CN113607626B - Research method for cancer promotion and radiation resistance of oral cancer-related fibroblasts - Google Patents

Research method for cancer promotion and radiation resistance of oral cancer-related fibroblasts Download PDF

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CN113607626B
CN113607626B CN202110786329.5A CN202110786329A CN113607626B CN 113607626 B CN113607626 B CN 113607626B CN 202110786329 A CN202110786329 A CN 202110786329A CN 113607626 B CN113607626 B CN 113607626B
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cancer
fibroblasts
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oral cancer
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CN113607626A (en
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周红梅
罗晶晶
刘扬帆
吴杨
杨津
吴芳龙
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Sichuan University
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    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N15/10—Investigating individual particles
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Abstract

The invention provides a research method for cancer promotion and radiation resistance of oral cancer-related fibroblasts, which comprises the following steps: irradiating the oral cancer-related fibroblasts under a first preset irradiation condition to construct zombie-like oral cancer-related fibroblasts; under a second preset irradiation condition, the influence of the zombie-like oral cancer-associated fibroblasts on the survival rate of oral squamous cell carcinoma cells and DNA damage is studied. The invention discloses a research method for the cancer promotion and radiation resistance of oral cancer-related fibroblasts, which is used for researching the influence of zombie-like oral CAFs on the radiation resistance of OSCC cells from the aspects of the influence of ionizing radiation on the biological functions of the oral cancer-related fibroblasts (CAFs) and the influence of the zombie-like oral CAFs after receiving radiotherapy on oral squamous carcinoma cells so as to find out factors which can possibly induce the OSCC relapse, and is beneficial to better researching the tumor relapse and metastasis caused by the oral CAFs in the future, thereby better exploring the direction for preventing the OSCC relapse and metastasis after the radiotherapy, which are related to the oral CAFs.

Description

Research method for cancer promotion and radiation resistance of oral cancer-related fibroblasts
Technical Field
The invention relates to the field of medical research, in particular to a research method for cancer promotion and radiation resistance of fibroblast related to oral cancer.
Background
Oral Squamous Cell Carcinoma (OSCC) is one of the most common malignancies of the head and neck, with an increasing incidence in recent years. Due to the specific oral maxillofacial physiological position of OSCC, the development of OSCC often causes great damage to the appearance and facial function of OSCC patients. In the existing treatment methods, the skilled person generally considers that radiotherapy has better short-term curative effect on the treatment of OSCC. So-called radiotherapy is the killing of cancer cells by direct induction of DNA double strand breaks by high energy radiation or by indirect oxidative DNA damage by Reactive Oxygen Species (ROS) accumulation.
However, it is common for patients with OSCC after radiotherapy to have radiation tolerance resulting in local recurrence or metastasis after significant inhibition of the primary tumor. The specific mechanism associated with radiation resistance to OSCC resulting in local recurrence or metastasis is not yet clear.
Disclosure of Invention
Based on the problem described in the background of the invention of OSCC recurrence or metastasis after receiving radiotherapy, the inventors have thought that radiotherapy may also play a "back-off" role under certain conditions: it is possible to promote proliferation or metastasis of cancer cells under specific conditions while killing the cancer cells. At the same time, the perturbation of the Tumor Microenvironment (TME) by radiation should also be of concern. Because TME is a complex system that contains a wide variety of cell types, mainly fibroblasts, endothelial cells, various immune cells and cancer stem cells. Among them, fibroblasts are the most predominant stromal cell type in the tumor microenvironment, which can promote tumorigenesis through paracrine actions and/or cross-talk with cancer cells.
Based on the above thought, the inventor of the present invention proposes to study the influence of oral cavity CAFs on the radiation resistance of OSCC cells from the angles of the biological function influence of ionizing radiation on oral cavity cancer-associated fibroblasts (oral cavity CAFs) and the influence of oral cavity CAFs after receiving radiotherapy on oral cavity squamous carcinoma cells (OSCC cells) so as to determine the factors that may induce the recurrence or metastasis of OSCC after radiotherapy, thereby facilitating better study of the tumor recurrence and metastasis mechanism regulated by oral cavity CAFs in the future, and further exploring the strategy of preventing and treating the recurrence and metastasis of OSCC after radiotherapy with the oral cavity CAFs target.
The invention provides a research method for influencing the radiation-resistant capacity of cancer by using oral cancer-related fibroblasts, which comprises the following steps:
irradiating the oral cancer-related fibroblasts under a first preset irradiation condition, and performing DNA damage detection and proliferation capacity detection on the irradiated oral cancer-related fibroblasts;
constructing zombie-like oral cancer related fibroblasts according to the DNA damage detection result and the proliferation capacity detection result, wherein the zombie-like oral cancer related fibroblasts are the oral cancer related fibroblasts which are not dead and have inhibited proliferation, increased senescence and reduced migration capacity;
studying the influence of the zombie-like oral cancer-associated fibroblasts on the survival rate of the oral squamous carcinoma cells and DNA damage under a second preset irradiation condition;
and determining that the corpse-like oral cancer related fibroblasts can reduce the radiation resistance of the irradiated oral squamous cancer cells according to the survival rate of the oral squamous cancer cells and the proportion of the DNA-damaged oral squamous cancer cells.
Preferably, the first preset irradiation conditions include ionizing radiation doses of single X-rays of 2Gy, 6Gy, 12Gy, and 18Gy, respectively.
Preferably, the method for detecting DNA damage of the oral cancer-associated fibroblasts after irradiation comprises the following steps: the irradiated oral cancer-associated fibroblasts were immunofluorescent stained with 53BP1 and the percentage of cells with a number of 53BP1 foci greater than 3 in 3-5 random fields of view was determined by fluorescence inverted microscopy.
