Method for identifying lung cancer brain metastatic tumor radiation necrosis and tumor recurrence based on tumor cell surface charges

Abstract

The invention relates to a method for identifying lung cancer brain metastatic tumor radiation necrosis and tumor recurrence based on tumor cell surface charges. Compared with the prior art, the method detects surface charges of tumor cells on the basis of a two-electrode electrochemical device and identifies tumors in a radiation necrosis state (RN) or a recurrent state (RT) through the surface charges of the tumor cells; according to the method, the two tumor forms of a radiation necrotic tumor (RN) and a recurrent tumor (RT) can be identified in the patient with the brain metastatic tumor of the lung cancer, and the method can be used for identifying the two tumor forms of the radiation necrotic tumor (RN) and the recurrent tumor (RT) in the patient with the brain metastatic tumor of the lung cancer. According to the method, a new technical method for tumor cell surface charge detection and RN / RT recognition with tumor electrophysiology as an entry point is developed from a new perspective.

Description

Technical Field

[0001] The present invention relates to the fields of tumor electrophysiology, tumor radiotherapy, tumor sugar metabolism and cell molecular biology, and in particular to a method for identifying radionecrosis of lung cancer brain metastases and tumor recurrence based on the surface charge of tumor cells. Background Art

[0002] Lung cancer is the most common cancer in China, with high morbidity and mortality rates. One of the major causes of death from lung cancer is brain metastasis. Statistics show that 20% to 65% of lung cancer patients will develop brain metastases during the course of the disease. Stereotactic radiosurgery (SRS) is a common treatment method that uses high radiation therapy doses to provide local control of tumors without the late neurocognitive sequelae associated with whole-brain radiotherapy. With SRS, the median survival of patients with brain metastases from lung cancer with EGFR mutations has been extended from 4 to 9 months to 46 months. However, 10% to 20% of SRS-treated patients will develop new parenchymal lesions, which are either necrosis caused by radiation therapy (radionecrosis) or recurrent tumors. These two types of lesions usually occur within a similar time frame but require very different treatment approaches. Radionecrosis can be treated conservatively to reduce treatment-related patient morbidity; in contrast, tumor recurrence is usually best treated early and with more aggressive control of lesion growth. To complicate matters, both chemotherapy and the increasingly used immunotherapy with SRS increase the incidence of radionecrosis. Therefore, clinical radiotherapy oncology needs to develop a reliable method to distinguish between radiation-necrotic tumors and recurrent tumors.

[0003] Biomarkers for distinguishing radiation necrosis from tumor recurrence include the following aspects: (1) Serum tumor markers: Specific proteins or substances in the blood can be used as markers to indicate the presence and status of tumors. For example, AFP (alpha-fetoprotein), CEA (carcinoembryonic antigen), etc. have certain sensitivity and specificity in certain tumor types. (2) Extracellular DNA (cfDNA): After radiotherapy, if tumor recurrence occurs, tumor-related mutations can be found by detecting extracellular DNA in the blood. (3) MicroRNA (miRNA): miRNA is a class of non-coding RNA molecules that can participate in gene regulation and play an important role in tumor formation and progression. Some specific miRNAs may be associated with radiation necrosis or tumor recurrence. The current technologies used to distinguish between radiation necrosis-type tumors and recurrent tumors mainly focus on proteins and differential genes.

[0004] The current technical limitations for identifying radiation-necrotic tumors and recurrent tumors are: (1) Lack of specificity: Certain tumor markers may also be elevated in other diseases or under normal conditions, resulting in low diagnostic specificity. (2) Individual differences: The expression of biomarkers may vary between individuals, and tumors of different stages and types may also have different biomarker patterns. (3) Technical limitations: The current technology used to detect biomarkers still has certain limitations, including sensitivity, specificity, and standardization. Therefore, in the clinical practice of differentiating radiation-necrotic tumors from brain metastases of lung cancer from tumor recurrence, the current application of biomarkers has certain limitations. The above status quo fully demonstrates that there is an urgent need to propose more technical means to identify radiation-necrotic tumors and recurrent tumors. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge in order to solve at least some of the technical problems in the prior art. The method is based on a two-electrode electrochemical device to detect the surface charge of tumor cells and identify tumors in a state of radiation necrosis (RN) or a recurrent state (RT) through the surface charge of tumor cells; the method can identify two tumor morphologies, radiation necrosis (RN) and recurrent tumor (RT), in patients with lung cancer brain metastases. The method develops a new technical method for tumor cell surface charge detection and RN / RT identification using tumor electrophysiology as an entry point from a new perspective.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, the specific steps are as follows:

[0008] S1. Construct a two-electrode electrochemical device to detect the surface charge intensity of tumor cells;

[0009] S2. Based on the differences in glucose metabolism levels between radionecrosis and tumor recurrence, two tumor biological models of radionecrosis and tumor recurrence were constructed;

[0010] S3, detecting the surface charge expression of tumor cells in the radionecrosis model and the tumor recurrence model constructed in step S2 by using the two-electrode electrochemical device constructed in step S1, wherein the low glycolysis level in the radionecrosis model causes the tumor cells to have a low surface charge intensity, while the high glycolysis level in the tumor recurrence model causes the tumor cells to have a high surface charge intensity;

[0011] S4. Add the tumor cells to be detected to the two-electrode electrochemical device constructed in step S1, determine the surface charge expression of the tumor cells to be detected, and identify whether the tumor cells are in a state of radiation necrosis or a state of tumor recurrence.

[0012] Furthermore, in step S1, the two-electrode electrochemical device includes a working electrode and a counter electrode,

[0013] The working electrode comprises a flexible polydimethylsiloxane substrate and carbon nanofibers arranged in sequence,

[0014] The counter electrode is a silver / silver chloride electrode.

[0015] Furthermore, the preparation method of the two-electrode electrochemical device is as follows:

[0016] S1-1, fixing the pretreated carbon nanofibers on a flexible polydimethylsiloxane substrate to obtain a carbon nanofiber electrode;

[0017] S1-2. Connect the carbon nanofiber electrode obtained in step S1-1 to one end of the working electrode of the electrochemical workstation, connect the silver / silver chloride electrode to one end of the counter electrode of the electrochemical workstation, and use phosphate-buffered saline as an electrolyte to obtain a two-electrode electrochemical device.

[0018] Furthermore, in step S1-1, the carbon nanofiber pretreatment method is:

[0019] The carbon nanofibers were heated to a molar concentration of 8-12 mol·L -1 The carbon nanofibers are immersed in nitric acid for 10-12 hours, rinsed with deionized water, immersed in deionized water for 8-12 hours, then rinsed with deionized water, and the water on the surface of the carbon nanofibers is absorbed with dust-free paper to obtain pretreated carbon nanofibers.

[0020] Furthermore, in step S1-1, the carbon nanofibers have a thickness of 5-15 μm and an area of ​​0.3-0.5 cm 2 .

[0021] Furthermore, in step S1-1, the preparation method of the flexible polydimethylsiloxane substrate is:

[0022] The polydimethylsiloxane and the curing agent are uniformly mixed in a mass ratio of (5-10):1, and heated and cured at 70-80°C for 40-60 minutes to obtain a flexible polydimethylsiloxane substrate.

[0023] Furthermore, in step S1-2, the volume of the electrolyte is 5-15 mL;

[0024] The molar concentration of the phosphate buffered saline (PBS) is 0.05-0.2 mmol·L -1 , preferably 0.067 mmol·L -1 .

[0025] Furthermore, in steps S3 and S4, the detection method used is a two-electrode linear sweep voltammetry test method.

[0026] The steps of the two-electrode linear sweep voltammetry test method are as follows: establishing the relationship between the surface charge of the tumor cells and the immediate response current, detecting the tumor cells to be detected in the two-electrode system and obtaining the surface charge intensity level of the tumor cells by calculation.

[0027] The above further establishes the relationship between the surface charge of tumor cells and the immediate response current as follows:

[0028] The tumor cells to be detected are placed in an electrolyte phosphate buffer, and an external voltage is applied to the two-electrode system. The amount of electron transfer on the working electrode during the reaction is tested using an electrochemical workstation, and the surface charge intensity level of the tumor cells is estimated based on the established relationship between the surface charge of the tumor cells and the immediate response current, thereby realizing the detection of the surface charge intensity of the tumor cells.

[0029] In the above, the scanning range of the two-electrode linear scanning voltammetry is 0 to 2V, and the scanning speed is 50-150mV·s -1 .

[0030] Furthermore, step S5 is provided after step S4, which uses transcriptome sequencing technology and bioinformatics analysis technology to explain why the surface charge of tumor cells can identify whether the tumor is in the RN or RT state, and verifies the reliability of the new method of identifying radionecrotic tumors and recurrent tumors by the surface charge of tumor cells.

