阅读说明：本技术 一种小鼠肉瘤放疗抵抗细胞株及其应用 (Mouse sarcoma radiotherapy resistant cell strain and application thereof ) 是由 彭星辰 苏勇林 何金兰 胡晓林 于 2021-08-13 设计创作，主要内容包括：本发明提供了一种小鼠肉瘤放疗抵抗细胞株S180-R,保藏于中国微生物菌种保藏管理中心CGMCC,保藏号CGMCC No.21412；它是由小鼠肉瘤细胞株S180经多次X射线照射和培养后获得的放疗抵抗细胞株,放射抵抗性强,在小鼠肉瘤放疗抵抗机制的细胞模型中具有良好应用前景。(The invention provides a mouse sarcoma radiation-resistant cell strain S180-R, which is preserved in China center for microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 21412; the cell strain is a radiotherapy resistant cell strain obtained by irradiating and culturing a mouse sarcoma cell strain S180 by X-rays for multiple times, has strong radiotherapy resistance, and has good application prospect in a cell model of a mouse sarcoma radiotherapy resistant mechanism.)

1. The mouse sarcoma radiation-resistant cell strain is characterized in that the mouse sarcoma radiation-resistant cell strain S180-R is preserved in China center for culture Collection of microorganisms (CGMCC) with the preservation number of CGMCC No. 21412.

2. The radiation therapy-resistant cell line according to claim 1, which is a cell and/or a daughter cell of a murine sarcoma cell line S180 that survives X-ray irradiation.

3. The radiation therapy-resistant cell line of claim 2, wherein the X-ray irradiation is three rounds of X-ray irradiation, wherein each round of X-ray irradiation is 15 shots at a dose of 2 Gy/shot.

4. The radiation therapy resistant cell line of claim 3, obtained by the method steps of:

(1) irradiating mouse sarcoma cell S180 with 2 Gy/time dose for 15 times to obtain viable cell;

(2) repeating the step (1) for three times to obtain the mouse sarcoma radiotherapy resistant cell strain S180-R.

5. The radiation therapy-resistant cell line of claim 4, wherein the sarcomas cell line S180 of step (1) is a sarcomas cell line S180 of logarithmic growth phase.

6. The radiation therapy resistant cell line of claim 4, wherein step (1) further comprises the steps of digesting, passaging, and culturing the viable cells.

7. Use of the radiation therapy resistant cell line of any one of claims 1 to 6 in a mouse sarcoma radiation therapy resistant cell model.

8. The use of claim 7, wherein the cellular model of radioresistance is a cellular model for screening for a radiosensitizing drug.

9. The use of claim 7, wherein the radiation therapy resistant cell model is a cell model for screening for radiation therapy resistant genes.

10. The use of claim 7, wherein said radiation therapy resistant cell model is a cell model for studying the radiation therapy resistant mechanisms of sarcoma in mice.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a mouse sarcoma radiotherapy resistant cell strain and application thereof.

Background

Radiotherapy is one of the main means for treating malignant tumors and is an indispensable key component in the comprehensive treatment of tumors. Clinically, over 70% of patients with tumors need to receive radiation therapy during the course of disease. Despite the great advances in radiotherapy technology, radiotherapy resistance remains a major obstacle to radiotherapy treatment, which ultimately can lead to tumor recurrence and metastasis. Finding out the reason for the resistance of malignant tumor to radiotherapy so as to increase the sensitivity of tumor to radiotherapy is an urgent problem to be solved in the field of malignant tumor treatment.

However, resistance to radiation therapy is a complex biological process associated with the co-action of multiple factors including DNA damage response abnormalities, apoptosis, autophagy, gene mutations, cell cycle checkpoints, and uncontrolled signaling pathways. The application of the radiotherapy resistant cell model can lay a foundation for the research of reversing radiotherapy resistance. Because the cell strains with different radiosensitivities in a specific tumor type cannot eliminate the influence caused by the genetic background difference of different cell strains, the cell strains with radiotherapy tolerance are obtained by repeatedly irradiating and screening cells in vitro through X-rays and are compared with parent cells for research, and the method is an important method for researching a radiotherapy resistance mechanism in vitro.

Patent applications CN110878284A and CN107779438B disclose methods for constructing radiation-resistant cell lines of human lung cancer cell strains A549 and PC9, respectively; patent applications CN109735495A and CN104017799A respectively disclose a method for constructing a radiation-resistant cell line of a human nasopharyngeal carcinoma cell line CNE-2; patent application CN106367391A discloses a method for constructing a cell line resistant to SW480 radiotherapy of human rectal cancer cells. In the prior art, the parent cells are irradiated by X-rays in different method modes, so that corresponding radiotherapeutic resistant cell lines are successfully constructed, and compared with the respective parent cells, the radiotherapeutic resistant cell lines obtained have different radiotherapeutic resistant effects along with different construction methods.

