CN113564126A

CN113564126A - In-vitro cell model for simulating proliferation after glioma radiotherapy and construction method and application thereof

Info

Publication number: CN113564126A
Application number: CN202111008755.2A
Authority: CN
Inventors: 冯骁
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-10-29

Abstract

The invention belongs to the technical field of medical biology, and discloses an in vitro cell model for simulating proliferation after glioma radiotherapy. The cell model is formed by co-culturing marked glioma cells and glioma cells irradiated by radiotherapy, wherein the marked glioma cells are human glioma cells marked by luciferase and green fluorescent protein together. The preparation method of the cell model comprises the steps of inoculating glioma cells irradiated by radiotherapy into a culture medium, culturing at 37 ℃ for 12-48h, inoculating glioma cells jointly marked by luciferase and green fluorescent protein into the culture medium, and performing co-culture. The cell model can be used for simulating the process of glioma re-proliferation after radiotherapy; in addition, certain compounds or drugs were added to this model to investigate whether these substances had inhibitory effects on repopulation following radiation therapy of glioma. Therefore, the model can be used for screening potential drugs for inhibiting the re-proliferation of glioma after radiotherapy.

Description

In-vitro cell model for simulating proliferation after glioma radiotherapy and construction method and application thereof

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to an in-vitro cell model for simulating the repopulation of glioma after radiotherapy, and a construction method and application thereof.

Background

Glioma refers to a malignant tumor originated from glial cells of the central nervous system, and accounts for about 80% of primary intracranial malignant tumors. Based on histological type, gliomas include astrocytomas, oligodendrogliomas, ependymomas, mixed cell types, and others. The World Health Organization (WHO) central nervous system tumor classification system classifies gliomas as grade I-IV, I, II as low grade gliomas, and III and IV as high grade gliomas. Different types and grades of glioma have different biological characteristics, clinical characteristics and prognostic profiles.

Glioblastoma is the most common primary brain malignancy, accounting for approximately 50%. Glioblastoma is the most malignant glioma, with WHO grading to grade IV. The basic treatment strategies for glioblastoma currently include maximal surgical resection, simultaneous postoperative chemoradiotherapy, and sequential chemotherapy. Although a comprehensive treatment mode based on operations, radiotherapy and chemotherapy is formed for glioblastoma, the clinical prognosis is still extremely undesirable, the annual survival rate is less than 40%, and the five-year survival rate is about 5%. Glioblastoma is almost inevitable to relapse after initial first-line treatment, which is a significant cause of poor prognosis.

The proliferation of glioma cells after radiotherapy refers to a biological process that glioma cells surviving in the radiotherapy accelerate proliferation at a higher speed in the intermittent period of the radiotherapy or after the end of the radiotherapy, and finally cause the reburning of the tumor. Exploring the mechanism of the re-proliferation after the glioma radiotherapy is favorable for disclosing the important molecular mechanism of the recurrence after the glioma radiotherapy, and providing more effective targets for researching and developing related medicaments capable of inhibiting the re-proliferation after the glioma radiotherapy.

The establishment of the model of simulating the in vitro cell proliferation after glioma radiotherapy, which has strong simulation, controllable conditions, good repeatability and convenient operation, not only can provide a research tool for exploring the mechanism of the model, but also can provide a screening model for screening drugs capable of inhibiting the proliferation after glioma radiotherapy. However, adequate literature search and mining still lack suitable cell models at home and abroad.

Disclosure of Invention

In view of the problems and deficiencies in the prior art, one object of the present invention is to provide an in vitro cell model simulating the re-proliferation of a glioma after radiotherapy, another object of the present invention is to provide a method for constructing an in vitro cell model simulating the re-proliferation of a glioma after radiotherapy, and yet another object of the present invention is to provide an application of the in vitro cell model simulating the re-proliferation of a glioma after radiotherapy.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides an in vitro cell model for simulating proliferation of glioma after radiotherapy, which is formed by co-culturing labeled glioma cells and glioma cells irradiated by radiotherapy, wherein the labeled glioma cells are human glioma cells jointly labeled by luciferase and green fluorescent protein.

