TWI720030B - Preparation method of multi-peptide composition stimulated by cytokine or growth factor in immune response path - Google Patents

Abstract

The present invention provides a method for preparing a multi-peptide composition, which comprises the following steps: (1) complete fibroblast-like cells and culture them in a culture medium; (2) administer the cytokines or growth factors in the immune response pathway And culture the fibroblasts for a period of time; and (3) washing the fibroblasts of step (2), add a serum-free medium and culture for a period of time to obtain a supernatant, which contains multiple peptides Composition. The multi-peptide composition prepared by the preparation method of the present invention can effectively inhibit the contents of IFN-γ and TNF-α secreted by peripheral blood lymphocytes in the inflammatory reaction, and can inhibit the inflammatory reaction caused by rose spots.

Description

Preparation method of multi-peptide composition stimulated by cytokine or growth factor in immune response path

The present invention relates to a method for preparing a multi-peptide composition, in particular a method for administering fibroblast-like cells with cytokines or growth factors in an immune response path to obtain the multi-peptide composition.

Different components in blood can be used in different fields of medicine. In modern medicine, it has been discovered that when blood is centrifuged, it is separated into an upper plasma layer, a middle buffy coat and a lower red blood cell layer. The turbid yellow layer is composed of platelets, stem cells and lymphocytes.

Peripheral blood mononuclear cell (PBMC) refers to all mononuclear cells in white blood cells, mainly including lymphocytes and monocytes. Among them, lymphocytes can be further divided into T cells, B cells, and natural killer cells (NK cells). Among them, monocytes are white blood cells in the human immune system, which are produced from bone marrow and spleen. After being stimulated, the monocytes will differentiate into macrophages and dendritic cells; for example, when there is an inflammatory response message, the monocytes will gather in the infected tissue and differentiate into macrophages. Phage cells and dendritic cells produce an immune response.

At present, for the treatment of inflammatory reactions, especially for inflammatory reactions with less clear pathogenesis such as seborrheic dermatitis, rose spots and eczema, the current treatment method can only adopt the administration of steroid drugs to produce the inflammatory reaction. Skin, to control the symptoms to avoid more serious. However, after long-term use, there are still many side effects. In mild cases, the skin becomes thinner and the wound is not easy to heal. In severe cases, the dosage of steroids may be higher and higher, and even cause liver damage.

In view of this, how to develop a more natural composition that can effectively inhibit the inflammatory response, the existing technology needs to be improved.

In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing a multi-peptide composition stimulated by cytokines or growth factors in the immune response pathway, so that the multi-peptide composition can inhibit the symptoms of inflammation.

In order to achieve the above-mentioned purpose of the invention, the present invention provides a preparation method of a multi-peptide composition, which comprises the following steps: (1) complete fibroblast like cells and cultured in a culture medium; (2) with an immune response The cytokines or growth factors in the route are administered to the fibroblasts and cultured for a period of time; and; (3) After washing the fibroblasts of this step (2), add a serum-free medium for a period of culture to obtain a The supernatant, which contains the multi-peptide composition.

Preferably, in the step (2), the cytokine or growth factor in the immune response path is tumor necrosis factor alpha (TNF-α).

More preferably, in the step (2), the concentration of tumor necrosis factor-α is 2 nanograms per milliliter (ng/ml) to 20 ng/ml.

Preferably, in the step (2), the time is 2 hours to 96 hours.

More preferably, in the step (2), the time is 24 hours.

Preferably, in the step (2), the culture temperature is 25°C to 45°C.

More preferably, in the step (2), the culture temperature is 37ºC or 39ºC.

Preferably, in the step (3), the culture temperature is 25°C to 45°C.

More preferably, in the step (3), the culture temperature is 37ºC.

Preferably, in the step (3), the time is 8 hours to 288 hours.

More preferably, in the step (3), the time is 72 hours.

The multi-peptide composition of the present invention includes, but is not limited to, anti-inflammatory peptides, pro-inflammatory peptides, immunomodulatory peptides and growth factors.

The "anti-inflammatory peptides" of the present invention include, but are not limited to, interleukin 4 (IL-4), IL-6, IL-10, and angiogenin (ANG).

