CN113444691A

CN113444691A - In-vitro cell model for simulating vascular regeneration after breast cancer radiotherapy and preparation method and application thereof

Info

Publication number: CN113444691A
Application number: CN202110687326.6A
Authority: CN
Inventors: 冯骁
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2021-09-28

Abstract

The invention belongs to the technical field of medical biology, and discloses an in vitro cell model for simulating vascular regeneration after breast cancer radiotherapy. The cell model is formed by co-culturing human umbilical vein vascular endothelial cells and breast cancer cells irradiated by radiotherapy, wherein the human umbilical vein vascular endothelial cells are labeled by luciferase and green fluorescent protein together. The preparation method of the cell model comprises the steps of inoculating the breast cancer cells irradiated by radiotherapy into a culture medium, culturing for 12-48h at 37 ℃, then inoculating human umbilical vein vascular endothelial cells jointly marked by luciferase and green fluorescent protein into the culture medium, and carrying out co-culture. The cell model can be used for simulating the process of promoting the regeneration of vascular endothelial cells by breast cancer cells after radiotherapy; in addition, certain compounds or drugs are added into the model to investigate whether the substances have inhibitory effect on the vascular regeneration after breast cancer radiotherapy. Therefore, the model can be used for screening drugs for inhibiting angiogenesis after breast cancer radiotherapy.

Description

In-vitro cell model for simulating vascular regeneration after breast cancer radiotherapy and preparation 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 vascular regeneration after breast cancer radiotherapy, and a preparation method and application thereof.

Background

Recent global cancer epidemiological studies (GLOBOCAN) have shown that: in all people, about 226 ten thousand cases of breast cancer of new women in 2020 account for 11.7 percent of all cancers, the incidence rate exceeds that of lung cancer, and the breast cancer becomes the first cancer in the world; the number of deaths was about 68.5 ten thousand, with the fifth digit. In women worldwide, breast cancer incidence and mortality are at the top. In China, the incidence of breast cancer is also at the head of female cancers, accounting for about 15% of all female cancers.

Radiotherapy (short for radiotherapy) is one of the important means for treating breast cancer. For patients who undergo breast sparing surgery and who have had an indication of radiotherapy for total mastectomy, breast radiotherapy is recommended to reduce the risk of local recurrence. Although radiotherapy can reduce the risk of local recurrence of breast cancer by about 15.7% in 10 years, there is still 19.3% of patients with local recurrence.

The support of tumor vessels cannot be released during the generation, development and recurrence and metastasis of the tumor, and the vessels can be regenerated after the breast cancer is subjected to radiotherapy. For example, Yu et al have shown that ionizing radiation can promote the activation of NF- κ B in breast cancer cells, thereby up-regulating the expression and release of growth factors such as VEGF, FGF-2, etc.; these pro-angiogenic factors mediate breast cancer recurrence by promoting angiogenesis. The mechanism of vascular regeneration after breast cancer radiotherapy is an important scientific problem to be solved urgently in the field of breast cancer radiotherapy, and is very important for solving the problem of breast cancer recurrence.

The establishment of the in vitro cell model which has strong simulation, controllable conditions, good repeatability and convenient operation and simulates the angiogenesis after the breast cancer radiotherapy can provide a convenient tool for exploring the mechanism of the model and also provide a screening model for screening drugs capable of inhibiting the angiogenesis after the breast cancer radiotherapy. However, there are limitations to using the currently available in vitro models of angiogenesis to simulate the process of angiogenesis after breast cancer radiotherapy. The tube formation assay (tube formation assay) is one of the widely used in vitro cell models for simulating angiogenesis. The following limitations are imposed if the model is applied to exploring the vascular regeneration mechanism after breast cancer radiotherapy. First, this model involves only vascular endothelial cells and does not reflect the interaction between breast cancer cells and vascular endothelial cells after radiotherapy. Secondly, even when vascular endothelial cells are treated with conditioned medium of breast cancer cells after radiotherapy, the effect of cell-cell contact (cell-cell contact) between breast cancer cells and vascular endothelial cells on angiogenesis cannot be demonstrated. Another widely used in vitro model method is to use a Transwell chamber, and to lay tumor cells in the upper chamber of the Transwell chamber and to lay vascular endothelial cells in the lower chamber of the Transwell chamber, thereby observing the effect of the tumor cells on the vascular endothelial cells. However, in vitro models using Transwell chambers spatially isolate tumor cells from vascular endothelial cells, preventing the direct contact of tumor cells with vascular endothelial cells from affecting angiogenesis. Therefore, in order to overcome the limitation of the existing in vitro model for simulating angiogenesis, an in vitro model capable of simulating breast cancer cells after radiotherapy to promote angiogenesis needs to be constructed, so that the direct effect of cell-cell contact between the breast cancer cells after radiotherapy and vascular endothelial cells can be reflected, and the indirect effect of the breast cancer cells on the vascular endothelial cells through secretion of soluble substances such as growth factors, cytokines and the like can be reflected.