Preferably, the glioma cells are human glioma cell line U87-MG (abbreviated U87) according to the in vitro cell model described above that mimics proliferation of glioma cells following radiation therapy.

According to the in vitro cell model simulating proliferation after glioma radiotherapy, preferably, the radiotherapy irradiation is X-ray irradiation, and the radiotherapy irradiation dose is 10 Gy. More preferably, the radiotherapy irradiation is irradiation using X-ray irradiation generated by a medical linear accelerator.

Preferably, the luciferase is a firefly luciferase according to the in vitro cell model which mimics proliferation after glioma radiotherapy.

The second aspect of the invention provides a method for constructing an in vitro cell model for simulating the proliferation of glioma after radiotherapy, which comprises the following steps: inoculating the glioma cells irradiated by radiotherapy into a culture medium, culturing for 12-48h at 37 ℃, then inoculating the marked glioma cells into the culture medium, carrying out co-culture, and obtaining the in vitro cell model simulating glioma proliferation after radiotherapy after the co-culture is finished.

According to the above construction method, the ratio of the amount of glioma cells inoculated after the irradiation with the radiation therapy to the amount of labeled glioma cells inoculated is preferably 150: 1.

According to the above construction method, preferably, the culture medium is 2% fetal bovine serum culture medium.

According to the above-mentioned construction method, it is preferable that the co-cultivation temperature is 37 ℃ and the co-cultivation time is 10 to 14 days.

Preferably, the glioma cell is human glioma cell line U87-MG according to the construction method.

According to the above construction method, preferably, the labeled glioma cell is a human glioma cell labeled by luciferase and green fluorescent protein together.

According to the above construction method, preferably, the radiation irradiation is X-ray irradiation, and the radiation irradiation dose is 10 Gy. More preferably, the radiotherapy irradiation is irradiation using X-ray irradiation generated by a medical linear accelerator.

In a third aspect, the invention provides an application of the in vitro cell model simulating the re-proliferation of glioma after radiotherapy in screening of a medicament for inhibiting the re-proliferation of glioma after radiotherapy.

Specifically, certain compounds or medicines are added into the model, and whether the substances have inhibition effect on the proliferation of the glioma after radiotherapy is further researched, so that the medicine with potential clinical value for inhibiting the proliferation of the glioma after radiotherapy is screened.

The invention applies a small molecule inhibitor NS-398 of a target Cyclooxygenase-2 (COX-2), and shows how to apply the in vitro cell model to screen a drug which can inhibit the re-proliferation of glioma after radiotherapy and has potential clinical value. The specific method comprises the following steps: inoculating glioma cells irradiated by radiotherapy to a 24-hole cell culture plate, culturing at 37 ℃ for 24 hours, inoculating marked glioma cells (the marked glioma cells are glioma cells marked by luciferase and green fluorescent protein together) to the cell culture plate, adding NS-398 to the culture hole at the same time, carrying out co-culture for 10-14 days, and detecting the proliferation condition of the marked glioma cells by using a cell living body imaging technology at the end of the co-culture. A control experiment was also set up with the addition of NS-398 as a control (methyl sulfoxide as the control and dimethyl sulfoxide as the solvent for dissolving NS-398). Through statistical analysis of luciferase activity difference between the NS-398 group and the control agent group, whether the NS-398 can inhibit the ability of re-proliferation of glioma after radiotherapy is judged.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) the invention establishes an in vitro cell model for simulating proliferation of glioma after radiotherapy, the cell model is established by direct coculture of marked glioma cells and glioma cells irradiated by radiotherapy, and the cell model can reflect the direct action of the glioma cells after the radiotherapy and the marked glioma cells without the radiotherapy and also reflect the indirect action of the glioma cells after the radiotherapy on the marked glioma cells by secreting soluble growth promoting substances. The in vitro cell model established by the invention can observe that compared with the glioma cells without radiotherapy, the glioma cells after radiotherapy promote the proliferation of the marked glioma cells at a higher speed. Therefore, certain compounds or medicines are added into the model, and whether the compounds or medicines have inhibition effect on the re-proliferation of the glioma after radiotherapy is further researched, so that the potential medicines capable of inhibiting the re-proliferation of the glioma after radiotherapy are screened.