The "pro-inflammatory peptides" of the present invention include, but are not limited to, interferon-γ (interferon gamma, IFN-γ), tumor necrosis factor alpha (TNF-α), and granular cell-giant Granulocyte-macrophage colony-stimulating factor (GM-CSF).

The "immune regulatory peptides" described in the present invention do not directly participate in anti-inflammatory response or pro-inflammatory response, but regulate immunity in an indirect manner. For example, ICAM-1 is related to the activation of T cells. The immunomodulatory peptides include, but are not limited to, intercellular adhesion molecule-1 (ICAM-1), IL-12p70, and hepatocyte growth factor (HGF).

The "growth factors" mentioned in the present invention include, but are not limited to, basic fibroblast growth factor (bFGF) and fibroblast growth factor-7 (FGF-7).

The advantage of the present invention is that the preparation method of the present invention can effectively inhibit the secretion of peripheral blood lymphocytes in the inflammatory reaction by stimulating the multi-peptide composition obtained by culturing fibroblasts by cytokine or growth factor in the immune response path. The content of IFN-γ and TNF-α; at the same time, it can also inhibit the inflammation caused by rose spots.

The following describes the technical means adopted by the present invention to achieve the objective in conjunction with the drawings and the preferred embodiments of the present invention.

Preparation Example 1 Preparation of peripheral blood lymphocyte mixture

(1) Human whole blood containing anticoagulant is centrifuged at 400 revolutions (g) for 5 minutes, and the buffy coat of the mononuclear cell is taken out.

(2) Add the same volume of phosphate buffered saline (PBS) to the brown layer and mix well to form a cell sap.

(3) Take 1.5 times the volume of Ficoll-paque (purchased from GE Healthcare Life Sciences, Ficoll-Paque PLUS, 17-1440-02) into a clean centrifuge tube. The cell fluid was slowly added to the interface of Ficoll-paque without destroying the interface, and centrifuged at 400 g for 35 minutes.

(4) Remove the supernatant, take the middle mononuclear cells into a new centrifuge tube, and wash with PBS three times the volume of the mononuclear cells, then centrifuge at 200 g for 10 minutes, repeat the above cleaning steps twice Times.

(5) Remove the supernatant and leave the precipitated mononuclear cells, and add 10 milliliters (mL) of the Roswell park memorial institute, RPMI- 1640 (Roswell park memorial institute, RPMI-) containing 10% fetal bovine serum by volume. 1640) The medium is re-dissolved.

(6) Add the re-dissolved mononuclear cell suspension to a 10 cm cell culture dish, place it in 37°C, 5% carbon dioxide (CO ₂ ) for 1 hour, and then remove the unattached mononuclear cells ( Mainly mixed lymphocytes), centrifuged at 200 g for 10 minutes.

(7) Remove the supernatant, and add RPMI-1640 medium containing 10% fetal bovine serum by volume and 1% penicillin-streptomycin-amphotericin B (PSA). The precipitated mononuclear cells were lysed to obtain a peripheral blood lymphocyte mixture, and the number of cells was counted with trypan blue.

Preparation Example 2 Preparation of multi-peptide composition

(1) Plant 2×10 ⁵ to 5×10 ⁵ fibroblast-like cells in a 10 cm2 culture dish. When the cells grow to 80% full, wash them twice with PBS.

(2) There are three groups here, namely Group A, Group B and Group C:

Group A: Add 10 mL of RPMI-1640 containing 1% PSA by volume to fibroblasts, and incubate them in a 37°C, 5% CO ₂ incubator for 24 hours;

Group B: Add 10 mL of RPMI-1640 containing 2 ng/mL to 20 ng/mL TNF-α and 1% PSA by volume to human fibroblasts, preferably 7 ng/mL TNF-α and 1% PSA's RPMI-1640, and incubate in a 37°C, 5% CO ₂ incubator for 24 hours;

Group C: Add 10 mL of RPMI-1640 containing 2 ng/mL to 20 ng/mL TNF-α and 1% PSA to fibroblasts, preferably RPMI of 7 ng/mL TNF-α and 1% PSA -1640, and incubate in a 39°C, 5% CO ₂ incubator for 24 hours;

(3) After the above three groups were washed twice with PBS (to remove the substances added in each group, such as TNF-α in groups B and C), 8 mL of RPMI-1640 containing 1% PSA was added. Group A and B at 37 ° C, 5% CO ₂ incubator for 8 to 288 hours, preferably 72 hours; group C at the same 39 ° C, 5% CO ₂ incubator for 8 hours 288 hours, preferably 24 hours.