Preferably, the method for detecting the proliferation capacity of the oral cancer-related fibroblasts after irradiation comprises the following steps: BrdU immunofluorescent staining experiments were performed on the irradiated oral cancer-associated fibroblasts and the percentage of cells that positively express BrdU in 3-5 random fields was determined with the aid of a fluorescence inverted microscope.
Preferably, the method for detecting the aging characteristics of the oral cancer-related fibroblasts after irradiation comprises the following steps: and (3) carrying out a senescence-associated beta-galactosidase staining experiment on the irradiated oral cancer-related fibroblasts, and determining the percentage of cells which are positively expressed in senescence-associated beta-galactosidase staining in 3 random visual fields by means of an inverted phase contrast microscope.
Preferably, the zombie-like oral cancer-associated fibroblasts are obtained under irradiation conditions with 18Gy ionizing radiation dose of single X-ray.
Preferably, the method for detecting the migration activity of the zombie-like oral cancer-related fibroblasts is as follows: performing a scratch experiment on the zombie-like oral cancer-related fibroblasts, shooting the scratch area and analyzing the cell crawling proportion by using ImageJ software.
Preferably, the method for detecting the migration activity of the zombie-like oral cancer-related fibroblasts is as follows: performing a scratch experiment on the zombie-like oral cancer-related fibroblasts, shooting the scratch area and analyzing the cell crawling proportion by using ImageJ software.
Preferably, the assay procedure for studying the effect of zombie-like oral cancer-associated fibroblasts on the viability of the oral squamous carcinoma cells and DNA damage comprises:
co-culturing the zombie-like oral cancer related fibroblasts and oral squamous carcinoma cells to serve as an experimental group, detecting the clone formation rate and DNA damage level of the irradiated oral squamous carcinoma cells in the experimental group under a second preset irradiation condition, and determining the survival rate of the oral squamous carcinoma cells in the experimental group and the proportion of the oral squamous carcinoma cells damaged by DNA.
Co-culturing the non-irradiated oral squamous cell carcinoma-associated fibroblasts and the oral squamous cell carcinoma cells to serve as a control group, detecting the clone formation rate and the DNA damage level of the irradiated oral squamous cell carcinoma cells in the control group under a second preset irradiation condition, and determining the survival rate of the oral squamous cell carcinoma cells in the control group and the proportion of the DNA damaged oral squamous cell carcinoma cells.
Preferably, the control group further comprises: conventional culture of the oral squamous carcinoma cells and co-culture of non-irradiated normally associated fibroblasts with the oral squamous carcinoma cells.
Preferably, the second preset irradiation condition includes ionizing radiation doses of single X-rays of 0Gy, 1Gy, 2Gy, 4Gy, 6Gy, respectively.
The invention has the beneficial effects that:
the inventor of the invention proposes that from the aspects of the influence of ionizing radiation on the biological functions of oral cancer-related fibroblasts (oral CAFs) and the influence of the oral CAFs after receiving radiotherapy on oral squamous cell carcinoma cells (OSCC cells), the influence of the zombie-like oral CAFs constructed after high-dose irradiation on the radiation resistance of the OSCC cells is researched, so that the factor which can possibly induce the recurrence or metastasis of OSCC is determined.
According to the research method provided by the invention, the influence of the zombie-like oral CAFs constructed after high-dose irradiation on the radiation resistance of the OSCC cells is researched, so that the zombie-like oral CAFs constructed after high-dose irradiation have an obvious inhibition effect on the radiation resistance of the OSCC cells. The conclusion is helpful for better researching the tumor recurrence and metastasis mechanism regulated by the oral CAFs in the future, and further exploring the strategy for preventing and treating OSCC recurrence and metastasis after radiotherapy by using the oral CAFs target.
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FIG. 1a is a schematic diagram showing the ICC staining results of CAFs in human primary oral cavities isolated according to the embodiment of the present invention; wherein, FAP (+), Vimentin (+), alpha-SMA (+), Pan-CK (-);
FIG. 1b is a schematic diagram showing the ICC staining results of NAFs in a human primary oral cavity isolated according to the embodiment of the present invention; wherein, FAP (-), Vimentin (+), alpha-SMA (-), Pan-CK (-);
FIG. 2a is a graph showing the results of 53BP1 nuclear focus immunofluorescent staining of oral CAFs after 24 hours after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation in accordance with an embodiment of the present invention;
FIG. 2b is a graph showing the results of 53BP1 nuclear focus immunofluorescent staining of oral CAFs after 6 days after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation in accordance with an embodiment of the present invention;
FIG. 3 is a statistical chart showing the mean value analysis of 53BP1 positive nuclear foci of single oral CAFs cells 53BP1 after 24 hours and 6 days by performing 53BP1 nuclear foci immunofluorescence staining on oral CAFs irradiated at 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient doses IR respectively according to the embodiment of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 4 is a statistical chart showing the analysis of the percentage of cells in oral CAFs (i.e., DNA-damaged CAFs) with 53BP1 nuclear focus immunofluorescent staining after 24 hours and 6 days, respectively, when the oral CAFs were irradiated at gradient doses of 0Gy, 2Gy, 6Gy, 12Gy and 18Gy, respectively, with 53BP1 positive nuclear focus greater than 3 according to an embodiment of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 5a is a schematic diagram showing BrdU staining results after 24h after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation in oral CAFs according to an embodiment of the present invention;
FIG. 5b is a graph showing BrdU staining after 6 days after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient IR irradiation of oral CAFs according to embodiments of the present invention;
FIG. 6 is a statistical plot showing the percentage of proliferating cells that are BrdU staining positive after 24 hours and after 6 days, respectively, after 0Gy, 2Gy, 6Gy, 12Gy, and 18Gy gradient dose IR irradiation for oral CAFs according to embodiments of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 7 is a schematic diagram showing the morphological changes of the oral CAFs after 18Gy IR irradiation according to the embodiment of the present invention;
