CN112023055A - Application of IL-6R inhibitor and IL-4R inhibitor combined medicine in breast cancer chemotherapy medicine - Google Patents
Application of IL-6R inhibitor and IL-4R inhibitor combined medicine in breast cancer chemotherapy medicine Download PDFInfo
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Abstract
The invention discloses an application of IL-6R inhibitor and IL-4R inhibitor combined medicine in breast cancer chemotherapy drugs, wherein IL-4 participates in promoting macrophage M2 polarization, IL-6 is a pleiotropic cytokine with wide function, both of which participate in regulating the growth and differentiation of various cells, and the pleiotropic cytokine has the functions of regulating immune response, acute phase reaction and hematopoiesis, and plays an important role in anti-infection immune response of organisms. In a tumor microenvironment, IL-4 and IL-6 participate in promoting macrophage polarization, and the two synergies in the late stage of tumor development promote macrophage M2 polarization, have anti-inflammatory effect and can promote tumor development. The effect of the inhibitor in breast cancer chemotherapy is researched through the synergistic effect of the IL-6R inhibitor and the IL-4R inhibitor.
Description
Technical Field
The invention relates to the technical field of chemotherapeutic drugs, in particular to application of a combined drug of an IL-6R inhibitor and an IL-4R inhibitor in breast cancer chemotherapeutic drugs.
Background
The immune system is a major participant in tumor microenvironment crosstalk. Tumor-associated macrophages (TAMs), which account for 50% of the total tumor mass, are the major immune cell population that constitutes the tumor microenvironment, and are characterized by the ability to promote tumor growth and angiogenesis, tissue remodeling, and suppress adaptive immunity. Macrophages have a high plasticity for a variety of external signals and are involved in innate and adoptive immune responses. Depending on the microenvironment signal present, macrophages are polarized into two distinct phenotypes, either classically activated (M1) or alternatively activated (M2) macrophages. Tumor Associated Macrophages (TAMs) are very similar to polarized M2. Clinical pathology studies have shown that accumulation of TAMs in tumors is associated with a poor clinical prognosis. In human breast cancer, high TAM density is associated with poor prognosis. Over the years, studies of the role of TAMs in breast cancer progression have established that TAMs are capable of inducing angiogenesis, remodeling the tumor extracellular matrix to aid invasion, modeling breast cancer cells to evade the host immune system and recruit immunosuppressive leukocytes to the tumor microenvironment. M1 macrophages are derived from interferon gamma (IFN-. gamma.) or Lipopolysaccharide (LPS) stimulation and secrete inflammatory cytokines (e.g., IL-6, IL-12, Reactive Oxygen Species (ROS), reactive nitrogen species (RN) and TNF-. alpha.). Validated macrophage surface markers of human M1 include high levels of CD14 and CD16, CD64, CD86, and HLA-DR α. M2 macrophages can be further classified into M2a, M2b and M2c macrophages. M2a macrophages are derived from IL-4 or IL-13 stimulation and release matrix remodeling cytokines. Elevated expression of CD206 and CD86 are validated phenotypic markers of macrophage M2 a. M2b macrophages are produced by immune complexes in combination with IL-1 β or LPS stimulation, and as with M2a macrophages, are involved in wound healing. Immunosuppressive M2c macrophages are products of IL-10, TGF- β (transforming growth factor β), glucocorticoids or an immune complex rich environment. Macrophage M2C further produces IL-10 and matrix remodeling factors such as Matrix Metalloproteinases (MMPs). High expression of CD163 expression is a valid marker for M2c polarization.
IL-4 is a multifunctional cytokine secreted mainly by type 2T helper cells (Th2), and can specifically bind to IL-4 receptor (IL-4R) on the surface of target cells to play corresponding biological role. IL-4 can bind to IL-4R on the surface of tumor cells, activating the JAK1/STAT6 signaling pathway. Affecting the polarization and function of macrophages around the tumor, thereby affecting tumor cells, causing the tumor to have immune escape, and finally causing the tumor to progress and metastasize.
In the breast cancer microenvironment, IL-6 is involved in promoting the polarization of tumor-associated macrophage M1, but IL-6 has also been shown to initiate epithelial-mesenchymal transition of tumor cells, activate cell membrane surface signal transducers (gp 130), induce signal transducer and activator of transcription 3 (STAT 3) activation, activate the IL-6/STAT 3 signaling pathway, and IL-6 binds to the IL-6 receptor alpha chain and transduces downstream signals through the gp130 receptor, which recruits Jak2 tyrosine kinase and signal transducer and activator of transcription 3 (STAT 3) transcription factors. Activated STAT3 induces inhibitors of cytokine signaling 3 in IL-6 target genes, such as macrophages. Activation of this pathway results in increased Twist expression, thereby decreasing E-cadherin expression, increasing N-cadherin expression, and promoting polarization of tumor-associated macrophage M2, thereby promoting tumor cell growth, proliferation, and migration.
