CN105200114A - Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell - Google Patents

Abstract

The invention discloses a method for predicting sensitivity to radiotherapy and chemotherapy by using cervical cancer side population cells, comprising the steps of: preparing cervical cancer cells, and in vitro adherent culture of cervical cancer cells; collecting cervical cancer cells in logarithmic growth phase, and Add Hoechst33342; use ultra-fast flow cytometry sorting system to sort SP cells, and the SP cells and NSP cells obtained after sorting are used for further experimental research; then add different concentrations of cisplatin and Annexin to NSP cells and SP cells respectively. V? FITC cell apoptosis detection kit staining, flow cytometry detection, SP cells and NSP cells given different doses of radiotherapy irradiation, Annexin? V? FITC cell apoptosis detection kit staining, flow cytometry detection, cell apoptosis rate=UR%+LR%. The present invention uses SP cells as an entry point to screen cervical cancer stem cells, analyzes the differences in sensitivity to radiotherapy and chemotherapy and their possible mechanisms, and provides a theoretical basis for the development of anti-tumor stem cell drugs and some cytokines and chemical substances that block signal pathways .

Description

A method for predicting radiotherapy and chemotherapy sensitivity using cervical cancer side population cells

technical field

The invention relates to the field of medical technology, in particular to a method for predicting sensitivity to radiotherapy and chemotherapy by using cervical cancer side population cells.

Background technique

Cervical cancer is the second most common malignant tumor in women worldwide, second only to breast cancer. There are about 100,000 new cases of cervical cancer in my country every year, accounting for about 1/5 of the new cases of cervical cancer in the world. In developing countries, the death rate of women's cervical cancer exceeds that of breast cancer, ranking first among the causes of death from malignant tumors in women. In recent years, the morbidity and mortality of cervical cancer in some areas of our country have been increasing, and some areas have also shown a trend of younger patients. The treatment of cervical cancer adopts a comprehensive treatment plan based on surgery and radiotherapy, supplemented by chemotherapy. The treatment effect of early cervical cancer is better, but the 5-year survival rate of stage IV and recurrent cervical cancer is only 3.2% to 13%, most of the recurrence occurs within 2 years after diagnosis, the prognosis is poor, and the median survival period is 7 months moon. Therefore, cervical cancer is one of the diseases that seriously threaten the health and life of women in the world.

The theory of cancer stem cells (cancer stem cells, CSC) is a research hotspot in the field of oncology at present. The theory holds that: cells in tumors are heterogeneous, most tumor cells do not have infinite proliferation ability, and only a few tumor cells have the similarity to stem cells. All tumor cells are derived from these tumor stem cells, which account for 0.1%-1% of the number of tumor cells, are the progenitor cells of all tumor cells, and are resistant to chemotherapy drugs and radiotherapy. It is the root cause of tumor drug resistance, recurrence and metastasis. Therefore, exploring the origin of cervical cancer cells and studying their biological characteristics can provide new ideas for the prevention and screening of cervical cancer, and point out a new direction for the treatment of patients with recurrent and metastatic cervical cancer.

The isolation and identification of cancer stem cells is the first step in the research of cancer stem cells. Separation of stem cells by CSC surface-specific marker screening method is highly reliable and is the most authoritative method for identifying CSCs. However, so far, only some tumor stem cell surface markers have been identified. In 1996, Goodell et al. used DNA dyes to stain hematopoietic stem cells Hoechst33342 and performed fluorescence activated cell sorting (fluorescence activated cell sorting, FACS) and found a group of cells with low staining and different from most other cells. The part of cells separated by this characteristic is called side population cells (Sidepopulation, SP), and this characteristic that can exclude Hoechest33342 is called SP phenotype. With the in-depth study of SP cells, it is found that the functions of SP cells are similar to normal stem cells, and can undergo asymmetric division, self-renewal, etc., so SP phenotype has become one of the common methods for sorting tumor stem cells. In recent years, studies have suggested that the SP phenotype of cervical cancer has the biological characteristics of tumor stem cell-like cells, which can be considered as an effective method for sorting cervical cancer stem cells. In 2010, domestic scholar Feng Dingqing reported for the first time that single cell suspensions isolated from 19 cases of cervical cancer tissues of different clinical stages were cultured in tumor cell spheroid medium (TSM) and eight cases had the formation of suspended tumor cell spheroids. Experiments confirmed these The cells fit the characteristics of stem cells. However, there are not many related experiments, and the current research in recent years is mainly focused on the isolation and identification of its biological function, while there are no reports on the sensitivity of radiotherapy and chemotherapy.

