CN112933238A

CN112933238A - Application of substance inhibiting TGF-beta R1 gene expression and cisplatin in treatment and/or prevention of esophageal cancer

Info

Publication number: CN112933238A
Application number: CN202110141889.5A
Authority: CN
Inventors: 胡建明; 谢玉芳; 李玲; 李锋; 李范平
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-06-11

Abstract

The invention discloses an application of a substance inhibiting TGF-beta R1 gene expression and cisplatin in treatment and/or prevention of esophageal cancer. The experiment of the invention proves that: the M2 type macrophage can promote dryness and proliferation of esophageal squamous cell carcinoma and inhibit apoptosis, and promote the drug resistance of esophageal squamous cell carcinoma to chemotherapy medicament cisplatin, while the substance inhibiting the expression of TGF-beta R1 can reverse the characteristic. The invention provides a new direction for further defining the malignant biological behavior mechanism of the esophageal squamous cell carcinoma and the targeted therapy.

Description

Application of substance inhibiting TGF-beta R1 gene expression and cisplatin in treatment and/or prevention of esophageal cancer

Technical Field

The invention relates to the application of a substance inhibiting TGF-beta R1 gene expression and cisplatin in treating and/or preventing esophageal cancer in the field of biomedicine.

Specifically designed to promote the sensitivity of esophageal squamous cell carcinoma chemotherapy, in particular to the specific application of a product for inhibiting dryness promotion, proliferation and apoptosis of M2 type macrophages in a tumor area.

Background

Esophageal cancer is the eighth most common malignant tumor in the world, the death rate is ranked sixth in the world, the esophageal cancer can be divided into esophageal adenocarcinoma and esophageal squamous cell carcinoma due to different pathological types, wherein the esophageal squamous cell carcinoma is the most common histological type of the esophageal cancer and accounts for 80 percent of the incidence rate of the esophageal cancer, the number of esophageal cancer deaths in China is the first in the world every year, the early symptoms of most esophageal cancer patients are not obvious, an early-stage sensitivity screening method is lacked, and once the symptoms appear, the patients are usually in the middle and late stages, and then chemical drug therapy becomes the main treatment means for the middle and late esophageal cancer patients. Cisplatin (cis-dichlorodiammineplatinum (II), cDDP) is the most commonly used first-line chemotherapeutic agent for the treatment of esophageal cancer, but as treatment continues, many patients develop resistance to cisplatin therapy, i.e., resistance to Chemotherapy, i.e., Chemotherapy resistance. Therefore, the development of a mechanism related to chemotherapy resistance of esophageal squamous cell carcinoma to search for a corresponding action target point so as to reverse the chemotherapy resistance of esophageal squamous cell carcinoma has important clinical significance.

Recently, some researchers found a cell subset, which is responsible for the occurrence, development, recurrence and metastasis of tumors and is closely related to chemotherapy resistance, i.e., Cancer Stem Cells (CSCs), which are a class of cells with self-renewal, immortalization, and multi-directional differentiation potential, are positively correlated with the malignancy of tumor tissues and can promote tumor invasion and metastasis, and are found in various tumor tissues and are closely related to chemotherapy resistance. And CSCs are a dynamically changing "state" rather than being fixed. It has been found that TGF-beta 1(Transforming growth factor beta 1) plays an important role in the dynamic regulation of CSCs in self-renewal and differentiation. TGF-. beta.R 1(Transforming growth factor beta receptor 1) is one of the major receptors for binding to TGF-. beta.1 on the surface of cancer cells, in addition to two classes of TGF-. beta.R 2 and TGF-. beta.R 3. TGF-beta 1 is one of the members of TGF-beta super family, is a multifunctional polypeptide cytokine existing in human tissue cells, and TGF-beta 1 is involved in various pathological processes of mammals, influences the biological processes of cell proliferation and differentiation, wound healing, angiogenesis, tissue fibrosis, extracellular matrix production and the like.

The tumor microenvironment is one of the research hotspots in recent years, and has a plurality of inflammatory cells, and macrophages playing immune and inflammatory roles account for a considerable proportion in the tumor microenvironment and are the first defense line for the body to regulate and control tumorigenesis. Macrophages have two phenotypes, type M1 and type M2. Specifically, the M1 type macrophage has high antigen presenting capability, can kill microorganisms and destroy the activity of tumor cells, thereby playing a role in proinflammatory antitumor. M2 type macrophage is substitute activated macrophage, and macrophage accumulated in most tumor region is M2 type macrophage, i.e. tumor-related macrophage, and has anti-inflammatory, tumor promotion and immunity suppression functions.

