A method for detecting tissue fibrosis

Abstract

The present invention discloses a method for detecting tissue fibrosis, comprising the following steps: (1) capturing a sample to be detected with a bare gold electrode pretreated with a gelatin solution; (2) adding an anti-α-SMA primary antibody to react with the α-SMA in the sample to be detected captured in step (1); (3) adding a secondary antibody against the primary antibody to react with the primary antibody bound to the cells to be detected, the secondary antibody having a biotin label; (4) adding streptavidin to react with the biotin on the secondary antibody bound to the primary antibody; (5) adding tetrahedral DNA nanomaterial and dichlorotris(1,10-o-phenanthroline)ruthenium(II) hydrate to react, and then performing ECL detection to obtain an α-SMA expression signal, thereby detecting the degree of tissue fibrosis. The present invention can directly detect the expression of α-SMA in tissue cells without destroying tissue and cell structure, without the need for homogenizing cells and tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and in particular relates to a method for detecting tissue fibrosis. Background Art

[0002] Fibrosis can occur in a variety of organs, with the main pathological changes being the proliferation of fibrous connective tissue and the reduction of parenchymal cells within organ tissues. If this condition continues to progress, it may lead to destruction of organ structure, functional impairment, and even failure, seriously threatening human health and life. Diseases involving fibrosis include pulmonary fibrosis, liver fibrosis, and renal fibrosis. Fibroblasts transform into myofibroblasts and express and secrete α-smooth muscle actin (α-SMA), the deposition of which in tissues is an important indicator for assessing tissue fibrosis.

[0003] Currently, the primary method for assessing tissue fibrosis is through histological staining, including Masson staining, Sirius red staining, immunohistochemistry, and immunofluorescence staining, to evaluate α-SMA expression. These methods enable morphological analysis of tissues and semi-quantitative analysis of the degree of fibrosis, but they are unable to achieve precise quantitative analysis. α-SMA expression can be semi-quantitatively analyzed using immunohistochemistry, immunofluorescence, and immunoblotting, while precise quantitative analysis requires enzyme-linked immunosorbent assay (ELISA). Summary of the Invention

[0004] The present invention aims to overcome the defects of the prior art and provide a method for detecting tissue fibrosis.

[0005] Another object of the present invention is to provide the use of tetrahedral DNA nanomaterials in the preparation of a tissue fibrosis detection kit.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for detecting tissue fibrosis comprises the following steps:

[0008] (1) Capturing the sample to be detected using a bare gold electrode pretreated with gelatin solution;

[0009] (2) adding an anti-α-SMA primary antibody to react with the α-SMA in the sample to be detected captured in step (1);

[0010] (3) adding a secondary antibody against the primary antibody to react with the primary antibody bound to the cells to be detected, wherein the secondary antibody is labeled with biotin;

[0011] (4) adding streptavidin to react with the biotin on the secondary antibody bound to the primary antibody;

[0012] (5) Tetrahedral DNA nanomaterials and dichlorotri(1,10-phenanthroline)ruthenium(II) hydrate were added for reaction, and then ECL detection was performed to obtain α-SMA expression signals, thereby detecting the degree of tissue fibrosis;

[0013] The tetrahedral DNA nanomaterial is self-assembled by hybridization of TA as shown in SEQ ID NO.01, TB as shown in SEQ ID NO.02, TC as shown in SEQ ID NO.03 and TD as shown in SEQ ID NO.04 through complementary base pairing.

[0014] In a preferred embodiment of the present invention, the concentration of the gelatin solution is 0.1%.

[0015] In a preferred embodiment of the present invention, the sample is a paraformaldehyde-fixed cell or a paraffin-embedded tissue section.

[0016] In a preferred embodiment of the present invention, the molar ratio of TA, TB, TC and TD is 1:1:1:1.

[0017] In a preferred embodiment of the present invention, the molar ratio of the tetrahedral DNA nanomaterial to dichlorotri(1,10-phenanthroline)ruthenium(II) hydrate is 2:1.

[0018] The invention relates to an application of a tetrahedral DNA nanomaterial in the preparation of a tissue fibrosis detection kit, wherein the tetrahedral DNA nanomaterial is self-assembled by hybridization of TA as shown in SEQ ID NO.01, TB as shown in SEQ ID NO.02, TC as shown in SEQ ID NO.03, and TD as shown in SEQ ID NO.04 through complementary base pairing.

[0019] In a preferred embodiment of the present invention, the molar ratio of TA, TB, TC and TD is 1:1:1:1.

