CN116990286A

CN116990286A - Method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

Info

Publication number: CN116990286A
Application number: CN202310965587.9A
Authority: CN
Inventors: 李玉民; 唐富天; 顾龙
Original assignee: Lanzhou University Second Hospital
Current assignee: Lanzhou University Second Hospital
Priority date: 2023-08-02
Filing date: 2023-08-02
Publication date: 2023-11-03

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a method for measuring the boron concentration in cells in brain tumor cell boron uptake medicine research. The application adopts a one-step method to directly digest the cultured adherent cells with mixed concentrated sulfuric acid and concentrated nitric acid, then uses ICP-AES to simultaneously determine the boron element intensity and the phosphorus element intensity in the cells, calculates the cell number from the linear equation of the phosphorus element intensity and the cell number, then uses a standard boron solution linear equation to calculate the boron concentration, and divides the boron concentration by the cell number to obtain the boron concentration in single cells.

Description

Method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

Technical Field

The application relates to the technical field of biological medicines, in particular to a method for measuring boron concentration in cells in research of taking boron medicine by brain tumor cells.

Background

Brain tumors seriously endanger human life health, and although great progress is made in tumor diagnosis and treatment, the treatment still does not reach the expected effect. Boron Neutron Capture Therapy (BNCT) is a binary radiotherapy for precisely and targeted killing of tumor cells, and the principle is that boron drugs taken in tumor cells react with neutrons to generate cell inner cores, so that the tumor cells are killed within a cell scale range, and the influence on normal cells is small. BNCT is the most advanced international accurate radiotherapy method for treating malignant brain tumor at present. BPA is currently used internationally and clinically, the high selectivity of BPA to tumors is the basis of BNCT treatment, and the determination of boron drug concentration in tumor cells is a key element in the formulation of treatment schemes.

In brain tumor cell uptake of BPA boron drug studies, it is necessary to determine the intracellular boron concentration and cell number to represent the boron concentration per cell. The prior art is divided into two steps, wherein the first step is to digest the cultured adherent cells with pancreatin and then count the cells by a cell counter, and the second step is to digest the counted cells with mixed concentrated sulfuric acid and concentrated nitric acid and then measure the boron concentration by ICP-AES. Dividing the obtained boron concentration by the number of cells is the boron concentration in each cell. However, each experiment requires re-culturing the cells, then digesting the cells, and counting the cells by using a cytometer, and the two-step technique of the prior art has the disadvantages of complex process and long time consumption.

Disclosure of Invention

In order to simplify the ICP-AES two-step method and obtain a substituted simple cell counting method, thereby simplifying the step of measuring the boron concentration in cells.

The application provides a method for measuring boron concentration in cells in research of taking boron drugs by brain tumor cells, which comprises the following steps:

s1, analyzing the linear relation between the cell number and the phosphorus element intensity, wherein the linear relation comprises the following steps:

culturing brain tumor cells, and performing cell count;

preparing brain tumor cell culture solutions with different cell numbers, respectively centrifuging to obtain cell precipitates of brain tumor cells, digesting with a mixture of concentrated nitric acid and sulfuric acid to obtain a digestive solution, measuring the phosphorus element intensity by using an inductively coupled plasma-atomic emission instrument (inductively coupled plasma-atomic emission spectroscopy, ICP-AES) at a wavelength of 213.618nm, analyzing the linear correlation between the phosphorus element intensity and the cell number, and calculating to obtain a linear equation;

s2, adding boron medicine into brain tumor cells for incubation, washing, then digesting, measuring the phosphorus element intensity at the wavelength of 213.618nm by utilizing ICP-AES, measuring the boron element intensity at the wavelength of 249.773nm, calculating the cell number by using a linear equation, calculating the boron concentration by using a standard boron solution linear equation, and dividing the boron concentration by the calculated cell number to obtain the boron concentration in single cells.

Further, in S2, the digestion process is to digest the washed brain tumor cells with an acid mixture.

Further, in S1, the preparation process of the brain tumor cell culture solution with different cell numbers comprises the following steps: cell culture medium was adjusted to 1.00×10 ⁶ Cell culture solution of cell number/mL, then aspirated 0.05X10 respectively ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ mL of cell culture broth.