Disclosure of Invention

The invention aims to provide any one of the following applications:

1. the application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in the preparation of products for treating and/or preventing esophageal cancer;

2. the application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in preparing dry products for inhibiting esophageal cancer cells;

3. the application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 in preparing products for inhibiting the balling of esophageal cancer cells;

4. the application of substances for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 in preparing products for inhibiting the proliferation capacity and/or the growth capacity of esophageal cancer cells;

5. the application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in the preparation of products for promoting the apoptosis of esophageal cancer cells;

6. the application of substances for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 in preparing products for reducing the drug resistance of esophageal cancer or esophageal cancer cells to chemotherapeutic drugs.

Above, the esophageal cancer may be esophageal squamous carcinoma, and the esophageal cancer cells may be esophageal squamous carcinoma cells.

The agent that inhibits expression of TGF- β R1 may be SB 431542.

The invention also provides a product with any one of the following functions, wherein the active ingredients of the product are a substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin:

x1, treatment and/or prevention of esophageal cancer;

x2, inhibiting esophageal cancer cell dryness;

x3, inhibiting esophageal cancer cell balling;

x4, ability to inhibit proliferation and/or growth of esophageal cancer cells;

x5, promoting the apoptosis of esophageal cancer cells.

In the above product, the substance may be SB 431542.

In one embodiment of the invention, the esophageal squamous carcinoma cells are human esophageal squamous carcinoma cells. The esophageal squamous carcinoma cells are EC109 and EC 9706.

In the present invention, the product may be a medicament or a vaccine. The inhibition of esophageal cancer cell sternness can be reflected in the inhibition of gene expression quantity of sternness marker proteins CD44 and/or OCT-4.

The experiment of the invention proves that: the M2 type macrophage can promote dryness and proliferation of esophageal squamous cell carcinoma and inhibit apoptosis, and promote the drug resistance of esophageal squamous cell carcinoma to chemotherapy medicament cisplatin, while the substance inhibiting the expression of TGF-beta R1 can reverse the characteristic. The invention provides a new direction for further defining the malignant biological behavior mechanism of the esophageal squamous cell carcinoma and the targeted therapy.

Drawings

FIG. 1 is the establishment and identification of a squamous cell carcinoma of esophagus spheroid model. EC109 and EC9706 spheres were spheres obtained from EC109 and EC9706 cells, respectively.

Wherein, A, the esophageal squamous carcinoma background cells EC109 and EC9706 have the microscopic morphological difference with the corresponding spheroblasts;

B. differential expression of protein levels of esophageal squamous cell carcinoma spheroblasts and background cell dryness markers CD44 and OCT-4;

C. differential expression of RNA levels of esophageal squamous cell carcinoma spheroblasts and background cell sternness markers CD44, OCT-4.

FIG. 2 shows that the esophageal squamous carcinoma spheroids have drug resistance to cisplatin as a chemotherapeutic drug.

Wherein, the proliferative activity of the esophageal squamous cell carcinoma spheroblasts under the action of the chemotherapeutic drug cisplatin is obviously higher than that of background cancer cells;

B. the apoptosis rate of esophageal squamous cell carcinoma spheroblasts under the action of cisplatin serving as a chemotherapeutic medicament is obviously lower than that of background cancer cells.

FIG. 3 shows the dryness of M2 type tumor-associated macrophages promoting esophageal squamous carcinoma cells.

Wherein, the A.M2 type tumor-associated macrophages promote the increase of the expression levels of the dryness markers CD44 and OCT-4 protein of esophageal squamous carcinoma cells; EC109 and EC9706 are background esophageal squamous carcinoma cells, EC109+ M2 and EC9706+ M2 represent esophageal squamous carcinoma cells after cocultivation with M2-type macrophages;

the M2 type tumor-associated macrophages promote the increase of the expression levels of esophageal squamous carcinoma EC109 cell dryness markers CD44 and OCT-4 RNA;

the M2 type tumor-associated macrophages promote the increase of the expression levels of esophageal squamous carcinoma EC9706 cell dryness markers CD44 and OCT-4 RNA.

FIG. 4 shows that M2 type tumor-associated macrophages promote the resistance of esophageal squamous carcinoma spheroblasts to cisplatin as a chemotherapeutic drug.

Wherein, the A.M2 tumor-related macrophages reduce the influence of cisplatin on the proliferation activity of esophageal squamous cell carcinoma spheroblasts;

the effect of cisplatin on apoptosis of esophageal squamous carcinoma spheroblasts is reduced under the participation of M2 tumor-associated macrophages.

FIG. 5 shows that TGF-beta R1 inhibitor SB431542 participates in reversing the dryness characteristic of M2 tumor-associated macrophages promoting esophageal squamous cell carcinoma.

