TWI493036B - Applications of an immunocyte containing a reporter system for cd54 expression and a novel cloned cell - Google Patents

Description

Application of immune cells containing a CD54 expression reporting system and Newly selected cells

The present invention relates to the use of immune cells comprising a reporter system for CD54 expression, and in particular to the use of immune cells comprising a reporter system for CD54 expression to identify an immunomodulatory material.

CD54 (Cluster of Differentiation 54) (also known as Intercellular Adhesion Molecule-1 (ICAM-1), which is a protein encoded by the ICAM1 gene in humans. The gene encodes a cell surface glycoprotein that is normally expressed on endothelial cells and cells of the immune system. CD54 binds to CD11a/CD18 type, or CD11b/CD18 integrins. And is also used as a receptor by rhinovirus. CD54 is currently known as a biomarker for immune responses, especially allergic reactions.

The immune response plays an important role in many diseases, such as inflammatory reactions, skin sensitivities, etc., so many drugs are developed to regulate the immune system. However, the immune system involves a large variety of biomolecules and cells, so it is often necessary to conduct experiments in vivo to conduct experimental evaluations, resulting in time and success. This increase. Although many cell experimental models are currently available for the regulation of immune responses, they often require ELISA or flow cytometry for analysis, and are more complex than normal biological experiments.

In addition, allergenicity is an important safety assessment project for cosmetics, various chemicals, agricultural products, environmental toxicants, industrial products and other products before they go on the market. In line with the trend of animal experiment ethics and reduction, research institutions and related companies in Europe, the United States and Japan have invested considerable resources in the development of alternative animal experimental methods, but there is no internationally accepted alternative to animal experiments. .

Therefore, there is an urgent need to develop new methods for confirming immunomodulatory substances or sensitizing substances that are not required for animal experiments to be suitable for high-throughput testing.

The invention provides a method for confirming an immunomodulatory substance, comprising: (a) providing a test object and an immune cell, wherein the immune cell comprises a CD54 performance reporting system, and the CD54 performance reporting system comprises: a promoter of the CD54 gene; and a reporter gene linked to the promoter of the CD54 gene for reporting the expression of the promoter of the CD54 gene; (b) treating the immune cell with the analyte; and (c) determining The product of the reporter gene produced by the immune cell treated by the test substance is evaluated to determine whether the test substance has an immunomodulatory effect.

The invention also provides a method for confirming a skin sensitizing substance, comprising: (a) providing a test object and an immune cell, wherein the immune cell comprises a CD54 performance reporting system, and the CD54 performance reporting system comprises a promoter of the CD54 gene; and a reporter gene linked to the promoter of the CD54 gene for reporting the expression of the promoter of the CD54 gene; (b) Measuring the immune cells; and (c) determining the product expression of the reporter gene produced by the immune cells treated with the analyte to determine whether the analyte is a skin sensitizer.

The invention further provides a novel cell which is selected and deposited in the Biological Resource Preservation and Research Center of the Republic of China Food Industry Development Institute on December 4, 102, and the registration number is BCRC No. 960475.

Therefore, the present invention provides a new method for confirming immunomodulatory substances or skin sensitizing substances without animal experiments to be suitable for high-throughput testing, and to develop a method for replacing animal experiments, which is consistent with the trend of related animal experiments ethics and reduction.

101, 103, 105, 107, 109, 109A, 109B, 201, 203, 205, 207, 209, 209A, 209B‧ ‧ steps

Figure 1 shows a CD54-promoter mLuc expression system in accordance with one embodiment of the present invention.

Fig. 2 is a view showing the flow of determining whether or not a substance to be tested is a skin sensitizer according to an embodiment of the present invention. And Fig. 3 shows a flow of determining whether or not the substance to be tested is an anti-inflammatory substance according to an embodiment of the present invention.

In an embodiment of the invention, the invention provides a method of identifying an immunomodulatory substance. The method of the present invention for confirming an immunomodulatory substance may include, but is not limited to, the steps described below.

First, an analyte is provided and an immune cell comprising a reporting system for CD54 expression.

Examples of the above-mentioned analytes may include, for example, cosmetics, drugs, chemicals, extracts of natural substances, and the like, but are not limited thereto. The above immune cells Examples of sources may include, but are not limited to, THP-1 cells, U937 cells, MUTZ-3 cells, and the like. In one embodiment, the source of the immune cells is THP-1 cells.

A reporting system for CD54 expression of the above immune cells, which may include However, it is not limited to a CD54 promoter and a reporter gene, wherein the reporter gene is linked to the promoter of the CD54 gene to report the expression of the promoter of the CD54 gene.

In one embodiment, the sequence of the promoter of the CD54 gene described above is The sequence of sequence identification number: 1 is included, and in another embodiment, the sequence of the promoter of the above CD54 gene may be the sequence of sequence number: 1.

