Cell capsaicin receptor activation rapid detection kit for high-throughput screening and application of cell capsaicin receptor activation rapid detection kit

Abstract

The invention discloses a rapid detection kit for zebra fish cell capsaicin receptor activation. The rapid detection kit comprises an RNA purification column and an RNA collection tube or an RNA capture plate, washing buffer solutions A and B, a DNase I digestive juice, a 5x reverse transcription premix solution, a 2x SYBR Green qPCR premix solution, and specific primer dry powder or premix solution. The invention also discloses an application of the kit. The invention also discloses a method for screening a compound or a substance for regulating and controlling the activation of the capsaicin receptor of the zebra fish cell by using the kit. The novel primer sequence provided by the invention can accurately reflect the activation state of the TRPV1 pathway; a large number of samples can be processed in parallel through plate type design and reagent premixing, and the screening efficiency is improved by dozens of times; meanwhile, a receptor gene and a downstream early response gene are detected, so that an activation event can be confirmed, indirect evidence of pathway activity can be provided, and the result is more convincing.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a rapid detection kit for high-throughput screening of capsaicin receptor activation in cells and its application. Background Technology

[0002] Capsaicin receptor, or transient receptor potential vanillic acid subtype 1, is a non-selective cation channel primarily expressed on sensory neurons, playing a crucial role in pain, inflammation, thermoregulation, and energy metabolism. Therefore, TRPV1 has become an important target for the development of analgesics, anti-inflammatory drugs, and weight-loss drugs.

[0003] Currently, common methods for detecting TRPV1 activation include:

[0004] (1) Calcium ion imaging technology: the "gold standard" for functional detection, which can directly reflect the calcium influx after the channel is opened. However, this method requires expensive equipment, is complicated to operate, has low throughput, and is difficult to analyze a large number of molecular events at the same time.

[0005] (2) Western Blotting: It can be used to detect the expression or phosphorylation level of TRPV1 protein, but the operation steps are complicated, time-consuming, and the quantitative accuracy is poor, making it unsuitable for large-scale screening.

[0006] (3) Conventional qPCR detection: Long-term or persistent activation of the receptor can be indirectly assessed by detecting the mRNA level of TRPV1 or its downstream early response genes. However, the primer sequences used to detect these genes in the existing technology often have problems such as low specificity and low amplification efficiency, resulting in high background and poor reproducibility of the detection results.

[0007] Therefore, there is an urgent need in the field for a detection method that is fast, accurate, and particularly suitable for high-throughput operation modes, in order to efficiently screen and evaluate candidate substances targeting TRPV1. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a rapid detection kit for cellular capsaicin receptor activation for high-throughput screening and its application, which has the advantages of fast detection speed, high accuracy and convenience.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a rapid detection kit for capsaicin receptor activation in zebrafish cells, comprising an RNA purification column and an RNA collection tube or RNA capture plate, washing buffers A and B, DNase I digestion solution, 5x reverse transcription premix, 2x SYBR Green qPCR premix, and specific primer powder or premix.

[0011] According to a preferred embodiment of the present invention, the specific primers include primers for detecting the TRPV1 gene, the c-Fos gene, and the internal reference gene GAPDH.

[0012] Furthermore, the specific primer sequences used to detect TRPV1 gene expression are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0013] Furthermore, the specific primer sequences used to detect c-Fos gene expression are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] Furthermore, the specific primer sequences used to detect the expression of the internal reference gene GAPDH are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0015] A second aspect of the present invention provides an application of the kit described above, wherein the kit is used to detect the activation level of capsaicin receptors in zebrafish cells by establishing a zebrafish model.

[0016] Furthermore, as described above, the application determines the activation level of capsaicin receptors in zebrafish cells by detecting upregulation of TRPV1 gene expression and / or c-Fos gene expression.

[0017] A third aspect of the present invention provides an application of the kit described above, wherein a zebrafish model is established and the kit is used to screen for compounds or substances that can regulate the activation level of capsaicin receptors in zebrafish cells.