It has been recognized that one of the characteristics of cancer stem cells is resistance to chemotherapy drugs. ABCG2 protein is one of the members of the ABC-binding cassette (ATP—binding cassette, ABC) superfamily. It is a P-glycoprotein that plays an important role in maintaining cell stability and normal physiological functions of the body. It can be seen that the decrease of cell sensitivity to drugs is caused by the expression of ABCG2. With the deepening of research on other molecules of the ABC transporter family, such as ABCB1 and ABCC2, researchers believe that other ABC transporters may also be the cause of the SP phenotype, and thus also the molecular basis of SP cell multidrug resistance.

Radiation therapy plays an important role in the treatment of cervical cancer. According to statistics, about 80% of cervical cancer patients need radiotherapy as one of the means of single treatment or comprehensive treatment. Radiotherapy has been used in various clinical stages of cervical cancer, but its curative effect has not been improved in recent years. Some tumor cells are not very sensitive to radiotherapy. The 5-year survival rate of clinical stage I and II patients after radiotherapy is 65% to 85%. ; Ⅲ, Ⅳ patients about 20% to 50%. The mechanism of radiation resistance of cancer stem cells may be related to the repair of damaged DNA, affecting cell cycle regulation, cell apoptosis, proliferation and other transduction signaling pathway changes. Existing evidence has shown that the insensitivity of breast cancer, glioma and other solid tumors to radiotherapy is related to the existence of CSCs. It can be boldly speculated that the radiotherapy insensitivity of advanced or recurrent cervical cancer is related to the radiation resistance of SP cells. The inherent radiosensitivity of the tumor can be understood by detecting radiosensitivity-related factors. Studies have shown that in malignant tumors, the overexpression of the tumor suppressor gene P53 is a common phenomenon, which can perform DNA repair or initiate apoptosis to cause cell death. It plays an important role and has a certain relationship with the radiosensitivity of cells. Epidermal growth factor receptor (EGFR) is the expression product of an activated proto-oncogene, a receptor for a transmembrane tyrosine kinase growth factor, expressed in many tumor cells, and closely related to the prognosis of tumors relation. The study also believes that the increase of EGFR is related to the curative effect of tumor radiotherapy. The EGFR signaling pathway is an important factor affecting the radiosensitivity of tumor cells. Its overexpression will lead to the resistance of tumor cells to radiation. It can be considered that EGFR is an important indicator for predicting radiosensitivity one.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for predicting sensitivity to radiotherapy and chemotherapy using cervical cancer side population cells.

In order to solve the above problems, the present invention provides a method for predicting sensitivity to radiotherapy and chemotherapy using cervical cancer side population cells, which is characterized in that it comprises the following steps:

(1) Obtain cervical cancer tissue from patients through surgery;

(2) Preparation of primary cervical cancer cell suspension:

Fresh cervical cancer tumor tissue removed by surgery was washed with sterile normal saline, cut into pieces of ^1mm3 size, added with a final concentration of 0.1% collagenase, 0.01% hyaluronidase, 0.002% DNase, 37 Digest at ℃ for 2 hours, filter with 100-mesh nylon mesh, centrifuge at 500rpm for 10min, discard the supernatant, add appropriate amount of Hanks solution, mix well and perform discontinuous density gradient centrifugation at 2000rpm for 20min, collect the suspension rich in tumor cells, wash twice, Adjust the number of cells with DMEM culture medium containing 10% serum until the test is ready for use;

(3) Cervical cancer cells were routinely cultured in DMEM medium in an incubator at 37°C, 5% CO ₂ , and 95% humidity;

(4) Collect cervical cancer cells in the logarithmic growth phase, adjust the number of cells to 1×10 ⁶ cells/mL, divide the cells into two groups, add Hoechst33342 to a final concentration of 5 mg/L to one group, and add 50 μmol/L to the other group at the same time. L Reserpine was used as a control; the mixed cells were resuspended in ice-cold DMEM medium containing 2% FBS, and the SP cells were sorted by an ultra-fast flow cytometry system, and the SP cells and NSP cells obtained after sorting were used for further experimental research;

(5) Collect SP and NSP cells in the logarithmic growth phase, prepare single cell suspension after digestion, adjust the cell concentration to 2×10 ⁵ cells/mL, inoculate on the culture plate, change the medium after culturing for 24 hours, NSP cells and SP The cells were added with different concentrations of cisplatin, digested after 24 hours of culture, washed with PBS, stained with AnnexinVFITC cell apoptosis detection kit, and detected by flow cytometry, cell apoptosis rate = UR% + LR%;

(6) Collect the SP and NSP cells in the logarithmic growth phase, prepare a single cell suspension after digestion, adjust the cell concentration to 2×10 ⁵ cells/mL, inoculate on the culture plate, change the medium after culturing for 24 hours, SP cells and NSP The cells were irradiated with different doses of radiotherapy, digested after 24 hours of culture, and washed once with PBS; stained with AnnexinVFITC cell apoptosis detection kit, and detected by flow cytometry, cell apoptosis rate = UR% + LR%.