IL-4 has been proved to be involved in promoting the polarization of tumor-associated macrophage M2, while IL-6 promotes the polarization of macrophage M1 type, secretes proinflammatory factors and chemokines and inhibits the development of tumors in the early stage of tumor development, and with the accumulation of IL-6, the polarization of macrophage M2 induced by IL-4 can be enhanced through the synergistic effect of STAT3, STAT6 and BATF transcription factors, and at present, no specific therapeutic drug aiming at IL-6 exists in breast cancer therapeutic drugs.
Disclosure of Invention
The invention provides application of IL-6R inhibitor and IL-4R inhibitor combined medicine in breast cancer chemotherapy drugs, and solves the technical problems of non-specificity IL-6-specific treatment drugs in the prior art and the like.
The invention adopts the following technical scheme: the IL-6R inhibitor and the IL-4R inhibitor are combined to be applied to the breast cancer chemotherapeutic drug, and the breast cancer chemotherapeutic drug is cisplatin.
As a preferred technical scheme of the invention: the IL-6R inhibitor is an interleukin-6 (IL-6) receptor inhibitor, the IL-4R inhibitor is an interleukin-4 (IL-4) receptor inhibitor, the IL-4 is involved in promoting the polarization of macrophage M2, the IL-6 is a pleiotropic cytokine with wide function, both of which are involved in regulating the growth and differentiation of various cells, have the functions of regulating immune response, acute phase reaction and hematopoiesis, and play an important role in the anti-infection immune response of the organism; in a tumor microenvironment, IL-4 and IL-6 participate in promoting macrophage polarization, and the two synergies in the advanced tumor development stage to promote macrophage M2 polarization and have anti-inflammatory effect.
As a preferred technical scheme of the invention: the method comprises the following steps:
firstly, establishing an in-vitro breast cancer cell and macrophage co-culture system to simulate the environment in a human body: wherein the breast cancer cell is MDA-MB-468 cell, and the macrophage is human monocyte THP-1;
in the second step, the effect of the IL-6R inhibitor and the IL-4R inhibitor in combination in the breast cancer chemotherapeutic drug is studied: in vitro verification of IL-4/IL-6 synergistic induction target genes;
the third step: mRNA expression analysis of indicator genes in macrophages treated with IL-4 and IL-6, alone or in combination for 24 hours;
the fourth step: dual stimulation of IL-6 and IL-4 enhances the expression of IL-4 targeted chemokines such as CCL17, CCL18, CCL23, and CCL 8;
the fifth step: extracting total RNA in macrophages by using a Trizol method, adding 1ml of Trizol into each sample, and transferring the sample into a centrifuge tube;
and a sixth step: adding 200 microliters of chloroform into each milliliter of Trizol, tightly covering a centrifugal tube, uniformly mixing by shaking for 15 seconds by hand, and standing for ten minutes at room temperature;
the seventh step: then, centrifuging for fifteen minutes at 12000g at four ℃ to absorb the upper aqueous phase, transferring the upper aqueous phase into another new centrifugal tube, adding 0.6ml of isoamyl alcohol into each ml of Trizol, uniformly mixing and standing for 5-10 minutes at room temperature;
eighthly, centrifuging for ten minutes at 12000g at four ℃, removing supernatant, adding 1ml of 75% ethanol into each ml of Trizol, and carrying out mild oscillation and suspension precipitation;
the ninth step: then, drying in the air or in vacuum for 5-10min at room temperature, and then measuring the absorbance value at 260nm to quantify the RNA concentration;
the tenth step: reverse transcription is carried out by utilizing a cDNA kit;
the eleventh step: performing real-time quantitative PCR by using a CFX96 system of Bio-Rad and iQ SYBR green Supermix (Bio-Rad), wherein an internal reference is beta-actin;
the twelfth step: treating macrophages with IL-6R and IL-4R inhibitors in subsequent experiments, wherein the IL-6R inhibitor is Tolizumab and is used at a concentration of 200 μ g/mL; the IL-4R inhibitor is dupilumab, and the use concentration is 1500 mug/mL;
step ten, in the process of co-culturing macrophage Thp-1 and breast cancer cell MDA-MB-468, the Thp-1 cell is on the upper layer, the MDA-MB-468 is on the lower layer, and two inhibitors are added on the upper layer to achieve the purpose of treating Thp-1 and co-culturing with the breast cancer cell, namely MDA-MB-468 cell: wherein macrophages are arranged on the upper layer, MDA-MB-468 cells are arranged on the lower layer, the middle layer is separated by a 0.4 mu m PET membrane to ensure that cytokines can pass but cells cannot pass, and then the apoptosis condition of tumor cells is detected by using a fluidic method;