[0018] A fourth aspect of the present invention provides a method for screening compounds or substances that regulate the activation of capsaicin receptors in zebrafish cells using the kit described above, comprising the following steps:

[0019] S1. Cultivate zebrafish and set up a model group, a control group, and a sample treatment group;

[0020] S2. Add the compounds or substances to be screened to the culture medium of the sample processing group respectively;

[0021] S3. Using the kit described above, RNA extraction, reverse transcription, and qPCR detection were performed on zebrafish in the model group, control group, and sample treatment group. The effects of the substances to be screened on the expression of cell capsaicin receptor-related genes mRNA were analyzed to screen for substances that can affect the activation of cell capsaicin receptors.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. The novel primer sequences fundamentally ensure the reliability of qPCR data and can accurately reflect the activation status of the TRPV1 pathway.

[0024] 2. The plate design and premixed reagents enable it to process a large number of samples in parallel, making it a perfect fit for automated equipment and improving screening efficiency by dozens of times.

[0025] 3. Simultaneous detection of receptor genes and downstream early response genes can not only confirm activation events but also provide indirect evidence of pathway activity, making the results more convincing. Attached Figure Description

[0026] Figure 1 This is a comparison of qPCR amplification curves for different primer pairs, where... Figure 1 Figure A shows a comparison of qPCR amplification curves for different primer pairs of the TRPV1 gene. Figure 1 B is a comparison of qPCR amplification curves for different primer pairs of the c-Fos gene.

[0027] Figure 2 This is a comparison of qPCR melting curves for different primer pairs. Figure 2 Figure A shows a comparison of qPCR melting curves for different primer pairs used with the TRPV1 gene. Figure 2 B is a comparison of qPCR melting curves for different primer pairs of the c-Fos gene.

[0028] Figure 3 This is a comparison diagram of the relative expression levels of the TRPV1 gene detected by different primer pairs.

[0029] Figure 4 This is a comparison diagram of the relative expression levels of the c-Fos gene detected by different primer pairs.

[0030] Figure 5 This figure shows the effect of different treatment groups on the expression of the c-Fos gene in zebrafish larvae.

[0031] Figure 6 This is a graph showing the effect of different treatment groups on TRPV1 gene expression in zebrafish larvae. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1: Kit Composition

[0036] The kit in this embodiment contains:

[0037] 1) RNA purification column and RNA collection tube or RNA capture plate;

[0038] 2) Washing buffers A and B;

[0039] 3) DNase I digestion solution (used to remove genomic DNA);

[0040] 4) 5x reverse transcription premix (containing reverse transcriptase, RNase inhibitor, and dNTPs);

[0041] 5) 2x SYBR Green qPCR premix.

[0042] 6) Specific primer powder or premixed solution:

[0043] ①The TRPV1 primer mixture has the following nucleotide sequence:

[0044] Forward primer SEQ ID NO:1: 5'-CCTTAGGTGGAGACCCGATG-3';

[0045] Reverse primer SEQ ID NO:2: 5'-CCTTTGCTCTTCAACCTCGC-3'.

[0046] ② The c-Fos primer mixture has the following nucleotide sequence:

[0047] Forward primer SEQ ID NO.3: 5'-CTAGGACTTCTGCACGGACC-3';

[0048] Reverse primer SEQ ID NO.4: 5'-GGCCCTTATGCTCAATCTCCA-3'.

[0049] ③The GAPDH primer mixture has the following nucleotide sequence:

[0050] Forward primer SEQ ID NO.5: 5'-AAAGGGCCCTGACAACTCTT-3';

[0051] Reverse primer SEQ ID NO.6: 5'-TACATGACAAGGTGCGGCTC-3'.

[0052] 7) Nuclease-free water.

[0053] Example 2: High-throughput screening of TRPV1 agonists using this kit

[0054] 2.1 Experimental Procedure:

[0055] (1) Cell treatment and culture: HEK293 cells expressing TRPV1 were cultured at a density of 1*10 cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates. Candidate compounds included cinnamaldehyde and panthenol, with capsaicin (a known agonist) also included as a positive control, and a solvent (0.1% DMSO) as a negative control. After 24 hours of incubation, the cells were replaced with 10 μM of the sample culture medium and stimulated for 30 minutes. Three replicates were performed for each condition.