Disclosure of Invention

In view of the problems and deficiencies in the prior art, one of the objectives of the present invention is to provide an in vitro cell model for simulating the vascular regeneration after breast cancer radiotherapy, another objective of the present invention is to provide a method for preparing an in vitro cell model for simulating the vascular regeneration after breast cancer radiotherapy, and a third objective of the present invention is to provide an application of the in vitro cell model for simulating the vascular regeneration after breast cancer 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 blood vessel regeneration after breast cancer radiotherapy, which is formed by co-culturing Human Umbilical Vein Endothelial Cells (HUVEC) and breast cancer cells irradiated by radiotherapy, wherein the human umbilical vein endothelial cells are co-labeled by luciferase and green fluorescent protein.

According to the in vitro cell model for simulating angiogenesis after breast cancer radiotherapy, preferably, the breast cancer cells are human breast cancer cell lines MDA-MB-231.

According to the in vitro cell model for simulating angiogenesis after breast cancer 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 firefly luciferase according to the in vitro cell model for simulating angiogenesis after breast cancer radiotherapy.

The invention provides a preparation method of an in vitro cell model for simulating vascular regeneration after breast cancer radiotherapy, which comprises the following steps: inoculating the breast cancer cells irradiated by the radiotherapy into a culture medium, culturing for 12-48h at 37 ℃, then inoculating human umbilical vein vascular endothelial cells jointly marked by luciferase and green fluorescent protein into the culture medium, and carrying out co-culture to obtain the in-vitro cell model simulating the regeneration of blood vessels after the radiotherapy of the breast cancer.

According to the preparation method, the ratio of the inoculation amount of the breast cancer cells after irradiation of the radiotherapy to the inoculation amount of the human umbilical vein vascular endothelial cells is preferably (50-100): 1.

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

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

According to the preparation method, preferably, the breast cancer cell is a human breast cancer cell line MDA-MB-231.

According to the preparation method, the human umbilical vein endothelial cells are preferably human umbilical vein endothelial cells labeled by luciferase and green fluorescent protein together.

According to the above preparation 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.

The third aspect of the present invention provides an application of the in vitro cell model described in the first aspect in screening drugs for inhibiting angiogenesis after breast cancer radiotherapy.

Specifically, certain compounds or medicines are added into the model, and whether the substances have an inhibiting effect on the angiogenesis after the breast cancer radiotherapy is explored, so that the medicine with potential clinical value for inhibiting the angiogenesis after the breast cancer radiotherapy is screened.

The invention applies a small molecule inhibitor Ki8751 of a targeting Vascular Endothelial Growth Factor Receptor 2 (VEGFR 2), and explains how to apply the model to screen a medicine with potential clinical value for inhibiting the Vascular regeneration after breast cancer radiotherapy. The specific method comprises the following steps: inoculating the breast cancer cells irradiated by radiotherapy into a 24-hole cell culture plate, culturing at 37 ℃ for 24 hours, inoculating a certain amount of human umbilical vein vascular endothelial cells jointly marked by luciferase and green fluorescent protein into the cell culture plate, then inoculating the human umbilical vein vascular endothelial cells jointly marked by the luciferase and the green fluorescent protein into culture holes for culturing the breast cancer cells irradiated by the radiotherapy, simultaneously adding a Ki8751 contrast agent into the culture holes, carrying out co-culture for 10-14 days, and detecting the proliferation condition of the marked vascular endothelial cells by using a cell living body imaging technology when the co-culture is finished. Finally, the luciferase activity difference between the Ki8751 group and the contrast agent group is analyzed through statistics, and whether Ki8751 can inhibit the proliferation capacity of the labeled vascular endothelial cells of the breast cancer cells after radiotherapy irradiation is judged.