In the preferred technical solution of the present invention, the order of step (5) and step (6) can be interchanged.

In the preferred technical solution of the present invention, in the step (5), NSP cells and SP cells are added with different concentrations of cisplatin 0 μg/mL, 0.475 μg/mL, 0.95 μg/mL and 1.9 μg/mL, respectively.

In the preferred technical solution of the present invention, in the step (6), NSP cells and SP cells are respectively given different doses of radiotherapy irradiation of 0, 2, 4, and 8 Gy.

The DMEM medium in the step (3) contains 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.

In the embodiments of the present invention, the cervical cancer cells are selected from HeLa cells.

The present invention combines the classic Hoechst33342 staining method reported by Goodell et al. with flow cytometry to sort SP cells and NSP cells in cervical cancer Hela cells, observe cell growth and in vitro clone formation, and further perform chemotherapy and The radiosensitivity experiment was conducted to observe the differences in their reactivity to the chemotherapeutic drug cisplatin and radiation, and to explore the possible related mechanisms, providing a research model and foundation for in-depth research on cervical cancer stem cells and a basis for clinical application.

Advantages of the present invention include as follows:

1. Cancer stem cells are closely related to tumor occurrence, development and metastasis, and have received more and more attention. These studies mainly focus on sorting cervical cancer stem cells and identifying their biological functions. Sensitivity studies have not been reported.

2. The present invention studies the correlation between tumor stem cells and resistance to radiotherapy and chemotherapy, shifts the focus of treatment to tumor stem cells, strives to eradicate tumor stem cells, reduces the overall tumor proliferation ability, and gradually degenerates and shrinks the tumor, thereby truly curing the tumor.

3. The present invention uses SP cells as an entry point to screen cervical cancer stem cells, analyzes the differences in sensitivity to radiotherapy and chemotherapy and their possible mechanisms, and provides information for the development of anti-tumor stem cell drugs and some cytokines and chemical substances that block signal pathways. Theoretical basis. Point out a new direction for individualized treatment of patients with recurrent and metastatic cervical cancer.

Description of drawings

Fig. 1 is a flow cytometric detection diagram of SP cells in cervical cancer Hela cells.

Fig. 2 is a comparison chart of SP and NSP cell proliferation ability in vitro.

Fig. 3 is an in vitro cloning test diagram of SP and NSP cells.

Figure 4 is a histogram of the in vitro clonogenicity of SP and NSP cells.

Fig. 5 is a flow cytometry detection graph of DDP added to SP and NSP cells at a concentration of 0.95 μg/ml.

Figure 6 shows the apoptosis rate of SP and NSP cells after adding different concentrations of cisplatin.

Fig. 7 is a diagram of flow cytometry detection after SP and NSP cells were irradiated with a dose of 2Gy.

Figure 8 shows the apoptosis rate of SP and NSP cells after being given different radiation doses.

Figure 9 shows the expression of ABCG2 and ABCC2 genes in SP and NSP cells before intervention.

Figure 10 shows the expression of P53 and EGFR genes in SP and NSP cells before intervention.

Figure 11 shows the expression of ABCG2 and ABCC2 genes in SP and NSP cells after chemotherapy.

Figure 12 is a histogram comparing the expression of P53 and EGFR genes in SP and NSP cells after radiotherapy.

Detailed ways

The specific embodiments of the present invention will be described below in conjunction with the accompanying drawings.

1. Materials and equipment

1. Sources and handling of experimental animals or materials

Human cervical cancer Hela cells were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai Institute of Biological Sciences Cell Resource Center).

2. Main instruments: CO2 constant temperature cell incubator, 96-well plate, FACS flow cytometer, inverted microscope, biological safety cabinet, ultrapure water system, centrifuge, water bath box, spectrophotometer, cell counting box-8 (CCK -8), automatic fluorescent quantitative PCR diagnostic system, linear accelerator.

3. Main reagents: 10% fetal bovine serum (FBS), DMEM/F121:1 medium, penicillin and streptomycin mixture, trypsin, Hochest33342, PI (propidium iodide), phosphate buffer, Giemsa Dye solution, Trizol reagent, RNase inhibitor, MMLV reverse transcriptase.

2. Experimental method

1. In vitro culture of cervical cancer Hela cells

HeLa cells were routinely adhered to culture in an incubator at 37°C, 5% CO ₂ , and 95% humidity in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.