Further, in S1, the centrifugation condition is 600-800g/min for 10-16min.

Further, in S1, the acid mixture is prepared from concentrated nitric acid and sulfuric acid according to a volume ratio of 1:1, and mixing.

Further, the brain tumor cells are any one of U87 cells and U251 cells.

Further, the meter of the linear correlation between the intensity of the U87 cell phosphorus and the cell numberThe linear equation is calculated as: y=72370x+1708, r ² ＝0.9995；

y represents the intensity of phosphorus element, x represents the number of cells, R ² Representing the correlation coefficient.

Further, the calculated linear equation of the linear correlation between the intensity of U251 cell phosphorus and the number of cells is: y=77250x+1846, r ² ＝0.9994；

Further, in S2, the standard boron solution linear equation is: y=162500x+651.3, r ² ＝0.9996；

y represents the intensity of boron element, x represents the boron concentration, R ² Representing the correlation coefficient.

Further, in S2, the boron drug is borylphenylalanine.

Compared with the prior art, the application has the beneficial effects that:

1. in the follow-up study of taking boron medicine by the same cell, only one-step method is needed to directly digest the cultured adherent cells with mixed concentrated sulfuric acid and concentrated nitric acid, then ICP-AES is utilized to simultaneously determine the boron element intensity and the phosphorus element intensity in the cells, then the cell number is calculated from the linear equation of the phosphorus element intensity and the cell number, then the standard boron solution linear equation is utilized to calculate the boron concentration, and then the boron concentration is divided by the calculated cell number to obtain the boron concentration in single cells;

the method only needs to obtain the linear equation of the phosphorus element intensity and the cell number in the first experiment, the subsequent related research of the same cell does not need to perform cell culture and counting, only needs to detect the phosphorus element intensity and the boron element intensity of the cell sample once, then calculates the cell number through the obtained linear equation of the corresponding cell, calculates the boron concentration of the same cell sample through the linear equation of the standard boron solution, and divides the calculated cell number by the boron concentration to obtain the boron concentration in single cell, thereby saving the experimental processes of cell culture, digestion and cell counter counting in a large number of experimental researches, obviously simplifying the experimental process and saving a large amount of time.

2. The method for measuring the boron concentration in the cells in the research of taking boron medicine by brain tumor cells provided by the application can directly detect the phosphorus intensity at the wavelength of 213.618nm and the boron intensity at the wavelength of 249.773nm in one-step experiment with respect to the subsequent experiments of the cells after determining the linear correlation and the linear equation between the phosphorus intensity and the cell number of the same brain tumor cells, then directly calculate the cell number by utilizing the linear equation of the cells, and then calculate the boron concentration in the cells by utilizing the linear equation of a standard boron solution. Compared with the prior art, the method has the advantages that in each experiment, two steps are needed, namely, cells are firstly cultured, cells are digested by pancreatin to be subjected to cell counting, then the boron concentration is measured by ICP-AES, and then the boron concentration in each cell is calculated, so that the method simplifies the steps and saves a large amount of experiment time.

3. The method for measuring the boron concentration in the cells in the research of taking the boron drug by the brain tumor cells is not only suitable for the brain tumor cells, but also suitable for other tumor cells, including cells such as head and neck cancer, melanoma, gastric cancer, liver cancer, breast cancer, colorectal cancer, pancreatic cancer and the like, and the method for measuring the boron concentration in the cells has high accuracy.

Drawings

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the linear correlation between phosphorus intensity and cell number of brain tumor cells;

in the figure, A is a linear correlation diagram between the phosphorus element intensity of U87 cells and the cell number;

b is a linear correlation diagram between the phosphorus element intensity of U251 cells and the cell number.

FIG. 2 shows the uptake of boron by brain tumor cells;

in the figure, A is the uptake condition of U87 cells on boron element;

b is the uptake condition of U251 cells on boron element.

Detailed Description

The following detailed description of specific embodiments of the application is, but it should be understood that the application is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. The experimental methods described in the examples of the present application are conventional methods unless otherwise specified, and materials, reagents, etc. used in the examples described below are commercially available.