fourteenth, cell collection: the Thp-1 suspension cells were collected directly into 10ml centrifuge tubes with 5X 10 cells per sample6Centrifuging at 1000r/min for 5min, and discarding the culture solution;
the fifteenth step: washing with incubation buffer solution (PBS buffer solution) for 1 time, and centrifuging at 1000r/min for 5 min;
sixteenth, step: resuspending the cells with 100ul of a labeling solution, and incubating for 15min at room temperature in the dark, wherein the labeling solution is SA-FLOUS labeling solution;
seventeenth step: centrifuging at 1000r/min for 5min, washing with PBS buffer solution for 1 time;
and eighteenth step: adding fluorescent SA-FLOUS solution, incubating at 4 deg.C for 20min, and shaking in dark at intervals;
the nineteenth step: flow cytometry analysis: the wavelength of exciting light of the flow cytometer is 488nm, FITC fluorescence is detected by a band-pass filter with the wavelength of 515nm, and PI is detected by a filter with the wavelength of more than 560 nm;
the twentieth step: and (5) judging a result: apoptotic cells are resistant to all dyes used for cell activity identification, such as PI, whereas necrotic cells are not; the DNA of the cell with damaged cell membrane can be stained by PI to generate red fluorescence, and the cell with intact cell membrane can not generate red fluorescence; thus, PI does not stain without red fluorescent signal in the early stages of apoptosis; normal living cells are similar thereto;
twenty-first step, study IL-6 and IL-4R inhibitor and use the effect jointly, design the experiment and prove whether IL-6R inhibitor and IL-4R inhibitor reduce the tumorigenicity of breast cancer cell line: injecting MDA-MB-231 cells cultured by IL-6R inhibitor or IL-4R inhibitor into the back of a nude mouse subcutaneously, respectively, after 10-14 days, 12 nude mice used for experiments all grow touchable subcutaneous tumors at the inoculated part, then starting drug treatment, selecting cisplatin as the drug, before starting drug treatment, aiming at a wild type, namely untreated MDA-MB-468 cell nude mouse subcutaneous tumor model, determining the drug concentration and the drug duration of the cisplatin, and finally selecting the cisplatin with the drug treatment mode as follows: 4mg of cisplatin powder is dissolved in normal saline, and the cisplatin powder is injected into the abdominal cavity, wherein the cisplatin powder is taken once every 5 days; in the combined use group, the tumor volume result shows that the tumor volume of the combined treatment group with the tumor inhibition effect is obviously lower than that of other groups (p is less than 0.01), and the significance of the combined medicine on the treatment effect of the breast cancer is prompted; the above groups had no significant effect on body weight, indicating that the treatment did not produce toxicity to mice.
As a preferred technical scheme of the invention: the IL-6R inhibitor is added with the IL-4R inhibitor, so that the sensitivity of tumor cells to chemotherapeutic drugs can be enhanced, and a new idea is provided for the chemotherapy and drug research of breast cancer.
As a preferred technical scheme of the invention: when the IL-6R inhibitor and the IL-4R inhibitor are used for the Thp-1 cells, the expression of CD86 is up-regulated, the proportion of the cells to be polarized to M1 is up-regulated, the proinflammatory function of the cells is enhanced, and the occurrence of tumor cell drug resistance is inhibited.
As a preferred technical scheme of the invention: after the IL-6 inhibitor (namely a co-culture group of macrophage and breast cancer cells using an IL-6R inhibitor) is treated by cisplatin serving as a chemotherapeutic drug, the early apoptosis rate of the breast cancer cells is increased compared with that of a normal group, the early apoptosis rate of the cells treated by the IL-6R inhibitor and the IL-4R inhibitor is increased, and the apoptosis rate of the cells is remarkably increased compared with that of the IL-4R inhibitor treated group after the IL-6R inhibitor and the IL-4R inhibitor are administered.
As a preferred technical scheme of the invention: the results of the drug experiments after the nude mice have subcutaneous tumor formation show that in a combined use group, the results of tumor volume show that the tumor volume of a combined treatment group with the tumor inhibition effect is obviously lower than that of an IL-6R inhibitor or IL-4R inhibitor single-use group. Therefore, the research result of the patent provides a new idea for the chemotherapy and drug research of breast cancer, and plays an important role in promoting the health of human beings.
As a preferred technical scheme of the invention: in the twentieth step, live cells are shown in the lower left quadrant of a scattergram of a bivariate flow cytometer as (FITC-/PI-); the upper right quadrant is a non-viable cell, i.e., a necrotic cell, and is (FITC +/PI +); whereas the lower right quadrant was apoptotic cells, visualized (FITC +/PI-).