(2) The marked glioma cells are jointly marked by firefly luciferase and green fluorescent protein, after a luminescent substrate is added into the marked glioma cells, the luciferase can catalyze the luminescent substrate to change to generate biochemical luminescence, the activity of the luciferase can be effectively evaluated by a cell living body imager in the follow-up process, and the luciferase activity and the cell number present a positive correlation relationship. Therefore, the proliferation condition of the marked glioma cells can be obtained by detecting the luciferase activity through a cell living body imaging instrument, the detection is quick and convenient, and the cells cannot be damaged.

Drawings

FIG. 1 is a map schematic of pLEX-GFP-luc2 plasmid;

FIG. 2 is a diagram showing the U87 (i.e., U87-Fluc) pattern of a fusion gene of stably transfected firefly luciferase (Fluc) and Green Fluorescent Protein (GFP);

FIG. 3 is a graph showing a positive correlation between luciferase activity and the number of cells in U87-Fluc;

FIG. 4 is a schematic diagram of an in vitro culture model of glioma cell lines U87 and U87-Fluc after irradiation by radiotherapy;

FIG. 5 is a graph showing the effect of U87 cells on U87-Fluc proliferation promotion after radiotherapy in an in vitro cell model that simulates proliferation after glioma radiotherapy;

FIG. 6 is a graph showing the effect of U87 cells on U87-Fluc proliferation promotion after radiotherapy when 10 μ M of the COX-2 inhibitor NS-398 is added to an in vitro cell model that mimics proliferation after glioma radiotherapy.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.

The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

In the present specification, the term "U87" represents "human glioma cell line U87-MG", which is a human glioma blastoma cell line that can be cultured in vitro.

In the present specification, the term "Fluc" represents "firefly luciferase (Fluc)".

In the present specification, the term "GFP" means "Green Fluorescent Protein (GFP)".

In the present specification, the term "U87-Fluc" represents "U87 cells stably expressing Fluc and GFP fusion proteins".

Example 1: construction of U87 cells stably expressing Fluc and GFP fusion proteins (U87-Fluc)

1. Lentiviral packaging

The specific operation of lentivirus packaging is as follows:

1) the procedure was started when the confluence of 293T cells cultured in 10 cm cell culture dishes reached 60-70%.

2) To 2ml of Opti-MEM medium were added 3.3. mu.g of pLEX-GFP-luc2 (the plasmid map is shown in FIG. 1), 3. mu.g of psPAX2, 1.2. mu.g of pMD2.G, and mixed well; to another 2ml of Opti-MEM medium, 50. mu.l of Lipofectamine 2000 was added and mixed well. The two mixtures were allowed to stand at room temperature for 5 minutes.

3) After about 5 minutes, the two mixtures were mixed well and allowed to stand at room temperature for 20 minutes.

4) After about 20 minutes, the 293T cell culture medium was aspirated, and the mixture from the previous step was slowly added to the 293T cell culture dish. The dish was gently shaken to allow the liquid to fully cover the cell surface.

5) After the 293T cells were cultured for 6 hours, the liquid covering the cell surface was aspirated, and 10% fetal bovine serum medium (without double antibody) was added to continue the culture.

6) After 48 hours, the expression of GFP in 293T cells was observed under a fluorescent microscope. If the GFP expression is good, the success of lentivirus packaging in 293T cells is basically demonstrated. The culture supernatant of 293T cells enriched with lentiviral particles was collected, centrifuged at 3000 rpm for 20 minutes at 4 ℃ and filtered through a 0.22 μm filter. The treated supernatant rich in lentiviral particles was dispensed into 1.5 ml EP tubes, 1ml per tube, and stored in a freezer at-80 ℃ until use.

2. Screening and construction of lentivirus infected U87 cell and U87-Fluc cell

The specific operation steps are as follows:

1) the cells of interest (U87 cells) were plated in 6-well plates, preferably at a cell number of 50% confluency overnight.