Example 1: a method for measuring cell number in brain tumor cell boron uptake medicine research.

1. Linear correlation between phosphorus intensity and cell number of brain tumor cell line U87 cells

Brain tumor cell line U87 was grown in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ The mL of the cell culture solution was placed in a centrifuge tube, centrifuged at 600g/min for 10min, the supernatant was discarded, the cell pellet was washed 1 time with 0.9% NaCl, and centrifuged at 600g/min for 10min, the supernatant was discarded. The cell pellet was digested with 50mL of the acid mixture for 2 hours to obtain a digested solution.

The acid mixed solution is prepared by mixing 68% of concentrated nitric acid and 98% of sulfuric acid according to a volume ratio of 1:1.

To the digest was added a total of 4.5mL of 10% nitric acid (dilution metering) and the final volume was 5mL. The digestion solution was measured for the phosphorus element intensity (phosphorus intensity for short) at a wavelength of 213.618nm using ICP-AES (PQ 9000, analytik Jena GmbH, jena, germany). The linear correlation between phosphorus intensity and cell number was analyzed, and the linear equation and the related parameters were calculated, and the results were shown in a of fig. 1.

The linear relationship between the number of U87 cells and the phosphorus intensity was good (a of fig. 1), and the linear equation was y=72370x+1708 (R ² =0.9995), y represents phosphorus element intensity, x represents cell number, R ² Representing the correlation coefficient. Correlation coefficient R ² =0.9995, demonstrating a good linear relationship, the U87 cell number can be calculated by the above equation.

2. Method for measuring boron concentration in cells in research on uptake of BPA by brain tumor cell line U87 cells

U87 cells were cultured in DMEM 25cm containing 10% FBS ² In a flask. After 90% of the cells are fused, the culture medium is replaced, BPA with the concentration of 12.5mg/mL and 25mg/mL is added into the U87 cells in groups, and the cells are incubated for 3 hours. The cells were washed 3 times with 0.9% nacl solution. The cells were digested with 1mL of the acid mixture at 60℃for 2 hours, then 4mL of 10% nitric acid was added, and the final volume was 5mL. The phosphorus element intensity was measured at a wavelength of 213.618nm using ICP-AES, and the boron element intensity was measured at a wavelength of 249.773nm using ICP-AES. The number of cells was calculated using a linear equation of phosphorus intensity and cell number, and then the boron concentration in the cells was calculated using a standard boron solution linear equation. Cells uptake BPA in μg/10 ⁷ The individual cells are shown and the results are shown in FIG. 2.

The standard boron solution linear equation is: y=162500x+651.3, r ² As shown in fig. 2 a, U87 cells ingest BPA in a concentration-dependent manner (concentrations of 12.5 and 25mg/mL, respectively) = 0.9996.

U87 cells ingested 5.01 μg/10 BPA after incubation at a concentration of 12.5mg/mL BPA ⁷ A cell; that is, after incubation of 12.5mg/mL BPA, single U87 cells ingest 5.01 x 10 BPA ^-7 μg。

U87 cells in concentrationBPA uptake after incubation at 25mg/mL BPA was 8.95 μg/10 ⁷ After incubation of individual cells, i.e., 25mg/mL BPA, individual U87 cells ingest 8.95X 10 BPA ^-7 μg。

In the subsequent measurement of boron concentration in U87, the cell is cultured and then the phosphorus element intensity and the boron element intensity are detected once, then the cell number is directly calculated according to the obtained linear equation of the phosphorus element intensity and the cell number, then the boron concentration (of all cells) is calculated by using the standard boron solution linear equation, and then the boron concentration is divided by the calculated cell number to obtain the boron concentration in single cells. The method of this example omits the whole cell processing and detection steps counted by the cell counter, measures the phosphorus and boron intensities only by one-step method at different wavelengths, calculates the cell number using the linear equation that has been obtained, and then calculates the boron concentration in the cell using the standard boron solution linear equation.

Example 2: a method for measuring boron concentration in cells in research of taking boron medicine by brain tumor cells.