Has the advantages that:
compared with the prior art, the application of the IL-6R inhibitor and the IL-4R inhibitor combined medicine in breast cancer chemotherapy drugs adopts the technical scheme, and the technical effects are as follows:
1. the effect of the inhibitor of IL-6 receptor (IL-6R) and the inhibitor of IL-4 receptor (IL-4R) in the breast cancer chemotherapy is studied by combining the inhibitor of IL-6 receptor and the inhibitor of IL-4 receptor, and a guidance scheme is provided for enhancing the effect of the chemotherapeutic cisplatin in the breast cancer chemotherapy process.
2. Establishing a tumor cell and macrophage in-vitro co-culture model, and growing the tumor cells in co-culture in a nude mouse after amplifying;
3. the series of experimental researches on the effect of the IL-6R inhibitor and the IL-4R inhibitor in the breast cancer chemotherapy are designed, and the obvious promotion effect on apoptosis by inhibiting IL-6 and adding the IL-4R inhibitor in the breast cancer chemotherapy drug resistance process is demonstrated, so that the drug resistance of tumor cells is twisted;
4. an in-vitro breast cancer cell and macrophage co-culture system is established to simulate the environment in a human body;
5. in vitro, an IL-6R inhibitor and an IL-4R inhibitor are respectively added into macrophages, the IL-4/IL-6 synergistically induces a target gene to participate in influencing the polarization process of the macrophages, and meanwhile, a series of in vivo experimental researches on the effect of the IL-6R inhibitor and the IL-4R inhibitor which are jointly used in breast cancer chemotherapy are designed, and the results also show that the inhibition of IL-6 and the inhibition of IL-4 have obvious promotion effect on apoptosis, can enhance the sensitivity of tumor cells to chemotherapeutic drugs, and provide a new idea for the chemotherapy and drug research of breast cancer.
6. When an IL-6R inhibitor and an IL-4R inhibitor are used for Thp-1 cells, the expression of CD86 is up-regulated, the proportion of the cells to M1 type polarization is up-regulated, the proinflammatory function of the cells is enhanced, and the occurrence of tumor cell drug resistance is inhibited.
7. After IL-6 inhibition (i.e. using IL-6R inhibitor macrophage and breast cancer cell co-culture group) is treated by chemotherapeutic drug cisplatin, the early apoptosis rate of breast cancer cells is increased compared with that of normal group, the early apoptosis rate of cells treated by IL-6R inhibitor and IL-4R inhibitor is increased, and the apoptosis rate of cells treated by IL-6R inhibitor and IL-4R inhibitor is obviously increased compared with that of IL-4R inhibitor.
8. The results of drug experiments after subcutaneous tumor formation of nude mice show that in the combined group, the results of tumor volume show that the tumor volume of the combined treatment group with tumor inhibition effect is significantly lower than that of the IL-6R inhibitor or IL-4R inhibitor single group. Therefore, the research result of the patent provides a new idea for the chemotherapy and drug research of breast cancer, and plays an important role in promoting the health of human beings.
Drawings
FIG. 1 is a diagram of mRNA expression analysis in the present application.
FIG. 2 is a graph of the quantitative analysis of breast cancer apoptosis in the present application.
Fig. 3 is a volume map of a tumor map in the present application.
Detailed Description
The present invention is further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the scope of the claims, and other alternatives which may occur to those skilled in the art are within the scope of the claims. .
Example 1:
the IL-6R inhibitor and the IL-4R inhibitor are combined to be applied to the breast cancer chemotherapeutic drug, and the breast cancer chemotherapeutic drug is cisplatin.
The IL-6R inhibitor is an interleukin-6 (IL-6) receptor inhibitor, the IL-4R inhibitor is an interleukin-4 (IL-4) receptor inhibitor, the IL-4 is involved in promoting the polarization of macrophage M2, the IL-6 is a pleiotropic cytokine with wide function, both of which are involved in regulating the growth and differentiation of various cells, have the functions of regulating immune response, acute phase reaction and hematopoiesis, and play an important role in the anti-infection immune response of the organism; in a tumor microenvironment, IL-4 and IL-6 participate in promoting macrophage polarization, and the two synergies in the advanced tumor development stage to promote macrophage M2 polarization and have anti-inflammatory effect.