Furthermore, examples of the above reporter genes may include, but are not limited to, long Metridia luciferase gene, firefly luciferase gene, green fluorescent protein (GFP) gene, β-glucuronidase gene, etc. In one embodiment, the reporter gene is a chilling enzyme gene of the genus Asparagus. The product of the cold-light enzyme gene of the genus Astragalus is directly secreted into the culture medium by the cells. Therefore, when the performance (for example, activity) is measured, the culture medium can be directly subjected to luminescence analysis without breaking the cells, which has the advantage of saving analysis time.

In one embodiment, the sequence of the promoter of the CD54 gene is the sequence of sequence number: 1, and the source of the above immune cells is THP-1 cells. In another embodiment, the sequence of the promoter of the CD54 gene is a sequence of sequence number: 1, and the reporter gene is a cold-light enzyme gene of the genus Asparagus. Further, in another embodiment, the sequence of the promoter of the CD54 gene is a sequence of sequence identification number: 1, and the source of the immune cell is THP-1 cells, and the above The reporter gene is the cold-light enzyme gene of the genus Asparagus.

An implementation of the method for confirming an immunomodulatory substance of the present invention In the example, the sequence of the CD54 expression reporting system comprising the promoter of the CD54 gene and the reporter gene may comprise the sequence of sequence number: 2. In a specific embodiment, the sequence of the CD54 expression reporting system can be a sequence of sequence identification number: 2, that is, the CD54 expression reporting system is a promoter of the CD54 gene - CD54 promoter-Metridia Luc. Form. In this particular embodiment, the immune cell used in the reporter system comprising CD54 expression may be a cell obtained by infecting a viral vector having the sequence of sequence number: 2 into a THP-1 cell. The cell may be a cell named CD54-mLuc-THP-1, which was deposited on December 4, 102, at the Food Industry Development Institute of the Corporation, under the registration number BCRC No. 960475.

Then, processing the above-mentioned report including the performance of the CD54 with the above-mentioned object to be tested Guide the immune cells of the system. In an embodiment, the processing time is about 1-2 days, but is not limited thereto. Moreover, in an embodiment, before the immune cells including the reporting system of the CD54 expression are processed by the above-mentioned analyte, the analyte may be subjected to cytotoxicity analysis on the immune cell, and the analyte is determined. The half growth inhibition concentration of this cell, and thereby the treatment concentration was obtained.

Then, the above-mentioned test including the CD54 is performed by the above-mentioned test object. After reporting the immune cells of the system, the product expression of the reporter gene produced by the above-mentioned immune cells treated with the above test substance is measured to determine whether the test substance has an immunomodulatory effect.

An implementation of the method for confirming an immunomodulatory substance of the present invention In the example, the reporter gene produced by measuring the immune cells treated by the above test substance is determined. The product is characterized in that the step of determining whether the test substance has an immunomodulatory effect is a report of the product expression of the reporter gene produced by the immune cell treated by the test substance and the above-mentioned immune cell treated without the test substance. The performance of the gene product is compared to determine if the analyte has immunomodulatory effects. If the product of the reporter gene produced by the immune cell treated by the test substance is higher than the performance of the reporter gene product of the immune cell not treated with the test substance, it is determined that the test substance has an immunomodulatory effect and is an immune regulation substance.

Another method for confirming an immunomodulatory substance of the present invention In one embodiment, in the step of treating the immune cells of the reporting system including the CD54 expression with the analyte, further comprising adding an immune-inducing substance to cause the immune cells to generate an immune response. In this embodiment, in the step of determining the product expression of the reporter gene produced by the immune cells treated by the test substance to determine whether the test substance has an immunomodulatory effect, the test substance and the immune-inducing substance are treated. The product of the reporter gene produced by the immune cell is compared with the performance of a reporter gene product of an immune cell treated only with the immune-inducing substance to determine whether the test substance has an immunomodulatory effect. If the product of the reporter gene produced by the immune cell treated with the analyte and the immuno-inducing substance exhibits lower performance than the reporter gene product of the immune cell treated only by the immuno-inducing substance, the analyte is determined to have an immunosuppressive effect. It is an immunosuppressive substance.

The above immune response inducing substance may include any immune reaction The substance to be used, for example, lipopolysaccharide (LPS), phorbol myristate acetate (PMA), concanavalin A (Con A), etc., is not limited thereto.

In one embodiment, an immunomodulatory substance is identified in the present invention The immune response inducing substance used in the method may be a lipopolysaccharide, a phorbol acetate or a concanavalin A, which is a consistent inflammatory substance.