At present, no report on a radiation-resistant cell strain of a mouse sarcoma cell line and a construction method thereof is found in the prior art. Provides a basis for further researching the molecular mechanism of sarcoma radiotherapy resistance, screening radiotherapy resistant targets and improving radiotherapy sensitivity, and needs to research the mouse sarcoma cell strain with excellent radiotherapy resistance.

Disclosure of Invention

The invention aims to provide a mouse sarcoma radiotherapy resistant cell strain with excellent radiotherapy resistant property, and a construction method and application of the cell strain.

The invention provides a mouse sarcoma radiotherapy resistant cell strain S180-R which is preserved in China center for culture Collection of microorganisms (CGMCC) with the preservation number of CGMCC No. 21412.

Biological material preservation:

the mouse sarcoma radiotherapy resistant cell strain S180-R has been preserved in China general microbiological culture Collection center (CGMCC, China, Beijing, China academy of sciences and microbiology research institute) in 12-11 months in 2020, and the registration number of the preservation center is as follows: CGMCC No. 21412.

Furthermore, the tumor cell line is a cell which survives the mouse sarcoma cell line S180 after being irradiated by X-rays and/or a progeny cell thereof.

Further, the above X-ray irradiation is three rounds of X-ray irradiation in which each round of X-ray irradiation is performed 15 times at a dose of 2 Gy/time.

Furthermore, the radiotherapeutic resistant cell strain is obtained by the following method steps:

(1) irradiating mouse sarcoma cell S180 with 2 Gy/time dose for 15 times to obtain viable cell;

(2) repeating the step (1) for three times to obtain the mouse sarcoma radiotherapy resistant cell strain S180-R.

Further, the mouse sarcomas cell line S180 in the step (1) is a mouse sarcomas cell line S180 in a logarithmic growth phase.

Further, the step (1) also comprises the steps of digesting, passaging and culturing the surviving cells.

The invention also provides application of the radiotherapeutic resistant cell strain in a mouse sarcoma radiotherapeutic resistant cell model.

Furthermore, the radiotherapy resistant cell model is a cell model for screening radiotherapy sensitizing drugs, or a cell model for screening radiotherapy resistant genes, or a cell model for researching a mouse sarcoma radiotherapy resistant mechanism.

Experimental results show that the mouse sarcoma radiotherapy resistant cell strain S180-R has strong radiation resistance, and the survival fraction of the S180-R reaches more than 22 times of that of a parent cell S180 under the irradiation dose of 10 Gy. The mouse sarcoma cell radiotherapeutic resistant cell strain provided by the invention provides a basis for further researching a molecular mechanism of mouse sarcoma radiotherapeutic resistance, screening radiotherapeutic resistant targets, improving radiotherapeutic sensitivity, screening radiotherapeutic sensitizing drugs and screening radiotherapeutic resistant genes.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the radiosurvival curves of mouse sarcoma parental cells S180 and mouse sarcoma radiotherapy resistant cells S180-R constructed by the method of the invention.

Detailed Description

Mouse sarcoma cells S180 were purchased from ATCC (American type culture collection ), subcultured and maintained by the laboratory of biotherapy nations of Sichuan university. The other experimental reagents and equipment are known products and are obtained by purchasing products sold in the market.

Example 1 construction of radiation therapy resistant cell lines of the invention

The radiotherapeutic resistant cell strain is established by adopting multiple times of routine segmentation, namely, the routine dose (2Gy) is adopted to carry out irradiation of the total dose of 2Gy multiplied by 45f by a three-wheel intermittent irradiation induction method, and the radiotherapeutic resistant cells are enriched by multiple times of low-dose radiotherapies. The specific operation method comprises the following steps:

(1) culturing mouse sarcoma cell S180, preparing single cell suspension from logarithmic phase cells, planting in culture bottle at appropriate concentration, and culturing for 8-12 hr while maintaining cell adherence and good state.

(2) Cells were irradiated using an X-ray biological irradiator (RS2000 series, Rad Source) at a voltage of 160kV, a current of 25mA, and a dose rate of 1.071 Gy/min.

(3) The first round of irradiation was carried out at a dose of 2Gy/f 5 times per week (Monday-Friday irradiation, Saturday-Sunday irradiation, and Sunday irradiation), 15 times in total, and a cumulative dose of 30Gy was carried out. The medium was changed every 48 hours during irradiation to remove dead cells from the culture supernatant.