The application of the polypeptide analogue in the medicine for treating breast cancer comprises the following steps:
firstly, establishing an in-vitro breast cancer cell and macrophage co-culture system to simulate the environment in a human body: wherein the breast cancer cell is MDA-MB-468 cell, and the macrophage is human monocyte THP-1;
in the second step, the effect of the IL-6R inhibitor and the IL-4R inhibitor in combination in the breast cancer chemotherapeutic drug is studied: in vitro verification of IL-4/IL-6 synergistic induction target genes;
the third step: mRNA expression analysis of indicator genes in macrophages treated with IL-4 and IL-6, alone or in combination for 24 hours;
the fourth step: dual stimulation of IL-6 and IL-4 enhances the expression of IL-4 targeted chemokines such as CCL17, CCL18, CCL23, and CCL 8;
the fifth step: extracting total RNA in macrophages by using a Trizol method, adding 1ml of Trizol into each sample, and transferring the sample into a centrifuge tube;
and a sixth step: adding 200 microliters of chloroform into each milliliter of Trizol, tightly covering a centrifugal tube, uniformly mixing by shaking for 15 seconds by hand, and standing for ten minutes at room temperature;
the seventh step: then, centrifuging for fifteen minutes at 12000g at four ℃ to absorb the upper aqueous phase, transferring the upper aqueous phase into another new centrifugal tube, adding 0.6ml of isoamyl alcohol into each ml of Trizol, uniformly mixing and standing for 5-10 minutes at room temperature;
eighthly, centrifuging for ten minutes at 12000g at four ℃, removing supernatant, adding 1ml of 75% ethanol into each ml of Trizol, and carrying out mild oscillation and suspension precipitation;
the ninth step: then, drying in the air or in vacuum for 5-10min at room temperature, and then measuring the absorbance value at 260nm to quantify the RNA concentration;
the tenth step: reverse transcription is carried out by utilizing a cDNA kit;
the eleventh step: performing real-time quantitative PCR by using a CFX96 system of Bio-Rad and iQ SYBR green Supermix (Bio-Rad), wherein an internal reference is beta-actin;
the twelfth step: treating macrophages with IL-6R and IL-4R inhibitors in subsequent experiments, wherein the IL-6R inhibitor is Tolizumab and is used at a concentration of 200 μ g/mL; the IL-4R inhibitor is dupilumab, and the use concentration is 1500 mug/mL;
step ten, in the process of co-culturing macrophage Thp-1 and breast cancer cell MDA-MB-468, the Thp-1 cell is on the upper layer, the MDA-MB-468 is on the lower layer, and two inhibitors are added on the upper layer to achieve the purpose of treating Thp-1 and co-culturing with the breast cancer cell, namely MDA-MB-468 cell: wherein macrophages are arranged on the upper layer, MDA-MB-468 cells are arranged on the lower layer, the middle layer is separated by a 0.4 mu m PET membrane to ensure that cytokines can pass but cells cannot pass, and then the apoptosis condition of tumor cells is detected by using a fluidic method;
fourteenth, cell collection: the Thp-1 suspension cells were collected directly into 10ml centrifuge tubes with 5X 10 cells per sample6Centrifuging at 1000r/min for 5min, and discarding the culture solution;
the fifteenth step: washing with incubation buffer solution (PBS buffer solution) for 1 time, and centrifuging at 1000r/min for 5 min;
sixteenth, step: resuspending the cells with 100ul of a labeling solution, and incubating for 15min at room temperature in the dark, wherein the labeling solution is SA-FLOUS labeling solution;
seventeenth step: centrifuging at 1000r/min for 5min, washing with PBS buffer solution for 1 time;
and eighteenth step: adding fluorescent SA-FLOUS solution, incubating at 4 deg.C for 20min, and shaking in dark at intervals;
the nineteenth step: flow cytometry analysis: the wavelength of exciting light of the flow cytometer is 488nm, FITC fluorescence is detected by a band-pass filter with the wavelength of 515nm, and PI is detected by a filter with the wavelength of more than 560 nm;
the twentieth step: and (5) judging a result: apoptotic cells are resistant to all dyes used for cell activity identification, such as PI, whereas necrotic cells are not; the DNA of the cell with damaged cell membrane can be stained by PI to generate red fluorescence, and the cell with intact cell membrane can not generate red fluorescence; thus, PI does not stain without red fluorescent signal in the early stages of apoptosis; normal living cells are similar thereto;
twenty-first step, study IL-6 and IL-4R inhibitor and use the effect jointly, design the experiment and prove whether IL-6R inhibitor and IL-4R inhibitor reduce the tumorigenicity of breast cancer cell line: injecting MDA-MB-231 cells cultured by IL-6R inhibitor or IL-4R inhibitor into the back of a nude mouse subcutaneously, respectively, after 10-14 days, 12 nude mice used for experiments all grow touchable subcutaneous tumors at the inoculated part, then starting drug treatment, selecting cisplatin as the drug, before starting drug treatment, aiming at a wild type, namely untreated MDA-MB-468 cell nude mouse subcutaneous tumor model, determining the drug concentration and the drug duration of the cisplatin, and finally selecting the cisplatin with the drug treatment mode as follows: 4mg of cisplatin powder is dissolved in normal saline, and the cisplatin powder is injected into the abdominal cavity, wherein the cisplatin powder is taken once every 5 days; in the combined use group, the tumor volume result shows that the tumor volume of the combined treatment group with the tumor inhibition effect is obviously lower than that of other groups (p is less than 0.01), and the significance of the combined medicine on the treatment effect of the breast cancer is prompted; the above groups had no significant effect on body weight, indicating that the treatment did not produce toxicity to mice.
