阅读说明：本技术 一种应用天然免疫激活剂抗病毒的方法 (Antiviral method using natural immune activator ) 是由 周翔 蔡苗苗 王柯 王晨光 蒋争凡 张同存 于 2020-07-14 设计创作，主要内容包括：本发明涉及一种应用小分子化合物氯喹那多抗病毒的方法,包括将有效浓度的氯喹那多应用于待处理的细胞中；本发明方法能有效激活I型干扰素信号通路,表达分泌大量的干扰素和干扰素刺激基因,从而强烈抑制病毒感染和复制,本发明方法安全、高效,使用简便易行,应用范围广泛。(The invention relates to an antiviral method for applying small molecular compound cloquindol, which comprises the steps of applying effective concentration of the cloquindol to cells to be treated; the method can effectively activate the I-type interferon signal pathway, express and secrete a large amount of interferon and interferon stimulating genes, and further strongly inhibit virus infection and replication.)

1. A method of using a small molecule compound, cloquindol, against a virus comprising applying an effective concentration of cloquindol to cells to be treated.

2. The method of claim 1, wherein the final concentration of chloroquinalder in the cell culture medium is 20 μ Μ.

3. The method of claim 2, wherein the cloquindol is diluted first to provide a 20mM working solution of cloquindol, as follows: cell culture fluid 1: 1000 to a final concentration of 20. mu.M in cell culture medium, shaking the culture, and culturing at 37 ℃ for 24 hours.

4. The method of any one of claims 1-3, wherein the chloroquinate is diluted with a sterile DMSO liquid.

5. The method according to any one of claims 1-3, wherein the method comprises the specific steps of:

1) culturing THP-1 cells, suspending and bringing them into a stable exponential division state:

cell recovery: rapidly taking out cells from a liquid nitrogen tank, rapidly shaking in water bath at 37 deg.C for 1-2min, and preventing water from entering into the freezing tube when the water surface is below the cover of the freezing tube;

transferring into a flat plate: taking a flat plate, adding 12ml of Gibco RPMI 1640 culture solution into the flat plate, paving the bottom of the flat plate, sucking all cells in a frozen tube into the flat plate, shaking up the cells in a shape like a character 8, observing the conditions in a microscopic mode, and then culturing at 37 ℃;

cell liquid change: after recovery overnight, observing that the culture solution turns yellow, at the moment, changing the solution, firstly sucking all the cell solution into a 15ml centrifuge tube, centrifuging for 5min at the speed of 900-;

the passage is carried out by adopting a direct passage method, namely ① natural sedimentation bottle bottoms of suspended cells, ② sucking supernatant 1/2-2/3 by using a suction pipe, ③ slightly blowing the supernatant into cell suspension, ④ equally filling the cell suspension into a plurality of culture bottles, and paying attention to the fact that the density of the cells is higher and is between 5 × 10⁵～10⁶Between ML, fast increment;

2) culturing HeLa cells, attaching to the wall, and bringing them into a stable exponential division state:

cell recovery: rapidly taking out cells from a liquid nitrogen tank, rapidly shaking in water bath at 37 deg.C for 1-2min, and preventing water from entering into the freezing tube when the water surface is below the cover of the freezing tube;

transferring into a flat plate: taking a flat plate, adding 12ml of Gibco DMEM culture solution into the flat plate, paving the bottom of the flat plate, sucking all cells in a frozen tube into the flat plate, shaking uniformly in a shape like a Chinese character '8', observing the condition in a microscopic mode, and then culturing at 37 ℃;

cell liquid change: after recovery overnight, observing the culture solution to turn yellow, at the moment, changing the culture solution, sucking the upper culture solution, adding new DMEM, and culturing at 37 ℃;

passage: adopting a trypsin digestion method: firstly, sucking supernatant by using a suction pipe; ② adding 1ml pancreatin to incubate for 2-4 min; adding new DMEM culture solution and lightly blowing and beating to form cell suspension; fourthly, according to the weight ratio of 1: 4-1: 6, equally dividing the cells into a plurality of new culture dishes, and carrying out passage once in 2-3 days generally, wherein the density of the cells is proper;