FIG. 8 is a graph showing the results of SA- β -gal staining of oral CAFs after 6 days after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation in accordance with an embodiment of the present invention;
FIG. 9 is a graph showing the results of percentage statistics of senescent cells that were positive for SA- β -gal staining after 6 days after irradiation of oral CAFs at gradient doses of 0Gy, 2Gy, 6Gy, 12Gy, and 18Gy IR in accordance with an embodiment of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 10 is a graph showing the scratch test results of ZCAFs of the present invention after 6 days after 18Gy IR irradiation at 0h, 24h, and 48h scratches, respectively;
FIG. 11 is a statistical graph showing the cell crawling area ratio at 24h and 48h of scratches after 6 days of scratch test after 18Gy IR irradiation in ZCAFs according to the embodiment of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 12 is a schematic diagram showing colony formation experiments on the survival of oral squamous cell carcinoma cells under different culture conditions after gradient irradiation of 1Gy, 2Gy, 4Gy and 6Gy according to the present invention;
FIG. 13 is a graph showing the statistics of percent colony formation for oral squamous cell survival under different culture conditions following 1Gy, 2Gy, 4Gy and 6Gy gradient irradiation of the present invention; wherein, represents p <0.05, represents p <0.01, represents p < 0.001;
FIG. 14 is a statistical plot of the number of survival rates of oral squamous cell carcinoma cells under different culture conditions after 1Gy, 2Gy, 4Gy and 6Gy gradient irradiation of the present invention;
FIG. 15 is a graph showing the results of immunofluorescent nuclear staining of squamous oral carcinoma cells 53BP1 in different culture conditions after 24 hours and 48 hours, respectively, after 6Gy IR irradiation according to the present invention;
FIG. 16 is a statistical plot showing the percentage analysis of DNA-damaged oral squamous carcinoma cells after 24 hours and 48 hours, respectively, after 6Gy IR irradiation of the present invention under different culture conditions.
Detailed Description
The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention falls within the protection scope of the present invention.
The specific experimental procedures or conditions are not indicated in the examples and can be performed according to the procedures or conditions of the conventional experimental procedures described in the prior art in this field. The reagents and other instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
In order to research the influence of oral CAFs on OSCC cells, the technical concept provided by the invention is as follows: firstly, the influence of Ionizing Radiation (IR) on oral cancer-related fibroblasts (oral CAFs) is researched, and the influence of ionizing radiation on the biological functions of the oral CAFs under different ionizing radiation doses is determined; in observing the effects of IR on the biological properties of oral CAFs, the inventors found that some CAFs did not die even when exposed to high doses of IR, but becomes a "dead" zombie-like state (named zombie-like oral cancer related fibroblasts by the inventor), so the inventor carries out further research experiments to co-culture the zombie-like oral cancer related fibroblasts and OSCC cells under the irradiation condition, setting corresponding control groups, detecting the clone formation rate and DNA damage level of the irradiated oral squamous cell carcinoma cells in the experimental group and the control groups, determining the survival rate of the oral squamous cell carcinoma cells in the experimental group and the control groups and the proportion of the oral squamous cell carcinoma cells damaged by DNA, according to the survival rate of the oral squamous cell carcinoma cells and the proportion of the oral squamous cell carcinoma cells damaged by DNA obtained by experiments, so as to determine the influence of the fibroblasts related to the zombie oral cancer on the radiation resistance of the oral squamous carcinoma cells. Based on the technical concept, the specific content of the embodiment of the invention is as follows:
irradiating the oral cancer-related fibroblasts under a first preset irradiation condition, and performing DNA damage detection and proliferation capacity detection on the irradiated oral cancer-related fibroblasts;
constructing zombie-like oral cancer related fibroblasts according to the DNA damage detection result and the proliferation capacity detection result, wherein the zombie-like oral cancer related fibroblasts are non-dead oral cancer related fibroblasts with inhibited proliferation, increased senescence and reduced migration capacity;
in specific implementation, the DNA damage detection result of the oral CAFs cells after irradiation shows that the CAFs after irradiation of other doses have enough capacity to repair DNA damage along with the prolonging of the time after irradiation except the high-dose IR irradiation of 18 Gy. DNA damage of oral CAFs irradiated by 18Gy IR cannot be recovered;
in specific implementation, the proliferation capacity detection result of the irradiated oral CAFs cells shows that when the IR reaches 6Gy or above, the cell proliferation is completely stopped; after 6 days, the proliferative capacity of the CAFs irradiated at 2Gy and 6Gy (relatively low dose IR) was partially restored, while the proliferation of the CAFs irradiated at 12Gy and 18Gy (relatively high dose IR) was still completely stopped.
In particular, the inventors observed that the morphology of oral CAFs after exposure to 18Gy IR doses changed to a "rigid" zombie-like state and exhibited an increased aging phenotype and decreased migratory capacity. Thus, the oral CAFs that show no death, impaired proliferation, difficulty in recovery from DNA damage, increased aging, and reduced migratory capacity after irradiation with 18Gy IR dose were named zombie-like oral cancer associated fibroblasts (zCAFs).
Under a second preset irradiation condition, researching the influence of the zombie-like oral cancer related fibroblasts on the survival rate of oral squamous cell carcinoma cells and DNA damage after ionizing radiation irradiation;
determining that the zombie-like oral cancer related fibroblasts can reduce the radiation-resistant capacity of the oral squamous carcinoma cells according to the survival rate of the oral squamous carcinoma cells and the proportion of the DNA-damaged oral squamous carcinoma cells.
In specific implementation, preferably, the first preset irradiation condition includes ionizing radiation doses of single X-rays of 2Gy, 6Gy, 12Gy and 18Gy, respectively.