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

(1) the in-vitro cell model for simulating the blood vessel regeneration after the breast cancer radiotherapy is established by directly co-culturing the marked human umbilical vein vascular endothelial cells and the breast cancer cells irradiated by the radiotherapy, and not only can direct effect of cell-cell contact between the breast cancer cells and the vascular endothelial cells on the vascular endothelial cells after the radiotherapy be reflected, but also can indirect effect of the breast cancer cells on the vascular endothelial cells through secretion of soluble angiogenesis promoting substances after the radiotherapy be reflected. Moreover, the in vitro cell model established by the invention can be observed as follows: compared with breast cancer cells without radiotherapy, the breast cancer cells after radiotherapy have stronger capacity of promoting the proliferation of vascular endothelial cells. Therefore, the cell model established by the invention can be used for simulating the process of promoting the vascular endothelial cells to accelerate proliferation of the breast cancer cells after radiotherapy.

(2) The labeled human umbilical vein vascular endothelial cells are labeled by adopting firefly luciferase and green fluorescent protein together, after a luminescent substrate is added into the labeled human umbilical vein vascular endothelial 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 positive correlation relationship is presented between the luciferase activity and the cell number. Therefore, the proliferation condition of the endothelial cells of the human umbilical vein can be obtained by detecting the activity of the luciferase by the cell living body imager, the detection is quick and convenient, and the cells cannot be damaged.

(3) One of the applications of the cell model established by the invention is that the cell model can be used for screening drugs with potential clinical value which can inhibit the regeneration of blood vessels after breast cancer radiotherapy. Specifically, certain compounds or medicines, such as certain growth factor receptor inhibitors and the like, are added into the model, and whether the compounds or medicines have an inhibitory effect on angiogenesis after breast cancer radiotherapy is further researched, so that the application of screening potential medicines capable of inhibiting angiogenesis after breast cancer radiotherapy is realized.

Drawings

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

FIG. 2 is a diagram showing the HUVEC (i.e., HUVEC-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 cell number in HUVEC-Fluc;

FIG. 4 is a model diagram of in vitro co-culture model of breast cancer cell line MDA-MB-231 and HUVEC-Fluc after irradiation by radiotherapy;

FIG. 5 shows the breast cancer cell line MDA-MB-231 and HUVEC-Fluc cells were seeded at a number of 2.5X 10⁴: at 500/hole, MDA-MB-231 cells after radiotherapy have a result graph of HUVEC-Fluc proliferation promoting effect;

FIG. 6 shows that the inoculation number of breast cancer cell lines MDA-MB-231 and HUVEC-Fluc cells in the in vitro cell model simulating angiogenesis after breast cancer radiotherapy is 5 × 10⁴: at 500/hole, MDA-MB-231 cells after radiotherapy have a result graph of HUVEC-Fluc proliferation promoting effect;

FIG. 7 is a graph showing the result of the proliferation effect of MDA-MB-231 cells on HUVEC-Fluc after radiotherapy when 10 μ M Ki8751 is added to an in vitro cell model simulating vascular regeneration after radiotherapy of breast cancer.

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 "MDA-MB-231" means "human breast cancer cell line MDA-MB-231", which is a human breast cancer cell that can be cultured in vitro.

In the present specification, the term "HUVEC" represents "Human Umbilical Vein Endothelial Cell (HUVEC)" which is a human vascular endothelial cell.

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 "HUVEC-Fluc" represents "HUVEC stably transfected with Fluc and GFP fusion genes".

Example 1: construction of human umbilical vein vascular endothelial cell (HUVEC-Fluc) stably transfected with fusion gene of firefly luciferase (Fluc) and Green Fluorescent Protein (GFP)

1. HUVEC cell resuscitation

The specific experimental procedure for HUVEC cell resuscitation was as follows:

1) the electric heating constant temperature water tank is preheated to 37 ℃.

2) Taking out the freezing tube from a liquid nitrogen tank or a-80 ℃ ultra-low temperature refrigerator, quickly placing the freezing tube in a 37 ℃ constant temperature water tank, and slightly shaking the freezing tube to quickly melt the cells in the freezing tube.