In the above embodiment, the immunological fines processed by the test object are measured. The step of determining whether the test substance has an immunomodulatory effect in the step of determining whether the test substance has an immunomodulatory effect in the step of determining whether the test substance has an immunomodulatory effect comprises determining whether the test object has an anti-inflammatory effect, thereby determining whether the test object has an anti-inflammatory effect. Whether the test is an anti-inflammatory substance.

The manner in which the expression of the product of the reporter gene produced by the above immune cells is determined depends on the nature of the product of the reporter gene (for example, the activity can be measured). Methods for determining the performance of products of different reporter genes are well known to those of ordinary skill in the art. For example, since the product of the cold-light enzyme gene of the genus Astragalus is directly secreted into the culture medium by the cells, the culture medium of the cultured cells can be directly subjected to cold light analysis to determine the product expression of the luminescent enzyme gene of the long-term ascites.

In another embodiment of the present invention, the present invention also provides a method of confirming a skin sensitizing substance, and the method of the present invention for confirming a skin sensitizing substance may include the following steps, but is not limited thereto.

An analyte and an immune cell comprising a reporting system for CD54 performance are provided.

The object to be tested may be any substance and is not particularly limited. Examples of the above-mentioned analytes may include, for example, cosmetics, drugs, chemicals, extracts of natural substances, and the like, but are not limited thereto. Examples of the source of the above immune cells may include, but are not limited to, THP-1 cells, U937 cells, MUTZ-3 cells, and the like. In one embodiment, the source of the immune cells is THP-1 cells.

In addition, the reporting system of CD54 expression contained in the above immune cells The promoter may include a CD54 gene and a reporter gene, but is not limited thereto, wherein the reporter gene is ligated to the promoter of the CD54 gene to report the expression of the promoter of the CD54 gene.

In one embodiment, the sequence of the promoter of the CD54 gene described above is The sequence of sequence identification number: 1 is included, and in another embodiment, the sequence of the promoter of the above CD54 gene may be the sequence of sequence number: 1.

And similar to the reporter gene used in the method for confirming an immunomodulatory substance of the present invention, examples of the reporter gene in the method for confirming a skin sensitizing substance of the present invention may include, but are not limited to, the genus luminescent enzyme Gene, firefly cold light enzyme gene, green fluorescent protein gene, β -glucuronidase gene, and the like. In one embodiment, the reporter gene is a chilling enzyme gene of the genus Asparagus. The product of the cold-light enzyme gene of the genus Astragalus is directly secreted into the culture medium by the cells. Therefore, when the performance (for example, activity) is measured, the culture medium can be directly subjected to luminescence analysis without breaking the cells, which has the advantage of saving analysis time.

In one embodiment, the sequence of the promoter of the CD54 gene is the sequence of sequence number: 1, and the source of the above immune cells is THP-1 cells. In another embodiment, the sequence of the promoter of the CD54 gene is a sequence of sequence number: 1, and the reporter gene is a cold-light enzyme gene of the genus Asparagus. Further, in another embodiment, the sequence of the promoter of the CD54 gene is the sequence of sequence number: 1, the source of the immune cell is THP-1 cells, and the reporter gene is a chilling enzyme gene of the genus Asarum.

In an embodiment of the method for identifying a skin sensitizing substance of the present invention, the aforementioned CD54 expression report comprising a promoter of the CD54 gene and a reporter gene The sequence of the guidance system can include a sequence of sequence identification number: 2. In a specific embodiment, the sequence of the CD54 expression reporting system can be a sequence of sequence identification number: 2, that is, the CD54 expression reporting system is a promoter of the CD54 gene - CD54 promoter-Metridia Luc. Form. In this particular embodiment, the immune cell used in the reporter system comprising CD54 expression may be a cell obtained by infecting a viral vector having the sequence of sequence number: 2 into a THP-1 cell. The cell may be a cell named CD54-mLuc-THP-1, which was deposited on December 4, 102, at the Food Industry Development Institute of the Corporation, under the registration number BCRC No. 960475.

Then, processing the above-mentioned report including the performance of the CD54 with the above-mentioned object to be tested Guide the immune cells of the system. In an embodiment, the processing time is about 1-2 days, but is not limited thereto.

Moreover, in an embodiment, the package is processed by the object to be tested Before the CD54 expresses the immune cells of the reporting system, the test substance can be subjected to cytotoxicity analysis on the immune cell, and the concentration of the semi-growth inhibition of the test substance on the cells is determined, thereby obtaining the treated concentration.

Finally, in the above-mentioned test object, the above-mentioned performance including CD54 is processed. After reporting the immune cells of the system, the product expression of the reporter gene produced by the above-mentioned immune cells treated with the test substance is determined to determine whether the test substance is a skin sensitizer.