In specific implementation, all oral CAFs subjected to IR irradiation do not die and proliferation is hindered; while CAFs irradiated at 12Gy and 18Gy have a proliferation-hindered and pronounced aging phenotype.
In specific implementation, preferably, the method for detecting DNA damage of the oral cancer-associated fibroblasts after irradiation comprises: the irradiated oral cancer-associated fibroblasts were subjected to 53BP1 immunofluorescent staining, and the percentage of cells with a number of 53BP1 foci greater than 3 in 3-5 random fields of view was determined by means of fluorescence inverted microscopy.
In specific implementation, DNA damage detection is carried out on the oral cancer-related fibroblasts after irradiation, and 53BP1 immunofluorescence staining experiments are carried out on the oral cancer-related fibroblasts which are continuously observed for 1 day and 6 days after irradiation.
In specific implementation, preferably, the method for detecting proliferation capacity of the oral cancer-associated fibroblasts after irradiation comprises: BrdU immunofluorescence staining experiments were performed on the irradiated oral cancer-associated fibroblasts and the percentage of cells that positively express BrdU in 3-5 random fields was determined with the aid of a fluorescence inverted microscope.
In specific implementation, proliferation capacity detection is carried out on the oral cancer-related fibroblasts after irradiation, and BrdU immunofluorescence staining experiments are carried out on the oral cancer-related fibroblasts continuously observed for 1 day and 6 days after irradiation.
In specific implementation, preferably, the method for detecting aging characteristics of the oral cancer-associated fibroblasts after irradiation comprises the following steps: aging-related beta-galactosidase staining experiments were performed on the irradiated oral cancer-related fibroblasts, and the percentage of cells positively expressed in aging-related beta-galactosidase staining in 3 random fields of view was determined by means of an inverted phase contrast microscope.
In specific implementation, aging characteristic detection is carried out on the irradiated oral cancer-related fibroblasts, and an aging-related beta-galactosidase staining experiment is carried out on the oral cancer-related fibroblasts continuously observed for 6 days after irradiation.
In specific implementation, preferably, the zombie-like oral cancer related fibroblasts are obtained under the irradiation condition that the ionizing radiation dose of single X-ray is 18 Gy.
In specific implementation, preferably, the method for detecting the migration activity of the zombie-like oral cancer-related fibroblasts comprises the following steps: scratching experiments were performed on zombie-like oral cancer-associated fibroblasts, the scratch area was photographed and the cell crawling proportion was analyzed using ImageJ software.
In particular embodiments, the performing an operation on the effect of zombie-like oral cancer-associated fibroblasts on the viability of oral squamous carcinoma cells and DNA damage after irradiation preferably comprises:
co-culturing zombie-like oral cancer related fibroblasts and oral squamous carcinoma cells to serve as an experimental group, detecting the clone formation rate and DNA damage level of the oral squamous carcinoma cells irradiated by the experimental group under a second preset irradiation condition, and determining the survival rate of the oral squamous carcinoma cells and the proportion of the oral squamous carcinoma cells damaged by DNA in the experimental group;
co-culturing non-irradiated oral cancer-related fibroblasts and oral squamous cell carcinoma cells to serve as a control group, detecting the clone formation rate and DNA damage level of the irradiated oral squamous cell carcinoma cells in the control group under a second preset irradiation condition, and determining the survival rate of the oral squamous cell carcinoma cells in the control group and the proportion of the DNA-damaged oral squamous cell carcinoma cells.
In specific implementation, under a second preset irradiation condition, the experimental results of the experimental group are compared with those of the control group, so that the experimental group can be obtained, the fibroblast related to the zombie-like oral cancer can reduce the promotion effect on the survival rate of the oral squamous cancer cell and the inhibition of DNA damage, and the radiation resistance of the irradiated oral squamous cancer cell reduced by the fibroblast related to the zombie-like oral cancer is determined.
In specific implementation, preferably, the control group further comprises: culturing in conventional culture medium and co-culturing with unirradiated normal fibroblasts and oral squamous cell carcinoma cells.
In specific implementation, preferably, the second preset irradiation condition includes that the ionizing radiation doses of single X-rays are respectively 0Gy, 1Gy, 2Gy, 4Gy and 6Gy,
in specific implementation, the detection of the clone formation rate is carried out after continuous observation for 10 days after irradiation, and the detection of the DNA damage level is respectively carried out after continuous observation for 1 day and 2 days after irradiation.
In order to make the present invention more understandable to those skilled in the art, the following description will illustrate a method for studying cancer-promoting and radiation-resistant capability of oral cancer-related fibroblasts according to the present invention by using specific examples.
The fresh OSCC tissues related to the embodiment of the invention are from OSCC patients who are treated by surgery, the normal oral mucosa tissues are from patients who receive orthognathic surgery or trauma plastic surgery, the clinical specimens comprise epithelial tissues and adjacent connective tissues (within 3mm from the epithelium), the human tissues used in the research are approved by the ethical committee of the human research in the oral hospital of Waxi, Sichuan university (number: WCHSIRB-D-2018-057), and all the patients sign informed consent. OSCC cell line Cal-27 was purchased from ATCC and has been identified by Short Tandem Repeat (STR).
Primary fibroblasts and OSCC cell lines at 37 ℃ and 5% CO2In a humidified atmosphere, the cells were cultured in a DMEM high-glucose medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin and streptomycin (Gibco, USA)
Example 1: isolation and culture of primary human CAFs
The oral CAFs were isolated from fresh OSCC tissue and normal tissue-associated fibroblasts (NAFs) were isolated from normal oral mucosal tissue.