3) And after the cells in the freezing tube are fully melted, taking out the freezing tube, and wiping the freezing tube by using a 75% alcohol cotton ball. The freezing tube is opened in a clean bench, the cells are transferred to a 15 ml centrifuge tube, 5 ml of 10% fetal calf serum culture medium is added, and the mixture is fully blown and uniformly mixed.

4) Centrifuge tubes of 15 ml in a centrifuge at 1000 rpm for 3 minutes.

5) After centrifugation, the supernatant was discarded, 2 ml of 10% fetal calf serum medium was added, and the cells were resuspended by pipetting.

6) Transferring the cell suspension to a culture dish, adding a certain amount of 10% fetal calf serum culture medium, fully and uniformly blowing, and placing in a constant-temperature incubator at 37 ℃ for culture.

2. HUVEC cell passage and culture

The specific experimental procedures for passage and culture of HUVEC cells were as follows:

1) when the cells grow to a certain confluency, the original culture medium in the culture dish is sucked out and discarded. Adding a proper amount of PBS into the culture dish, and then slowly shaking the culture dish to achieve the purpose of washing cells by using the PBS. This process was repeated twice.

2) After the PBS was aspirated, an appropriate amount of pancreatin was added to the dish to cover the cells properly, and the dish was placed again in the cell incubator.

3) After 2-5 minutes, the petri dish was placed under a microscope for observation. If cytoplasmic retraction, cell morphology rounding, and disruption of intercellular junctions are observed, pancreatin digestion is terminated by adding an appropriate amount of 10% fetal bovine serum medium to the culture dish.

4) The cell suspension was transferred to a 15 ml centrifuge tube and gently pipetted evenly. Followed by centrifugation at 1000 rpm for 3 minutes.

5) The supernatant in the centrifuge tube was discarded and fresh 10% fetal bovine serum medium was added to resuspend the cells. The cell suspension is transferred to a new culture dish in a certain ratio (e.g., 1:3 or 1: 4) according to the cell growth habit and the subsequent experimental schedule. After the culture medium is replenished, the mixture is placed in a cell culture box for continuous culture.

6) And in the cell culture process, the culture medium is replaced according to the cell growth state and the experiment requirement.

3. HUVEC cells cryopreserved

The specific procedure for cryopreservation of HUVEC cells was as follows:

1) cells in log phase were selected for cryopreservation. Cells in the dish were first digested and the detailed procedure was as for cell passage above.

2) While centrifuging the cell suspension, a cell cryopreservation solution is prepared.

3) And after the centrifugation is finished, removing supernatant in the centrifuge tube, adding a proper amount of cell cryopreservation solution, and fully blowing and beating the cells to be uniformly mixed. After counting the cells, the cell suspension concentration was adjusted to 5X 10 with the frozen stock solution⁶/ml-1×10⁷/ml。

4) 1 ml of cell-enriched cryopreservation solution was added to each cryopreservation tube. The information of the frozen cell name, date, operator, etc. is marked on the wall of the frozen tube for subsequent use.

5) Placing the freezing tube in a freezing box, and placing the freezing box in a refrigerator at the temperature of minus 80 ℃. After 12 hours, the vial was transferred from a-80 ℃ freezer to liquid nitrogen for longer preservation of the cells. If the freezing box is not used, a gradient cooling freezing method can be used. In short, the mixture is placed in a refrigerator at 4 ℃ for 1 hour, then transferred to a refrigerator at-20 ℃ and stored for about 2 hours, and then transferred to a refrigerator at-80 ℃ and stored for more than 12 hours, and then directly transferred into liquid nitrogen for storage.

4. Plasmid amplification

1) DH 5. alpha. competent E.coli was taken out of a-80 ℃ refrigerator, and the objective plasmids to be amplified (pLEX-GFP-luc 2, psPAX2, pMD2. G) were taken out of a-20 ℃ refrigerator, and these were placed on ice and allowed to melt naturally. Wherein the map structure of the pLEX-GFP-luc2 plasmid is shown in figure 1.

2) The electric heating constant temperature water tank is preheated to 42 ℃.

3) Mu.l of the plasmid of interest was added to 30-50. mu.l of DH 5. alpha. competent E.coli, mixed well and then placed on ice for 30 minutes.