1. Research on correlation of phosphorus element intensity and cell number of brain tumor cell line U251 cells

Brain tumor cell line U251 cells in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ The mL of the cell culture solution was placed in a centrifuge tube, centrifuged at 600g/min for 10min, the supernatant was discarded, the cell pellet was washed 1 time with 0.9% NaCl, and centrifuged at 600g/min for 10min, the supernatant was discarded. The cell pellet was digested with 50mL of the acid mixture for 2 hours to obtain a digested solution.

To the digest was added a total of 4.5mL of 10% nitric acid (dilution metering) and the final volume was 5mL. The digestion solution was assayed for the intensity of elemental phosphorus using ICP-AES (PQ 9000, analytik Jena GmbH, jena, germany) at a wavelength of 213.618 nm. The linear correlation between phosphorus intensity and cell number was analyzed, and the linear equation and the related parameters were calculated, and the results were shown in B of fig. 1.

The linear relationship between the number of U251 cells and the phosphorus intensity was good (B of fig. 1), the linear equation being y=77250x+1846 (R ² =0.9994), y represents phosphorus element intensity, x represents cell number, R ² Representing the correlation coefficient. The correlation coefficient was 0.9994, which shows that the linear relationship is good, and the number of U251 cells can be calculated by the above equation.

2. Method for measuring boron concentration in cells in research on uptake of BPA by brain tumor cell line U251 cells

U251 cells were cultured in DMEM 25cm containing 10% FBS ² In a flask. After 90% of the cells were fused, the medium was changed, BPA was added to U251 cells in groups at concentrations of 12.5mg/mL and 25mg/mL, respectively, and incubated for 3 hours. The cells were washed 3 times with 0.9% nacl solution. The cells were digested with 1mL of the acid mixture at 60℃for 2 hours, then 4mL of 10% nitric acid was added, and the final volume was 5mL. The phosphorus intensity was measured at a wavelength of 213.618nm using ICP-AES, and the boron intensity was measured at a wavelength of 249.773nm using ICP-AES. The number of cells was calculated using a linear equation of phosphorus intensity and cell number, and then the boron concentration in the cells was calculated using a standard boron solution linear equation. Cells uptake BPA in μg/10 ⁷ The individual cells represent, and the results are shown in FIG. 2B.

The standard boron solution linear equation is: y=162500x+651.3, r ² ＝0.9996

As a result, as shown in FIG. 2B, the U251 cells ingested BPA in a concentration-dependent manner (concentrations of 12.5 and 25mg/mL, respectively).

U251 cells ingested 7.25 μg/10 BPA after incubation at a concentration of 12.5mg/mL BPA ⁷ After incubation of individual cells, i.e.12.5 mg/mL BPA, individual U251 cells ingest 7.25 x 10 BPA ^-7 μg；

The uptake of BPA by U251 cells after incubation at 25mg/mL BPA was 13.25μg/10 ⁷ After incubation of individual cells, i.e., 25mg/mL BPA, individual U251 cells ingest 13.25X 10 BPA ^-7 μg。

In the subsequent measurement process of the boron concentration in U251 cells, only the cell culture is needed to detect the phosphorus element intensity and the boron element intensity once, then the cell number is directly calculated according to the obtained linear equation of the phosphorus element intensity and the cell number, then the boron concentration (of all cells) is calculated by using the standard boron solution linear equation, and then the boron concentration is divided by the calculated cell number to obtain the boron concentration in single cells.

The method of this example omits the whole cell processing and detection steps counted by the cytometer, measures the phosphorus and boron intensities only at different wavelengths, calculates the cell number using the linear equation that has been obtained, and then calculates the boron concentration in the cell using the standard boron solution linear equation.

Example 3: method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

1. Research on correlation between intensity and cell number of U87 cell phosphorus of brain tumor cell line

Brain tumor cell line U87 was grown in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ The mL of the cell culture solution was placed in a centrifuge tube, the supernatant was discarded after centrifugation at 700g/min for 13min, the cell pellet was washed 1 time with 0.9% NaCl, and the supernatant was discarded after centrifugation at 700g/min for 13 min. The cell pellet was digested with 55mL of the acid mixture for 2 hours to obtain a digested solution.