Separating and culturing primary oral CAFs and NAFs by tissue adherence method. The method comprises the following simple steps: shearing the tissue blocks as much as possible until the diameter of the fragments is not more than 3 mm; transferring the tissue fragments to a T25 cell culture bottle to adhere to the wall, adding 1-2ml of culture medium, placing the culture bottle in an incubator in a reverse direction for 2 hours, adding 3ml of culture medium, and placing the culture bottle in the forward direction until the full degree of the creeping cells reaches 80%.
Immunocytochemistry (ICC) staining was performed on isolated human primary oral CAFs and NAFs to further identify their morphology and growth pattern and observed by inverted phase contrast microscopy. Among them, an SP-9000 detection kit and a DAB kit (Beijing Zhongshan Jinqiao Biotech Co., Ltd.) were used according to the instructions. The primary antibodies used included anti-alpha-smooth muscle actin (alpha-SMA) antibody (Abcam,1:100), anti-Fibroblast Activation Protein (FAP) antibody (Abcam,1:100), anti-Vimentin (Vimentin) antibody (Abcam,1:250), and anti-cytokeratin (Pan-CK) antibody (Abcam,1: 100). PBS was used as negative control.
FIG. 1a and FIG. 1b show ICC staining results of CAFs and NAFs in human primary oral cavity isolated by the present invention. Wherein, fig. 1a represents the ICC staining result of human primary oral cavity CAFs separated according to the embodiment of the present invention, and fig. 1b represents the ICC staining result of NAFs separated according to the embodiment of the present invention. It is observed from fig. 1 that the present invention successfully isolated primary oral CAFs and normal tissue associated fibroblasts (NAFs). Also, compared to normal tissue associated fibroblasts (NAFs), primary oral CAFs showed positive expression of Vimentin, FAP and α -SMA, negative expression of Pan-CK (fig. 1a), whereas oral NAFs showed only positive expression of Vimentin, negative expression of other markers (fig. 1b), indicating that the isolated oral CAFs of the invention are in an activated state.
Example 2: detecting the effects of Ionizing Radiation (IR) on DNA damage of oral CAFs
The cells were irradiated with X-rays using an X-ray 160 irradiator (Precision X-ray (PXi), USA) at a dose rate of 1.92Gy/min, with parameters set to 160KV, 18.70mA of current, and a source target distance of 50 cm. During irradiation, the orifice plate is placed right below the X-ray source to avoid ray scattering. The irradiated cells were returned to the 37 ℃ incubator as soon as possible within 1 hour. Morphological changes of irradiated cells were observed under an inverted phase contrast microscope (LEICA, germany) at regular intervals to confirm that ionizing radiation was effective.
2Gy, 6Gy, 12Gy and 18Gy single-dose X-ray irradiation is carried out on CAFs, 53BP1 immunofluorescence staining is carried out on the CAFs after irradiation after 24h and 6 days respectively, and the influence of Ionizing Radiation (IR) on the CAFs DNA damage is detected. Cells were fixed with 10% neutral formaldehyde for 15 minutes at room temperature before staining and then disrupted with 0.1% Triton X-100 solution for 10 minutes at room temperature. The primary antibody was an anti-53 bp1 antibody (Abcam, UK, 1: 250). The secondary antibody was fitc-labeled goat anti-rabbit IgG (sequoia jeansii, china, 1:100) diluted with 5% BSA. DAPI was used for nuclear staining. The 200 × field image was scanned under a german come card fluorescence inverted microscope. The cell percentage of 53BP1 foci greater than 3 and the 53BP1 nuclear foci were measured in 3-5 random fields, and the mean and standard error of the measurements were calculated.
Fig. 2a and 2b show graphs of the results of 53BP1 focus staining after 24 hours and 6 days respectively after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation of oral CAFs according to embodiments of the present invention, as shown in fig. 2a and 2b, all doses of IR resulting in an increase in nuclear focus compared to unirradiated cells (IR irradiation dose of 0 Gy). This indicates the effectiveness of the radiation and the DNA damage response of oral CAFs to IR irradiation.
FIG. 3 is a statistical chart showing the mean value analysis of 53BP1 positive nuclear foci of single oral CAFs cells 53BP1 after 24 hours and 6 days by immunofluorescence staining of 53BP1 nuclear foci after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation in oral CAFs according to the embodiment of the present invention; as shown in fig. 3, the number of nuclear foci of individual oral CAFs cells increased with increasing IR irradiation dose (fig. 3, left), and the differences between groups except the high dose groups (12Gy and 18Gy groups) were statistically significant, confirming the effectiveness of irradiation.
Furthermore, the inventors have observed that the number of nuclear foci in unirradiated CAFs does not exceed 3. Therefore, the present invention sets 53BP1 nuclear foci >3 cells as DNA-damaged cells and makes statistics on the percentage of DNA-damaged cells, and fig. 4 shows a statistical graph of the analysis of the percentage of oral CAFs (i.e., DNA-damaged CAFs) cells with 53BP1 nuclear foci immunofluorescent staining after 24 hours and 6 days after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation, respectively, and 53BP1 positive nuclear foci greater than 3 for the oral CAFs (i.e., DNA-damaged CAFs) according to the example of the present invention, as shown in fig. 4, the results of which have the same trend as the average nuclear foci count statistics (fig. 4, left).
Comparing the average nuclear focus number after 6 days of irradiation (fig. 3, right) in fig. 3 with the DNA damaged cell ratio after 6 days of irradiation (fig. 4, right) in fig. 4, it was found that the nuclear focus number and the DNA damaged cell ratio still increased in dependence of the IR dose after 6 days of irradiation, but were significantly decreased compared to 24 hours. Furthermore, the oral CAFs DNA damage irradiated at 18Gy dose is almost unrecoverable. This indicates that, in addition to 18Gy of high dose IR irradiation, the irradiated CAFs have sufficient capacity to repair DNA damage with time after irradiation. DNA damage of oral CAFs irradiated with 18Gy IR cannot be recovered.