4) A1.5 ml EP tube containing a mixture of DH 5. alpha. competent Escherichia coli and the objective plasmid was placed in a water bath thermostatically controlled at 42 ℃ and subjected to thermal shock. The thermal shock time was strictly controlled at 90 seconds, during which time the 1.5 ml EP tube was not removed at will.

5) After the thermal shock, 500. mu.l of LB medium without ampicillin was added to a 1.5 ml EP tube. This EP tube was placed on a constant temperature shaker at 37 ℃ and 200 rpm for 1 hour to pre-amplify E.coli.

6) The liquid in the 1.5 ml EP tube was transferred to a 15 ml centrifuge tube and 4 ml LB medium with an ampicillin concentration of 100. mu.g/ml was added. The 15 ml centrifuge tube was placed on a constant temperature shaker at 37 ℃ for 10-12 hours at 200 rpm to amplify the E.coli having resistance.

7) The liquid in the 15 ml centrifuge tube was transferred to a 250 ml Erlenmeyer flask and about 100 ml of LB medium with an ampicillin concentration of 100. mu.g/ml was added. The Erlenmeyer flask was placed on a constant temperature shaker at 37 deg.C, 200 rpm, overnight, to allow large scale amplification of the E.coli containing the resistance.

5. Plasmid extraction

The plasmid extraction uses an endotoxin-free plasmid large-extraction kit of Tiangen Biotechnology (Beijing) Co., Ltd, and comprises the following steps:

1) the overnight culture was poured into a centrifuge tube, centrifuged at 8000rpm at room temperature for 3 minutes to precipitate the bacteria, and the supernatant was aspirated. If the bacteria liquid is more, the process can be repeated. However, it should be noted that the amount of the microbial solution is preferably sufficient to achieve sufficient lysis, but not too much, which may reduce the extraction efficiency due to insufficient lysis.

2) After centrifugation, the supernatant should be sucked off as much as possible, and the droplets on the tube wall can be sucked off by using clean absorbent paper.

3) To the centrifuge tube containing the bacterial pellet was added 8 ml of solution P1 (ensuring RNase A had been added to P1) and the bacterial pellet was resuspended thoroughly using a vortex shaker. The bacterial pellet should be resuspended thoroughly, otherwise lysis effects will be affected and the amount and purity of plasmid extraction will be reduced.

4) Adding 8 ml of the solution P2 into a centrifuge tube, then turning the centrifuge tube up and down gently for a plurality of times, fully cracking the thalli, and standing for 5-10 minutes at room temperature. Care was taken not to shake vigorously to avoid contamination with genomic DNA.

5) Adding 8 ml of the solution P4 into a centrifuge tube, turning the centrifuge tube up and down gently for a plurality of times, and fully mixing until white flocculent precipitate appears in the solution. Standing at room temperature for about 10 minutes. Centrifuge at 8000rpm for 5-10 minutes (optionally increasing the centrifugation time) to allow the pellet to settle to the bottom of the tube. The centrifuged supernatant was carefully poured into the filter CS1, the push handle was slowly pushed, and the filtrate was collected in a new centrifuge tube.

6) Column balancing: the adsorption column was placed in a 50 ml centrifuge tube, and 2.5 ml of equilibration solution BL was added to adsorption column CP 6. Centrifuging at room temperature 8000rpm for 2 min, discarding waste liquid in the collecting tube, and replacing the adsorption column in the collecting tube.

7) 0.3 times the volume of the filtrate of isopropyl alcohol was added to the filtrate, and the mixture was turned upside down and mixed, and then transferred to an adsorption column CP 6.

8) Centrifuging at room temperature 8000rpm for 2 min, discarding the waste liquid in the collection tube, and placing the adsorption column CP6 in the collection tube again. Since the adsorption column CP6 had a maximum volume of 15 ml, two column passes were required.

9) 10 ml of the rinsing solution PW (ensuring that the corresponding volume of absolute ethanol has been added) are introduced into the adsorption column CP6, centrifuged at 8000rpm for 2 minutes at room temperature, the waste liquid is discarded and the adsorption column is placed again in the collection tube.

10) And repeating the previous step.

11) 3 ml of absolute ethanol was added to the adsorption column CP6, and centrifuged at 8000rpm at room temperature for 2 minutes. The waste liquid is discarded, and the adsorption column is placed in the collecting pipe again.