(4) Stopping irradiation after the first round of irradiation is finished, digesting the surviving clone-like cell mass into a single cell for culture and passage, and performing the second round of irradiation after the cell is in a recovery state after 2-8 weeks of culture. During cell recovery, cells are immediately passaged and cryopreserved according to cell density.

(5) The second round was still irradiated at a dose of 2Gy/f 5 times a week for a total of 15 times, and the second round was irradiated at a cumulative dose of 30 Gy. The medium was changed every 48 hours during irradiation to remove dead cells from the culture supernatant.

(6) Stopping irradiation after the second round of irradiation is finished, digesting the surviving clone-like cell mass into a single cell for culture and passage, and performing the third round of irradiation after the cell recovers the state after the culture is carried out for 2-10 weeks. During cell recovery, cells are immediately passaged and cryopreserved according to cell density.

(7) The clone-like cell mass which still survives after the third round of irradiation is the radiotherapy resistant cell and is named as S180-R.

As a result of detection, the S180-R cell strain prepared by the method has very excellent radiation resistance, so that the S180-R cell strain is preserved in China general microbiological culture Collection center (CGMCC, address: China, Beijing, institute of microbiology, China academy of sciences) at 12-11 th of 2020, and the registration accession number of the preservation center is as follows: CGMCC No. 21412.

The experimental examples below demonstrate the radiotherapeutic resistance effect of the S180-R cell line of the present invention.

Experimental example 1 detection of radiotherapeutic resistance characteristics of radiotherapeutic resistance cell lines constructed by the method of the present invention

1. Experimental methods

Clone formation experimental detection:

preparation of radiotherapy-resistant cells: the S180-R obtained in example 1 was cultured for 5 generations and then subjected to the experiment, and was frozen in time during the culture.

Preparation of parental cells: the unirradiated S180 cells are used as parent cells, planted in culture flasks according to a relatively dilute concentration, cultured synchronously with the cells of example 1, and subjected to cell passage and cryopreservation in time.

The method comprises the following specific steps:

(1) cells (including parent cells and radiotherapeutic resistant cells) in a logarithmic growth phase are digested and then inoculated into a six-well plate, different numbers of cells are respectively inoculated according to different X-ray irradiation doses, and 3 multiple wells are arranged at each dose point.

(2) The cells were irradiated the next day with an X-ray biological irradiator at dose gradients of 0Gy, 2Gy, 4Gy, 6Gy, 8Gy, 10 Gy. After irradiation, the cells were immediately returned to CO₂The incubator continues to culture for about 2 weeks, and the cell culture medium is replaced every 3-5 days.

(3) The culture was terminated when macroscopic cell clones appeared in the six-well plate, the medium in the six-well plate was discarded, and the cells were washed twice with 1 × DPBS.

(4) Adding appropriate amount of pure methanol, fixing at room temperature for 15min, discarding the fixing solution, adding crystal violet dye solution, standing at room temperature for 20min, and dyeing.

(5) The crystal violet staining solution was discarded, slowly rinsed with double distilled water until the residual violet solution was washed clean, left to air dry at room temperature, and the cell clone number was counted under a microscope (a cell mass of more than 50 cells was considered as one clone).

(6) Calculating inoculation rate and survival score

The inoculation rate was [ colony formation number (0 Gy)/cell inoculation number (0Gy) ] × 100%

Survival score-colony formation number (X Gy)/[ (number of seeded cells (X Gy) × seeding rate) ]

(7) Survival curves were plotted using graphpadprism7.0 and the differences in radiotherapeutic resistance between the two groups of cells were compared.

2. Results of the experiment

The radiosensitivity of cells was analyzed by colony formation experiments as well as cell survival curves. And (4) prompting by a result: survival of S180-R after irradiation with different dose gradients (0Gy, 2Gy, 4Gy, 6Gy, 8Gy, 10Gy) was clearly higher than that of the parental cell S180 (as shown in table 1 and fig. 1):

TABLE 1

The above results indicate that the survival score of S180-R reaches more than 22 times that of S180 of its parent cells at irradiation doses up to 10 Gy. S180-R cells were demonstrated to have significantly improved radioresistance compared to the parental cells.

In conclusion, the invention adopts an intermittent irradiation induction method to induce the mouse sarcoma cell S180, and selects a cell S180-R with very strong radiation resistance, and the survival fraction of the S180-R reaches more than 22 times of the parent cell S180 under the irradiation dose of 10 Gy. The mouse sarcoma cell radiotherapeutic resistant cell strain provided by the invention provides a basis for further researching a molecular mechanism of mouse sarcoma radiotherapeutic resistance, screening radiotherapeutic resistant targets and improving radiotherapeutic sensitivity.