3) collecting mouse abdominal cavity macrophages, adhering to the wall, and using for antiviral experiment;

injecting 5ml of PBS precooled at 4 ℃ into the abdominal cavity of a BALBC mouse, gently softening the abdominal cavity of the BALBC mouse for 2-3min, standing for 5-7min, dislocating the cervical vertebra of the mouse to be killed, placing the mouse on a dissection plate, opening the abdominal cavity under aseptic conditions, extracting the abdominal cavity liquid by using an injector, centrifuging for 5min at 1000rpm/min, removing the supernatant, washing for 2 times by using the PBS, and then adjusting to 2 × 10 by using RPMI-1640 culture solution containing 10% fetal calf serum and 0.1% double antibody solution⁶Perml, inoculated in 6-well plate, placed at 37 ℃ with 5% CO₂Culturing in an incubator;

4) preparing a working solution of the chloroquinalder and applying the working solution to cells to be treated;

preparing a working solution: according to a 10-fold dilution method, a stock solution of the chloroquinate is diluted by a sterile DMSO liquid to reach a working concentration: 20 mM;

according to the working solution: cell culture fluid 1: 1000, adding the working solution of the chloroquinate into a cell culture medium to ensure that the final concentration of the chloroquinate in the cell culture medium is 20 mu M, shaking the culture evenly in a shape of 8, standing at 37 ℃ for culturing for 24 hours, and after the chloroquinate fully enters cells and activates an intracellular I-type interferon signal channel, expressing a large amount of I-type interferon and generating an antiviral effect.

Technical Field

The invention relates to an antiviral method by using a natural immune activator, in particular to a method for activating a natural immune type I interferon signal pathway by using a small molecular compound with high-efficiency antiviral capacity.

Background

Innate immunity is the first line of defense in the body against viral infection. Infection by the virus can activate the innate immune antiviral response. There are a series of Pattern Recognition Receptors (PRRs) in the innate immune system that recognize pathogen-associated molecular patterns of viral origin and recruit specific adaptor proteins to mediate downstream signaling pathways, leading to the expression and secretion of large amounts of type I interferons and inflammatory factors. Type I interferons include a single interferon beta (IFN β) and interferon alpha (IFN α) family of proteins. The secreted I-type interferon is combined with an interferon receptor positioned on the surface of a cell membrane, activates a downstream JAK-STAT signal channel, starts the expression of a large number of interferon stimulating genes ISGs, and plays a role in inhibiting the replication, proliferation and propagation of viruses. Therefore, the induced production of type I interferons plays a key role in the antiviral innate immune response.

Many stimulating factors can induce the body to produce type I interferon, and play the role of antivirus. These stimuli include: viral infection, stimulation of pathogen-associated molecular patterns of viral origin, overexpression of key protein molecules of the type I interferon signaling pathway, and the like. However, none of these stimuli has been applied to clinical antiviral therapy. Although interferon itself has an antiviral function, its action as a protein is influenced by activity and half-life, and has a certain limitation. How to find a medicine which can activate immune cells to generate type I interferon, does not depend on the substances of viruses, can meet the requirements of medicine activity and stability, and can be very effectively applied to the clinical treatment of virus infection.

Disclosure of Invention

The invention aims to overcome the defects of the existing antiviral clinical treatment medicines and provides an application small molecular compound of chloroquinaldol (C)₁₀H₇C_l2NO), which has a wide application range and controllable safety, strongly activates a type I interferon signaling pathway in cells, and generates an antiviral natural immune response.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of using a small molecule compound, cloquindol, against a virus comprising applying an effective concentration of cloquindol to cells to be treated.

Preferably, the final concentration of the chloroquinalder in the cell culture medium is 20. mu.M. More preferably, the cloquindol is diluted by a sterile DMSO solution to obtain a 20mM cloquindol working solution, and the ratio of the working solution: cell culture fluid 1: 1000 (while setting a concentration gradient within this range), add chloroquinate working solution to the cell culture medium so that the final concentration of chloroquinate in the cell culture medium is 20 μ M (a concentration gradient containing this concentration), shake the culture, and incubate at 37 ℃ for 24 hours while standing.

Further, the method comprises the following specific steps:

1. THP-1 cells were cultured (suspended) and brought into a stable state of exponential division.

Cell recovery: the cells were removed rapidly from the liquid nitrogen tank and then shaken rapidly in a 37 ℃ water bath for about 1-2min, with the water surface below the vial lid to prevent water from entering the vial.

Transferring into a flat plate: taking a flat plate, adding 12ml of Gibco RPMI 1640 culture solution into the flat plate, paving the bottom of the flat plate, sucking all cells in a frozen tube into the flat plate, shaking up the cells in a shape like a character 8, observing the condition in a microscopic mode, and then culturing the cells at 37 ℃;

cell liquid change: after the culture medium is recovered overnight, the culture medium is observed to turn yellow (2-3 days), at the moment, the culture medium is changed, all the cell sap is firstly sucked into a 15ml centrifuge tube, centrifuged for 5min at 900-1000r/min (the rotating speed can not be more than 1000), the upper culture medium is sucked off, new RPMI 1640 is added, the mixture is uniformly blown, all the mixture is sucked into a culture plate, and the culture is carried out at 37 ℃.