[0031] In addition, the present invention also provides a pharmaceutical composition for regulating the tumor PI3K-Akt signaling pathway, wherein the pharmaceutical composition comprises an inhibitor for inhibiting the surface charge of tumor cells.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. The present invention uses the electrophysiological index of tumors, the surface charge of tumor cells, for identification, and provides detection technology and theoretical basis for the method. The present invention provides new ideas for the detection technology of tumor cell surface charge and the new method for identifying radionecrotic tumors and recurrent tumors.

[0034] 2. The detection method of the tumor cell surface charge intensity adopted in the present invention is an electrochemical two-electrode system test method. Compared with the three-electrode system test method, in the two-electrode system, the reference electrode socket and the counter electrode are plugged together, the reference electrode is eliminated, and the reference circuit and the working circuit are merged.

[0035] 3. A key to the non-destructive detection of tumor cell surface charge in the present invention is to instantly complete the detection of cell surface charge without leaving the physiological environment of the cell. The present invention uses phosphate buffered saline (PBS), a cell buffer most widely used in biochemical research, as an electrolyte.

[0036] 4. The originality of the present invention lies in the fact that the development of new technology is based on a new perspective to solve the problem of difficulty in distinguishing between radionecrosis-type tumors and recurrent tumors. The surface charge of tumor cells is an electrophysiological indicator of tumors. There are currently no reports on the use of tumor cell surface charge for the identification of radionecrosis and tumor recurrence. Bioelectricity is an important part of all life activities. In the past few decades, advances in electrophysiology have shown that cell surface charge is an important attribute of cell characteristics and plays a vital role in regulating cell function. The present invention is based on a new perspective as a technical solution.

[0037] 5. The beneficial effect of the present invention is that the surface charge of tumor cells, as a broad-spectrum biomarker of tumors, has the potential to be expanded to more cancer types. The electrochemical device, transcriptome sequencing technology and bioinformatics analysis involved in the present invention further realize the detection of surface charge of tumor cells and the expanded discovery of the role of surface charge of tumor cells in regulating cell function.

[0038] 6. The beneficial effect of the present invention is that as a new technology for identifying RN / RT, it is expected to have stronger specificity. This is because in the brain parenchyma, that is, normal tissue, there is sometimes a higher level of glucose metabolism, which makes it difficult to distinguish from tumor tissue through the glucose content; however, even at a high level of glucose metabolism, normal cells make the cell surface close to electrical neutrality due to the oxidative phosphorylation pathway, while tumor cells carry a strong negative charge on the cell surface due to the Warburg effect, making them easier to identify. Therefore, although glucose is also a metabolite related to tumor glucose metabolism, the expression of charge on the surface of tumor cells is more suitable as a marker for characterizing tumor glucose metabolism levels than the glucose content of the tumor, and is expected to have stronger recognition specificity for identifying indicators of radiation necrosis and tumor recurrence in patients with lung cancer brain metastases. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] A more complete understanding of the present invention and its attendant advantages and features will be more readily appreciated by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0040] Figure 1 A schematic diagram of the working principle of a method for identifying radionecrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge in the present invention;

[0041] Figure 2 Identify radiation necrosis (RN) and tumor recurrence (RT) of lung cancer brain metastases for tumor cell surface charge. This new technology includes an overall development flow chart of the technical effects;

[0042] Figure 3 is a structural diagram of the two-electrode electrochemical device in Example 1;

[0043] Figure 4 Schematic diagram of the two-electrode electrochemical device for detecting the surface charge of tumor cells in Example 2;

[0044] Figure 5 This is a schematic diagram of the technical principle by which DCA and 3BP can regulate tumor glucose metabolism levels;

[0045] Figure 6 Schematic diagram of the surface charge of the radionecrotic tumor and recurrent tumor biological model constructed by the two-electrode electrochemical device detection in Example 3;

[0046] Figure 7 This is a schematic diagram showing that the change in the surface charge of tumor cells will lead to the change in gene expression of tumor cells in Example 4, wherein Up is the number of up-regulated genes with significant differences, and Down is the number of down-regulated genes with significant differences;

[0047] Figure 8 Schematic diagram of gene expression values ​​of all samples analyzed by PCA;

[0048] Fig. 9 Schematic diagram of KEGG pathway analysis results.

[0049] It should be noted that the drawings are used to illustrate the present invention, rather than to limit the present invention. Note that the drawings showing the structures may not be drawn to scale. In addition, in the drawings, the same or similar elements are marked with the same or similar reference numerals. DETAILED DESCRIPTION

[0050] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0051] In the following examples, the sources of the materials are as follows:

[0052] The polydimethylsiloxane and curing agent were both purchased from Dow Corning Company and used together. The model of the curing agent is 184.