Wherein, the A.SB431542 reduces the protein level expression of M2 tumor-related macrophages promoting esophageal squamous carcinoma cell dryness markers CD44 and OCT-4 under the participation;

SB431542 reduces the expression of dry markers CD44 and OCT-4RNA level of M2 tumor-associated macrophages promoting esophageal squamous carcinoma EC109 cells under the participation;

c. SB431542 reduces the expression of RNA level of M2 tumor-associated macrophages promoting esophageal squamous carcinoma EC9706 cell dryness markers CD44 and OCT-4 under the participation.

FIG. 6 shows that the TGF-beta R1 inhibitor SB431542 participates in reversing M2 tumor-associated macrophages to promote the proliferation of esophageal squamous cell carcinoma spheroblasts (stem cell-like cells) and inhibit apoptosis under the action of cisplatin.

Wherein, under the action of A, cis-platinum, SB431542 participates in reducing the proliferation activity of M2 type tumor-related macrophages for promoting esophageal squamous cell carcinoma (plate clone experiment);

B. under the action of cisplatin, SB431542 participates in reducing the proliferation activity of M2 type tumor-related macrophages for promoting esophageal squamous cell carcinoma (CCK8 proliferation experiment);

C. under the action of cisplatin, SB431542 participates in reducing M2 type tumor-associated macrophages and promoting apoptosis inhibition of esophageal squamous cell carcinoma cells.

SB denotes SB431542 and TAM denotes M2-type macrophages. The EC 109-ball + TAM experiments on the bar graphs in the figures also contained cDDP.

FIG. 7 shows that M2 tumor-associated macrophages promote the resistance of esophageal squamous cell carcinoma-bearing mice to chemotherapeutic drugs in animals.

Wherein, the graph A shows that the reactivity of the M2 tumor-associated macrophage group tumor to cisplatin is obviously lower than that of the control group tumor, and is mainly reflected in the tumor size;

panel b shows that M2 tumor-associated macrophage group tumors were significantly less reactive to cisplatin than the control group, mainly due to tumor volume and tumor weight;

the protein expression of tumor participation dry markers CD44 and OCT-4 of the M2 tumor-associated macrophage group is higher than that of the control group;

and D, the RNA expression of tumor participation dry markers CD44 and OCT-4 of the M2 tumor-associated macrophage group is higher than that of the control group.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The experimental nude mice used in the following embodiments are BALB/C female nude mice, 4-6 weeks old, and 15-20g in weight, Beijing Wittingle laboratory animal technology Ltd, which were bred at the animal experiment center of Xinjiang medical university.

The human esophageal squamous carcinoma cell lines EC109 and EC9706 and the human monocyte THP-1 in the following examples are all products of Shanghai Reineckia Biotech limited.

The culture medium for culturing the human esophageal squamous carcinoma cell line comprises the following components: the culture Medium RPMI Medium 1640basic and DMEM basic are both products of American GIBCO company; the fetal bovine serum is a product of Israel Biological Industries; penicillin streptomycin mixed solution (100 x), Solambio company, Beijing, was diluted 100-fold at the time of use.

Cisplatin (cDDP): shandong Qilu pharmaceutical works.

PMA inducer: beijing Sizhengbai Biotech Co.

In the examples below, the TGF-. beta.R 1 inhibitor SB431542 is available from Selleck, Inc. USA under catalog number: s1067, and the IC50 value was 94 nM. In use, the mixture is dissolved in DMSO and diluted with water to the corresponding concentration. The chemical structural formula of SB431542 is as follows:

the information of the Western blot main antibody used in the following examples is shown in Table 1.

TABLE 1 Bioantibody information for Western blot experiments

Name of antibody	Source	Using dilution ratios	Company(s)
				Beta-actin antibodies	Mouse anti-human	1:1000	China fir bridge in Beijing
CD44 antibody	Mouse anti-human	1:1000	CST
				OCT-4 antibodies	Rabbit anti-human	1:500	Abcam

The reagents used in the following embodiments are as follows: cell culture and induction reagents: RPMI 1640 liquid medium, F12 liquid medium, beta-mercaptoethanol (GIBCO Co.), 20 XPBS (Shanghai Biotech Co.), FBS fetal bovine serum (BI Co.), 0.25% EDTA pancreatin, double antibody (Solarbio Co.), DMSO (SIGMA Co., Ltd.), 25cm²Culture flask, freezing tube (Corning Corp.), 15ml, 50ml centrifugeHeart tube (Corning), Transwell chamber, cell culture plate (Corning);