The passage is carried out by adopting a direct passage method, namely ① natural sedimentation bottle bottoms of suspended cells, ② sucking supernatant 1/2-2/3 by using a suction tube, ③ slightly blowing the supernatant into cell suspension, ④ equally filling the cell suspension into a plurality of culture bottles (dishes), and paying attention to the fact that the density of the cells is higher and is between 5 × 10⁵～10⁶Between ML, fast increment.

2. HeLa cells were cultured (adherent) and brought into a stable state of exponential division.

Cell recovery: the cells were removed rapidly from the liquid nitrogen tank and then shaken rapidly in a 37 ℃ water bath for about 1-2min, with the water surface below the vial lid to prevent water from entering the vial.

Transferring into a flat plate: taking a flat plate, adding 12ml of Gibco DMEM culture solution into the flat plate, paving the bottom of the flat plate, sucking all cells in a frozen tube into the flat plate, shaking uniformly in a shape like a Chinese character '8', observing the condition in a microscopic mode, and then culturing at 37 ℃;

cell liquid change: after overnight recovery, the culture medium was observed to yellow, at which time the medium was changed, the upper layer was aspirated, fresh DMEM was added, and the culture was incubated at 37 ℃.

Passage: adopting a trypsin digestion method: firstly, sucking supernatant by using a suction pipe; ② adding 1ml pancreatin to incubate for 2-4 min; adding new DMEM culture solution and lightly blowing and beating to form cell suspension; fourthly, according to the weight ratio of 1: 4-1: 6 aliquots were loaded into several new dishes. Note that the density of the cells is suitable, and the cells are generally passaged once in 2-3 days.

3. Mouse peritoneal macrophages (adherent) were collected for antiviral experiments.

Intraperitoneal injecting PBS 5ml precooled at 4 ℃ into BALBC mice, gently softening the abdomen of the mice for 2-3min, standing for 5-7min, dislocating cervical vertebrae of the mice to be killed, placing the mice on a dissection plate, opening the abdominal cavity under aseptic conditions, extracting the abdominal cavity liquid by using a syringe, centrifuging for 5min (1000rpm/min), removing the supernatant, washing for 2 times by using PBS, adjusting to 2 × 10 by using RPMI-1640 culture solution (0.1% double antibody solution) containing 10% fetal calf serum⁶Perml, inoculated in 6-well plate, placed at 37 ℃ with 5% CO₂Culturing in an incubator.

4. And preparing a chloroquinalder working solution and applying the chloroquinalder working solution to cells to be treated.

Preparing a working solution: according to a 10-fold dilution method, a stock solution of the chloroquinate is diluted by a sterile DMSO liquid to reach a working concentration: 20 mM.

According to the working solution: cell culture fluid 1: 1000 (while setting a concentration gradient within this range), a working solution of cloquindol was added to the cell culture medium so that the final concentration of cloquindol in the cell culture medium was 20 μ M (a concentration gradient containing this concentration). Shaking the 8-shaped culture evenly, standing at 37 ℃ for culturing for 24 hours, and after the cloquindol fully enters cells, activating intracellular I-type interferon signal channels and expressing a large amount of I-type interferon to generate antiviral effect.

5. Various biochemical, molecular biological, bioimaging methods identify the antiviral response of cells.

(1) Immunofluorescence imaging and flow cytometry verify that the chloroquinate strongly inhibits replication and amplification of virus particles with green fluorescent protein in cells.

After a certain period of infection, the virus particles entering the cells express GFP, and the infected cells are clearly seen to be green under the 488nm channel by a fluorescence microscope.

In order to prove that the chloroquinate can effectively inhibit the infection and the replication of the virus, a chloroquinate working solution with a certain concentration gradient is prepared, the chloroquinate working solution is added into a cell culture medium which grows in an exponential phase, after incubation for 24 hours at 37 ℃, different viruses expressing green fluorescent protein are added to infect cells, and after infection for 24-48 hours, the cells which are not incubated by the chloroquinate are taken as a control:

observing the number of cells expressing the green fluorescent protein of the virus after the co-incubation with the chloroquinalder and the number of cells infected with the virus in cells without the chloroquinalder under a fluorescence microscope;

secondly, analyzing the cells by using a GFP green fluorescent protein channel on a flow cytometer. Thereby, it was semi-quantitatively determined whether cloquindol could cause significant differences in the number of cells infected with the virus.