To the digest was added a total of 4.5mL of 10% nitric acid (dilution metering) and the final volume was 5mL. The digestions were assayed for elemental phosphorus intensity using ICP-AES (PQ 9000, analytik Jena GmbH, jena, germany) at a wavelength of 213.618 nm. And analyzing the linear correlation between the phosphorus element intensity and the cell number, and calculating a linear equation and related parameters.

U87 cells were cultured in DMEM 25cm containing 10% FBS ² In a flask. After 90% of the cells are fused, the culture medium is replaced, BPA with the concentration of 12.5mg/mL and 25mg/mL is added into the U87 cells in groups, and the cells are incubated for 3 hours. The cells were washed 3 times with 0.9% nacl solution. The cells were digested with 1mL of the acid mixture at 60℃for 2 hours, then 4mL of 10% nitric acid was added, and the final volume was 5mL. The phosphorus element intensity was measured at a wavelength of 213.618nm using ICP-AES, and the boron element intensity was measured at a wavelength of 249.773nm using ICP-AES. Calculating the cell number by using a linear equation of phosphorus element intensity and cell number, then calculating the boron concentration (of all cells) by using a linear equation of a standard boron solution, and dividing the boron concentration by the calculated cell number to obtain the boron concentration in single cells.

The standard boron solution linear equation is: y=162500x+651.3, r ² ＝0.9996。

Example 4: method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

Brain tumor cell line U87 was grown in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ Placing the cell culture solution into a centrifuge tube, centrifuging at 700g/min for 13min, and discarding the supernatantThe cell pellet was washed 1 time with 0.9% NaCl and centrifuged at 800g/min for 15min, and the supernatant was discarded. The cell pellet was digested with 50mL of the acid mixture for 2 hours to obtain a digested solution.

Example 5: method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

1. Study on correlation of intensity and cell number of phosphorus in U251 cell of brain tumor cell line

Brain tumor cell line U251 cells in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ The mL of the cell culture solution was placed in a centrifuge tube, centrifuged at 700g/min for 15min, the supernatant was discarded, the cell pellet was washed 1 time with 0.9% NaCl, and centrifuged at 700g/min for 15min, the supernatant was discarded. The cell pellet was digested with 55mL of the acid mixture for 2 hours to obtain a digested solution.

U251 cells were cultured in DMEM 25cm containing 10% FBS ² In a flask. After 90% of the cells were fused, the medium was changed, BPA was added to U251 cells in groups at concentrations of 12.5mg/mL and 25mg/mL, respectively, and incubated for 3 hours. The cells were washed 3 times with 0.9% nacl solution. The cells were digested with 1mL of the acid mixture at 60℃for 2 hours, then 4mL of 10% nitric acid was added, and the final volume was 5mL. The phosphorus element intensity was measured at a wavelength of 213.618nm using ICP-AES, and the boron element intensity was measured at a wavelength of 249.773nm using ICP-AES. Calculating the cell number by using a linear equation of phosphorus element intensity and cell number, then calculating the boron concentration (of all cells) by using a linear equation of a standard boron solution, and dividing the boron concentration by the calculated cell number to obtain the boron concentration in single cells. Cells uptake BPA in μg/10 ⁷ Individual cells are indicated. The standard boron solution linear equation is: y=162500x+651.3, r ² ＝0.9996。

Example 6: method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells

Brain tumor cell line U251 cells in 25-cm of DMEM containing 10% FBS ² After culturing in the flask for 48 hours, the cells were digested with pancreatin and counted by a cell counter. The culture medium for cells was adjusted to 1.00×10 ⁶ Cell culture solutions with cell number of 0.05X10 were taken per mL ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ The mL of the cell culture solution was placed in a centrifuge tube, centrifuged at 800g/min for 13min, the supernatant was discarded, the cell pellet was washed 1 time with 0.9% NaCl, and centrifuged at 800g/min for 13min, the supernatant was discarded. The cell pellet was digested with 50mL of the acid mixture for 2 hours to obtain a digested solution.