RNA extraction main reagent and real-time fluorescent quantitative PCR detection reagent: trizol (Invitrogen), chloroform, isopropanol, primers (Shanghai Processo Co.), an enzyme-free pipette tip (Shanghai Processo Co.), an enzyme-free 1.5ml, 200ul small EP tube (Shanghai Processo Co.), a reverse transcription Kit TIANCcript RT Kit (TIANGEN Co.), an enzyme-free PCR 96-well plate (Shanghai Processo Co.), a PCR in-silico Kit UltraSYBR mix (available to century Biotech Co.); western blot main reagent: PVDF membranes (0.45 μm and 0.22 μm, millipore corporation), high performance RIPA cell lysate (Solarbio corporation), 5 × loading buffer (kang century biotechnology corporation), PMSF (Solarbio corporation), rainbow protein Marker (Thermo corporation), fully defatted milk powder (illite corporation), ECL chemiluminescence assay kit (Thermo corporation); reagents required for animal experiments: normal saline (Shandong Qilu pharmaceutical factory).

The main instruments used in the following embodiments are as follows: 371 type CO₂Constant temperature incubator (Thermo corporation), -20 ℃ refrigerator (hel ltd), -80 ℃ refrigerator (nurir corporation), SW-CJ-IF type clean bench (shanghai bosun corporation), liquid nitrogen biocontainer (sika, chuanxiong), uv spectrophotometer Nanodrop 2000(Thermo corporation), OLYMPUS TH4-200 microscope (OLYMPUS corporation, japan), electrophoresis apparatus (BIO-RAD corporation), Trans-Blot SD Cell type electric transfer apparatus (BIO-RAD corporation), GEL-2000 GEL imager (BIO-RAD corporation), autoclave (Thermo Scientific corporation), magnetic heating stirrer (jiangsu jintan medical instrument factory), Ph 213Microprocessor Ph Meter (HANNA corporation, italy), PFS-400 rapid bench sealer (zhejiang south china ltd), vortex mixer (XW-80A) (which is manufactured by beilin berl corporation, tsu corporation), low temperature high speed centrifuge (Thermo corporation), Grant XB70 ice maker (usa), 7500FAST fluorescent quantitative PCR instrument (ABI corporation), Htpot 80 dry bath (ABSON corporation).

The method of preparing the cell culture medium in the following embodiments is as follows:

100ml of 1640 complete medium (10% serum, 1% double antibody, 1640 complete medium) was prepared, namely: 89ml of 1640basic medium, 10ml of fetal bovine serum (BI), and 1ml of a mixture of streptomycin and streptomycin were stored in a refrigerator at 4 ℃ until use.

F12(DMEM/F12) serum-free medium (0.1% EGF, 0.1% FGF, 1% B27 serum-free medium) was prepared, i.e.: 98ml of F12(DMEM/F12) basal medium, a mixture of 2mlB27, 0.02ml of EGF and 0.02ml of FGF, sealed and stored in a refrigerator at 4 ℃ until use.

In the following embodiments, Gelpro32 was used to measure the gray scale values of expression of the dry marker protein as CD44 and OCT-4 proteins in Western Blot experiments, the expression levels being described as mean ± standard deviation; the cell experiments were repeated at least 3 times, and the t-test was applied differentially between the two groups. All relevant experimental data analysis was processed and analyzed using SPSS 22.0 statistical software, and differences were considered statistically significant with P < 0.05. "+" indicates P < 0.05, "+" indicates P < 0.01, and "+" indicates P < 0.001. The following chart was made using graphpad5.0 software.

Example 1 use of SB431542 with cisplatin for the treatment and/or prevention of esophageal cancer

Serum-free balling culture of esophageal squamous cell carcinoma stem cell-like cells and identification

The cells to be detected are human esophageal squamous carcinoma cell lines EC109 and EC9706, and the serum-free balling experiment comprises the following specific steps:

1. taking out the cells to be detected from a constant temperature incubator at 37 ℃, adding 3ml of 1 XPBS into the bottle, slightly shaking the culture bottle, washing, pouring out, and repeating for 3 times;

2.1 ml of trypsin was added to the flask and digested for 1-2min, and 3ml of F12 serum-free medium was added to the flask to stop the digestion.

3. Transferring the liquid into a centrifuge tube, centrifuging at 800rpm/5min, and removing the supernatant;

4. after being resuspended by PBS, centrifuging at 800rpm/5min, pouring out supernatant in the tube after centrifugation is finished, sucking out a small amount of residual liquid by a pipette, only remaining cell sediment, and repeating the operation again;

5. adding 2ml of F12 serum-free culture medium into the centrifuge tube, gently blowing and beating the cell sediment at the bottom of the centrifuge tube to uniformly distribute the cells in the culture medium, and countingAdjusting the cell concentration to 8 × 10⁴Obtaining cell suspension;

6. after the step 5 is finished, adding the cell suspension into a low-adhesion 6-hole plate, wherein the volume of the cell suspension is 2ml per hole;

7. strictly aseptic operation, placing into incubator, and observing cell state the next day.

