error and accuracy met the experimental requirements.

[0034] The results are shown in Table 2. It was found that the plutonium content in the hepatocytes of both groups decreased over time, regardless of the presence or absence of DTPA. The percentage of residual content in the DTPA group was lower than that in the control group. The ratio of the residual plutonium content in the plutonium-loaded hepatocytes of the control group and the DTPA group was 1.12±0.11 to 1.37±0.03 (Table 3), indicating that DTPA can significantly promote the excretion of plutonium in hepatocytes, which is consistent with the theoretical results. The study of the effect of DTPA (diethylenetriaminepentaacetic acid) on the transfer process of plutonium in hepatocytes has been verified, indicating that the plutonium-loaded hepatocyte model is feasible.

[0035] Table 3 Ratio of plutonium content in cells of DTPA-group, DTPA+group and control group at different time points (n=8)

[0036]

[0037]

[0038] #, compared with the group without DTPA at the same time point, #: p < 0.05; ##: p < 0.01.

Claims

1. A plutonium-loaded hepatocyte model for plutonium biokinetics research, characterized in that: A plutonium-loaded hepatocyte model was obtained by plutonium exposure on the 4th to 5th day after hepatocyte inoculation. The number of hepatocytes inoculated in a 6-well plate was 5×10 5 pcs / hole~10×10 5 The concentration of plutonium cell poisoning solution is 800pg / mL~1200pg / mL.

2. A plutonium-loaded hepatocyte model for plutonium biokinetics research according to claim 1, characterized in that: The liver cell infection time is 2h-24h.

3. A plutonium-loaded hepatocyte model for plutonium biokinetics research according to claim 1 or 2, characterized in that: The hepatocytes are L-02 human hepatocytes.

4. A plutonium-loaded hepatocyte model for plutonium biokinetics research according to claim 1 or 2, characterized in that: The preparation method of plutonium cell poisoning solution is as follows: use Na2CO3 solution to adjust the pH of 2 mol / L plutonium nitrate solution to neutral, add plutonium nitrate solution to the cell culture medium inoculated with hepatocytes so that its final concentration is the plutonium poisoning concentration set by the poisoning solution, which is the plutonium cell poisoning solution.

5. A plutonium-loaded hepatocyte model for plutonium biokinetics research according to claim 3, characterized in that: The preparation method of plutonium cell poisoning solution is as follows: use Na2CO3 solution to adjust the pH of 2 mol / L plutonium nitrate solution to neutral, add plutonium nitrate solution to the cell culture medium inoculated with hepatocytes so that its final concentration is the plutonium poisoning concentration set by the poisoning solution, which is the plutonium cell poisoning solution.

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