2. Sorting SP cells and NSP cells by flow cytometry

HeLa cells in the logarithmic growth phase were collected, digested and prepared into a single cell suspension, centrifuged, washed twice with PBS, resuspended in DMEM medium containing 2% FBS, and adjusted to 1×106 cells/mL. The cells were divided into two groups, one group was added with Hoechst33342 to a final concentration of 5 mg/L, and the other group was simultaneously added with 50 μmol/L reserpine as a control; the cells were mixed and incubated in a constant temperature water bath shaker at 37 °C for 90 min in the dark. Wash once with PBS containing 2% FBS, and resuspend in ice-cold DMEM medium containing 2% FBS. According to the instructions of the cell apoptosis detection kit, HeLa cells were sorted by an ultra-fast flow cytometry sorting system. The sorted SP cells and NSP cells were used for further experimental research.

3. Comparing the proliferation ability and in vitro clone formation ability of SP and NSP cells

3.1 Compare the proliferation ability of SP and NSP cells (ATP-TCP method):

SP and NSP cells in the logarithmic growth phase were collected and digested to prepare a single cell suspension. Each cell was inoculated into a 96-well plate with 1000 cells in 100 μL per well. Four parallel wells were made for each cell, and a total of 8 cells were made. group, DMEM (10% FBS) was used as the blank control group. Follow the instructions of the ATP detection kit to obtain the detection value. The cell proliferation rate was calculated according to the reading value of cells in each group every day, the cell proliferation rate on the nth day=the reading value on the nth day/the reading value on the first day*100%. The cell proliferation curve was drawn with the proliferation rate as the vertical axis and time as the horizontal axis.

3.2 Comparing the in vitro clone formation ability of SP and NSP cells (plate cell clone formation method):

The SP and NSP cells in the logarithmic growth phase were collected and digested to prepare a single cell suspension. Each cell was inoculated into a 6-well plate with 100 cells in 5 mL per well, and three parallel wells were made for each cell. When colonies visible to the naked eye appeared, the culture was terminated. Wash 2 times with PBS after discarding the solution. 1 mL of 4% paraformaldehyde fixed the cells for 15 minutes. After discarding the solution, add 1mL giemsa for staining, wash off slowly with running water after 30 minutes, and dry at 37°C for 30 minutes. Colonies larger than 50 cells were counted under a microscope. Ig clone formation rate=Ig (cloning number/inoculation cell number)×100%.

4. Detection of expression differences of related genes in SP cells and NSP cells

4.1 RNA extraction

Collect about 107 SP and NSP cells, put them into a grinding tube, add 1 mL of Biozol, mix by pipetting, incubate at room temperature for 7 minutes, and then add 200 μL of chloroform for extraction. Centrifuge at 12000g for 15min at 4°C, pipette the supernatant into a new ep tube, add an equal volume of pre-cooled isopropanol, blow well, and incubate at room temperature for 20min. Centrifuge at 12,000 g for 10 min at 4°C, remove the supernatant, add 1 mL of 75% pre-cooled ethanol, resuspend and wash. Centrifuge at 10,000 g for 5 min at 4°C, and remove the supernatant. Add 50 μL DEPC water to dissolve, and store at -80°C after measuring the concentration.

4.2 cDNA synthesis

Take a clean ep tube and add 1 μL random6mers, 1 μL dNTP, 7 μL RNasefreedH ₂ O, 1 μL TempRNA, blow evenly, put it into a PCR machine, set the program: 65°C, 5min. Add 4 μL 5*buffer, 0.5 μL RNase inhibitor, 1 μL RTase, 4.5 μL RNasefreedH2O, blow evenly, put it in a PCR instrument, set the program: 30°C, 10min→42°C, 50min→95°C, 5min. After the inversion is completed, store at -80°C after measuring the concentration.

4.3 RT-QPCR reaction

30 μL system: 15 μL SYBRMIX+0.6 μL primer-F+0.6 μL primer-R+11.8 μL ddH2O+2 μL cDNA. The standard two-step reaction procedure is: pre-denaturation at 95°C for 10 minutes→40cycles95°C for 15s→60°C for 1min. See Table 1 Primer Sequence

Expression difference fold=△CT(SP)/△CT(NSP);△CT=CT(target gene)-CT(GAPDH)

5. Radiotherapy and chemotherapy sensitivity test

5.1 Chemosensitivity experiment

The SP and NSP cells in the logarithmic growth phase were collected, digested and prepared into a single cell suspension, the cell concentration was adjusted to 2×105 cells/mL, and seeded in a 6-well culture plate. After 24 hours of culture, the medium was changed, and different concentrations of cisplatin 0 μg/mL, 0.475 μg/mL, 0.95 μg/mL and 1.9 μg/mL were added to NSP cells and SP cells, respectively. After 24 hours of incubation, the cells were digested and washed once with PBS. AnnexinVFITC cell apoptosis detection kit was stained and detected by flow cytometry. Apoptosis rate=UR%+LR%.