[0053] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. All raw materials without specifying the synthesis method were purchased from manufacturers such as CORNING, Aladdin, and Sigma-Aldrich.

[0054] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0055] Example 1

[0056] like Figure 3 As shown, this embodiment provides a method for constructing a two-electrode electrochemical device for detecting the surface charge intensity of tumor cells, and the specific steps are as follows:

[0057] (1) Pretreatment of carbon nanofibers:

[0058] The carbon nanofibers were heated to a molar concentration of 8 mol·L -1 The carbon nanofibers were immersed in nitric acid for 10 h, rinsed with deionized water, immersed in deionized water for 12 h, and then rinsed with deionized water. The surface moisture of the carbon nanofibers was dried with dust-free paper to obtain pretreated carbon nanofibers. The thickness of the pretreated carbon nanofibers was 5 μm and the area was 0.5 cm 2 .

[0059] (2) Preparation of flexible polydimethylsiloxane substrate:

[0060] Polydimethylsiloxane and curing agent were uniformly mixed in a mass ratio of 10:1, and heated and cured at 70°C for 40 minutes to obtain a flexible polydimethylsiloxane substrate.

[0061] (3) fixing the pretreated carbon nanofibers on a flexible polydimethylsiloxane substrate to obtain a carbon nanofiber electrode;

[0062] (4) Metallic silver with a diameter of about 0.5 mm and a length of about 8 cm is immersed in a saturated potassium chloride solution to obtain a silver / silver chloride electrode.

[0063] (5) The carbon nanofiber electrode was connected to one end of the working electrode of the electrochemical workstation, the silver / silver chloride electrode was connected to one end of the counter electrode of the electrochemical workstation, and 10 mL of phosphate buffered saline was used as the electrolyte to obtain a two-electrode electrochemical device ( Figure 3 ).

[0064] Example 2

[0065] This embodiment provides a method for detecting the surface charge of any tumor cell to be tested by a two-electrode electrochemical device constructed based on Embodiment 1, wherein the surface charge of the cell is detected by a two-electrode linear sweep voltammetry test method, and the specific steps are as follows:

[0066] The PC-9 cells to be tested were collected and added to the electrolyte (PBS volume was 10 mL). The linear sweep voltammetry was applied with a scan range of 0 to 2 V and a scan rate of 100 mV·s -1 The electric field drives the tumor cells with charges on the surface to migrate, causing charge accumulation on the electrode surface. The LSV curve test of the electrochemical workstation is used to test the change in response current.

[0067] Figure 4 After tumor cells are added to the two-electrode electrochemical device, the constructed two-electrode electrochemical device detects the cell surface charge through linear scanning voltammetry to generate a response current.

[0068] This example evaluates the intensity of the surface charge of tumor cells based on the change in the response current after adding different tumor cells.

[0069] Example 3

[0070] According to the "Guidelines for Clinical Management of Glioma in China" issued by the Glioma Professional Committee of the Chinese Anti-Cancer Association, radionecrosis-type tumors have low glycolysis levels, while recurrent tumors have high glycolysis levels. Glycolysis refers to the process in which glucose is broken down into pyruvate in the cytoplasm under anaerobic conditions. In this embodiment, the glucose metabolism of tumor cells is regulated by adjusting the concentration of glucose or using the glycolysis inhibitor DCA / 3BP. Figure 5 The schematic diagram illustrates why DCA and 3BP can regulate glucose metabolism levels.

[0071] This embodiment provides a method for constructing two tumor biological models of radiation necrosis and tumor recurrence, and the specific steps are as follows:

[0072] (1) Regulating glucose concentration

[0073] High-glucose DMEM ([+] 4.5g / L D-Glucose) and low-glucose DMEM ([+] 1.0g / L D-Glucose) are already available on the market. In order to study the effect of glucose concentration in the culture medium on the surface charge of tumor cells, different glucose concentrations were prepared by adding D-Glucose to low-glucose DMEM. The test cells were cultured for 48 hours in 5 different glucose concentrations of 1.0g / L, 2.0g / L, 3.0g / L, 4.0g / L and 4.5g / L. Subsequently, Hep-3B cells were collected by trypsin digestion and centrifugation, and the cell surface charge was measured.