12) Centrifugation is carried out at room temperature of 8000rpm for 5 minutes in order to remove the residual rinsing liquid from the adsorption column.

13) Placing the adsorption column CP6 in a new 50 ml centrifuge tube, hanging and dripping 0.5-1 ml of elution buffer TB into the middle position of the adsorption membrane, and standing for 5 minutes at room temperature. Centrifuge at 8000rpm for 2 minutes at room temperature. The eluate from the 50 ml centrifuge tube was transferred to a new 1.5 ml EP tube and stored at-20 ℃.

6. 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 2 ml of Opti-MEM medium were added 3.3. mu.g of pLEX-GFP-luc2, 3. mu.g of psPAX2, and 1.2. mu.g of pMD2.G, followed by thorough mixing; to another 2 ml 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, 1 ml per tube, and stored in a freezer at-80 ℃ until use.

7. Lentiviral infection HUVEC cell and screening and construction of stable transfection cell strain

The specific operation steps are as follows:

1) target cells (HUVEC) were plated in 6-well plates, and the number of plated cells was calculated to reach confluency after overnight

Preferably 50%.

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

3) 1 ml of virus solution, 1 ml 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. Cells stably transfected with the gene of interest survive, while the remaining cells are killed.

7) HUVEC cells stably transfected with pLEX-GFP-luc2 were obtained after a period of puromycin selection culture (final puromycin concentration could be increased to 3-4. mu.g/ml).

According to the operation steps, the HUVEC cells which stably transfect firefly luciferase (Fluc) and Green Fluorescent Protein (GFP) fusion genes and stably express the Fluc-GFP fusion proteins are successfully constructed and named as HUVEC-Fluc. The HUVEC-Fluc pattern is shown in FIG. 2. The effect of luciferase in HUVEC-Fluc cells was: after the luminescent substrate is added into the HUVEC-Fluc cells, the firefly luciferase can catalyze the luminescent substrate to change, and biochemical luminescence is generated. The instrument for detecting the biochemical luminescence can effectively evaluate the activity of the luciferase.

Example 2: study on correlation between luciferase activity of HUVEC-Fluc cells and cell number

In a subsequent in vitro co-culture model, it is desirable to reflect HUVEC-Fluc proliferation by detecting the strength of the luciferase activity of HUVEC-Fluc firefly. Therefore, it is necessary to confirm whether the firefly luciferase activity in HUVEC-Fluc exhibits a positive correlation with the number of cells. We spread the specified number of HUVEC-Fluc in 96-well plates, and after the cells attached to the walls, the luciferase activity of HUVEC-Fluc in 96-well plates was detected by means of a cell living biochemical luminescence imager.

The specific experimental operations were as follows:

HUVEC-Fluc cells in good growth state were digested to prepare single cell suspensions. HUVEC-Fluc was plated into 96-well plates at 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 HUVEC-Fluc in the 96-well plate.

The specific operation of using the cell living body imager to image the cells is as follows: discarding the culture solution in the well plate to be imaged, adding a proper volume (24 well plate: 150. mu.l/well, 96 well plate: 80. mu.l/well) of D-luciferin potassium to the well plate to obtain a final concentration of 0.15 mg/ml; and then carrying out HUVEC-Fluc cell biochemical luminescence in-vivo imaging by using a cell in-vivo imaging instrument 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 HUVEC-Fluc cells showed a very close positive correlation with the cell number, R²Is 0.9923. Thus, subsequent in vitro co-culture modelsIn vitro proliferation can be reflected by detecting the luciferase activity of the HUVEC-Fluc cells.

Example 3: construction of cell model for simulating vascular regeneration after breast cancer radiotherapy

1. Radiotherapy of breast cancer cell line MDA-MB-231 by medical linear accelerator

The specific operation steps are as follows:

1) cell preparation: the X-ray irradiation can be carried out on the breast cancer cell line MDA-MB-231 which grows in cell culture dishes with various specifications.

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 cell model for simulating vascular regeneration after breast cancer radiotherapy

In the invention, MDA-MB-231 cells after being subjected to radiotherapy by a medical linear accelerator and HUVEC-Fluc cells are co-cultured to obtain a cell model (the mode diagram of the cell model is shown in figure 4) for simulating the regeneration of blood vessels of breast cancer cells after the radiotherapy, and the cell model is used for simulating the biological process of promoting the regeneration of blood vessels of breast cancer cells after the radiotherapy.