The IL-6R inhibitor is added with the IL-4R inhibitor, so that the sensitivity of tumor cells to chemotherapeutic drugs can be enhanced, and a new idea is provided for the chemotherapy and drug research of breast cancer.
When an IL-6R inhibitor and an IL-4R inhibitor are used for Thp-1 cells, the expression of CD86 is up-regulated, the proportion of the cells to M1 type polarization is up-regulated, the proinflammatory function of the cells is enhanced, and the occurrence of tumor cell drug resistance is inhibited.
After the IL-6 inhibitor (namely a co-culture group of macrophage and breast cancer cells using an IL-6R inhibitor) is treated by cisplatin serving as a chemotherapeutic drug, the early apoptosis rate of the breast cancer cells is increased compared with that of a normal group, the early apoptosis rate of the cells treated by the IL-6R inhibitor and the IL-4R inhibitor is increased, and the apoptosis rate of the cells is remarkably increased compared with that of the IL-4R inhibitor treated group after the IL-6R inhibitor and the IL-4R inhibitor are administered.
The results of the drug experiments after the nude mice have subcutaneous tumor formation show that in a combined use group, the results of tumor volume show that the tumor volume of a combined treatment group with the tumor inhibition effect is obviously lower than that of an IL-6R inhibitor or IL-4R inhibitor single-use group. Therefore, the research result of the patent provides a new idea for the chemotherapy and drug research of breast cancer, and plays an important role in promoting the health of human beings.
In the twentieth step, live cells are shown in the lower left quadrant of a scattergram of a bivariate flow cytometer as (FITC-/PI-); the upper right quadrant is a non-viable cell, i.e., a necrotic cell, and is (FITC +/PI +); whereas the lower right quadrant was apoptotic cells, visualized (FITC +/PI-).
FIG. 1 shows an mRNA expression analysis chart; dual stimulation of IL-6 and IL-4 enhances the expression of several chemokines targeted by IL-4 (CCL 17, CCL18, CCL23 and CCL 8).
As shown in fig. 2: quantitative analysis chart of breast cancer cell apoptosis; after IL-6 inhibition (namely a co-culture group of macrophage and breast cancer cells by using an IL-6R inhibitor) is treated by cisplatin serving as a chemotherapeutic medicament, the early apoptosis ratio of the breast cancer cells is increased compared with that of a normal group, and the early apoptosis ratio of the cells treated by using the IL-6R inhibitor and then the IL-4R inhibitor is increased, so that the IL-6R inhibitor and the IL-4R inhibitor can effectively promote the apoptosis of the breast cancer cells.
As shown in fig. 3: tumor volume plot, MDA-MB-468 breast cancer cells treated with IL-6R inhibitor in combination with IL-4R inhibitor, the nude mice were much less resistant to cis-platin subcutaneously than the single group, i.e., the tumor volume was significantly less than the rest of the groups, indicating that the combination of-6R inhibitor and IL-4R inhibitor with chemotherapy helped to inhibit the growth of breast cancer.
The experimental study on the effect of the IL-6R inhibitor and the IL-4R inhibitor in the breast cancer chemotherapy shows that the IL-4R inhibitor is added to the IL-6R inhibitor, so that the sensitivity of tumor cells to chemotherapeutic drugs can be enhanced, and a new thought is provided for the breast cancer chemotherapy and drug research. The result shows that when an IL-6R inhibitor and an IL-4R inhibitor are used for Thp-1 cells, the expression of CD86 is up-regulated, the proportion of the cells to be polarized to M1 is up-regulated, so that the proinflammatory function of the cells is enhanced, and the occurrence of tumor cell drug resistance is inhibited; after IL-6 inhibition (namely a co-culture group of macrophage and breast cancer cells by using an IL-6R inhibitor) is treated by cisplatin serving as a chemotherapeutic medicament, the early apoptosis rate of the breast cancer cells is increased compared with that of a normal group, the early apoptosis rate of the cells treated by the IL-6R inhibitor and the IL-4R inhibitor is increased, and the apoptosis rate of the cells is remarkably increased compared with that of the IL-4R inhibitor treated group after the IL-6R inhibitor and the IL-4R inhibitor are further administered; the results of drug experiments after subcutaneous tumor formation of nude mice show that in the combined group, the results of tumor volume show that the tumor volume of the combined treatment group with tumor inhibition effect is significantly lower than that of the IL-6R inhibitor or IL-4R inhibitor single group. Therefore, the research result of the patent provides a new idea for the chemotherapy and drug research of breast cancer, and plays an important role in promoting the health of human beings.