[0074] (2) Glycolysis disruptor-DCA

[0075] Dichloroacetic acid (DCA) is an indirect inhibitor of glycolysis. Its function is not to directly inhibit any enzymes in the glycolysis pathway, but to promote the oxidation of pyruvate in mitochondria, thereby inhibiting the conversion of pyruvate to lactate. Tumor cells were treated with 5 different DCA concentrations of 0, 20, 40, 60 and 120 mM to inhibit the glycolysis metabolic pathway. The test cells were cultured at 37°C for 24 hours. The growth status of tumor cells was observed, and the surface charge of tumor cells was detected.

[0076] (3) Glycolysis disruptor-3BP

[0077] 3-Bromopyruvate (3BP) is a direct inhibitor of glycolysis. Its function is to block the activity of hexokinase, the first step in the glycolysis pathway, thereby inhibiting the main sugar metabolism pathway of tumor cells and minimizing the secretion of lactate. Tumor cells were treated with 5 different 3BP concentrations of 0, 10, 25, 50 and 100 μM to inhibit the glycolysis metabolic pathway. The test cells were cultured at 37°C for 24 hours. The growth status of tumor cells was observed and the surface charge of tumor cells was detected.

[0078] Through Example 3, the construction of two tumor models, radionecrosis and tumor recurrence, was achieved based on the regulation of the glycolytic pathway, and the surface charge detection of the two tumor models was performed through Example 2. Figure 6 The results showed that the radionecrosis type tumor had a low surface charge (ranging from 135-178 mA), while the recurrent type tumor had a high surface charge (ranging from 230-244 mA), and the charge expression levels of the two tumor morphologies were significantly different, demonstrating the utility of the technical method of the present invention. Since the surface charge expression of the two tumor phenotypes (radionecrosis / tumor recurrence) was significantly different, it can be used as a differential diagnostic indicator.

[0079] Example 4

[0080] Based on the results of Example 2 and Example 3, it can be seen that the low sugar metabolism level of radionecrosis causes the tumor cells to have a low surface charge intensity, while the high sugar metabolism level of tumor recurrence causes the tumor cells to have a high surface charge intensity; this example provides a method for analyzing the molecular signaling pathway involved in mediating radionecrosis and tumor recurrence by transcriptome sequencing and bioinformatics analysis technology, and verifies the feasibility of tumor cell surface charge as a method for distinguishing radionecrosis from tumor recurrence, as follows:

[0081] (1) Differential gene analysis was performed on samples after surface charge regulation of tumor cells to screen genes with significant differences in gene expression levels between the cell surface charge up-regulation group (named cas1), the cell surface charge down-regulation group (named cas3) and the control group (named con). The screening conditions were: q-value < 0.05 & |log2FC| > 1.0.

[0082] The number of up-regulated genes (marked as up) and down-regulated genes (marked as down) is shown in Figure 7 , comparing the common and unique differentially expressed genes between different groups, the test results showed that changes in the surface charge of tumor cells can lead to changes in the gene expression of tumor cells.

[0083] (2) Principal component analysis: PCA was used to analyze the gene expression values ​​of all samples. The three replicates of the experimental group (cell surface charge increased group, cell surface charge decreased group) were clustered into one category and were significantly different from the control group. If the results showed that there were significant differences in gene expression among the three groups of cells, it was in line with expectations.

[0084] The principal component analysis results are shown in Figure 8 , PCA analysis of gene expression values ​​of all samples is as follows Figure 8 The three replicates of the experimental groups (cell surface charge increased group, cell surface charge decreased group) were clustered into one category and were significantly different from the control group, indicating that there were significant differences in gene expression among the three groups of cells, indicating that the experimental scheme described in this example met the experimental expectations.

[0085] (3) KEGG pathway enrichment analysis: KEGG is a database that systematically analyzes gene functions and links genomic information and functional information, exploring which pathways the differentially expressed genes detected by the present invention may be related to. KEGG is one of the most important public databases in Pathway. The KEGG database (and combined with the KEGG annotation results) was used to perform Pathway analysis on differentially expressed protein-coding genes, and Cluster Profiler software was used to perform KEGG analysis on differentially expressed genes. The hypergeometric distribution method was used to calculate the significance of the richness of different protein-coding genes in each Pathway project.

[0086] Fig. 9 The enriched pathways with significant differences were listed. From the results, it can be seen that the differentially expressed genes were significantly enriched in the PI3K-Akt signaling pathway (Top 20) after cell surface charge regulation, which proves that the changes in the surface charge of tumor cells described in the present invention can mediate the changes in the PI3K-Akt signaling pathway, and the changes in the PI3K-Akt signaling pathway are closely related to the regulation of tumor survival and death, suggesting that changes in the surface charge of tumor cells may affect the progression of tumor survival / death by regulating the PI3K-Akt signaling pathway. This result explains the basis of the new technical method for the tumor cell surface charge proposed in the present invention to identify RN / RT.