In one embodiment, the above-mentioned treatment by the above-mentioned test object is measured. The product of the reporter gene produced by the immune cell is used to determine whether the test substance is a skin sensitizer, and the product of the reporter gene produced by the immune cell treated by the test substance is Through the above test object The performance of the reported gene product of the treated immune cells is compared to determine whether the test substance has an immunomodulatory effect. If the product of the reporter gene produced by the immune cell treated by the analyte is higher than the performance of the reporter gene product of the immune cell not treated with the analyte, the analyte is determined to be a skin sensitizer.

The manner in which the expression of the product of the reporter gene produced by the above immune cells is determined depends on the nature of the product of the reporter gene (for example, the activity can be measured). Methods for determining the performance of products of different reporter genes are well known to those of ordinary skill in the art. For example, since the product of the cold-light enzyme gene of the genus Astragalus is directly secreted into the culture medium by the cells, the culture medium of the cultured cells can be directly subjected to cold light analysis to determine the product expression of the luminescent enzyme gene of the long-term ascites.

In addition, in still another embodiment of the present invention, the present invention further provides a novel cell which is selected and named CD54-mLuc-THP-1, which is deposited on December 04, 102 in the food of the corporation. Industrial Development Institute, the registration number is BCRC No.960475.

This cell has a CD54 expression reporting system in the form of the promoter of the CD54 gene, CD54 promoter-Metridia Luc, and the sequence of the CD54 expression reporting system is the sequence of sequence number: 2.

Example

Example 1

Establishment of CD54-mLuc-THP-1 cells

LVX-MetLuc plastids were treated with BstB I and BamH I restriction enzymes according to the manufacturer's instructions for LVX-MetLuc (Clontech, PT4422-5) plastids to allow transcription of the CD54 promoter sequence (SEQ ID NO: 1). To the LVX-MetLuc plastid to form the CD54-promoter mLuc expression system (Fig. 1), wherein the sequence of the CD54 promoter-mLuc is the sequence of sequence number: 2 in this expression system. After the CD54-promoter mLuc performance of the system is formed using the ^{Lenti-X TM Ready-To-} Glow TM Secreted Luciferase Reporter System (Clontech, 631746) was transfected into the Lentivirus 293FT cells to produce with the CD54-mLuc. This virus is then used to infect THP-1 cells. The THP-1 cell line was cultured in THP-1 medium, which was composed of RPMI medium (GIBCO, 31800) containing 10% FBS (GIBCO, 10437), 4.5 g/L glucose (Sigma, G7021), 10 mM HEPES ( Sigma, H4034), 1x penicillin and streptomycin (Biowest, L0022), 1 mM sodium pyruvate (Biowest, L0642), 0.05 mM 2-mercaptoethanol, 2-mercaptoethanol, 2- ME) (GIBCO, 21985-023).

Then, with 0.5~1μg/mL puromycin (Invivogen, ant-pr-1) was used to screen for THP-1 cells. After 2 weeks of screening, 100 uL of cell-culture medium (concentration: 5 cells/mL) was added to a 96-well plate (Corning Incorporation COSTAR, 3599). After the number of cells was increased, the cells were gradually utilized in a 24-well plate (Corning). Incorporation COSTAR, 3516), 6-well plate (Corning Incorporation COSTAR, 3524) and T25 flask (Corning Incorporation COSTAR, 430639) were scaled up. Each cell strain is known as a sensitizer 1-chloro-2,4-dinitrobenzene (DNCB) and a non-sensitizer. Hexadecyltrimethylammonium bromide (CTAB) was tested and the best cell line was selected and named CD54-mLuc-THP-1, which was deposited on December 4, 102. Republic of China Food Industry Development Institute Biological Resources Deposit and Research Center, registration number BCRC No.960475.

Example 2

Whether the substance to be tested is a skin sensitizer (or immunomodulatory substance)

The procedure for determining whether the substance to be tested is a skin sensitizer in this experiment is shown in Fig. 2. See Figure 2 for a detailed description of the steps below.

1. Toxicity test of test substance to CD54-mLuc-THP-1 cells

CD54-mLuc-THP-1 cells were cultured in THP-1 medium, which was composed of RPMI medium (GIBCO, 31800) containing 10% fetal bovine serum (GIBCO, 10437) and 4.5 g/L glucose (Sigma, G7021). ), 10 mM HEPES (Sigma, H4034), 1x penicillin and streptomycin (Biowest, L0022), 1 mM sodium pyruvate (Biowest, L0642), 0.05 mM 2-mercaptoethanol (2) -mercaptoethanol) (GIBCO, 21985-023) and 0.5 μg/mL puromycin.

The reference chemical samples of known sensitization listed in Table 1 or the herbal extracts commonly found in cosmetics listed in Table 2, in a suitable solvent (DMSO or THP-1 broth without 2-ME), After dissolution, a stock solution is formed. The stock solution is then serially diluted 3 to 8 times 6 to 8 times to form working solutions of different dilution factors.