The cells which are not subjected to the spherulization culture are used as a control, and the results of serum-free spherulization experiments show that compared with the adherent fusiform morphology of background cells of esophageal squamous carcinoma, the cells subjected to the serum-free spherulization culture grow in a suspension manner to form macroscopic spheres (shown as A in figure 1), and the spheres obtained from EC109 and EC9706 cells are respectively marked as EC109sphere and EC9706 sphere.

Western blot is used for detecting the expression quantity of the dry marker proteins CD44 and OCT-4 in the cells (marked as spheroblasts) of the spheres obtained in the step, beta-actin is used as an internal reference, the used primary antibodies are respectively a CD44 antibody, an OCT-4 antibody and a beta-actin antibody, and the used secondary antibodies are products with the product number of SAP-9100 of Beijing China fir gold bridge biotechnology limited.

Western blot detection results show that compared with esophageal squamous cell carcinoma background cells (namely cells which are not subjected to a balling experiment), the expression levels of the dry marker proteins CD44 and OCT-4 in the spheroid cells are remarkably increased (P is less than 0.001) (shown as B in figure 1).

Detecting the expression quantity of the dry marker in the sphere obtained in the step on the RNA level by utilizing qRT-PCR, wherein the used primers are shown in Table 2; and (3) reading a result: CT values were read in 7500Fast run software v2.2.1.

TABLE 2 primer sequences

The qRT-PCR detection result shows that compared with the esophageal squamous cell carcinoma background cell group, the CD44 and OCT-4 of the bulbar cell group are obviously increased, which shows that the in-vitro esophageal squamous cell carcinoma bulbar cell model is successfully established (as shown in C in figure 1).

Secondly, the esophageal squamous carcinoma spheroblasts (stem cell-like cells) promote drug resistance

The CCK8 kit detection comprises the following specific steps, wherein the cells to be detected are two esophageal squamous carcinoma background cells (EC109 cells and EC9706 cells) and globoid cells (namely, the cells of the sphere induced by the EC109 cells and the EC9706 cells obtained in the step one are respectively marked as EC109 spheres and EC9706 spheres):

1. adding cells to be tested into a 96-well plate, wherein 4000 cells are added in each well, and grouping conditions are as follows: EC109 spheres (experimental group), EC109 (control group), EC9706 spheres (experimental group), EC9706 (control group), each group set 5 multiple wells;

2. respectively adding cisplatin medicinal solution (obtained by dissolving cisplatin in normal saline) with specific concentration into a 96-well plate, and placing the 96-well plate into an incubator at 37 ℃ for 48h, wherein the concentration of cisplatin in the system is set to be 4 ug/ml; and 0ug/ml (i.e. no cisplatin added) was used as a blank;

3. the 96-well plate was removed at 0 hour, 24 hours, 36 hours, and 72 hours of incubation, respectively, and the following operations were performed: adding 10ul CCK8 reagent (Japanese Dojindo, Cat: 5000T, CCK8) into each well with cells, respectively (light-shielding operation), wrapping 96-well plate with sterilized tinfoil paper, and placing into incubator for light-shielding incubation for 2 hr;

4. after incubation, detecting the absorbance value (OD value) at 450nm by using an enzyme-labeling instrument;

5. the cell survival rate was calculated according to the following calculation formula:

the results of CCK8 show that the survival rate (i.e., proliferation index) of spheroblasts under the action of cisplatin is significantly higher than that of background cells (P < 0.05) (see A in FIG. 2).

Detecting the specific steps of the experiment by using a flow cytometer, wherein the cell to be detected is the cell which is obtained by detecting the cell obtained in the step 2 and is incubated for 48 hours by using a CCK8 kit, and the EC109, EC9706 and EC9706 spheres treated by cisplatin are respectively marked as EC109+ cDDP, EC109sphere + cDDP, EC9706+ cDDP and EC9706sphere + cDDP:

1. collecting cells, rinsing the cells with 1 × PBS pre-cooled in a refrigerator at 4 deg.C for 2 times, and discarding the supernatant;

2. 1.5ml precooled 1 × Binding Buffer resuspended the untreated cells, divided equally into three tubes, one tube blank control tube, one tube added with 5ul Annexin V-FITC, one tube added with 10ul PI;

3. incubating at room temperature in dark for 5min, and detecting on a machine. The sample detection induces apoptosis according to an experimental scheme, and the experiment is divided into the following groups: EC109+ cDDP group, EC109sphere + cDDP group, EC9706+ cDDP group, EC109sphere + cDDP group;

4. respectively collecting culture solution supernatants in different holes, collecting cells by using Accutase cell digestive juice, centrifuging at 800rpm for 5min, and removing the supernatants;

5. precooling 1 × PBS for centrifugal washing, and collecting 1-10 × 10⁵500ul of 1 XBinding Buffer was taken to resuspend the cells (including the cells in the culture supernatant);

6. 5ul FITC and 10ul PI were added to the sample tube;

7. after gentle vortex mixing, incubating for 5min at room temperature in the dark;

8. and (6) performing detection on the machine.