2) 1ml of the virus solution was taken out from a-80 ℃ refrigerator and placed on ice to be naturally thawed.

3) 1ml of virus solution, 1ml of 10% fetal bovine serum medium (without double antibody), and 2. mu.l of polybrene were thoroughly mixed to obtain a mixed solution.

4) And (4) sucking out the cell culture medium in the 6-well plate, adding the mixed solution in the previous step, and continuing to culture the cells.

5) After 24 hours, GFP expression was observed in the cells of interest under a fluorescent microscope. If the proportion of GFP positive cells is higher, the culture medium in the 6-well plate is replaced by 10% fetal bovine serum culture medium. If the percentage of GFP positive cells is low (less than 10%), the culture medium is changed after further culturing for 24 hours.

6) After the confluency of the target cells in the 6-well plate reached 100%, the cells were subcultured in a 10 cm cell culture dish. After the desired cells reached 50% confluency in the 10 cm dish, cell culture was started using a medium containing puromycin at a concentration of 1. mu.g/ml. U87 cells stably transfected with pLEX-GFP-luc2 survived, while the remaining cells were killed.

7) After a period of puromycin screening culture (10-20 days), the final puromycin concentration can be increased to 3-4. mu.g/ml, and U87 cells stably transfected with pLEX-GFP-luc2 can be obtained.

According to the operation steps, the U87 cells for stably expressing firefly luciferase (Fluc) and Green Fluorescent Protein (GFP) are successfully constructed and named as U87-Fluc cells. The U87-Fluc cell pattern is shown in FIG. 2. The luciferase functions in U87-Fluc cells are: after a luminescent substrate is added into the U87-Fluc cells, the firefly luciferase can catalyze the luminescent substrate to change, generate biochemical luminescence, and can efficiently evaluate the luciferase activity by detecting the biochemical luminescence through a cell living body imager.

Example 2: correlation between luciferase activity of U87-Fluc cells and cell number

In a subsequent in vitro co-culture model, it is desirable to reflect the proliferation of U87-Fluc by detecting the intensity of the luciferase activity of U87-Fluc firefly. Therefore, it was necessary to confirm whether the firefly luciferase activity in U87-Fluc exhibits a positive correlation with the number of cells. A specified number of U87-Fluc cells were plated in a 96-well plate, and after the cells adhered to the wall, the luciferase activity of U87-Fluc cells in the 96-well plate was detected by means of a cell living body biochemical luminescence imager.

The specific experimental operations were as follows:

U87-Fluc cells in good growth state were digested to prepare a single cell suspension. U87-Fluc was plated in 96-well plates for 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000 cells per well, respectively. And after the cells are fully attached to the wall, performing cell imaging on the 96-well plate by using a cell living body imaging instrument, and detecting the luciferase activity of the U87-Fluc cells in the 96-well plate.

The specific operation of using the cell living body imager to image the cells is as follows: the culture medium in the cell culture plate to be imaged was discarded, and an appropriate volume (96 well plate: 80. mu.l/well, 24 well plate: 150. mu.l/well) was added to give a final concentration of 0.15 mg/ml D-luciferin potassium. And then carrying out U87-Fluc cell biochemical luminescence in-vivo imaging by using a cell in-vivo imager to finish image acquisition, and carrying out data analysis by using matched software.

The data analysis is specifically operated as: data are presented as Mean ± SEM. We performed statistical analysis using statistical software SPSS 20.0 (IBM, USA). In the parametric test, we used One-way ANOVA (One-way analysis of variance) and Least Significant Difference (LSD) for pairwise comparison of sets of independent data. P values less than 0.05 are considered statistically different.

The luciferase activity was plotted against the number of cells, and the results are shown in FIG. 3.

As can be seen from FIG. 3, the luciferase activity in U87-Fluc cells showed a close positive correlation with the number of cells, R²Is 0.9864. Therefore, in a subsequent in vitro co-culture model, the in vitro proliferation condition of the U87-Fluc cells can be reflected by detecting the luciferase activity of the U87-Fluc cells.