U251 cells were cultured in DMEM 25cm containing 10% FBS ² In a flask. After 90% of the cells were fused, the medium was changed, BPA was added to U251 cells in groups at concentrations of 12.5mg/mL and 25mg/mL, respectively, and incubated for 3 hours. The cells were washed 3 times with 0.9% nacl solution. The cells were digested with 1mL of the acid mixture at 60℃for 2 hours, then 4mL of 10% nitric acid was added, and the final volume was 5mL. The phosphorus element intensity was measured at a wavelength of 213.618nm using ICP-AES, and the boron element intensity was measured at a wavelength of 249.773nm using ICP-AES. Linear square using phosphorus intensity and cell numberThe cell number is calculated, then the boron concentration (of all cells) is calculated by using a standard boron solution linear equation, and then the boron concentration is divided by the calculated cell number to obtain the boron concentration in the single cell. Cells uptake BPA in μg/10 ⁷ Individual cells are indicated. The standard boron solution linear equation is: y=162500x+651.3, r ² ＝0.9996。

While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for measuring boron concentration in cells in research of boron drug uptake by brain tumor cells, which is characterized by comprising the following steps:

culturing brain tumor cells, and performing cell count;

preparing brain tumor cell culture solutions with different cell numbers, respectively centrifuging to obtain cell precipitates of brain tumor cells, digesting with a mixture of concentrated nitric acid and sulfuric acid to obtain digestive juice, measuring the phosphorus element intensity, analyzing the linear correlation between the phosphorus element intensity and the cell number, and calculating to obtain a linear equation;

s2, adding boron drugs into brain tumor cells for incubation, washing, then digesting, measuring the phosphorus element intensity, calculating the cell number through a linear equation, measuring the boron element intensity, and then calculating the boron concentration through a standard boron solution linear equation; and dividing the boron concentration by the calculated cell number to obtain the boron concentration in the single cell.

2. The method for measuring the boron concentration in cells in a study on the uptake of boron drug by brain tumor cells according to claim 1, wherein in S2, the digestion process is to digest the washed brain tumor cells by using an acid mixture.

3. The method for measuring the boron concentration in cells in the study of taking boron medicine by brain tumor cells according to claim 1, wherein in the S1, the preparation process of brain tumor cell culture solutions with different cell numbers is as follows: cell culture medium was adjusted to 1.00×10 ⁶ Cell culture solution of cell number/mL, then aspirated 0.05X10 respectively ⁶ mL、0.10×10 ⁶ mL、0.20×10 ⁶ mL、0.40×10 ⁶ mL、0.60×10 ⁶ mL、0.80×10 ⁶ mL、1.00×10 ⁶ mL、1.20×10 ⁶ mL、1.40×10 ⁶ mL and 1.60×10 ⁶ mL of cell culture broth.

4. The method for measuring the boron concentration in cells in the study of taking boron medicine by brain tumor cells according to claim 3, wherein in S1, the centrifugation condition is 600-800g/min for 10-16min.

5. The method for measuring the boron concentration in cells in the study of taking boron drugs by brain tumor cells according to claim 4, wherein in S1, the acid mixture is prepared from concentrated nitric acid and sulfuric acid according to a volume ratio of 1:1, and mixing.

6. The method for measuring the boron concentration in cells in a study on the uptake of boron drugs by brain tumor cells according to claim 5, wherein the brain tumor cells are any one of U87 cells and U251 cells.

7. The method for measuring the boron concentration in cells in the study of taking boron drugs by brain tumor cells according to claim 6, wherein the calculated linear equation of the linear correlation between the phosphorus element intensity of the U87 cells and the cell number is: y=72370x+1708, r ² ＝0.9995；

8. The method for measuring the boron concentration in cells in the study of taking boron drugs by brain tumor cells according to claim 6, wherein the calculated linear equation of the linear correlation between the phosphorus intensity of the U251 cells and the number of cells is: y=77250x+1846, r ² ＝0.9994；

9. The method for measuring the boron concentration in cells in the study of taking boron drugs by brain tumor cells according to claim 1, wherein in S2, the linear equation of the standard boron solution is: y=162500x+651.3, r ² ＝0.9996；

10. The method for measuring the concentration of boron in cells in a study on the uptake of boron drug by brain tumor cells according to claim 1, wherein in S2, the boron drug is borylphenylalanine.