[0087] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, characterized in that: The specific steps are as follows: S1. Construct a two-electrode electrochemical device to detect the surface charge intensity of tumor cells; S2. Based on the differences in glucose metabolism levels between radionecrosis and tumor recurrence, two tumor biological models of radionecrosis and tumor recurrence were constructed; S3, detecting the surface charge expression of tumor cells in the radionecrosis model and the tumor recurrence model constructed in step S2 by using the two-electrode electrochemical device constructed in step S1, wherein the low glycolysis level in the radionecrosis model causes the tumor cells to have a low surface charge intensity, while the high glycolysis level in the tumor recurrence model causes the tumor cells to have a high surface charge intensity; S4. Add the tumor cells to be detected to the two-electrode electrochemical device constructed in step S1, determine the surface charge expression of the tumor cells to be detected, and identify whether the tumor cells are in a state of radiation necrosis or a state of tumor recurrence.

2. A method for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge according to claim 1, characterized in that: In step S1, the two-electrode electrochemical device includes a working electrode and a counter electrode, The working electrode comprises a flexible polydimethylsiloxane substrate and carbon nanofibers arranged in sequence, The counter electrode is a silver / silver chloride electrode.

3. The method of identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge according to claim 2, characterized in that: The preparation method of the two-electrode electrochemical device is as follows: S1-1, fixing the pretreated carbon nanofibers on a flexible polydimethylsiloxane substrate to obtain a carbon nanofiber electrode; S1-2. Connect the carbon nanofiber electrode obtained in step S1-1 to one end of the working electrode of the electrochemical workstation, connect the silver / silver chloride electrode to one end of the counter electrode of the electrochemical workstation, and use phosphate-buffered saline as an electrolyte to obtain a two-electrode electrochemical device.

4. The method of identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge according to claim 3, characterized in that: In step S1-1, the carbon nanofiber pretreatment method is: The carbon nanofibers were heated to a molar concentration of 8-12 mol·L -1 The carbon nanofibers are immersed in nitric acid for 10-12 hours, rinsed with deionized water, immersed in deionized water for 8-12 hours, then rinsed with deionized water, and the water on the surface of the carbon nanofibers is absorbed with dust-free paper to obtain pretreated carbon nanofibers.

5. The method of identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge according to claim 3, characterized in that: In step S1-1, the preparation method of the flexible polydimethylsiloxane substrate is: The polydimethylsiloxane and the curing agent are uniformly mixed in a mass ratio of (5-10):1, and heated and cured at 70-80°C for 40-60 minutes to obtain a flexible polydimethylsiloxane substrate.

6. The method of claim 3 for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, characterized in that: In step S1-2, the volume of the electrolyte is 5-15 mL; The molar concentration of the phosphate buffered saline is 0.05-0.2 mmol·L -1 .

7. The method of claim 1 for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, characterized in that: In steps S3 and S4, the detection method used is a two-electrode linear sweep voltammetry test method. The steps of the two-electrode linear sweep voltammetry test method are as follows: establishing the relationship between the surface charge of the tumor cells and the immediate response current, detecting the tumor cells to be detected in the two-electrode system and obtaining the surface charge intensity level of the tumor cells by calculation.

8. The method of claim 7 for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, characterized in that: The relationship between the tumor cell surface charge and the immediate response current was established as follows: The tumor cells to be detected are placed in an electrolyte phosphate buffer, and an external voltage is applied to the two-electrode system. The amount of electron transfer on the working electrode during the reaction is tested using an electrochemical workstation, and the surface charge intensity level of the tumor cells is estimated based on the established relationship between the surface charge of the tumor cells and the immediate response current, thereby realizing the detection of the surface charge intensity of the tumor cells.

9. The method of claim 7 for identifying radiation necrosis and tumor recurrence of lung cancer brain metastases based on tumor cell surface charge, characterized in that: The scanning range of the two-electrode linear scanning voltammetry is 0 to 2 V, and the scanning speed is 50-150 mV·s -1 .

10. A pharmaceutical composition for regulating the tumor PI3K-Akt signaling pathway, characterized in that: The pharmaceutical composition includes an inhibitor that inhibits the surface charge of tumor cells.

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