[0056] (2) RNA extraction from the plate: Discard the culture medium, add lysis buffer directly to the cells, and perform RNA binding, DNase I digestion, washing, and elution steps on a 96-well RNA binding plate according to the instructions. The entire process is carried out using a multichannel pipette or an automated workstation and takes approximately 45 minutes.

[0057] (3) Reverse transcription: The eluted RNA solution was directly mixed with 5x reverse transcription premix and nuclease-free water, and the reverse transcription reaction was performed on a PCR instrument (50℃ for 15 minutes, 85℃ for 5 minutes).

[0058] (4) qPCR detection:

[0059] ① Prepare the qPCR reaction system (10μL): 5μL 2x qPCR premix, 0.5μL specific primer mixture (such as TRPV1 or c-Fos), 2μL diluted cDNA, and 2.5μL nuclease-free water.

[0060] ② The reaction was performed using a qPCR plate with the following program: 95℃ for 30 seconds; 40 cycles (95℃ for 5 seconds, 60℃ for 30 seconds); melting curve analysis was performed.

[0061] (5) Data Analysis: Using 2 -ΔΔCt The method calculates the relative gene expression levels. Using GAPDH as an internal reference, the fold change in TRPV1 and c-Fos gene expression in the candidate compound treatment group relative to the solvent control group is calculated.

[0062] 2.2 Group settings:

[0063] (1) Primer effectiveness determination: The primers in Example 1 were named primer pair 1 and primer pair 5. The previously disclosed primers were named primer pair 2, primer pair 3, primer pair 4, primer pair 6, primer pair 7, and primer pair 8. The specific primer sequences are as follows:

[0064] TRPV1:

[0065] Primer pair 1: Forward primer: 5'-CCTTAGGTGGAGACCCGATG-3';

[0066] Reverse primer: 5'-CCTTTGCTCTTCAACCTCGC-3'.

[0067] Primer pair 2: Forward primer: 5'-GGAAGACAGATAGCCTGAAG-3';

[0068] Reverse primer: 5'-GAGAATGTAGGCCAAGACC-3'.

[0069] Primer pair 3: Forward primer: 5'-ACACATCATCAGCCAGGTGC-3';

[0070] Reverse primer: 5'-GCCAGGATGCTTGACAGATG-3'.

[0071] Primer pair 4: Forward primer: 5'-TCCAGAAGAGCCAAGCAGAC-3';

[0072] Reverse primer: 5'-CTGATGATGTGTGGACCCGTT-3'.

[0073] c-Fos:

[0074] Primer pair 5: Forward primer: 5'-AAAGGGCCCTGACAACTCTT-3';

[0075] Reverse primer: 5'-TACATGACAAGGTGCGGCTC-3'.

[0076] Primer pair 6: Forward primer: 5'-TGAAACTGACCAGCTTGAGGAT-3';

[0077] Reverse primer: 5'-GTGTGCGGCGAGGATGAA-3'.

[0078] Primer pair 7: Forward primer: 5'-CGTGGCAGGATCGTTTCTCT-3';

[0079] Reverse primer: 5'-CCACAGAGTACCTACCGCTTG-3'.

[0080] Primer pair 8: Forward primer: 5'-AGGGGGAGACCTTTCATCCA-3';

[0081] Reverse primer: 5'-GCAACCCACAGAGTACCTACC-3'.

[0082] (2) Screening of cellular capsaicin receptor activators:

[0083] The primers from Example 2.2 were used for substance screening, including the control group, capsaicin group, cinnamaldehyde group, and panthenol group.

[0084] Control group: Cells were cultured in normal cell culture medium (DMEM medium) containing 0.1% DMSO.

[0085] Capsaicin group: Cells were cultured in a medium containing 10 μM capsaicin and 0.1% DMSO.