5.2 Radiotherapy sensitivity test

The SP and NSP cells in the logarithmic growth phase were collected, digested and prepared into a single cell suspension, the cell concentration was adjusted to 2×105 cells/mL, and seeded in a 6-well culture plate. After 24 hours of culture, the medium was changed, and SP cells and NSP cells were irradiated with 0, 2, 4, and 8 Gy, respectively. After 24 hours of incubation, the cells were digested and washed once with PBS. AnnexinVFITC cell apoptosis detection kit was stained and detected by flow cytometry. Apoptosis rate = UR% + LR%

6 statistical processing

SPSS17.0 software was used to process data. data adoption Said that the t-test was used between the doses, and the x2 test was used for the count data, and P<0.05 was considered statistically significant.

3. Experimental results

(1) Sorting results of SP cells in cervical cancer Hela cell line

The cultured cells were stained with Hoechest33342 fluorescence, and the results of flow cytometry analysis showed that there were a small amount of SP cells in the human cervical cancer Hela cell line, and the sorting ratio was 1.47±0.85%. After verapamil antagonistic The content of SP cells was reduced to 0.02%. (see picture 1).

(2) Comparison of the proliferation ability of SP and NSP cells

The two cell lines isolated from Hela were cultured for 8 days respectively. It was found that the proliferation difference between the cells on the 5th day and the 6th day was statistically significant, respectively p=0.0057 and p=0.0163, but as the culture There was no statistically significant difference between cells over time. The proliferation rate of SP cells was significantly faster than that of NSP cells on the 5th day, indicating that the cell activity of SP cells was significantly better than that of NSP cells. However, with the prolongation of the culture time, the continuous shrinkage of the remaining living space in the culture dish and the continuous consumption of the nutrient solution, the speed of cell proliferation slowed down significantly. The difference between the two has narrowed significantly, see Figure 2.

(3) Comparing the in vitro clonogenicity of SP and NSP cells

The SP and NSP cells in the logarithmic growth phase were collected and digested to prepare a single cell suspension. Each cell contained 100 cells in 5 mL per well. When clones visible to the naked eye appeared, the culture was terminated. Cells were fixed with formaldehyde, stained with Giemsa, and dried. Colonies larger than 50 cells were counted under a microscope. Ig clone formation rate=Ig (cloning number/inoculation cell number)×100%. The results are shown in Figure 3 and Figure 4.

The colony formation ability of SP and NSP cells was studied in vitro, and the two different types of cell clones were observed after 5 days. Compared with NSP, SP cells increased significantly, and the colony formation efficiency was calculated by lg. It was found that SP cells and NSP cells were statistically The analysis (2.407±0.1509 vs 1.160±0.1989, p=0.009) was statistically significant.

(4) Chemosensitivity experiment

After giving chemotherapy with different concentrations of DDP, it was found that the apoptosis rate of SP cells was lower than that of NSP cells under different drug concentrations, which was statistically significant. The difference between them was the largest (p=0.005). The analysis found that with the increase of drug concentration, the apoptosis rate of cells gradually increased, but the apoptosis rate of NSP cells was faster than that of SP cells. It can be seen that NSP cells are more sensitive to the chemotherapeutic drug DDP than SP cells. See Figure 5, Figure 6, and Table 2.

Table 2 Apoptosis rate of SP and NSP cells after adding different concentrations of cisplatin

The Rate of Apoptosis by DDP

p<0.05

(5) Radiotherapy sensitivity experiment

Taking SP cells and NSP cells that did not receive radiotherapy as controls, the apoptosis of cells after receiving different doses of radiotherapy was analyzed. The results of the study showed that after giving different radiation doses, there was a statistically significant difference between the apoptosis rates of SP and NSP under 2Gy irradiation (p=0.003), and there was no statistical difference between the two under other irradiation doses. See Figure 7, Figure 8,

table 3.

Table 3 Apoptosis rate of SP and NSP cells given different radiation doses

The Rate of Apoptosis by RT

p<0.05

(6) Detection of expression differences of related tumor genes in SP cells and NSP cells

1. Expression of related genes in SP cells and NSP cells before intervention

The expression levels of ABCG2, ABCC2, P53, and EGFR genes in SP cells and NSP cells without the influence of external factors. The results showed that the expression differences of ABCG2, ABCC2, P53, and EGFR genes in SP cells and NSP cells were 1.2, 1.0, 1.9, and 2.0, respectively. In SP cells and NSP cells without the influence of external factors, the gene expression of SP cells is higher than that of NSP cells, the ratios of ABCG2, ABCC2, P53, and EGFR are not statistically significant, and there is no significant difference in molecular typing, indicating that the two cell lines are of the same origin , derived from the same Hela cell. See Figure 9, Figure 10, Table 4, Table 5.