(2) The RT-PCR method verifies that the chloroquinalder activates the up-regulation of the transcription level of the I-type interferon gene in the cells.

Cloquindol exerts an antiviral immune response in cells, relying on its strong activation of the intracellular type I interferon signaling pathway. Thus, cloquindol can stimulate the up-regulation of the type I interferon gene at the transcriptional level in cells.

In order to verify that the cloquindol activates the up-regulation of the transcription level of the type I interferon gene in the cell, a working solution of the cloquindol with a certain concentration gradient is prepared and added into a cell culture medium which grows in an exponential phase, the cell is collected by removing the culture medium after incubation for 24 hours at 37 ℃, the total RNA in the cell is extracted by using a Trizol method, a cDNA template is obtained by using an oligo-dT primer, and then the primer of the type I interferon gene is used for amplification. The RT-PCR method can intuitively obtain the transcriptional up-regulation of the I-type interferon gene in the chloroquinalder stimulated cells.

(3) Western blotting confirmed that chloroquinate strongly inhibited the expression of intracellular viral proteins.

The intracellular antiviral response is accomplished by the activation of the innate immune signaling pathway type I interferon. Therefore, by detecting the activation state of the type I interferon signaling pathway in cells and the expression of interferon-stimulated genes, the effect of chloroquinate on the activation of the type I interferon signaling pathway can be responded.

In order to verify that the cloquindol can efficiently activate a type I interferon signal channel in a cell, a working solution containing a certain concentration gradient is prepared, the working solution is added into a cell culture medium which grows in an exponential phase, the cell culture medium is removed after incubation for 24 hours at 37 ℃, the cell is collected by removing the culture medium, the cell is lysed by a RIPA lysate, and the phosphorylation level of a key transcription factor IRF3 which is used for representing activation of the type I interferon in the cell and the expression quantity change of an interferon stimulating gene are detected by a Western blot method.

The invention has the advantages that:

1. the core reagent of the invention, namely the micromolecular chloroquinadol, is a high-efficiency activator of natural immunity, and can generate the effect of resisting virus infection very obviously.

2. The small molecular compound of the invention, namely the chloroquinadol, is a safe antiviral agent. In the experimental process, no matter the observation is carried out by a common microscope or a fluorescence microscope, no change of the cell state after the treatment of the chloroquinadol is found, and the micromolecule compound is proved to be a safe antiviral agent without obvious toxic and side effects to a great extent.

3. The micromolecule immune activator disclosed by the invention can rapidly penetrate through the cell surface to enter cells, is very simple and easy to use, and can induce the cells to generate antiviral reaction by direct co-incubation. Avoids the limitations of limited activity, short half-life and the need of blood injection administration of other types of interferon agonists.

4. The small molecular compound cloquindol can be suitable for various cultured cells, has a wide application range and is beneficial to the use of various antiviral stress occasions.

Description of the drawings:

FIG. 1 is a structural diagram of chloroquinalder,

FIG. 2 shows that chloroquinate inhibits replication and proliferation of DNA viruses,

FIG. 3 shows that the chloroquinate can inhibit the replication and proliferation of virus in THP-1 cell,

FIG. 4 shows that chloroquinate stimulates mouse cells to produce type I interferon,

FIG. 5 shows that cloquindol activates human THP-1 cells to produce type I interferon,

FIG. 6 is a graph of the antiviral effect of cloquindol at various concentrations.

Detailed Description

In the following examples, the structure of the used chloroquinaldol is shown in fig. 1, and the chloroquinaldol stock solution is prepared: the dry powder in the EP tube was centrifuged at a high speed at 4 ℃ and dissolved sufficiently in sterile DMSO in the amount shown in Table 1 to prepare a stock solution of the corresponding concentration. After the powder is completely dissolved, the EP tube is reversed and mixed evenly, and after short centrifugation at 4 ℃, the mixture is subpackaged in a sterile EP tube and can be stored for 2 years at-80 ℃.

TABLE 1 formulation of stock solution of chloroquinalder

Preparing a chloroquinate working solution:

taking out the packaged stock solution of the chloroquinader from a refrigerator at the temperature of-80 ℃, fully melting at normal temperature, diluting in a sterile DMSO liquid according to the use concentration, and immediately using. The working solution of the chloroquinadol is prepared and used at present.