Tissue fibrosis detection method

Abstract

The invention discloses a method for detecting tissue fibrosis. The method comprises the following steps: (1) capturing a sample to be detected by using a bare gold electrode pretreated by a gelatin solution; (2) adding an anti-alpha-SMA primary antibody to react with alpha-SMA in the to-be-detected sample captured in the step (1); (3) adding a secondary antibody for resisting the primary antibody to react with the primary antibody combined on the cell to be detected, wherein the secondary antibody has a biotin label; (4) adding streptavidin to react with biotin on the secondary antibody combined with the primary antibody; and (5) adding the tetrahedral DNA nano material and a dichlorotris (1, 10-phenanthroline) ruthenium (II) hydrate for reaction, and then carrying out ECL detection to obtain an alpha-SMA expression signal, thereby detecting the fibrosis degree of the tissue. According to the method, the expression of alpha-SMA in tissue cells can be directly detected under the condition that tissue and cell structures are not damaged, and cells and tissues do not need to be homogenized.

Description

Technical Field

[0001] The 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, and its main pathological changes are the proliferation of fibrous connective tissue and the reduction of parenchymal cells in 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] At present, the main method for evaluating tissue fibrosis is through histological staining analysis, including Masson staining, Sirius red staining, immunohistochemistry and immunofluorescence staining, etc., to evaluate the expression of α-SMA. These methods can perform morphological analysis of tissues and semi-quantitative analysis of the degree of fibrosis, but cannot achieve accurate quantitative analysis. The expression of α-SMA can be detected by semi-quantitative analysis through immunohistochemistry, immunofluorescence and immunoblotting, while accurate quantitative analysis requires enzyme-linked immunosorbent assay. Summary of the invention

[0004] The purpose of the present invention is 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 solution of the present invention is 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) adding tetrahedral DNA nanomaterials and dichlorotri(1,10-phenanthroline)ruthenium(II) hydrate to react, and then performing ECL detection 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] Application of 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 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 dichlorotri(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 cells fixed with paraformaldehyde 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, and does not require homogenization of cells and tissues.

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

[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 The gold electrode in Example 2 of the present invention captures cells.

[0029] Figure 3 The electrochemical analysis method in Example 3 of the present invention is shown to detect the expression of α-SMA on the cell surface.

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

[0031] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination 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 a tetrahedral DNA nanomaterial. The ratio of the DNA sequences TA:TB:TC:TD was 1:1:1:1. Finally, the synthesized tetrahedral DNA nanomaterial was stored at 4°C for further use. In this example, the self-assembly of the tetrahedral DNA nanomaterial was evaluated by gel electrophoresis, as shown in Figure 1 As shown in the figure, the four oligonucleotide chains TA (red), TB (blue), TC (purple), and TD (green) hybridize into double-stranded DNA through the principle of complementary base pairing, and self-assemble into a DNA tetrahedron structure. In terms of electrophoresis, as more chains are hybridized, the molecular weight gradually increases and the electrophoresis speed gradually slows down.

[0034] Table 1 Nucleotide sequences of tetrahedral DNA nanomaterials

[0035]

[0036] Example 2 Gold electrodes directly capture cells

[0037] In this embodiment, 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 dripped 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 A, the scanning electron microscope can observe the existence of cell clusters on the surface of the electrode after the cell is captured, proving the success of cell capture. Secondly, the electrochemical impedance spectroscopy (EIS) method is used to verify the modification process of the electrode surface, and then verify the successful capture of the cell. The impedance of the gold electrode that captures the cell is greatly increased compared to the gold electrode that does not capture the cell. This is because the presence of cells on the electrode surface weakens the electron transfer at the electrode interface, which can indirectly prove that the gold electrode successfully captures the cell.