A cytotoxicity test is then performed (step 101). 10 μL/well of the working solution was added to the 96-well plate. Next, CD54-mLuc-THP-1 cells were suspended in THP-1 medium containing no 2-ME and puromycin, and then seeded in the above 96-well cells at a density of 5 × 10 ⁴ cells / 90 μL / well. Train in the dish. The plate was then placed in a cell culture incubator (NUAIR nu-5510) and incubated for 1 day. It should be noted that if the analyte is dissolved in DMSO, the concentration of DMSO in the culture solution when the final analyte is in contact with the cells is controlled to 0.2%.

Then, 10 μL of 5 mg/mL MTT culture solution was added to the culture. The plate was placed in a carbon dioxide cell incubator for 2 hours. Next, 100 μL of 10% SDS was added to the plate and allowed to stand overnight. The purple precipitate was then dissolved and the absorbance of the cells at a wavelength of 560 nm was measured in a continuous wavelength microplate assay system (Molecular Devices, SPECTRAMAX 5).

The average absorbance of the control group represents the survival rate of 100% of the cells. (%), to calculate the survival rate (%) of the experimental group cells to which various concentrations of the samples were added. The cell survival rate was calculated as: (experimental group absorbance value / control group absorbance value) × 100%. The cell growth inhibition rate of each concentration of the test substance was calculated, and the cell growth inhibition rate was calculated as: 1-cell survival rate.

Thereafter, the cell growth inhibition rate of each experimental group was used as an xy scattergram, and the sample concentration (CC ₅₀ ) of 50% cell growth inhibition of each test substance was determined by the IC ₅₀ equation using Grafit software (step 103).

If the drug interferes with the color interpretation of the MTT, use Cell-Glo instead. Kit for cytotoxicity testing. The procedure was as follows: 35 μL of the cell solution was added to a 96-well white plate for luminescence, and then 35 μl of Cell-Glo kit (Promega, G7571) was added to the above 96-well white plate, and the white plate was shaken for 10 minutes in an oscillator. Then, the substrate was added to the above 96-well white plate, and the cold light intensity (Rlu/s) produced by the cells was measured immediately by a microplate luminometer (Berthold, MPL4) as the test cell for each concentration of the experiment. Toxicity reference value.

The cell survival rate was calculated as: (experimental group cold light value / control group cold Light value) × 100%. The cell growth inhibition rate of each concentration of the test substance was calculated, and the cell growth inhibition rate was calculated as: (1-cell survival rate).

The cell growth inhibition rate of each experimental group was used as an xy scattergram, and the concentration of 50% cell growth inhibition (CC ₅₀ ) of each test substance was determined by the IC ₅₀ equation using Grafit software.

In the subsequent experiments, CC ₅₀ ± 25% of each test substance was selected as the reference test starting concentration of mLuc luminescence activity of CD54-mLuc-THP-1 cells.

If the accurate CC ₅₀ cannot be measured in the first experiment, the concentration of the analyte and the dilution ratio are changed with reference to the experimental results. To get the best trend line to get the CC ₅₀ .

And the measured concentration of 50% cell growth inhibition of each test substance as shown in Table 3.

2. Test of the effect of the test substance on m-Luc activity of CD54-mLuc-THP-1 cells

According to the CC ₅₀ of each test substance obtained above, the test starting concentration of each test object is selected (about CC ₅₀ ±25% concentration, and if the accurate CC ₅₀ cannot be obtained, the highest soluble concentration of the test object is obtained. Or the final concentration is 4000 μM for testing), and the stock solution of the test substance is prepared at this concentration. Thereafter, the stock solution of the above test substance was diluted twice in two times to form an operation solution of the other two concentrations, and a total of three concentrations of the solution were obtained (step 105).

10 μL/well of the working solution was added to the 96-well plate. Next, CD54-mLuc-THP-1 cells were suspended in THP-1 medium containing no 2-ME and puromycin, and then seeded in the above 96-well cells at a density of 5 × 10 ⁴ cells / 90 μL / well. Train in the dish. The plate was then placed in a cell culture incubator (NUAIR nu-5510) for 1 day with 50 ng/mL lipopolysaccharide (LPS) as a positive control. It should be noted that if the analyte is dissolved in DMSO, the concentration of DMSO in the culture solution when the final analyte is in contact with the cells is controlled to 0.2%.

Thereafter, 10 μL of the cell liquid was taken out from the above culture plate and added to a 96-well white plate for cold light. Next, add 40 μL of Luciferase Assay reagent (GeneCopoeia, SPGA-G100) to the above white plate, and immediately use the microdisk. A cold light meter (Berthold, MPL4) was used to measure the cold light intensity (Rlu/s) of the cells (step 107).