Example 3: detecting the effect of Ionizing Radiation (IR) on the proliferative capacity of oral CAFs
The cells were irradiated with X-rays using an X-ray 160 irradiator (Precision X-ray (PXi), USA) at a dose rate of 1.92Gy/min, with parameters set to 160KV, 18.70mA of current, and a source target distance of 50 cm. During irradiation, the orifice plate is placed right below the X-ray source to avoid ray scattering. The irradiated cells were returned to the 37 ℃ incubator as soon as possible within 1 hour. Morphological changes of irradiated cells were observed under an inverted phase contrast microscope (LEICA, germany) at regular intervals to confirm that ionizing radiation was effective.
2Gy, 6Gy, 12Gy and 18Gy single-dose X-ray irradiation is carried out on CAFs, BrdU immunofluorescence staining is carried out on the CAFs after irradiation 24h and 6 days respectively, and the influence of Ionizing Radiation (IR) on the proliferation capacity of the CAFs is detected. Cells were fixed with 10% neutral formaldehyde for 15 minutes at room temperature before staining and then disrupted with 0.1% Triton X-100 solution for 10 minutes at room temperature. The primary antibody was an anti-BrdU antibody (Abcam, UK, 1: 250). The secondary antibody was fitc-labeled goat anti-rabbit IgG (sequoia jeansii, china, 1:100) diluted with 5% BSA. DAPI was used for nuclear staining. The 200 Xfield image was scanned under a fluorescence inverted microscope in Leica, Germany. The percentage of BrdU positive cells was determined in 3-5 random fields, and the mean and standard error of three determinations were calculated.
FIGS. 5a and 5b are graphs showing BrdU staining after 24h and after 6 days, respectively, after 0Gy, 2Gy, 6Gy, 12Gy and 18Gy gradient dose IR irradiation, in oral CAFs according to embodiments of the present invention; FIG. 6 is a statistical plot showing the percentage of proliferating cells that are BrdU staining positive after 24 hours and after 6 days, respectively, after 0Gy, 2Gy, 6Gy, 12Gy, and 18Gy gradient dose IR irradiation for oral CAFs according to embodiments of the present invention; as shown in FIGS. 5a, 5b and 6, the proliferation of oral CAFs 24 hours and 6 days after irradiation with immunofluorescence-quantified BrdU markers was examined as a function of the gradient dose of IR, after 24 hours, the proliferation of oral CAFs decreased significantly with increasing IR dose, and when IR reached 6Gy and above, cell proliferation stopped completely (FIG. 5 a). After 6 days, the proliferative capacity of the CAFs irradiated at 2Gy and 6Gy (relatively low dose IR) was partially restored, while the proliferation of the CAFs irradiated at 12Gy and 18Gy (relatively high dose IR) was still completely stopped (FIG. 5 b).
FIG. 7 is a schematic diagram showing the morphological changes of the oral CAFs after 18Gy IR irradiation according to the embodiment of the present invention; as shown in fig. 7, the oral CAFs after being irradiated by 18Gy IR dose have larger volume and more complex shape (shown by arrows), and show a "dead" state.
Based on this, the inventors named zombie-type CAFs (zcafs) for oral CAFs that showed no death, impaired proliferation, and failure to recover from DNA damage after irradiation with 18Gy IR dose.
To further determine the phenotypic and functional changes of zombie-type cafs (zcafs) following 18Gy IR dose irradiation, the inventors performed the following experiments:
(1) galactosidase assay:
endogenous senescence-associated beta-galactosidase (SA-beta-gal) expression was detected. The CAFs cells subjected to single-dose X-ray irradiation of 2Gy, 6Gy, 12Gy and 18Gy were inoculated in 6-well plates at a density of 3X 10 after 6 days4A hole. Detection was performed using galactosidase (SA- β -gal) staining kit (bi yun, china) according to the method provided by the manufacturer. Cells were fixed for 15 minutes in 1 × fixative and then incubated overnight at 37 ℃ in a carbon dioxide free environment using 1 × staining solution. 40X and 100X images were taken under an inverted phase contrast microscope, respectively, and the SA-. beta. -gal positive expression cell ratio was counted in 3 random fields. Cells with green or dark green staining of the cytoplasm were positive.
FIG. 8 is a graph showing the results of SA- β -gal staining of oral CAFs after 6 days after 0Gy, 2Gy, 6Gy, 12Gy, and 18Gy gradient IR irradiation in accordance with embodiments of the present invention; FIG. 9 is a graph showing statistics of the percentage of cells that are positively expressed following SA- β -gal staining after 6 days after 0Gy, 2Gy, 6Gy, 12Gy, and 18Gy gradient IR irradiation of oral CAFs in accordance with embodiments of the present invention; as shown in FIGS. 8 and 9, SA- β -gal staining was performed on irradiated oral CAFs, and it was observed that after 6 days, SA- β -gal-positively expressed oral CAFs increased in dependence of IR dose.
(2) Scratch test:
the oral CAFs subjected to 18Gy single-dose X-ray irradiation and the oral CAFs not subjected to irradiation are respectively treated by 4 × 104Density per well was seeded in 6-well plates and allowed to grow to almost 100% binding. The monolayer cells were scratched using 100ul of the tip of a gun, and then the medium was changed to DMEM medium containing 2% fetal bovine serum. The scratched area was observed and photographed at 0 hour, 24 hours and 48 hours after scratching, respectively, andthe proportion of cell crawl was analyzed using ImageJ software (national institutes of health and laboratory, Bethesda, MD, usa).