The specific operation steps of the co-culture cell model construction method are as follows:

1) the breast cancer cell line MDA-MB-231 after radiotherapy is digested in the same day and then is added according to a certain number (2.5 multiplied by 10)⁴Per well or 5X 10⁴One/well) were re-inoculated into 24-well plates and cultured using 2% fetal bovine serum medium at 37 ℃ for 24 hours. In addition, MDA-MD-231 cells that were not subjected to radiotherapy were also seeded in the same number into 24-well plates.

2) After the breast cancer cell line MDA-MB-23137 ℃ is cultured for 24h after radiotherapy, HUVEC-Fluc cells are inoculated into culture wells containing MDA-MB-231 cells according to a certain number (500 cells/well), and 2% fetal bovine serum culture medium is adopted for culture at 37 ℃. In addition, one group was set to be plated only into HUVEC-Fluc cells as one of the negative controls.

3) Changing 2% fetal bovine serum culture medium once every 2 days, after 12 days of co-culture, carrying out cell imaging on a 24-well plate by using a cell living body imager, and detecting the luciferase activity of the HUVEC-Fluc cells in the co-culture system. The specific operations of cell imaging and data analysis processing by using the cell living body imager are the same as those in embodiment 2, and detailed descriptions thereof are omitted.

3. The inoculation number of MDA-MB-231 cells and HUVEC-Fluc cells after radiation treatment is 2.5 multiplied by 10⁴: study on proliferation effect of MDA-MB-231 cells after radiotherapy on HUVEC-Fluc cells at 500/well

Constructing a cell model for simulating angiogenesis after breast cancer radiotherapy according to the co-culture cell model construction method, wherein the MDA-MB-231 cells after radiotherapy irradiation are MDA-MB-231 cells after radiotherapy with the dose of 10Gy, and the inoculation amount is 2.5 multiplied by 10⁴The number of HUVEC-Fluc cells inoculated per well was 500 per well. After the co-culture is carried out for 12 days, a cell living body imager is used for carrying out cell imaging on the 24-hole plate, the luciferase activity of the HUVEC-Fluc cells in the co-culture system is detected, and the proliferation condition of vascular endothelial cells is reflected.

The specific experimental results are shown in fig. 5. As can be seen from FIG. 5, compared with the HUVEC-Fluc cell single culture group, MDA-MB-231 cells after being irradiated by 10Gy can significantly stimulate the HUVEC-Fluc cell proliferation; compared with MDA-MB-231 cells without radiotherapy, MDA-MB-231 cells subjected to radiotherapy of 10Gy can also obviously stimulate HUVEC-Fluc cells to proliferate; furthermore, the biochemical luminescence signal values of the HUVEC-Fluc cell &10 Gy MDA-MB-231 cell co-culture group were about 4.2 times and 23 times or more as those of the HUVEC-Fluc cell single culture group and the HUVEC-Fluc cell & non-radiotherapy (0 Gy) MDA-MB-231 cell co-culture group.

4. The inoculation number of MDA-MB-231 cells and HUVEC-Fluc cells after radiation treatment is 5 multiplied by 10⁴: study on proliferation effect of MDA-MB-231 cells after radiotherapy on HUVEC-Fluc cells at 500/well

Constructing a cell model for simulating angiogenesis after breast cancer radiotherapy according to the co-culture cell model construction method, wherein the MDA-MB-231 cells after radiotherapy irradiation are MDA-MB-231 cells after radiotherapy with the dose of 10Gy, and the inoculation amountIs 5.0X 10⁴The number of HUVEC-Fluc cells inoculated per well was 500 per well. After the co-culture is carried out for 12 days, a cell living body imager is used for carrying out cell imaging on the 24-hole plate, the luciferase activity of the HUVEC-Fluc cells in the co-culture system is detected, and the proliferation condition of vascular endothelial cells is reflected.

The specific experimental results are shown in fig. 6. As can be seen from FIG. 6, compared with the HUVEC-Fluc cell single culture group, MDA-MB-231 cells after being irradiated by 10Gy can significantly stimulate the HUVEC-Fluc cell proliferation; compared with MDA-MB-231 cells without radiotherapy, MDA-MB-231 cells subjected to radiotherapy of 10Gy can also obviously stimulate HUVEC-Fluc cells to proliferate; moreover, the biochemical luminescence signal values of the HUVEC-Fluc cell &10 Gy MDA-MB-231 cell co-culture group were about 4.2 times and 23 times of those of the HUVEC-Fluc cell single culture group and the HUVEC-Fluc cell & non-radiotherapy (0 Gy) MDA-MB-231 cell co-culture group.