The experimental group of each sample was calculated based on the luminescence intensity of the control group. It promotes the induction rate of CD54 promoter activity. The calculation formula of the induction rate is: (cold light value of the experimental group / cold light value of the control group). If the result indicates that a test substance promotes mLuc expression, it is determined that the test substance is a sensitizer (step 109 (including step 109A and step 109B)). The results of the induction rate of the CD54 promoter activity measured by each of the above analytes are shown in Table 4, and the results of determining whether each analyte is a sensitizer are shown in Table 5.

In addition, add 35 μL of cell fluid to 96-well white for cold light. Plate, then 35 μL of Cell-Glo kit (Promega, G7571) was added to the above 96-well white plate, and the white plate was placed in an oscillator for 10 minutes. Then, the substrate was added to the above 96-well white plate, and the cold light intensity (Rlu/s) produced by the cells was measured immediately by a microplate luminometer (Berthold, MPL4) as the test cell for each concentration of the experiment. Toxicity reference value. The cell survival rate was calculated as: (experimental group cold light value / control group cold light value) × 100%.

Example 3

Whether the substance to be tested is an anti-inflammatory substance (or immunomodulatory substance)

The procedure for determining whether the substance to be tested is a skin sensitizer in this experiment is shown in Fig. 3. See Figure 3 and the detailed step instructions below.

1. Toxicity test of test substance to CD54-mLuc-THP-1 cells

CD54-mLuc-THP-1 cells were cultured in THP-1 medium, which was composed of RPMI medium (GIBCO, 31800) containing 10% fetal bovine serum (GIBCO, 10437) and 4.5 g/L glucose (Sigma, G7021). ), 10 mM HEPES (Sigma, H4034), 1x penicillin and streptomycin (Biowest, L0022), 1 mM sodium pyruvate (Biowest, L0642), 0.05 mM 2-mercaptoethanol (2) -mercaptoethanol) (GIBCO, 21985-023) and 0.5 μg/mL puromycin.

The test substance known to have anti-inflammatory ability listed in Table 6 was dissolved in a suitable solvent (DMSO or THP-1 medium without 2-ME) to form a stock solution. The stock solution is then serially diluted 3 times to 6 times to form a working solution of different dilution factors.

A cytotoxicity test is then performed (step 201). 10 μL/well of the working solution was added to the 96-well plate. Next, CD54-mLuc-THP-1 cells were suspended in THP-1 medium containing no 2-ME and puromycin, and then seeded in the above 96-well cells at a density of 5 × 10 ⁴ cells / 90 μL / well. Train in the dish. The plate was then placed in a cell culture incubator (NUAIR nu-5510) and incubated for 1 day. It should be noted that if the analyte is dissolved in DMSO, the concentration of DMSO in the culture solution when the final analyte is in contact with the cells is controlled to 0.2%.

Then, 10 μl of 5 mg/mL MTT culture solution was added to the culture. The plate was placed in a carbon dioxide cell incubator for 2 hours. Next, 100 μl of 10% SDS was added to the plate and allowed to stand overnight. The purple precipitate was then dissolved and the absorbance of the cells at a wavelength of 560 nm was measured in a continuous wavelength microplate assay system (Molecular Devices, SPECTRAMAX 5).

The average absorbance of the control group represents the survival rate of 100% of the cells. (%), to calculate the survival rate (%) of the experimental group cells to which various concentrations of the samples were added. The cell survival rate was calculated as: (experimental group absorbance value / control group absorbance value) × 100%. The cell growth inhibition rate of each concentration of the test substance was calculated, and the cell growth inhibition rate was calculated as: 1-cell survival rate.

Thereafter, the cell growth inhibition rate of each experimental group was used as an xy scattergram, and the sample concentration (CC ₅₀ ) of 50% cell growth inhibition of each test substance was determined by the IC ₅₀ equation using Grafit software (step 203).

If the drug interferes with the color interpretation of the MTT, use Cell-Glo instead. Kit for cytotoxicity testing. The procedure was as follows: 35 μL of the cell liquid was added to a 96-well white plate for cold light, and then 35 μL of Cell-Glo kit (Promega, G7571) was added to the above 96-well white plate, and Place the white plate in the shaker for 10 minutes. Then, the substrate was added to the above 96-well white plate, and the cold light intensity (Rlu/s) produced by the cells was measured immediately by a microplate luminometer (Berthold, MPL4) as the test cell for each concentration of the experiment. Toxicity reference value.

The cell survival rate was calculated as: (experimental group cold light value / control group cold Light value) × 100%. The cell growth inhibition rate of each concentration of the test substance was calculated, and the cell growth inhibition rate was calculated as: (1-cell survival rate).