(4) Collect the supernatants of the above three groups and put them in a 15 mL centrifuge tube, and centrifuge at 400 g for 10 minutes.

(5) Collect the supernatants of the above three groups and put them in a 15 mL centrifuge tube. Each of the supernatants contains the multi-peptide composition and can be stored at -80°C.

Example 1 Analysis of the content of multi-peptide composition under different conditions

Analyze the supernatants of group A and group B in Preparation Example 2. As shown in Figure 1, both groups A and B can analyze anti-inflammatory peptides IL-4, IL-6, IL-10 and ANG , And use the semi-quantitative method as the benchmark of relative content in group A without any stimulus. Compared with the IL-4 content of group A, the IL-4 content of group B increased by 23%; compared to the IL-6 content of group A, the IL-6 content of group B increased by 165%; compared to the IL-6 content of group A IL-10 content, the IL-10 content of group B increased by 15%; compared to the ANG content of group A, the ANG content of group B increased by 222%. Therefore, group B treated with TNF-α can collect the most anti-inflammatory peptides IL-4, IL-6, IL-10 and ANG.

Please refer to Figure 2. Both groups A and B can analyze the pro-inflammatory peptides IL-1β, IL-16, IFN-γ, TNF-α and GM-CSF, and group A without any stimulation Use semi-quantitative method as the basis of relative content. Compared with the IL-1β content of group A, the IL-1β of group B increased by 109%; compared with the IL-16 content of group A, the IL-16 of group B increased by 177%; compared with the IFN- of group A γ content, the IFN-γ content of group B increased by 47%; compared with the TNF-α content of group A, the TNF-α content of group B increased by 5%; compared with the GM-CSF content of group A, the GM of group B -CSF increased by 15%. Therefore, group B treated with TNF-α can collect the most pro-inflammatory peptides IL-1β, IL-16, IFN-γ, TNF-α and GM-CSF.

As shown in Figure 3, the immunomodulatory peptides ICAM-1, IL-12p70, and HGF can be analyzed in group A and group B, and group A without any stimulation is used as the benchmark for relative content in a semi-quantitative manner. Compared with the ICAM-1 content of group A, the ICAM-1 of group B increased by 650%; compared to the IL-12p70 content of group A, the IL-12p70 of group B increased by 338%; compared to the HGF content of group A , HGF in group B increased by 72%. Therefore, group B treated with TNF-α can collect the most immunomodulatory peptides ICAM-1, IL-12p70 and HGF.

Please refer to Figure 4, both groups A and B can analyze the growth factors bFGF and FGF-7, and group A without any stimulation is used as the benchmark for relative content in a semi-quantitative manner. Compared with the bFGF content of group A, the bFGF of group B decreased by 11%; compared with the FGF-7 content of group A, the FGF-7 of group B increased by 533%. Therefore, group B treated with TNF-α can collect the most growth factor FGF-7. Table 1. The content of multi-peptide composition in each group

As shown in Table 1 above, the multi-peptide composition of each group contains 14 kinds, and the content of the multi-peptide composition of each group is mostly different.