[0020] In a preferred embodiment of the present invention, the tissue fibrosis detection kit further comprises a bare gold electrode, a gelatin solution, an anti-α-SMA primary antibody, a biotin-labeled secondary antibody against the primary antibody, streptavidin and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate, and the detection method used is the detection method described in any one of claims 1 to 5.

[0021] Further preferably, the concentration of the gelatin solution is 0.1%.

[0022] In a preferred embodiment of the present invention, the detection object of the tissue fibrosis detection kit is paraformaldehyde-fixed cells or paraffin-embedded tissue sections.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention can directly detect the expression of α-SMA in tissue cells without destroying the tissue and cell structure, without the need to homogenize cells and tissues.

[0025] 2. The present invention can accurately quantify α-SMA expression in cells and tissues, thereby assessing the degree of tissue fibrosis. The accuracy of the results is consistent with traditional immunoblotting, immunohistochemical staining, immunofluorescence staining, and Masson staining. Compared with traditional Masson staining and immunohistochemical staining methods, the present invention is simpler to operate.

[0026] 3. The present invention has high sensitivity and requires very few sample cells, with a minimum of only 10 cells required for detection and quantification, making the quantitative data more reliable and accurate, and providing an efficient and sensitive new tool for the study and diagnosis of tissue fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The synthesis and gel electrophoresis characterization of the tetrahedral DNA nanomaterial in Example 1 of the present invention are shown.

[0028] Figure 2 This shows the gold electrode capturing cells in Example 2 of the present invention.

[0029] Figure 3 This shows that the electrochemical analysis method in Example 3 of the present invention detects the expression of α-SMA on the cell surface.

[0030] Figure 4 The electrochemical analysis method in Example 4 of the present invention was used to detect α-SMA expression in tissue sections. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0032] Example 1 Synthesis and Characterization of Tetrahedral DNA Nanomaterials

[0033] A mixture of four oligonucleotides (the sequences of the four oligonucleotides are shown in Table 1, where the oligonucleotide chain TA is modified with biotin) was prepared in 1×TE-Mg buffer (10 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, pH 8.0) and heated at 95°C for 5 min. The mixture was then directly cooled to 4°C to form tetrahedral DNA nanomaterials. The ratio of DNA sequences TA:TB:TC:TD was 1:1:1:1. Finally, the synthesized tetrahedral DNA nanomaterials were stored at 4°C for further use. In this example, the self-assembly of tetrahedral DNA nanomaterials was evaluated by gel electrophoresis, as shown in FIG. Figure 1 As shown in Figure 1, four oligonucleotide chains—TA (red), TB (blue), TC (purple), and TD (green)—hybridize into double-stranded DNA through the principle of complementary base pairing, self-assembling into a DNA tetrahedron. Electrophoresis shows that as more chains are hybridized, the molecular weight increases and the electrophoretic speed slows.

[0034] Table 1 Nucleotide sequences of tetrahedral DNA nanomaterials

[0035]

[0036] Example 2 Direct cell capture by gold electrodes

[0037] In this embodiment, the surface of the bare gold electrode is treated with 0.1% gelatin, and then the cells to be detected (which can be fixed with 4% paraformaldehyde) are directly dropped onto the surface of the bare gold electrode and dried in a drying oven at 37°C. In this embodiment, two methods are used to detect whether the gold electrode surface has successfully captured cells. First, a scanning electron microscope is used to take a photo of the electrode surface to directly detect whether the gold electrode surface has successfully captured cells. Figure 2 As shown in Figure A, scanning electron microscopy (SEM) can detect cell clusters on the electrode surface after cell capture, confirming successful cell capture. Next, electrochemical impedance spectroscopy (EIS) is used to verify the electrode surface modification process and, in turn, the successful cell capture. The impedance of the gold electrode with captured cells is significantly higher than that of the gold electrode without captured cells. This is because the presence of cells on the electrode surface reduces electron transfer at the electrode interface, indirectly confirming the successful cell capture.