FIG. 10 is a graph showing the scratch test results of ZCAFs of the present invention after 6 days after 18Gy IR irradiation at 0h, 24h, and 48h scratches, respectively; FIG. 11 is a graph showing the ratio of the cell crawling area at 24h and 48h of scratches after 6 days of scratching test after 18Gy IR irradiation in ZCAFs according to the embodiment of the present invention; as shown in fig. 10 and 11, the zCAFs migration activity was reduced compared to non-irradiated CAFs.
According to galactosidase experiments and scratch experiments, it was confirmed that oral CAFs irradiated with 18Gy IR have a pronounced aging phenotype and reduced migratory activity. Based on this, the inventors further clarified that the presence of epizombie cafs (zcafs) after irradiation with 18Gy IR dose is characterized by non-death after irradiation, hindered proliferation, failure to recover from DNA damage, increased senescence, and decreased migratory capacity.
Example 4: detecting the influence of ZCAFs on the radiation resistance of OSCC cells
To explore the effect of zCAFs on the radiation resistance of OSCC cells, the inventors performed a clone formation experiment and a 53BP1 immunofluorescence staining experiment on Cal-27 cells under different culture conditions. Co-culturing non-irradiated oral cancer-related fibroblasts and oral squamous cell carcinoma cells to serve as a control group, detecting the clone formation rate and DNA damage level of the irradiated oral squamous cell carcinoma cells in the control group under a second preset irradiation condition, and determining the survival rate of the oral squamous cell carcinoma cells in the control group and the proportion of the DNA-damaged oral squamous cell carcinoma cells.
The specific experimental process is as follows:
(1) clone formation experiments:
unirradiated oral squamous carcinoma cells were seeded in 6-well plates at densities of 800, 1600, 3200, 6400 and 10000 cells/well, respectively, and exposed to 0, 1, 2, 4 and 6Gy of ionizing radiation, respectively. Culturing under different conditions, including conventional medium (Cal-27+ DMEM), ZCAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ ZCAFs), non-irradiated NAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ NAFs), non-irradiated CAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ CAFs); the culture was terminated when macroscopic colony formation appeared in 6-well plates after 10-14 days. Cells were fixed with 10% neutral formaldehyde for 15 min and stained with crystal violet for 30 min. Clonal colonies containing more than 50 cells were counted and the colony formation rate and survival score were calculated.
FIG. 12 is a schematic diagram showing colony formation experiments on the survival of oral squamous cell carcinoma cells under different culture conditions after gradient irradiation of 1Gy, 2Gy, 4Gy and 6Gy according to the present invention; FIG. 13 is a graph showing the statistics of percent colony formation for oral squamous cell survival under different culture conditions following 1Gy, 2Gy, 4Gy and 6Gy gradient irradiation of the present invention; as shown in fig. 12 and 13, zCAFs have a reduced ability to promote colony formation after irradiation of oral squamous cell carcinoma cells compared to non-irradiated CAFs. FIG. 14 is a statistical plot of the number of survival rates of oral squamous cell carcinoma cells under different culture conditions after 1Gy, 2Gy, 4Gy and 6Gy gradient irradiation of the present invention; as shown in figure 14, zCAFs have a reduced ability to promote survival of oral squamous carcinoma cells after irradiation compared to non-irradiated CAFs.
(2)53BP1 immunofluorescent staining experiment:
the cells were irradiated with X-rays using an X-ray 160 irradiator (Precision X-ray (PXi), USA) at a dose rate of 1.92Gy/min, with parameters set to 160KV, 18.70mA of current, and a source target distance of 50 cm. During irradiation, the orifice plate is placed right below the X-ray source to avoid ray scattering. The irradiated cells were returned to the 37 ℃ incubator as soon as possible within 1 hour. Morphological changes of irradiated cells were observed under an inverted phase contrast microscope (LEICA, germany) at regular intervals to confirm that ionizing radiation was effective.
Unirradiated oral squamous carcinoma cells are treated at 2.5 × 104The density of each well is inoculated in a 6-well plate and placed at 37 ℃ and 5% CO2Culturing in a saturation constant temperature incubator for 1-2 days, and then changing to different culture conditions for culture, wherein the culture conditions comprise a conventional culture medium (Cal-27+ DMEM), a ZCAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ ZCAFs), an unirradiated NAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ NAFs) and an unirradiated CAFs-Cal27 transwell Co-culture environment (Co-Cal-27+ CAFs); and then exposed to 6Gy of ionizing radiation, after 24 hours and 48 hours of irradiation,53BP1 immunofluorescent staining is carried out on the oral squamous carcinoma cells which are not irradiated and are irradiated, and the influence of Ionizing Radiation (IR) on DNA damage of the oral squamous carcinoma cells is detected. Cells were fixed with 10% neutral formaldehyde for 15 minutes at room temperature before staining and then disrupted with 0.1% Triton X-100 solution for 10 minutes at room temperature. The primary antibody was an anti-53 bp1 antibody (Abcam, UK, 1: 250). The secondary antibody was fitc-labeled goat anti-rabbit IgG (sequoia jeansii, china, 1:100) diluted with 5% BSA. DAPI was used for nuclear staining. The 200 × field image was scanned under a german come card fluorescence inverted microscope. The percentage of cells with a focus number of 53BP1 greater than 3 was measured in 3-5 random fields and the mean and standard error of the measurements were calculated.
FIG. 15 is a graph showing the results of nuclear-focal immunofluorescence staining of oral squamous carcinoma cells 53BP1 under different culture conditions after 24 hours and 48 hours, respectively, after irradiation of 6Gy IR (maximum dose of ionizing radiation tolerated by oral squamous carcinoma cells) in accordance with the present invention; FIG. 16 is a statistical plot showing the percentage analysis of oral squamous carcinoma cells with a 53BP1 positive nuclear focus greater than 3 (DNA-damaged oral squamous carcinoma cells) under different culture conditions after 6Gy IR (maximum dose of ionizing radiation tolerated by oral squamous carcinoma cells) irradiation of the present invention; as shown in fig. 12 and 13, zCAFs have a reduced ability to inhibit DNA damage after irradiation of oral squamous cell carcinoma cells compared to non-irradiated CAFs.