[0086] Cinnamaldehyde group: Cells were cultured in a medium containing 10 μM cinnamaldehyde and 0.1% DMSO.

[0087] Panthenol group: Cells were cultured in a medium containing 10 μM panthenol and 0.1% DMSO.

[0088] The RNA extraction procedure was performed according to Example 2, using the kit described in Example 1. Subsequent gene expression level detection was performed using the sequences of preferred primer pair 1 and primer pair 5, and 2 -ΔΔCt The method calculates the relative expression levels of genes.

[0089] 2.3 Experimental Results:

[0090] like Figure 1 and Figure 2 As shown, all wells exhibited excellent qPCR amplification and melting curves, demonstrating high amplification efficiency and the absence of nonspecific products. Furthermore, primer pair 1 showed an earlier peak and better specificity than primer pairs 2, 3, and 4; primer pair 5 showed an earlier peak and better specificity than primer pairs 6, 7, and 8.

[0091] like Figure 3 and Figure 4 As shown, c-Fos and TRPV1 showed significant changes in gene expression when detected using primer pairs 1, 2, 3, and 4. Comparatively, primer pair 1 showed the most significant results.

[0092] Compared to the control group (containing only 0.1% DMSO), the capsaicin group significantly induced upregulation of c-Fos gene expression (more than 10-fold), while the expression of TRPV1 itself showed relatively small changes.

[0093] like Figure 5 and Figure 6As shown, the effects of cinnamaldehyde and panthenol on c-Fos and TRPV1 gene expression were examined. Compared to the control group (containing only 0.1% DMSO), the cinnamaldehyde group showed an approximately 8-fold upregulation of c-fos gene expression and an approximately 1.3-fold upregulation of TRPV1, consistent with the changes in capsaicin gene expression, while panthenol showed no significant change. This indicates that cinnamaldehyde is a novel molecule with potential TRPV1 agonist activity.

[0094] The above description is only a partial embodiment of the present invention and is not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made based on the description of the present invention fall within the protection scope of the present invention. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A rapid detection kit for capsaicin receptor activation in zebrafish cells, characterized in that, Includes RNA purification column and RNA collection tube or RNA capture plate, washing buffers A and B, DNase I digestion solution, 5x reverse transcription premix, 2x SYBR Green qPCR premix, and specific primer powder or premix.

2. The reagent kit according to claim 1, characterized in that, The specific primers include primers for detecting the TRPV1 gene, c-Fos gene, and internal reference gene GAPDH.

3. The reagent kit according to claim 2, characterized in that, The specific primer sequences used to detect TRPV1 gene expression are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The reagent kit according to claim 2, characterized in that, The specific primer sequences used to detect c-Fos gene expression are shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. The reagent kit according to claim 2, characterized in that, The specific primer sequences used to detect the expression of the internal reference gene GAPDH are shown in SEQ ID NO.5 and SEQ ID NO.

6.

6. The application of a reagent kit as described in any one of claims 1-5, characterized in that, By establishing a zebrafish model, the activation level of capsaicin receptors in zebrafish cells was detected using the kit described above.

7. The application according to claim 6, characterized in that, The activation level of capsaicin receptor in zebrafish cells was determined by detecting upregulation of TRPV1 gene expression and / or c-Fos gene expression.

8. The application of a reagent kit as described in any one of claims 1-5, characterized in that, By establishing a zebrafish model, the kit was used to screen for compounds or substances that could regulate the activation level of capsaicin receptors in zebrafish cells.

9. A method for screening compounds or substances that regulate capsaicin receptor activation in zebrafish cells using a kit as described in any one of claims 1-5, comprising the following steps: S1. Cultivate zebrafish and set up a model group, a control group, and a sample treatment group; S2. Add the compounds or substances to be screened to the culture medium of the sample processing group respectively; S3. Using the kit described above, RNA extraction, reverse transcription, and qPCR detection were performed on zebrafish in the model group, control group, and sample treatment group. The effects of the substances to be screened on the expression of cell capsaicin receptor-related genes mRNA were analyzed to screen for substances that can affect the activation of cell capsaicin receptors.

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