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

The in vitro cell model for simulating angiogenesis after breast cancer radiotherapy constructed by the invention can be applied to screening potential drugs for inhibiting angiogenesis after breast cancer radiotherapy. Ki8751 is an inhibitor of vascular endothelial cell growth factor receptor 2 (VEGFR 2). The Ki8751 is used for explaining how the model is applied to screening potential drugs for inhibiting angiogenesis after breast cancer radiotherapy. The specific implementation is as follows.

The breast cancer cell line MDA-MB-231 irradiated by 10Gy radiotherapy has a weight ratio of 2.5 × 10⁴The number of the cells/well is inoculated in a 24-well cell culture plate, 2 groups are paved, and 2% fetal bovine serum culture medium is adopted for culture, the culture temperature is 37 ℃, and the culture time is 24 hours. After the breast cancer cell line MDA-MB-23137 ℃ is cultured for 24h after radiotherapy, HUVEC-Fluc cells are inoculated into culture wells containing MDA-MB-231 cells according to 500 cells/well, Ki8751 is added into the culture wells at the same time, and 2% fetal bovine serum culture medium is adopted for culture at 37 ℃. The 2% fetal bovine serum culture medium was replaced every 2 days, and after 14 days of co-culture, HUVEC-Fluc luciferase activity in the cell culture plate was detected by a cell biopsy imager. In addition, a negative control group without Ki8751 addition and HUV-only plating were performedBlank control group of EC-Fluc cells. The results are shown in FIG. 7.

As can be seen from FIG. 7, the HUVEC-Fluc cell independent culture group added with 10. mu.M Ki8751 did not inhibit the self-proliferation ability of the HUVEC-Fluc cells; in the group co-cultured by 10Gy MDA-MB-231 cells and HUVEC-Fluc cells, 10 mu M Ki8751 obviously inhibits the proliferation of the HUVEC-Fluc cells in the group. Therefore, Ki8751 has no inhibition effect on the HUVEC-Fluc cells, and the effect of inhibiting the HUVEC-Fluc cell proliferation is realized by inhibiting the interaction of the breast cancer cells and the HUVEC-Fluc cells after radiotherapy. Ki8751 can be used as a potential future drug for inhibiting the regeneration of blood vessels after breast cancer 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 for simulating the vascular regeneration after the radiotherapy of breast cancer is characterized in that the in vitro cell model is formed by co-culturing human umbilical vein vascular endothelial cells and breast cancer cells irradiated by the radiotherapy, wherein the human umbilical vein vascular endothelial cells are human umbilical vein vascular endothelial cells jointly marked by luciferase and green fluorescent protein.

2. The in vitro cell model for simulating angiogenesis after radiotherapy of breast cancer according to claim 1, wherein the breast cancer cell is a human breast cancer cell line MDA-MB-231.

3. The in vitro cell model for simulating angiogenesis after radiation therapy of breast cancer 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 preparing an in vitro cell model for simulating blood vessel regeneration after breast cancer radiotherapy as claimed in any one of claims 1-3, wherein the breast cancer cells irradiated by the radiotherapy are inoculated into a culture medium, cultured at 37 ℃ for 12-48h, then inoculated with human umbilical vein vascular endothelial cells jointly labeled by luciferase and green fluorescent protein, and co-cultured, and the in vitro cell model simulating blood vessel regeneration after breast cancer radiotherapy is obtained after co-culture is finished.

5. The in vitro cell model for simulating angiogenesis after radiotherapy of breast cancer according to claim 4, wherein the ratio of the inoculation amount of the breast cancer cells after irradiation of the radiotherapy to the inoculation amount of the endothelial cells of the umbilical vein of the human is (50-100): 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-cultivation temperature is 37 ℃ and the co-cultivation time is 10 to 14 days.

8. Use of the in vitro cell model of any one of claims 1 to 3 for screening a medicament for inhibiting angiogenesis after radiation therapy of breast cancer.