The cell growth inhibition rate of each experimental group was used as an xy scattergram, and the concentration of 50% cell growth inhibition (CC ₅₀ ) of each test substance was determined by the IC ₅₀ equation using Grafit software.

In the subsequent experiments, CC ₅₀ ± 25% of each test substance was selected as the reference test starting concentration of mLuc luminescence activity of CD54-mLuc-THP-1 cells.

If the accurate CC ₅₀ cannot be measured in the first experiment, the concentration of the analyte and the dilution ratio are changed with reference to the experimental results. To get the best trend line to get the CC ₅₀ .

And the measured concentration of 50% cell growth inhibition of each test substance As shown in Table 7.

2. Test of the effect of analytes on lipopolysaccharide (LPS)-induced m-Luc activity in CD54-mLuc-THP-1 cells

According to the CC ₅₀ of each test substance obtained above, the test starting concentration of each test object is selected (about CC ₅₀ ±25% concentration, and if the accurate CC ₅₀ cannot be obtained, the highest soluble concentration of the test object is obtained. Or the final concentration is 4000 μM for testing), and the stock solution of the test substance is prepared at this concentration. Thereafter, the stock solution of the above test substance was diluted twice in two times to form an operation solution of the other three concentrations, and a total of four concentrations of the solution were obtained (step 205).

10 μL/well of the working solution and 10 μL of 100 ng/mL lipopolysaccharide (Sigma, L2880) were added to a 96-well plate. Next, CD54-mLuc-THP-1 cells were suspended in THP-1 medium containing no 2-ME and puromycin, and then seeded in the above 96-well cells at a density of 5 × 10 ⁴ cells / 80 μL / well. Train in the dish. The plate was then placed in a cell culture incubator (NUAIR nu-5510) for 1 day. It should be noted that if the analyte is dissolved in DMSO, the concentration of DMSO in the culture solution when the final analyte is in contact with the cells is controlled to 0.2%.

Thereafter, 10 μL of the cell liquid was taken out from the above culture plate and added to a 96-well white plate for cold light. Next, 40 μL of Luciferase Assay reagent (GeneCopoeia, SPGA-G100) was added to the above white plate, and the cold light intensity (Rlu/s) of the cells was immediately measured by a microplate luminometer (Berthold, MPL4). (Step 207).

The inhibition rate of the lipopolysaccharide-induced CD54 promoter activity of each sample was calculated based on the luminescence activity of the control group and the lipopolysaccharide-inducing group. The calculation formula for the induction rate is: (The luminescence value of the lipopolysaccharide-induced group - the lipopolysaccharide-induced luminescence value after co-treatment of the test substance) / (the luminescence value of the lipopolysaccharide-induced group - the luminescence value of the untreated group) x 100%. If the inhibition rate is positive, it means that the analyte has an inhibitory immune response induced by lipopolysaccharide (step 209 (including step 209A and step 209B)).

Then, the inhibition rate of the CD54 promoter activity of each experimental group was used as an xy scattergram, and the concentration of the 50% lipopolysaccharide-induced mLuc activity (IC ₅₀ ) of each test substance was determined by the IC ₅₀ equation using Grafit software. See Table 7 for the results.

【Biomaterial Storage】

Domestic registration information [please note according to the registration authority, date, number order]

Bioresource Collection and Research Center (BCRC), Republic of China Food Industry Development Institute

December 04, 102

BCRC No.960475

<110> Institute of Industrial Technology

<120> Application of immune cells containing a CD54 expression reporting system and novel cells once selected

<130> 0965-A24490-TW

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1323

<212> DNA

<213> Human

<400> 1

<210> 2

<211> 2029

<212> DNA

<213> Artificial sequence

<220>

<223> CD54 Performance Reporting System

<400> 2

101, 103, 105, 107, 109, 109A, 109B‧‧‧ steps

Claims (24)