Example 3: in-vitro cell model construction for simulating repopulation process after glioma radiotherapy

1. Glioma cell U87 radiotherapy by using medical linear accelerator

The specific operation steps are as follows:

1) cell preparation: u87 cells grown on various cell culture dishes can be irradiated with X-rays.

2) Placing the cells to be irradiated on a piece of transparent organic glass with the thickness of 15 cm, and covering a thin transparent plastic plate above the cell culture dish for limiting the irradiation field; the irradiation was carried out using a medical linear accelerator generating X-rays at a radiation dose rate of 3.6 Gy/min.

2. Construction of in vitro cell model for simulating proliferation after glioma radiotherapy

In the invention, U87-Fluc cells and U87 cells subjected to radiotherapy by a medical linear accelerator are co-cultured to obtain an in-vitro cell model simulating proliferation after radiotherapy of glioma (a mode diagram of the in-vitro cell model is shown in figure 4).

The method for constructing the in vitro cell model for simulating the proliferation of the glioma after the radiotherapy comprises the following steps:

1) u87 cells after radiotherapy by using a medical linear accelerator are digested on the same day and then are counted according to a certain number (1.5 multiplied by 10)⁴And/well) were inoculated into 24-well plates and cultured in 2% fetal bovine serum medium at 37 ℃ for 24 hours.

2) Then, inoculating a certain number (100 cells/hole) of U87-Fluc cells into culture holes containing U87 cells cultured in the step 1) after radiotherapy irradiation, and culturing by adopting a 2% fetal bovine serum culture medium at 37 ℃ to obtain the in-vitro cell model simulating tumor cell re-proliferation after glioma radiotherapy.

According to the co-culture cell model construction method, a co-culture system of U87 cells (namely 0Gy U87 cells) and U87-Fluc cells without radiotherapy is constructed; a group was also set to be plated with only U87-Fluc cells (U87-Fluc cell culture alone system) as a negative control.

Respectively culturing an in-vitro cell model simulating tumor cell re-proliferation after glioma radiotherapy, a co-culture system of U87 cells and U87-Fluc cells without radiotherapy and a U87-Fluc cell independent culture system, replacing 2% fetal bovine serum culture medium once every 2 days, performing cell imaging on a 24-well plate by using a cell living body imager after 12 days of culture, detecting luciferase activity of the U87-Fluc cells, and reacting the re-proliferation condition of the U87-Fluc cells. The specific operations of performing cell imaging and data analysis processing on the constructed in vitro cell model by using the cell living body imager are the same as those in embodiment 2, and are not described in detail. The specific experimental results are shown in fig. 5.

As can be seen from FIG. 5, compared with the group of U87-Fluc cells cultured alone, U87 cells (i.e., 10Gy U87 cells) after 10Gy radiotherapy can significantly stimulate the proliferation of U87-Fluc cells; compared with U87 cells without radiotherapy (namely 0Gy U87 cells), U87 cells after 10Gy radiotherapy can obviously stimulate U87-Fluc cells to proliferate. Specifically, the biochemical luminescence signal values of the 10Gy U87 cell + U87-Fluc cell co-culture group are about 3.5 times and 5.8 times or more of the U87-Fluc cell single culture group and the 0Gy U87 cell + U87-Fluc cell co-culture group.

Example 4: application of in-vitro cell model constructed by the invention in screening of drugs for inhibiting proliferation of glioma after radiotherapy

The in vitro cell model for simulating the proliferation after glioma radiotherapy can be applied to screening potential drugs for inhibiting the proliferation after glioma radiotherapy. NS-398 is a specific Cyclooxygenase-2 (COX-2) inhibitor. In the present invention, NS-398 is used to demonstrate how the in vitro cell model constructed in example 3 can be used to screen drugs that inhibit the proliferation of glioma cells after radiation therapy. The specific operation steps are as follows:

1) the glioma cell line U87 (namely 10Gy U87 cells) irradiated by 10Gy radiotherapy is 1.5 multiplied by 10⁴The number of the cells/well is inoculated in a 24-well cell culture plate, and the cells are cultured by adopting a 2% fetal bovine serum culture medium, wherein the culture temperature is 37 ℃, and the culture time is 24 hours.