The above steps are detected by using a flow apoptosis kit (Biyunyan, C1062S).

The apoptosis result of flow detection shows that the apoptosis rate of the globulocyte is obviously lower than that of a background cell group (P is less than 0.05) under the action of cisplatin (as shown in B in figure 2).

III, M2 type macrophage for promoting dryness of esophageal squamous cell carcinoma cells

Plating and inducing human monocyte THP-1:

(1) taking THP-1 with proper growth density, transferring the culture solution to a centrifuge tube, and centrifuging at 800rpm/5 min;

(2) discarding the supernatant after centrifugation, collecting cell precipitate, resuspending the cell precipitate by using RPMI-1640 culture medium, and counting;

(3) after the step (2) is finished, planting 1.5 multiplied by 10 cells in each hole of the 6-hole plate⁶Adding 20ul PMA inducer, supplementing 2ml of RPMI-1640 culture medium in each hole, and placing in an incubator for continuous culture;

(4) after the culture is continued for 36h, the culture medium is changed to RPMI-1640 medium containing IL-4 and IL-13 with the concentration of 20ng/ml for further induction for 48h, and M2 type macrophages (recorded as M2-TAM) are obtained for subsequent experiments.

Establishing a non-contact co-culture system:

(1) pancreatin digesting the M2 type macrophage obtained above, spreading in the upper chamber of a 6-pore plate small chamber, performing strict aseptic operation, and placing in an incubator for 12h to adhere to the wall;

(2) simultaneously, trypsinizing EC109 or EC9706 cells, planting in a lower chamber of a 6-well plate, and culturing at 37 ℃ under the condition of 5% CO2 for 12h to adhere to the wall; (cancer cell: M2-TAM: 1: 3: 40X 10)⁴：1.2×10⁶)

(3) Establishing co-culture: washing cells in upper and lower chambers with 1 × PBS for 2-3 times, placing the upper chamber in esophageal squamous carcinoma hole, adding 2ml of RPMI-1640 culture medium into the upper and lower chambers, respectively, placing at 37 deg.C and 5% CO₂The culture is carried out in an incubator for 48h, and esophageal squamous carcinoma cells in a 6-well plate of a lower chamber are collected to detect the expression of CD44 and OCT-4.

Control group: human esophageal squamous carcinoma cell lines EC109 and EC9706 in 5% CO₂The cancer cells after 48h of culture were performed alone in the incubator.

Western blot detection results show that compared with esophageal squamous cell carcinoma cells alone, the expression of the sternness marker proteins CD44 and OCT-4 is obviously reduced (P is less than 0.001) compared with an M2 macrophage co-culture group (shown as A in figure 3).

The qRT-PCR detection result shows that the expression of the dry markers RNA CD44 and OCT-4 is obviously reduced compared with the esophageal squamous carcinoma cell alone compared with the M2 macrophage co-culture group (P is less than 0.05) (as B-C in figure 3).

Fourth, M2 type macrophage promoting esophagus squamous carcinoma spheroblast to resist chemotherapy medicament cisplatin

And adding an F12 serum-free culture medium into the M2 type macrophages obtained in the third part for culture for 36h (the phenotype of the M2 type macrophages is maintained stably), and collecting a cell-free culture solution, namely an extraction condition culture medium (marked as an F12/M2-TAM condition culture medium) for detecting the subsequent co-culture of the M2 type macrophages participating in the function test of the lower esophageal squamous cell carcinoma spheroblasts.

Detection with CCK8 kit:

the detection is carried out according to the steps of the CCK8 kit in the second part, and the cells to be detected are obtained according to the following steps: adding F12-M2 type macrophage conditioned medium (F12/M2-TAM conditioned medium) into the first part of the obtained spheroblasts, incubating at 37 ℃ for 12h, adding a medicine cisplatin to act for 48h, wherein the concentrations of the cisplatin in the system are 11.343ug/ml (EC109 spheres) and 2.863ug/ml (EC9706 spheres), and replacing the F12/M2-TAM conditioned medium containing the same concentration of cisplatin every 3 days; each set was set with 5 duplicate wells and incubation time was 48 hours.