Expression of ABCG2 and ABCC2 genes in SP and NSP cells before table 4 intervention

Gene expression

p<0.05

Expression of P53 and EGFR genes in SP and NSP cells before table 5 intervention

Gene expression

p<0.05

2. Expression of ABCG2 and ABCC2 genes in SP and NSP after chemotherapy

We selected the IC50 value of 0.95 μg/ml of NSP cells to DDP as the observed concentration, and further analyzed the changes of the target gene under the same concentration of chemotherapy drugs. After 0.95 μg/mL DDP and Hela cells were co-cultured for 24 hours, RT-PCR detected the expression of ABCG2 and ABCC2 genes in SP and NSP after treatment, and found that there was a statistical difference between the expression of ABCG2 gene in SP cells and the expression of NSP ( p=0.013), there was a statistical difference between the expression of ABCC2 gene and the expression of NSP in SP cells (p=0.024). It shows that the relatively high expression of ABCG2 and ABCC2 genes in SP cells has a certain resistance to the chemotherapeutic drug DDP. See Figure 11, Table 6.

Table 6 Expression of ABCG2 and ABCC2 genes in SP and NSP cells after chemotherapy

Gene expression after chemotherapy by DDP

p<0.05

3. Expression of SP and NSP-related P53 and EFGR genes after radiotherapy

After the cultured Hela cells were irradiated with 2Gy of radiation, RT-PCR detected the gene expression of P53 and EGFR in SP and NSP after radiotherapy, and found that there was a statistical difference between the expression of P53 gene and the expression of NSP in SP cells (p= 0.044), there was a statistical difference between the expression of VEGF gene in SP cells and the expression of NSP (p=0.036). It shows that the relatively high expression of P53 and VEGF genes in SP cells has a certain resistance to radiation. See Figure 12, Table 7.

Table 7 Expression of P53 and EGFR genes in SP and NSP cells after radiotherapy

Gene expression after RT by 2Gy

p<0.05

Cervical cancer SP cells have the biological characteristics of tumor stem cells

Recurrence and metastasis are two major problems in the treatment of malignant tumors. In recent years, with the in-depth study of malignant tumors, it has been found that there are a small number of tumor cells in tumor tissues that have characteristics similar to stem cells, which is the cause of tumor metastasis and recurrence. . Subsequently, the theory of cancer stem cells was proposed. The theory holds that not all tumor cells can grow infinitely, but only a small part of them can infinitely self-renew, highly proliferate, and multidirectionally differentiate, called tumor stem cells, and this small group of cells is the initiation of tumor formation. cells and maintain tumor growth. At the same time, this part of cells is resistant to radiotherapy and chemotherapy through various mechanisms, which is the root cause of tumor recurrence and metastasis. Therefore, the existence of tumor stem cells is the main reason for treatment failure. Research on cervical cancer stem cells is still in its infancy. SP sorting has become a common method in cancer stem cell research. Cells isolated by this method are called SP cells. Many scholars believe that compared with NSP cells, SP cells have stronger self-renewal, multidirectional differentiation, high proliferation, and resistance to radiotherapy and chemotherapy. Similar to tumor stem cells, SP cells can be used as an ideal model for stem cell research. SP cells were first isolated and confirmed in hematological tumors. With the deepening of research, SP cells have been found to widely exist in a variety of tumor cell lines and tumor tissues.

In this paper, a small amount of SP cells were sorted from the human cervical cancer Hela cell line by FACS method, and the sorting ratio was 1.47±0.85%, which was similar to that reported in the literature. In order to explore whether the sorted SP cells have the characteristics of tumor stem cells, the cell proliferation ability and in vitro clone formation ability of the sorted SP cells were studied. These two experiments confirmed that SP cells sorted from the cervical cancer Hela cell line have stronger proliferation activity and clone formation ability than NSP cells in vitro, which conforms to the characteristics of high proliferation and clone formation of tumor stem cells, and preliminarily confirms that SP cells It does have some characteristics of tumor stem cells, and can be used as a model for cervical cancer stem cell research, providing an experimental basis for further research on cervical cancer stem cells.

2. Cervical cancer side population cells are associated with chemotherapy resistance

One of the characteristics of tumor stem cells is resistance to chemotherapy drugs. Some studies have used As2O3 to reverse the drug-resistant strain SiHa/CDDP of human cervical cancer cells. It was found that the drug resistance of SiHa/CDDP cells decreased significantly with the weakening of its stem cell characteristics, and at the same time The expression of ABCG2 in SiHa/CDDP cells was significantly reduced, suggesting that the drug resistance of cervical cancer cells to CDDP is closely related to the characteristics of stem cells. In order to know whether SP cells in cervical cancer Hela cell line have chemotherapy resistance, the present invention uses SP cells and NSP cells sorted from cervical cancer Hela cells to conduct in vitro chemotherapy sensitivity test. After giving SP cells and NSP cells different concentrations of DDP chemotherapy, it was found that the apoptosis rate of SP cells was lower than that of NSP cells at different drug concentrations, and both were statistically significant. Among them, the apoptosis rate was different after the DDP concentration of 1.9 μg/ml The analysis found that with the increase of drug concentration, the apoptosis rate of cells gradually increased, but the apoptosis rate of NSP cells was faster than that of SP cells. It can be seen that NSP cells are more sensitive to the chemotherapeutic drug DDP than SP cells. Our findings are consistent with literature reports.