The invention establishes an in vitro breast cancer cell and macrophage co-culture system to simulate the environment in a human body. In vitro, an IL-6R inhibitor and an IL-4R inhibitor are respectively added into macrophages, the IL-4/IL-6 synergistically induces a target gene to participate in influencing the polarization process of the macrophages, and meanwhile, a series of in vivo experimental researches on the effect of the IL-6R inhibitor and the IL-4R inhibitor which are jointly used in breast cancer chemotherapy are designed, and the results also show that the inhibition of IL-6 and the inhibition of IL-4 have obvious promotion effect on apoptosis, can enhance the sensitivity of tumor cells to chemotherapeutic drugs, and provide a new idea for the chemotherapy and drug research of breast cancer.
Claims (8)
- The application of the IL-6R inhibitor and the IL-4R inhibitor in the combination of breast cancer chemotherapy drugs is characterized in that: the breast cancer chemotherapeutic drug is cisplatin.
- 2. The use of a polypeptide analog of claim 1 in a medicament for treating breast cancer, wherein: the IL-6R inhibitor is an interleukin-6 (IL-6) receptor inhibitor, the IL-4R inhibitor is an interleukin-4 (IL-4) receptor inhibitor, the IL-4 is involved in promoting the polarization of macrophage M2, the IL-6 is a pleiotropic cytokine with wide function, both of which are involved in regulating the growth and differentiation of various cells, have the functions of regulating immune response, acute phase reaction and hematopoiesis, and play an important role in the anti-infection immune response of the organism; in a tumor microenvironment, IL-4 and IL-6 participate in promoting macrophage polarization, and the two synergies in the advanced tumor development stage to promote macrophage M2 polarization and have anti-inflammatory effect.
- 3. The use of the polypeptide analog of claim 1 in a medicament for treating breast cancer, comprising the steps of:firstly, establishing an in-vitro breast cancer cell and macrophage co-culture system to simulate the environment in a human body: wherein the breast cancer cell is MDA-MB-468 cell, and the macrophage is human monocyte THP-1;in the second step, the effect of the IL-6R inhibitor and the IL-4R inhibitor in combination in the breast cancer chemotherapeutic drug is studied: in vitro verification of IL-4/IL-6 synergistic induction target genes;the third step: mRNA expression analysis of indicator genes in macrophages treated with IL-4 and IL-6, alone or in combination for 24 hours;the fourth step: dual stimulation of IL-6 and IL-4 enhances the expression of IL-4 targeted chemokines such as CCL17, CCL18, CCL23, and CCL 8;the fifth step: extracting total RNA in macrophages by using a Trizol method, adding 1ml of Trizol into each sample, and transferring the sample into a centrifuge tube;and a sixth step: adding 200 microliters of chloroform into each milliliter of Trizol, tightly covering a centrifugal tube, uniformly mixing by shaking for 15 seconds by hand, and standing for ten minutes at room temperature;the seventh step: then, centrifuging for fifteen minutes at 12000g at four ℃ to absorb the upper aqueous phase, transferring the upper aqueous phase into another new centrifugal tube, adding 0.6ml of isoamyl alcohol into each ml of Trizol, uniformly mixing and standing for 5-10 minutes at room temperature;eighthly, centrifuging for ten minutes at 12000g at four ℃, removing supernatant, adding 1ml of 75% ethanol into each ml of Trizol, and carrying out mild oscillation and suspension precipitation;the ninth step: then, drying in the air or in vacuum for 5-10min at room temperature, and then measuring the absorbance value at 260nm to quantify the RNA concentration;the tenth step: reverse transcription is carried out by utilizing a cDNA kit;the eleventh step: performing real-time quantitative PCR by using a CFX96 system of Bio-Rad and iQ SYBR green Supermix (Bio-Rad), wherein an internal reference is beta-actin;the twelfth step: treating macrophages with IL-6R and IL-4R inhibitors in subsequent experiments, wherein the IL-6R inhibitor is Tolizumab and is used at a concentration of 200 μ g/mL; the IL-4R inhibitor is dupilumab, and the use concentration is 1500 mug/mL;step ten, in the process of co-culturing macrophage Thp-1 and breast cancer cell MDA-MB-468, the Thp-1 cell is on the upper layer, the MDA-MB-468 is on the lower layer, and two inhibitors are added on the upper layer to achieve the purpose of treating Thp-1 and co-culturing with the breast cancer cell, namely MDA-MB-468 cell: wherein macrophages are arranged on the upper layer, MDA-MB-468 cells are arranged on the lower layer, the middle layer is separated by a 0.4 mu m PET membrane to ensure that cytokines can pass but cells cannot pass, and then the apoptosis condition of tumor cells is detected by using a fluidic method;fourteenth, cell collection: the Thp-1 suspension cells were collected directly into 10ml centrifuge tubes with 5X 10 cells per sample6Centrifuging at 1000r/min for 5min, and discarding the culture solution;the fifteenth step: washing with incubation buffer solution (PBS buffer solution) for 1 time, and centrifuging at 