2) After 24 hours of culture, inoculating U87-Fluc cells (the inoculation amount is 100 per well) into culture wells inoculated with U87 cells after radiotherapy irradiation; the culture wells inoculated with U87-Fluc cells were then divided into two groups, one group to which NS-398 was added (so that the concentration of NS-398 in the medium was 10. mu.M), and the other group to which a control agent DMSO for NS-398 (DMSO is dimethyl sulfoxide which is a solvent for dissolving NS-398) was added; culturing with 2% fetal calf serum culture medium at 37 deg.C, changing 2% fetal calf serum culture medium every 2 days, and finishing co-culture after 12 days.

Meanwhile, for comparison, a control experiment containing only U87-Fluc cells is set, and the specific operation steps of the control experiment are as follows: inoculating U87-Fluc cells into a 24-well cell culture plate according to 100 cells per well, culturing for 24 hours at 37 ℃ by adopting a 2% fetal bovine serum culture medium, then dividing the culture plate into two groups through a culture well inoculated with the U87-Fluc cells, adding NS-398 (enabling the concentration of the NS-398 in the culture medium to be 10 mu M) into one group of the culture well, and adding a contrast agent DMSO of the NS-398 into the other group of the culture well; culturing with 2% fetal calf serum culture medium at 37 deg.C, changing 2% fetal calf serum culture medium every 2 days, and finishing co-culture after 12 days.

And after the culture is finished, detecting the activity of the U87-Fluc luciferase in each system of the cell culture plate by a cell living body imager. The results are shown in FIG. 6.

As can be seen from FIG. 6, after 10. mu.M NS-398 is added into the single U87-Fluc cell culture system, the self proliferation capacity of the U87-Fluc cells is not obviously inhibited; and 10 mu M NS-398 is added into a 10Gy radiotherapy irradiation U87 cell (recorded as 10Gy U87) + U87-Fluc cell co-culture system, so that the proliferation of U87-Fluc cells in the system is obviously inhibited. Thus, NS-398 has no inhibitory effect on U87-Fluc cells per se, but rather achieves the effect of inhibiting U87-Fluc cell proliferation by inhibiting U87 cells from interacting with U87-Fluc cells after radiotherapy. NS-398 can be used as a potential future drug for inhibiting the proliferation of glioma after radiotherapy.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.

Claims

1. An in vitro cell model simulating proliferation after glioma radiotherapy is characterized in that the in vitro cell model is formed by co-culturing marked glioma cells and glioma cells irradiated by radiotherapy, wherein the marked glioma cells are human glioma cells marked by luciferase and green fluorescent protein together.

2. The in vitro cell model for simulating reimplantation of glioma following radiation therapy of claim 1, wherein said glioma cells after irradiation with radiation therapy are human glioma cell line U87-MG.

3. The in vitro cell model simulating the repopulation of glioma following radiation therapy according to claim 1 or 2, wherein said radiation therapy irradiation is X-ray irradiation and said radiation therapy irradiation dose is 10 Gy.

4. A method for constructing an in vitro cell model simulating glioma proliferation after radiotherapy according to any one of claims 1 to 3, wherein glioma cells irradiated by radiotherapy are inoculated into a culture medium, cultured at 37 ℃ for 12-48h, then labeled glioma cells are inoculated into the culture medium, and co-culture is carried out, so that the in vitro cell model simulating glioma proliferation after radiotherapy is obtained after the co-culture is finished.

5. The method of claim 4, wherein the ratio of the amount of glioma cells inoculated after the irradiation with the radiation therapy to the amount of labeled glioma cells inoculated is 150: 1.

6. The method according to claim 5, wherein the culture medium is 2% fetal bovine serum culture medium.

7. The method according to claim 6, wherein the co-culture temperature is 37 ℃ and the co-culture time is 10 to 14 days.

8. Use of an in vitro cell model mimicking glioma repopulation following radiation therapy as defined in any one of claims 1 to 3 for the screening of a medicament for inhibiting glioma repopulation following radiation therapy.