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

[0039] In this embodiment, after the gold electrode captures the cells, the anti-α-SMA antibody (Wuhan Seville Biotechnology Co., Ltd., GB13044, final concentration 500ng / mL) is added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Then, the biotin-labeled secondary antibody of the anti-α-SMA antibody (Hangzhou Huaan Biotechnology Co., Ltd., HA1108, final concentration 500ng / mL) is added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Then, streptavidin (Beijing Solebold Technology Co., Ltd., S9171, final concentration 1.25μg / mL) is 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 dichlorotri(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, rinsed with PBS buffer, and 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 AB, ECL can detect the α-SMA expression signal on the surface of fibroblast NIH-3T3. This example detects the α-SMA expression of 0, 10, 50, 100, 250, 500, 1000, 2500 and 5000 cells, and finds that the α-SMA expression signal increases with the increase in the number of cells. In addition, this method can detect the expression of α-SMA in extremely low cell amounts, and only 10 cells are needed to detect the α-SMA expression signal. This sensitivity is much higher than the detection sensitivity of traditional immunofluorescence, immunoblotting, Elisa and immunohistochemistry.

[0041] like Figure 3 As shown in CD, this example uses the traditional immunofluorescence method to detect the TGF-β signal-induced NIH-3T3 fibroblasts to myofibroblasts. It can be seen from the fluorescence imaging that cells begin to transform 36 hours after induction, and the transformation is more obvious after 72 hours. 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 the transformation of NIH-3T3 fibroblasts to myofibroblasts had significant statistical differences after 72 hours. At the same time, the immunoblotting method was used to detect the TGF-β signal-induced NIH-3T3 fibroblasts to myofibroblasts ( 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 the immunoblotting method was more sensitive, and a significant increase in α-SMA expression could be 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, and 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 compared to the expression of 48-72h α-SMA, there was no statistical difference. 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 signals 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 embodiment does not require homogenization of cells, does not destroy the structure of cells, 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 the signal amplified by tetrahedral DNA nanomaterials to detect the expression of α-SMA in tissue sections captured by gold electrodes

[0052] In this embodiment, ionizing radiation is used to induce pulmonary fibrosis in mice. Pulmonary fibrosis can occur in mouse lung tissue 3-5 months after irradiation, and the tissue is harvested. Paraffin-embedded pulmonary fibrosis tissue and control tissue slices are captured with a gold electrode (bare gold electrode treated with 0.1% gelatin) and then dried after capturing 10μm tissue slices. Anti-α-SMA antibody (Wuhan Seville Biotechnology Co., Ltd., GB13044, final concentration 500ng / mL) is added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Then, biotin-labeled secondary antibody (Hangzhou Hua'an Biotechnology Co., Ltd., HA1108, final concentration 500ng / mL) of anti-α-SMA antibody is added dropwise to the electrode surface, incubated at 37°C for 0.5h, and then rinsed with PBS buffer. Streptavidin (Beijing Solebold Technology Co., Ltd., S9171, 1.25μg / mL) is 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 dichlorotri(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, rinsed with PBS buffer, and 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 AC, conventional Masson staining and immunohistochemical analysis showed that ionizing radiation successfully induced pulmonary fibrosis. Collagen deposition and α-SMA expression began to increase in lung tissue 3 months after irradiation, and the phenomenon worsened further 5 months after irradiation. The ECL analysis method also detected the same trend. α-SMA expression began to increase in lung tissue 3 months later, and the increase was further aggravated after 5 months ( Figure 4 D) Compared with the traditional Masson staining and immunohistochemical analysis, the method of this embodiment does not require the dewaxing, dehydration, antigen repair, rehydration and sealing of paraffin sections, and the operation method is simpler. And by comparison, the detection method of this embodiment is also applicable to tissue detection and has reliability.

[0053] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent 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 cells 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) adding tetrahedral DNA nanomaterials and dichlorotri(1,10-phenanthroline)ruthenium(II) hydrate to react, and then performing ECL detection to obtain α-SMA expression signals, thereby detecting the degree of tissue fibrosis; 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.

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

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

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

1.

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

1.

6. Application of tetrahedral DNA nanomaterials in the preparation of a tissue fibrosis detection kit, characterized in that: 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.

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 dichlorotri(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 paraffin-embedded tissue sections.

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