The results of CCK8 show that the survival rate of esophageal squamous cell carcinoma spheroblasts co-cultured with M2 type macrophages is obviously higher than that of the untreated group (P < 0.001) under the action of cisplatin (see A in figure 4).

Flow detection of apoptosis:

the method is carried out according to the steps of the flow cytometry detection experiment in the second part, and the cells to be detected are obtained according to the following steps: adding F12-M2 type macrophage conditioned medium (F12/M2-TAM conditioned medium) into the first part of the obtained spheroblasts, incubating at 37 ℃ for 12h, adding a medicine cisplatin to act for 48h, wherein the concentrations of the cisplatin in the system are 11.343ug/ml (EC109 spheres) and 2.863ug/ml (EC9706 spheres), and replacing the F12/M2-TAM conditioned medium containing the same concentration of cisplatin every 3 days; each set was set with 5 duplicate wells and incubation time was 48 hours.

The apoptosis result of flow detection shows that the apoptosis rate of esophageal squamous carcinoma spheroblasts co-cultured with M2 type macrophages under the action of cisplatin is obviously lower than that of esophageal squamous carcinoma spheroblasts (P is less than 0.05) of a single culture group (as shown in B in figure 4).

Fifthly, the TGF-beta R1 inhibitor SB431542 reverses M2 type macrophage to promote dryness characteristic of esophageal squamous cell carcinoma cells

Control group: the third part of the obtained human esophageal squamous carcinoma cells EC109 and EC9706 in non-contact co-culture with M2 type macrophages;

experimental groups: a third fraction of the resulting human esophageal squamous carcinoma cells EC109 and EC9706 co-cultured in non-contact with M2-type macrophages were pretreated with SB431542(40 μ M) (pretreatment time 24 hours), i.e., the cells were incubated in SB431542(40 μ M) at 37 ℃ for 24 hours.

Western blot detection results show that the expression of the dry marker proteins CD44 and OCT-4 in the esophageal squamous carcinoma cell group after the co-culture of SB431542 pretreated M2 type macrophages is obviously reduced (P is less than 0.05) compared with an untreated control group (as shown in A in figure 5).

The qRT-PCR detection result shows that the expression of dry markers RNA CD44 and OCT-4 in the esophageal squamous carcinoma cell group after the SB431542 pretreatment M2 type macrophage co-culture is obviously reduced (P is less than 0.05) compared with the untreated control group (as B-C in figure 5).

Sixthly, under the action of TGF-beta R1 inhibitor SB431542 reversing cis-platinum action, M2 type macrophage promotes proliferation and apoptosis inhibition of esophageal squamous cell carcinoma spheroblasts

Control group: adding F12-M2 type macrophage conditioned medium (F12/M2-TAM conditioned medium) into the first part of the obtained spheroblasts, incubating at 37 ℃ for 12h, adding a medicine cisplatin to act for 48h, wherein the concentrations of the cisplatin in the system are 11.343ug/ml (EC109 spheres) and 2.863ug/ml (EC9706 spheres), and replacing the F12/M2-TAM conditioned medium containing the same concentration of cisplatin every 3 days; each group is provided with 5 compound holes, and the incubation time is 48 hours;

experimental groups: adding F12-M2 type macrophage conditioned medium (F12/M2-TAM conditioned medium) into the spheroblasts obtained in the first part, incubating at 37 ℃ for 12h, adding medicaments of cis-platinum and SB431542 for 48h, wherein the concentrations of the cis-platinum in the system are 11.343ug/ml (EC109 spheres) and 2.863ug/ml (EC9706 spheres), the concentration of the SB431542 in the system is 40 mu M, and replacing the F12/M2-TAM conditioned medium containing the cis-platinum and the SB431542 with the same concentration every 3 days; each set was set with 5 duplicate wells and incubation time was 48 hours.

According to the plate cloning experiment in the second step, the plate cloning experiment result shows that the proliferation capacity of the esophageal squamous carcinoma spheroblasts after being co-cultured with M2 type macrophages under the action of cisplatin is obviously higher than that of the SB431542 treatment group (P is less than 0.05) (as shown in A in figure 6).

According to the detection procedure of the CCK8 kit in the second step, the CCK8 result shows that the survival rate of the esophageal squamous cell carcinoma spheroblasts cultured together with M2 type macrophages is obviously higher than that of the SB431542 treated group (P is less than 0.05) under the action of cisplatin (as shown in B in figure 6).