Example 2 Multipeptide composition administered under simulating inflammation of peripheral blood lymphocytes

(1) Prepare the following four sets of test conditions, and add 1 mL to the 24-well culture plate, repeat each test condition twice:

Control group: taken from the peripheral blood lymphocyte mixture obtained in step (7) of Preparation Example 1 (with medium RPMI-1640, 10% fetal bovine serum, 1% PSA, and 2×10 ⁵ cells/mL peripheral hemolymph Cells), and add 5 mg/L phytohemagglutinin;

Group 1: Take the group A supernatant (containing the multi-peptide composition) from step (5) in Preparation Example 2, and add it in order to contain 10% fetal bovine serum, 2×10 ⁵ cells/mL peripheral blood Lymphocyte mixture (taken from Step 7 of Preparation Example 1) and 5 mg/L phytohemagglutinin;

Group 2: Take the group B supernatant (containing the multi-peptide composition) in step (5) of Preparation Example 2, and add it in order to contain 10% fetal bovine serum, 2×10 ⁵ cells/mL peripheral blood Lymphocyte mixture (taken from Step 7 of Preparation Example 1) and 5 mg/L phytohemagglutinin;

Group 3: Take the group C supernatant (containing the multi-peptide composition) from step (5) in Preparation Example 2, and add it in order to contain 10% fetal bovine serum, 2×10 ⁵ cells/mL peripheral blood Lymphocyte mixture (taken from Step 7 of Preparation Example 1) and 5 mg/L phytohemagglutinin.

(2) Place the 24-well culture plate in a 37°C, 5% CO ₂ incubator, and collect the supernatant after 72 hours.

(3) Add 100 microliters per well (μL/well) of the supernatant from step (2) to the 96-well reaction plate of the enzyme-linked immunosorbent assay (ELISA), and add IFN-γ or and TNF-α antibody reacts overnight at 4°C.

(4) After washing the reaction wells with 300 μL/well buffer for 4 times, add 100 μL/well of biotinylated antibody to the 96-well reaction plate of ELISA, and react at room temperature for 1 hour.

(5) After washing the reaction wells with 300 μL/well buffer for 4 times, add 100 μL/well streptavidin solution to the 96-well reaction plate of ELISA, and react at room temperature for 45 minutes.

(6) Add 100 μL/well of enzyme substrate reagent [TMB (3,3',5,5'-tetramethylbenzidine) one-step substrate reagent] to the 96-well reaction plate of ELISA, and react at room temperature without light. 30 minutes.

(7) Add 50 μL/well of stop solution to the 96-well reaction plate of ELISA.

(8) Detect the absorbance at 450 nanometer (nm) wavelength by machine analysis, and calculate the concentration of IFN-γ and TNF-α in the supernatant of each group in the analysis step (2).

As shown in Figure 5, the content of IFN-γ in the supernatant of the control group was as high as 1335 pg/mL, indicating that the inflammatory response was simulated after stimulation by phytohemagglutinin; however, whether it was group 1 or group 2, each group The content of IFN-γ in the supernatant decreased to 267 pg/mL and 121 pg/mL, respectively, and the second group had the best inhibitory effect on IFN-γ. Therefore, the multi-peptide composition obtained by stimulating human fibroblasts by TNF-α can effectively inhibit the content of IFN-γ secreted by peripheral blood lymphocytes during inflammation.

As shown in Figure 6, the content of TNF-α in the supernatant of the control group was as high as 1891 pg/mL, indicating that the inflammatory response was simulated after stimulation by phytohemagglutinin; however, whether it was group 1 or group 2, each group The content of TNF-α in the supernatant decreased to 877 pg/mL and 596 pg/mL respectively, and the second group was the group that could inhibit the most TNF-α content. Therefore, the multi-peptide composition obtained by stimulating human fibroblasts by TNF-α can effectively inhibit the content of TNF-α secreted by peripheral blood lymphocytes during inflammation.

Please refer to Figure 7. This result is based on the human peripheral blood lymphocytes of Preparation Example 1. The content of IFN-γ in the supernatant of the control group is as high as 1012 pg/mL, which shows that it is simulated after stimulation by phytohemagglutinin Inflammation reaction; however, the content of IFN-γ in the supernatant of group 2 dropped to 727 pg/mL, and the content of IFN-γ in the supernatant of group 3 dropped to 691 pg/mL, respectively. Therefore, whether it is a multi-peptide composition obtained by culturing porcine fibroblasts at TNF-α and 37ºC (group 2), or a multi-peptide composition obtained by culturing porcine fibroblasts at TNF-α and 39ºC The peptide composition (group 3) can effectively inhibit the level of IFN-γ secreted by human peripheral blood lymphocytes during inflammation.