1000r/min for 5 min;sixteenth, step: resuspending the cells with 100ul of a labeling solution, and incubating for 15min at room temperature in the dark, wherein the labeling solution is SA-FLOUS labeling solution;seventeenth step: centrifuging at 1000r/min for 5min, washing with PBS buffer solution for 1 time;and eighteenth step: adding fluorescent SA-FLOUS solution, incubating at 4 deg.C for 20min, and shaking in dark at intervals;the nineteenth step: flow cytometry analysis: the wavelength of exciting light of the flow cytometer is 488nm, FITC fluorescence is detected by a band-pass filter with the wavelength of 515nm, and PI is detected by a filter with the wavelength of more than 560 nm;the twentieth step: and (5) judging a result: apoptotic cells are resistant to all dyes used for cell activity identification, such as PI, whereas necrotic cells are not; the DNA of the cell with damaged cell membrane can be stained by PI to generate red fluorescence, and the cell with intact cell membrane can not generate red fluorescence; thus, PI does not stain without red fluorescent signal in the early stages of apoptosis; normal living cells are similar thereto;twenty-first step, study IL-6 and IL-4R inhibitor and use the effect jointly, design the experiment and prove whether IL-6R inhibitor and IL-4R inhibitor reduce the tumorigenicity of breast cancer cell line: injecting MDA-MB-231 cells cultured by IL-6R inhibitor or IL-4R inhibitor into the back of a nude mouse subcutaneously, respectively, after 10-14 days, 12 nude mice used for experiments all grow touchable subcutaneous tumors at the inoculated part, then starting drug treatment, selecting cisplatin as the drug, before starting drug treatment, aiming at a wild type, namely untreated MDA-MB-468 cell nude mouse subcutaneous tumor model, determining the drug concentration and the drug duration of the cisplatin, and finally selecting the cisplatin with the drug treatment mode as follows: 4mg of cisplatin powder is dissolved in normal saline, and the cisplatin powder is injected into the abdominal cavity, wherein the cisplatin powder is taken once every 5 days; in the combined use group, the tumor volume result shows that the tumor volume of the combined treatment group with the tumor inhibition effect is obviously lower than that of other groups (p is less than 0.01), and the significance of the combined medicine on the treatment effect of the breast cancer is prompted; the above groups had no significant effect on body weight, indicating that the treatment did not produce toxicity to mice.
- 4. The use of the IL-6R inhibitor and IL-4R inhibitor combination according to claim 3 in a breast cancer chemotherapeutic agent, wherein: the IL-6R inhibitor is added with the IL-4R inhibitor, so that the sensitivity of tumor cells to chemotherapeutic drugs can be enhanced, and a new idea is provided for the chemotherapy and drug research of breast cancer.
- 5. The use of the IL-6R inhibitor and IL-4R inhibitor combination according to claim 3 in a breast cancer chemotherapeutic agent, wherein: when an IL-6R inhibitor and an IL-4R inhibitor are used for Thp-1 cells, the expression of CD86 is up-regulated, the proportion of the cells to M1 type polarization is up-regulated, the proinflammatory function of the cells is enhanced, and the occurrence of tumor cell drug resistance is inhibited.
- 6. The use of the IL-6R inhibitor and IL-4R inhibitor combination according to claim 3 in a breast cancer chemotherapeutic agent, wherein: after the IL-6 inhibitor (namely a co-culture group of macrophage and breast cancer cells using an IL-6R inhibitor) is treated by cisplatin serving as a chemotherapeutic drug, the early apoptosis rate of the breast cancer cells is increased compared with that of a normal group, the early apoptosis rate of the cells treated by the IL-6R inhibitor and the IL-4R inhibitor is increased, and the apoptosis rate of the cells is remarkably increased compared with that of the IL-4R inhibitor treated group after the IL-6R inhibitor and the IL-4R inhibitor are administered.
- 7. The use of the IL-6R inhibitor and IL-4R inhibitor combination according to claim 3 in a breast cancer chemotherapeutic agent, wherein: the results of the drug experiments after the nude mice has subcutaneous tumor formation show that in the combined use group, the results of the tumor volume show that the tumor volume of the combined treatment group with the tumor inhibition effect is obviously lower than that of the IL-6R inhibitor or the IL-4R inhibitor,therefore, the research result of the patent provides a new idea for the chemotherapy and drug research of breast cancer, and plays an important role in promoting the health of human beings.
- 8. The use of the IL-6R inhibitor and IL-4R inhibitor combination according to claim 3 in a breast cancer chemotherapeutic agent, wherein: in the twentieth step, live cells are shown in the lower left quadrant of a scattergram of a bivariate flow cytometer as (FITC-/PI-); the upper right quadrant is a non-viable cell, i.e., a necrotic cell, and is (FITC +/PI +); whereas the lower right quadrant was apoptotic cells, visualized (FITC +/PI-).
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