[0038] Example 3: Electrochemical analysis of the signal amplified by tetrahedral DNA nanomaterials to detect α-SMA expression in cells captured by gold electrodes

[0039] In this embodiment, after the gold electrode captures the cells, an anti-α-SMA antibody (Wuhan Seville Biotechnology Co., Ltd., GB13044, a final concentration of 500 ng / mL) is added dropwise to the electrode surface, incubated at 37°C for 0.5 h, and then rinsed with PBS buffer. Then, a biotin-labeled secondary antibody (Hangzhou Huaan Biotechnology Co., Ltd., HA1108, a final concentration of 500 ng / mL) of the anti-α-SMA antibody is added dropwise to the electrode surface, incubated at 37°C for 0.5 h, and then rinsed with PBS buffer. Streptavidin (Beijing Solaibao Technology Co., Ltd., S9171, a final concentration of 1.25 μg / mL) is then added dropwise, incubated at 37°C for 0.5 h, and then rinsed with PBS buffer. Finally, the tetrahedral DNA nanomaterial of Example 1 (final concentration 10 μmol / L) and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate ([Ru(phen)32]Cl2) (Sigma-Aldrich Co., Ltd. (Shanghai, China), final concentration 0.5 mmol / L) were added dropwise, incubated at 37°C for 0.5 h, and then rinsed with PBS buffer. The electrode was placed in a mixed solution of 2.0 mL PBS (10 mM, pH 7.4) and 20 mM co-reactant TPA for ECL detection (the detection cell was placed in a dark room).

[0040] like Figure 3 As shown in Figures AB, ECL can detect α-SMA expression signals on the surface of NIH-3T3 fibroblasts. This example detected α-SMA expression in 0, 10, 50, 100, 250, 500, 1000, 2500, and 5000 cells, and found that the α-SMA expression signal increased with increasing cell number. This method can also detect α-SMA expression in extremely low cell numbers, with as few as 10 cells required to detect α-SMA expression signals. This sensitivity is far higher than that of traditional immunofluorescence, immunoblotting, ELISA, and immunohistochemistry.

[0041] like Figure 3 As shown in CD, this embodiment uses the traditional immunofluorescence method to detect the TGF-β signal-induced NIH-3T3 fibroblasts to myofibroblast transformation. It can be seen from the fluorescence imaging that after 36 hours of induction, cells begin to transform, and after 72 hours, the transformation is more obvious. The positive percentage of α-SMA expression was quantitatively analyzed using Image J software, and it was found that the quantitative analysis of the immunofluorescence method showed that after 72 hours, the transformation of NIH-3T3 fibroblasts to myofibroblasts had a significant statistical difference. At the same time, the immunoblotting method was used to detect the TGF-β signal-induced NIH-3T3 fibroblasts to myofibroblast transformation ( Figure 3EF), and found that immunoblotting chemiluminescence imaging observation also showed that α-SMA expression began to increase after 36 hours of induction, and the increase in α-SMA expression became more obvious as the induction time prolonged. Relative quantitative analysis of α-SMA expression was performed using Image J software, and it was found that the quantitative analysis of immunoblotting method was more sensitive, and the increase in α-SMA expression was significantly detected after 48 hours. ECL method was used to analyze α-SMA expression ( Figure 3 G) quantitative analysis was performed, and it was found that the increased expression of α-SMA was significantly different after 24h. And as shown in Tables 2 to 4, the quantitative analysis of the immunofluorescence method showed that there was no statistical difference in the expression of 72h α-SMA compared to the expression of 12-60h. The quantitative analysis of the immunoblotting method showed that the expression of 72h α-SMA was statistically different from the expression of 12-36h α-SMA, but there was no statistical difference compared to the expression of 48-72h α-SMA. However, the ECL method showed that the expression of 72h α-SMA was statistically different from the expression of 12-60h, indicating that the method of this embodiment is more sensitive and accurate, and can distinguish small differences in α-SMA expression.

[0042] Table 2 Statistical analysis of α-SMA expression in NIH-3T3 cells induced by TGF-β signaling at different time points using ECL analysis

[0043]

[0044]

[0045] Table 3 Statistical analysis of α-SMA expression in NIH-3T3 cells induced by TGF-β signaling at different time points by immunofluorescence analysis

[0046]

[0047]

[0048] Table 4 Statistical analysis of α-SMA expression in NIH-3T3 cells induced by TGF-β signaling at different time points by immunoblotting

[0049]

[0050] In summary, compared with immunoblotting and ELISA, this example does not require homogenization of cells, does not destroy the cell structure, requires fewer cells, and is more accurate in quantification; compared with immunofluorescence staining, the quantification is more sensitive and accurate.