Statistical analysis
Statistical analysis was performed using GraphPad Prism software. When the sample size is less than 8, the normality is determined by the Shapiro-Wilk normality test. Data are presented as mean ± standard deviation. The difference between the two groups was measured by Student's t test, and the difference between the two or more groups was analyzed by one-way anova using Tukey's multiple comparison test. Two-sided P <0.05 was considered statistically significant.
The embodiment of the invention is researched to obtain: compared with unirradiated CAFs, the zCAFs have reduced ability to promote the survival of OSCC cancer cells after IR irradiation and reduced effect of inhibiting DNA damage under the interaction of the zCAFs and OSCC cells. Therefore, the research results show that the high-dose IR-induced ZCAFs are hindered in proliferation, incapable of restoring DNA damage repair, in an aging state and weakened in migration activity, have a certain cancer promotion effect, but are remarkably weakened in the promotion effect on the radiation resistance of OSCC, and can be used as a new prevention and treatment target for postoperative recurrence or metastasis after oral squamous cell carcinoma radiotherapy.
The method for researching the cancer-promoting and radiation-resisting capability of the fibroblast related to oral cancer provided by the invention is described in detail above, and the principle and the implementation mode of the invention are explained by applying specific examples, and the description of the examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims (10)

1. A method for studying the cancer-promoting and radiation-resistant capability of an oral cancer-associated fibroblast, wherein the method comprises the following steps:
irradiating the oral cancer-related fibroblasts under a first preset irradiation condition, and performing DNA damage detection and proliferation capacity detection on the irradiated oral cancer-related fibroblasts;
according to the DNA damage detection result and the proliferation capacity detection result, constructing zombie-like oral cancer related fibroblasts, and carrying out migration activity detection on the zombie-like oral cancer related fibroblasts, wherein the zombie-like oral cancer related fibroblasts are the oral cancer related fibroblasts which are not dead and have inhibited proliferation, increased senescence and reduced migration capacity;
under a second preset irradiation condition, researching the influence of the zombie-like oral cancer related fibroblasts on the survival rate of oral squamous cell carcinoma cells and DNA damage;
and determining that the corpse-like oral cancer related fibroblasts can reduce the radiation resistance of the irradiated oral squamous cancer cells according to the survival rate of the oral squamous cancer cells and the proportion of the DNA-damaged oral squamous cancer cells.
2. The method according to claim 1, wherein the first preset irradiation condition comprises ionizing radiation doses of single X-rays of 2Gy, 6Gy, 12Gy and 18Gy, respectively.
3. The method of claim 1, wherein the method for detecting DNA damage of the oral cancer-associated fibroblast cells after irradiation comprises: the irradiated oral cancer-associated fibroblasts were immunofluorescent stained with 53BP1 and the percentage of cells with a number of 53BP1 foci greater than 3 in 3-5 random fields of view was determined by fluorescence inverted microscopy.
4. The method of claim 1, wherein the method for detecting proliferation capacity of the oral cancer-associated fibroblasts after irradiation comprises: BrdU immunofluorescent staining experiments were performed on the post-irradiation oral cancer-associated fibroblasts and the percentage of cells positively expressing BrdU in 3-5 random fields of view was determined by means of fluorescence inverted microscopy.
5. The method of claim 1, wherein the aging characteristics of the irradiated oral cancer-associated fibroblasts are detected by: and (3) carrying out a senescence-associated beta-galactosidase staining experiment on the irradiated oral cancer-related fibroblasts, and determining the percentage of cells which are positively expressed in senescence-associated beta-galactosidase staining in 3 random visual fields by means of an inverted phase contrast microscope.
6. The method of claim 1, wherein the zombie-like oral cancer-associated fibroblasts are obtained under irradiation with 18Gy of ionizing radiation for a single X-ray.
7. The method of claim 1, wherein the detection of the migratory activity of the zombie-like oral cancer-associated fibroblasts is performed by: performing a scratch experiment on the zombie-like oral cancer-related fibroblasts, shooting the scratch area and analyzing the cell crawling proportion by using ImageJ software.
8. The method of claim 1, wherein said testing procedure to study the effect of said zombie-like oral cancer-associated fibroblasts on the viability of said oral squamous carcinoma cells and DNA damage comprises:
co-culturing the zombie-like oral cancer related fibroblasts and oral squamous carcinoma cells to serve as an experimental group, detecting the clone formation rate and the DNA damage level of the irradiated oral squamous carcinoma cells in the experimental group under a second preset irradiation condition, and determining the survival rate of the oral squamous carcinoma cells and the proportion of the DNA damaged oral squamous carcinoma cells in the experimental group;
co-culturing the non-irradiated oral squamous cell carcinoma-associated fibroblasts and the oral squamous cell carcinoma cells to serve as a control group, detecting the clone formation rate and the DNA damage level of the irradiated oral squamous cell carcinoma cells in the control group under a second preset irradiation condition, and determining the survival rate of the oral squamous cell carcinoma cells in the control group and the proportion of the DNA damaged oral squamous cell carcinoma cells.
9. The method of claim 1, wherein the control group further comprises: conventional culture of the oral squamous carcinoma cells and co-culture of non-irradiated normally associated fibroblasts with the oral squamous carcinoma cells.
10. The method according to claim 1, wherein the second preset irradiation condition comprises ionizing radiation doses of single X-rays of 0Gy, 1Gy, 2Gy, 4Gy and 6Gy, respectively.
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