3. Cervical cancer side population cells are associated with radiotherapy resistance

The resistance of tumor stem cells to radiotherapy may be related to the following reasons: 1. Cancer stem cells can effectively promote the repair of cellular DNA damage after radiotherapy ^[10] ; 2. Cancer stem cell cycle arrest; 3. The relationship between tumor stem cells and their surrounding microenvironment Interaction ^[20-21] . Bao et al ^[22] found that malignant glioma stem cells exhibited radioresistance, which may lead to failure of clinical radiotherapy. It can be speculated that the insensitivity of some cervical cancer patients to radiotherapy may also be related to the radioresistance of tumor stem cells. The present invention found that before X-ray irradiation, the apoptosis rate of SP cells was lower than that of NSP cells. After 2Gy dose irradiation, the apoptosis rates of SP and NSP all increased, and the difference was statistically significant. After 4Gy, 8Gy dose irradiation , the apoptosis rate further increased, but the difference was not statistically significant. It shows that giving high-dose radiotherapy can lead to the intolerance of both types of cells and increased apoptosis, while giving a lower dose of radiotherapy, the apoptosis rate of SP cells is lower than that of NSP cells, and there is a statistical difference, which further shows that SP cells have Stronger radiation resistance and better survival in radiotherapy may be the root cause of tumor progression, recurrence and metastasis.

4. Expression of ABCG2, ABCC2, P53 and EGFR in SP cells and NSP cells

Studies have found that the abnormal expression levels of chemoradiotherapy-sensitive factors and their products in some tumor cells are related to the chemotherapeutic and radiotherapy sensitivity of tumor cells.

Most of the cell membranes of tumor stem cells express ABC transporter family membrane proteins, especially ABCG2, which can transport and excrete metabolites, drugs and other substances, so that many anti-tumor chemotherapy drugs cannot exert their killing effect on tumor stem cells, or the effect is significantly weakened. Studies by ChenZ et al. have shown that ABCG-2, in addition to its transport function, also plays an important role in regulating the proliferation, differentiation and protection of SP cells. With the deepening of research on other molecules of the ABC transporter family, such as ABCB1 and ABCC2, researchers believe that other ABC transporters may also be the cause of the SP phenotype, and thus also the molecular basis of SP cell multidrug resistance. The results of the present invention show that the expression of ABCG2 and ABCC2 genes in SP cells and NSP cells is higher than that in NSP cells without the influence of external factors, but the ratio has no statistical significance. After DDP chemotherapy, it was found that the expression of ABCG2 and ABCC2 genes in SP cells was higher than that in NSP cells, with statistical difference (p=0.013, P=0.024). It shows that the high expression of ABCG2 and ABCC2 in SP cells has a certain resistance to the chemotherapeutic drug DDP. The gene expression results before DDP treatment of the present invention are consistent with foreign reports, but are inconsistent with Qi Wenjuan's research report showing that ABCG2 is highly expressed in SP cells after HeLa and SiHa cells are sorted, while it is almost not expressed in NSP cells. According to the analysis in this paper, there is no obvious difference in the molecular typing of SP and NSP cells, indicating that the two cell lines have the same root and homology. It may be that the subtle difference between the two cells under the action of DDP drugs causes the two cell lines to produce diametrically opposite phenomena. Previously, we found that the growth rate of SP cells was faster than that of NSP cells, and the level of apoptosis was lower than that of NSP cells. Under the action of DDP drugs, the cells continued to die. The difference in the expression of ABCG2 and ABCC2 led to the expression of ABCG2 and ABCC2 in SP cells The relative expression level was gradually higher than that of NSP cells, which eventually led to qualitative changes, which led to the generation of drug-resistant cell lines. We boldly speculate that with the continuous progress of chemotherapy, SP cells with high expression of ABCG2 and ABCC2 will accumulate in the tumor tissue, and the quantitative change will eventually lead to qualitative change, resulting in the emergence of drug-resistant cell lines. One of the causes of chemotherapeutic drug resistance.