Example 3 Rose Spot Test

Take the supernatant containing the multi-peptide composition in the step (5) of Preparation Example 2 and apply it once a day, 1 mL once a day, for a total of 2 months, on the inflamed skin part of the rosette patient. Figure 8 shows the nose and eyebrows of the patient before use. The skin has large swelling and inflammation, which is one of the symptoms of rosette; the supernatant containing the multi-peptide composition of group B was administered for 2 months. As shown in 9, the original red, swollen and inflamed skin has shown a very light red color, which is almost the same as the color of normal skin, and the original swollen part has disappeared. Therefore, the multi-peptide composition of the group B of the present invention helps to suppress the symptoms of the inflammatory reaction of rosette.

The various modifications and changes that can be made according to the present invention will obviously not deviate from the scope and spirit of the present invention for those skilled in the art. Although the present invention has described specific preferred specific facts, it must be understood that the present invention should not be unduly limited to these specific specific facts. In fact, in terms of implementing the described modes of the present invention, different modifications that are obvious to those familiar with the technology are also covered in the scope of the following patent applications.

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Figure 1 is a histogram of the relative contents of IL-4, IL-6, IL-10 and ANG in each group of multi-peptide compositions of the present invention. Figure 2 is a histogram of the relative contents of IL-1β, IL-16, IFN-γ, TNF-α and GM-CSF of each group of multi-peptide compositions of the present invention. Figure 3 is a histogram of the relative contents of ICAM-1, IL-12p70 and HGF of each group of multi-peptide compositions of the present invention. Figure 4 is a histogram of the relative contents of bFGF and FGF-7 in each group of multi-peptide compositions of the present invention. Figure 5 is a histogram of the IFN-γ content of peripheral blood lymphocytes in the control group, the first group and the second group of the present invention; picograms per milliliter (pg/mL). Fig. 6 is a bar graph of the TNF-α content of peripheral blood lymphocytes in the control group, the first group and the second group of the present invention; picograms/ml (pg/mL). Figure 7 is a histogram of the IFN-γ content of peripheral blood lymphocytes in the control group, the second group and the third group of the present invention; picograms/ml (pg/mL), where the source of fibroblasts is pigs . Fig. 8 is a photograph of the nose and eyebrows of a patient with rosette before using the multi-peptide composition of the present invention. Figure 9 is a photograph of the nose and eyebrows of a patient with rosette after using the multi-peptide composition of group B in step (5) of Preparation Example 2 of the present invention for 2 months.

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Claims (3)

A preparation method of a multi-peptide composition comprising the following steps: (1) complete fibroblast-like cells and culture them in a culture medium; (2) administer the fibroblast-like cells with cytokines or growth factors in the immune response pathway And the culture time is 2 hours to 96 hours, and the culture temperature is 25°C to 45°C. The cytokine or growth factor in the immune response path is tumor necrosis factor alpha (TNF-α) And the concentration is 2 nanograms (ng/ml) to 20ng/ml per milliliter; and, (3) After washing the fibroblasts of this step (2), add serum-free medium and culture for 8 hours to 288 hours, and the culture temperature At 25°C to 45°C, a supernatant is obtained, the supernatant contains the multi-peptide composition, and the multi-peptide composition contains anti-inflammatory peptides, pro-inflammatory peptides, immunomodulatory peptides and growth Factor, wherein the anti-inflammatory peptide contains interleukin 4 (IL-4), IL-6, IL-10 and angiogenin (ANG), and the pro-inflammatory peptide contains interleukin-γ ( interferon gamma, IFN-γ), tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-α) and granulocyte-macrophage colony-stimulating factor (granulocyte-macrophage colony-stimulating factor, GM-CSF), the immune system Regulatory peptides include intercellular adhesion molecule-1 (ICAM-1), IL-12p70 and hepatocyte growth factor (HGF), and the growth factor includes basic fibroblast growth factor (basic fibroblast growth factor, bFGF) and fibroblast growth factor-7 (fibroblast growth factor-7, FGF-7). The method according to claim 1, wherein in the step (2), the culture time is 24 hours. The method according to claim 1, wherein in the step (2), the temperature of the culture is 37°C or 39°C.

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