A method for confirming an immunomodulatory substance, comprising: (a) providing a test substance and an immune cell, wherein the immune cell comprises a CD54 expression reporting system, and the CD54 performance reporting system comprises: a CD54 gene a promoter; and a reporter gene linked to the promoter of the CD54 gene for reporting the expression of the promoter of the CD54 gene; (b) treating the immune cell with the analyte itself; and (c) determining the The product of the reporter gene produced by the immune cell treated by the analyte itself determines whether the analyte itself has an immunomodulatory effect. The method for confirming an immunomodulatory substance according to claim 1, wherein in the step (c), the product of the reporter gene produced by the immune cell treated by the analyte itself is expressed as a The performance of the reporter gene product of the immune cell that is not treated by the analyte itself is compared to determine whether the analyte itself has an immunomodulatory effect. A method for confirming an immunomodulatory substance according to claim 1, wherein the product of the reporter gene produced by the immune cell treated by the analyte itself is higher than that of the sample itself The performance of the reported gene product of the treated immune cell determines that the test substance itself has an immunomodulatory effect and is an immunomodulatory substance. The method for confirming an immunomodulatory substance according to claim 1, wherein the step (b) further comprises adding an immune-inducing substance to make the fine The cell produces an immune response. A method for confirming an immunomodulatory substance according to claim 4, wherein in step (c), the reporter gene produced by the immune cell treated by the test substance itself and the immune-inducing substance is The product performance is compared to the performance of the reporter gene product of the immune cell treated only by the immune-inducing substance to determine whether the test substance itself has an immunomodulatory effect. The method for confirming an immunomodulatory substance according to claim 5, wherein the product of the reporter gene produced by the immune cell treated by the analyte itself and the immune-inducing substance exhibits a performance lower than the The expression of the reporter gene product of the immune cell treated by the immune-inducing substance determines that the test substance itself has an immunosuppressive action and is an immunosuppressive substance. A method for confirming an immunomodulatory substance according to claim 4, wherein the immunologically inducing substance comprises lipopolysaccharide (LPS), phorbol myristate acetate (PMA) or concanavalin. A (concanavalin A, Con A). The method for confirming an immunomodulatory substance according to claim 7, wherein in the step (c), determining whether the test substance itself has an immunomodulatory effect comprises determining whether the test object itself has an anti-inflammatory effect, To determine whether the test object itself is an anti-inflammatory substance. A method for confirming an immunomodulatory substance according to claim 1, wherein the source of the immune cell comprises THP-1 cells, U937 cells or MUTZ-3 cells. A method for confirming an immunomodulatory substance according to claim 1, wherein the sequence of the promoter of the CD54 gene comprises a sequence identification number: sequence. The method for confirming an immunomodulatory substance according to the first aspect of the patent application, wherein the reporter gene comprises a Metridia luciferase gene, a firefly luciferase gene, and a green fluorescent protein (green). Fluorescent protein, GFP) gene or β-glucuronidase gene. A method for confirming an immunomodulatory substance according to claim 1, wherein the sequence of the CD54 expression reporting system is a sequence identification number: 2. The method for confirming an immunomodulatory substance according to claim 12, wherein the immune cell is deposited at the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute on December 4, 102, and the registration number is BCRC. No.960475 of THP-1 cells. A method for confirming an immunomodulatory substance according to claim 1, wherein the analyte comprises an extract of a cosmetic, a pharmaceutical, a chemical or a natural product. A method for confirming a skin sensitizing substance, comprising: (a) providing a test substance and an immune cell, wherein the immune cell comprises a CD54 expression reporting system, and the CD54 performance reporting system comprises: a CD54 gene a promoter; and a reporter gene linked to the promoter of the CD54 gene for reporting the expression of the promoter of the CD54 gene; (b) treating the immune cell with the analyte itself; and (c) measuring the The immune cell produced by the test object itself is produced by the immune cell The product of the gene is reported to determine whether the analyte itself is a skin sensitizer. A method for confirming a skin sensitizing substance according to claim 15, wherein the product of the reporter gene produced by the immune cell treated by the analyte itself is not associated with the analyte The performance of the reporter gene product of the immune cell treated by itself is compared to determine whether the analyte itself is a skin sensitizer. The method for confirming a skin sensitizing substance according to claim 16, wherein the product of the reporter gene produced by the immune cell treated by the test object itself is higher than the sample without the test object The expression of the reporter gene product of the immune cell treated by itself determines that the analyte itself is a skin sensitizer. A method for confirming a skin sensitizing substance according to claim 15, wherein the source of the immune cell comprises THP-1 cells, U937 cells or MUTZ-3 cells. A method for confirming a skin sensitizing substance according to claim 15, wherein the sequence of the promoter of the CD54 gene comprises the sequence of sequence number: 1. A method for confirming a skin sensitizing substance according to claim 15, wherein the reporter gene comprises a chilling enzyme gene, a firefly cold light enzyme gene, a green fluorescent protein gene or a β-glucuronidase gene. A method for confirming a skin sensitizing substance according to claim 15 wherein the sequence of the CD54 expression reporting system is a sequence identification number: 2. A method for confirming a skin sensitizing substance as described in claim 21, wherein the immune cell is deposited at the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute on December 4, 102, the registration number is THP-1 cells of BCRC No. 960475. A method for confirming a skin sensitizing substance as described in claim 15 wherein the test substance comprises an extract of a cosmetic, a pharmaceutical, a chemical or a natural product. A novel cell that has been selected and deposited in the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute on December 4, 2012. The registration number is BCRC No.960475.