At present, studies on the tumor suppressor gene P53 are extensive and in-depth, showing that the wild-type p53 gene inhibits the repair of tumor cells after radiation damage, promotes the apoptosis of tumor cells, and has a radiosensitizing effect. However, once the P53 gene is mutated, it is conducive to tumor cell proliferation, canceration, progression, and radioresistance. The overexpression of EGFR (Epithelial Growth Factor Receptor) may lead to tumor radioresistance by stimulating abnormal growth, inhibiting apoptosis, and repairing DNA damaged by radiotherapy. Myllynen et al. found that EGFR's regulation of double-strand break (DSB) repair involves both non-homologous end joining (NHEJ) and homologous recombination repair (HRR). When EGFR is activated, the repair function of the entire DSB is significantly improved. , resulting in radiation resistance. The results of this study showed that in SP and NSP cells without the influence of external factors, the gene expression of P53 and VEGF in SP cells was higher than that in NSP cells, but the ratio was not statistically significant. After 2Gy irradiation, it was found that the expression of P53 and VEGF in SP cells was significantly different from that of NSP. According to our analysis, there was no significant difference in the molecular typing of SP and NSP cells before radiotherapy, indicating that the two cells were of the same origin, and the gene level changed significantly after 2Gy X-ray irradiation. The radiation resistance of SP cells has a certain relationship. SP cells are more resistant to radiation, which leads to the failure and recurrence of tumor treatment.

In summary, the present invention uses the classic Hoechst33342 staining method reported by Goodell et al. in combination with flow cytometry to sort SP cells and NSP cells in cervical cancer Hela cells, and observe cell growth and in vitro clone formation , and further chemotherapy and radiotherapy sensitivity experiments were carried out, and the sorted SP cells had strong proliferative activity and clone formation ability in vitro, which was in line with the biological characteristics of tumor stem cells; the apoptosis rate of SP cells was significantly uniform under different drug concentrations. Lower than that of NSP cells, with the increase of drug concentration, the apoptosis rate of cells gradually increased, but the apoptosis rate of NSP cells was faster than that of SP cells; the apoptosis rate of SP cells was significantly lower after a lower dose of radiotherapy NSP cells, and high-dose radiotherapy can lead to both cell intolerance and increased apoptosis; radiochemotherapy-sensitive factors ABCG2, ABCC2P53, and EGFR in cervical cancer SP cells are slightly higher than those in NSP cells without the influence of external factors. However, there was no significant difference. After DDP chemotherapy and radiation irradiation, the gene expression of the above-mentioned related factors in SP cells was significantly higher than that in NSP cells. As a result, tumor cells resisted chemotherapy and radiotherapy, and SP cells were more sensitive to radiotherapy and chemotherapy. Not as good as NSP cells.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned examples. What are described in the above-mentioned examples and descriptions are only to illustrate the principles of the present invention. The present invention also has various changes without departing from the spirit and scope of the present invention. These changes and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

1. apply a method for cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity, it is characterized in that, comprise the steps:

(1) patient's cervical cancer tissues is obtained by operation source;

(2) primary cervical cancer cell suspension is prepared:

Get the fresh Cervical Tumor tissue of excision, stroke-physiological saline solution is rinsed well, is cut into 1mm ³the fragment of size, adds the collagenase that final concentration is 0.1%, 0.01% Unidasa, the DNA enzymatic of 0.002%, 37 DEG C digest 2 hours, 100 order nylon net filters, the centrifugal 10min of 500rpm, abandon supernatant, add appropriate Hanks liquid, after mixing, row discontinuous density gradient is centrifugal, 2000rpm20min, collect the suspension being rich in tumour cell, after washing 2 times, for subsequent use to testing by the DMEM nutrient solution adjustment cell count containing 10% serum;

(3) by cervical cancer cell in DMEM substratum, in 37 DEG C, 5%CO ₂, conventional adherent culture in incubator under 95% humidity condition;

(4) cervical cancer cell of logarithmic phase is collected, adjustment cell count to 1 × 10 ⁶individual/mL, is divided into two groups by cell, one group adds Hoechst33342 to final concentration is 5mg/L, and another group adds 50 μm of ol/L reserpines in contrast simultaneously; Mixing cell is resuspended in the ice-cold DMEM substratum containing 2%FBS, and with hypervelocity fluidic cell separation system sorting SP cell, the SP cell obtained after sorting and NSP cell are for further experimental study;

(5) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10 ⁵individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, NSP cell and SP cell add the cis-platinum of different concns respectively, digest after cultivating 24h, and PBS washs, AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%;

(6) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10 ⁵individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, and the radiotherapy that SP cell and NSP cell give various dose is irradiated, and digest after cultivating 24h, PBS washs 1 time; AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%.

2. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, described step (5) can be exchanged with step (6) tandem.

3. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, in described step (5), NSP cell and SP cell add cis-platinum 0 μ g/mL, 0.475 μ g/mL, the 0.95 μ g/mL and 1.9 μ g/mL of different concns respectively.

4. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, is characterized in that, in described step (6), NSP cell and SP cell give the radiotherapy irradiation that various dose 0,2,4,8Gy is irradiated respectively.

5. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, in described step (3), DMEM substratum contains 10%FBS, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.