According to the flow detection step in the second step, the flow detection apoptosis result shows that the apoptosis rate of the esophageal squamous carcinoma spheroblasts cultured together with M2 type macrophages under the action of cisplatin is obviously lower than that of the SB431542 treatment group (P is less than 0.05) (as shown in C in figure 6).

Seventhly, in vivo animal experiments detect the reactivity of M2 type macrophages to the growth condition of nude mouse esophageal squamous cell carcinoma transplantable tumors and the action of chemotherapeutic drugs cisplatin

BALB/C nude mice (4-6 weeks old, 15-20g in body weight) were divided into 4 groups: EC109+ PBS control liposome + cDDP group, EC109+ PBS control liposome + saline group, EC109+ M2 type macrophage + cDDP group, EC109+ M2 type macrophage + saline group. Each group had 5.

(1) Culturing 70% -80% of esophageal squamous carcinoma cell line EC109 cells, digesting the cells into single cells by pancreatin, counting the cells, washing the cells with 1 XPBS for multiple times to remove residual pancreatin, and finally diluting the tumor cells to 1 XPBS by 1 XPBS⁷Each dose/ml (all liquid is preheated in a 37 deg.C water bath), and is injected subcutaneously into left anterior axilla of nude mouse, 100 ul/mouse, i.e. 1 × 10⁶One/one; for the co-injected group, TAM: EC109 ═ 1:1, i.e. tumor-associated macrophages 1 × 10⁶ 1X 10 EC109 tumor cells/cell⁶One/mouse, also injected subcutaneously in the left anterior axilla of nude mice.

(2) After injecting EC109 cells, the general condition of the nude mice was observed every 3-4 days and the body weight and the tumor volume of the nude mice were recorded, and the major diameter (a) and the minor diameter (b) of the tumor were measured with a vernier caliper, and the subcutaneous tumor volume formula V is a. sub.a. times.b²0.5 until the end of the experiment.

(3) When the diameter of the transplanted tumor reaches 0.2-0.4cm, the nude mice are correspondingly grouped and injected with chlorophosphate liposome (Liposomes, 14E0218) and PBS control liposome (Liposomes, CP-005) in the tail vein for the first time, 200ul/20g (5mg/ml) for the second time, after 4 days, the nude mice are correspondingly grouped and injected with corresponding liposome in the tumor for the same first time, after 4 days, 50 ul/mouse are injected once every 4 days, and the injection in the tumor is performed for 3 times until the experiment is finished.

(4) Cisplatin injection was started two weeks after neoplasia, 2mg/kg body weight 2 times per week, i.p. for a total of 5 times.

(5) Tumor cells were injected at 32d, and nude mice were sacrificed. And taking out the tumor for photographing, and measuring the volume and weight of the tumor.

(6) One part of the tumor tissue of the nude mice is frozen in liquid nitrogen, and the other part is preserved in 4% formaldehyde solution and then embedded in paraffin.

The nude mouse transplanted tumor experiment shows that the growth rate of the transplanted tumor of M2 type macrophage and esophageal squamous carcinoma cell co-injection group is obviously higher than that of the untreated group and the macrophage consumption group (P is less than 0.05); meanwhile, the sensitivity of the co-injected group to cisplatin treatment was significantly lower than that of the other two groups, i.e., the weight and volume of the transplanted tumors were still higher in the co-injected group than in the other two groups after cisplatin treatment, and the difference was statistically significant (P < 0.05) (see a-D in fig. 7).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. The application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in the preparation of products for treating and/or preventing esophageal cancer.

2. The application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in preparing dry products for inhibiting esophageal cancer cells.

3. The application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 in preparing products for inhibiting the balling of esophageal cancer cells.

4. The application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 in the preparation of products for inhibiting the proliferation capability and/or the growth capability of esophageal cancer cells.

5. The application of the substance for inhibiting the expression of TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin in the preparation of products for promoting the apoptosis of esophageal cancer cells.

7. Use according to any one of claims 1 to 6, characterized in that: the esophageal cancer is esophageal squamous carcinoma, and the esophageal cancer cells are esophageal squamous carcinoma cells.

8. Use according to any one of claims 1 to 7, characterized in that: the substance inhibiting the expression of TGF-beta R1 is SB 431542.

9. The product has any one of the following functions, and the active ingredients of the product are a substance for inhibiting the expression of a TGF-beta R1 gene or reducing the content or activity of TGF-beta R1 and cisplatin:

x1, treatment and/or prevention of esophageal cancer;

x2, inhibiting esophageal cancer cell dryness;

x3, inhibiting esophageal cancer cell balling;

x4, ability to inhibit proliferation and/or growth of esophageal cancer cells;

x5, promoting the apoptosis of esophageal cancer cells.

10. The product of claim 9, wherein: the substance is SB 431542.