[0051] Example 4: Electrochemical analysis of α-SMA expression in tissue sections captured by gold electrodes using tetrahedral DNA nanomaterials to amplify signals

[0052] In this example, ionizing radiation was used to induce pulmonary fibrosis in mice. Pulmonary fibrosis occurred in the mouse lung tissue 3-5 months after irradiation, and the tissue was harvested. Paraffin-embedded pulmonary fibrosis tissue and control tissue slices were captured with a gold electrode (bare gold electrode treated with 0.1% gelatin) and then dried. Anti-α-SMA antibody (Wuhan Seville Biotechnology Co., Ltd., GB13044, final concentration 500 ng / mL) was added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Then, a biotin-labeled secondary antibody (Hangzhou Huaan Biotechnology Co., Ltd., HA1108, final concentration 500 ng / mL) of the anti-α-SMA antibody was added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Streptavidin (Beijing Solaibao Technology Co., Ltd., S9171, 1.25 μg / mL) was added dropwise, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Finally, the tetrahedral DNA nanomaterial of Example 1 (final concentration 10 μmol / L) and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate ([Ru(phen)32]Cl2) (Sigma-Aldrich Co., Ltd. (Shanghai, China), concentration 0.5 mmol / L) were added dropwise, incubated at 37°C for 0.5 h, and then rinsed with PBS buffer. The electrode was placed in a mixed solution of 2.0 mL PBS (10 mM, pH 7.4) and 20 mM co-reactant TPA for ECL detection (the detection cell was placed in a dark room). Figure 4 As shown in Figures AC, conventional Masson staining and immunohistochemistry analysis showed that ionizing radiation successfully induced pulmonary fibrosis. Three months after irradiation, collagen deposition began to appear in lung tissue, and α-SMA expression increased. This phenomenon worsened five months after irradiation. ECL analysis also detected the same trend. α-SMA expression began to increase in lung tissue three months after irradiation, and the increase worsened five months later. Figure 4 D) Compared to traditional Masson staining and immunohistochemical analysis, the method of this embodiment does not require the dewaxing, dehydration, antigen retrieval, and post-staining rehydration and mounting of paraffin sections, making it much simpler to use. Furthermore, by comparison, the detection method of this embodiment is also applicable to tissue testing and is reliable.

[0053] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for detecting tissue fibrosis, characterized in that: The steps include: (1) Capturing the sample to be detected using a bare gold electrode pretreated with gelatin solution; (2) adding an anti-α-SMA primary antibody to react with the α-SMA in the sample to be detected captured in step (1); (3) adding a secondary antibody against the primary antibody to react with the primary antibody bound to the sample to be detected, wherein the secondary antibody is labeled with biotin; (4) Adding streptavidin to react with the biotin on the secondary antibody bound to the primary antibody; (5) Tetrahedral DNA nanomaterials and dichlorotri(1,10-phenanthroline)ruthenium(II) hydrate were added to react, and then ECL detection was performed to obtain α-SMA expression signals, thereby detecting the degree of tissue fibrosis; The above-mentioned tetrahedral DNA nanomaterial is self-assembled by TA as shown in SEQ ID NO. 01, TB as shown in SEQ ID NO. 02, TC as shown in SEQ ID NO. 03 and TD as shown in SEQ ID NO. 04 through base complementary pairing hybridization.

2. The method for detecting tissue fibrosis according to claim 1, wherein: The concentration of the gelatin solution is 0.1%.

3. The method for detecting tissue fibrosis according to claim 1, wherein: The samples are cells fixed with paraformaldehyde or tissue sections embedded in paraffin.

4. The method for detecting tissue fibrosis according to claim 1, wherein: The molar ratio of TA, TB, TC and TD is 1:1:1:

1.

5. The method for detecting tissue fibrosis according to claim 1, wherein: The molar ratio of the tetrahedral DNA nanomaterial to dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate is 2:

1.

6. Use of tetrahedral DNA nanomaterials in the preparation of a tissue fibrosis detection kit, characterized in that: The tetrahedral DNA nanomaterial is self-assembled by base complementary pairing hybridization of TA as shown in SEQ ID NO. 01, TB as shown in SEQ ID NO. 02, TC as shown in SEQ ID NO. 03 and TD as shown in SEQ ID NO.

04.

7. The use according to claim 6, characterized in that: The molar ratio of TA, TB, TC and TD is 1:1:1:

1.

8. The use according to claim 6, characterized in that: The tissue fibrosis detection kit also includes a bare gold electrode, a gelatin solution, an anti-α-SMA primary antibody, a biotin-labeled secondary antibody against the primary antibody, streptavidin, and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate, and the detection method used is the detection method described in any one of claims 1 to 5.

9. The use according to claim 8, characterized in that: The concentration of the gelatin solution is 0.1%.

10. The use according to any one of claims 6 to 9, characterized in that: The detection object of the tissue fibrosis detection kit is cells fixed with paraformaldehyde or tissue sections embedded in paraffin.

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