CN110607362A - Biological diagnosis marker for chronic pain diseases and application of CXCL10 in preparation of chronic pain medicines - Google Patents

Abstract

The present invention provides a biological diagnostic marker for chronic pain diseases, which is characterized by comprising at least CXCL 10. The application of CXCL10 in preparing chronic pain medicine. A large number of experiments prove that the CXCL10 has obvious up-regulation expression in the blood and the cerebrospinal fluid of patients suffering from neuropathic pain, and the content of the CXCL10 in the cerebrospinal fluid and the blood is obviously related to the occurrence of chronic pain; animal experiments prove that the expression of CXCL10 in spinal cords and DRGs of neuropathic pain model mice is remarkably increased, and the effect target of knocking down CXCL10 or inhibiting CXCL10 in spinal cords can remarkably reduce pain behaviors of the mice. The invention also carries out quantitative analysis on blood and cerebrospinal fluid clinical samples of chronic pain patients, shows that the CXCL10 content change in the clinical samples can accurately reflect the pathological condition of neuropathic pain, can provide basis for prevention and prognosis judgment of neuropathic pain, and has good application prospect.

Description

Biological diagnosis marker for chronic pain diseases and application of CXCL10 in preparation of chronic pain medicines

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a biological diagnostic marker for chronic pain diseases and application of CXCL10 in preparation of chronic pain medicines.

Background

Neuropathic pain is a common chronic pain, which is newly defined by the international society for pain: it is a pain that is directly caused by injury and disease to the somatosensory system. Neuropathic Pain (NP) affects approximately 6% to 8% of the general population. In the past decades, despite the great progress made in the study of neuropathic pain, there has been a lack of effective analgesic drugs. Clinically, the drugs commonly used for treating neuropathic pain, such as opioids, tricyclic antidepressants, non-steroidal anti-inflammatory drugs, antiepileptics and the like, have certain treatment effect on some patients, but many patients still cannot obtain effective analgesia for a long time, so that the patients suffer from pain, and heavy burden is caused to families and society. With the continuous and intensive research, it is also recognized that there are many unknown factors for the development mechanism of neuropathic pain, and the search for relevant diagnostic markers before onset of neuropathic pain becomes very important. Not all patients with the same disease can eventually develop neuropathic pain, for example, some herpes zoster patients lose pain after they have disappeared, and some patients develop neuropathic pain that afflicts life-long. Therefore, the development of neuropathic pain needs to be understood, and a diagnosis molecule capable of accurately reflecting the development process of chronic pain needs to be found out, so that a detection kit is developed to intervene in advance, and the condition of a patient can be understood as soon as possible.

Chemokine 10(C-X-C motif ligand 10, CXCL10), also known as Interferon gamma-induced protein 10 (Interferon-gamma-induced protein, IP10), belongs to the CXC chemokine family. CXCL10 and CXCR3 have high affinity, and the CXCL10/CXCR3 axis formed by the CXCL10 and the CXCR3 plays an important role in the processes of immune-mediated virus infection, immune reaction, infiltration of tumorigenesis and the like. The application of CXCL10 as a gene for inhibiting chronic pain and as a diagnostic marker of chronic pain is not reported yet.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a biological diagnosis marker for chronic pain diseases and application of CXCL10 in preparation of chronic pain medicines.

In order to solve the above technical problems, an embodiment of the present invention provides a biological diagnostic marker for chronic pain diseases, which is characterized by comprising at least CXCL 10.

The embodiment of the invention also provides application of CXCL10 in preparation of a chronic pain medicine.

Wherein the medicament comprises CXCL 10.

The technical scheme of the invention has the following beneficial effects: a large number of experiments prove that the CXCL10 has obvious up-regulation expression in the blood and the cerebrospinal fluid of patients suffering from neuropathic pain, and the content of the CXCL10 in the cerebrospinal fluid and the blood is obviously related to the occurrence of chronic pain; animal experiments prove that the expression of CXCL10 in spinal cords and DRGs of neuropathic pain model mice is remarkably increased, and the effect target of knocking down CXCL10 or inhibiting CXCL10 in spinal cords can remarkably reduce pain behaviors of the mice. The invention also carries out quantitative analysis on blood and cerebrospinal fluid clinical samples of chronic pain patients, shows that the CXCL10 content change in the clinical samples can accurately reflect the pathological condition of neuropathic pain, can provide basis for prevention and prognosis judgment of neuropathic pain, and has good application prospect.

Drawings

FIG. 1 is a graph of the expression of mice CXCL9, CXC10 and CXCL11 in the spinal cord of the present invention;

wherein, FIG. 1A is the expression profile of CXCL9, CXC10 and CXCL11 in the spinal cord of mice analyzed by the semi-quantitative PCR result; FIG. 1B shows the expression of CXCL9, CXC10 and CXCL11 in spinal cord by fluorescence quantitative RT-PCR analysis, the ordinate represents Ct value, the lower the Ct value shows the higher gene expression, the CXC10Ct value is significantly lower than CXCL9 and CXCL10, which shows that the base expression of CXCL10 in spinal cord is significantly lowerIs higher than CXCL9 and CXCL11,^*P<0.05, one way ANOVA; FIG. 1C shows the basal expression levels of CXC10 in Spleen, popliteal Lymph node, DRG, Spinal cord and Brain of immune organs by fluorescent quantitative RT-PCR analysis, and the results show that the basal expression of CXCL10 in the above tissues is sequentially from high to low in Spleen (Spleen), Lymph node (Lymph node), Spinal cord (Spinal cord), Brain (Brain) and Dorsal Root Ganglion (DRG); FIG. 1D shows the semi-quantitative PCR analysis of CXC10 expression in Spleen (Spleen), Lymph node (Lymph node), Spinal cord (Spinal cord), Brain (Brain) and Dorsal Root Ganglion (DRG), with CXCL10 expression in these tissues being consistent with the results of FIG. C and β -actin being an internal reference gene.

FIG. 2 is a graph showing the tissue expression profile of mouse CXCR3 according to the present invention;

wherein, fig. 2A shows the basal expression content of CXCR3 in Spleen, popliteal Lymph node, DRG, Spinal cord and Brain of immune organs analyzed by fluorescent quantitative RT-PCR, and the results show that the basal expression of CXCR3 in the above tissues is Lymph node (lymphaden), Spleen (Spleen), Spinal cord (Spinal cord), Brain (Brain) and Dorsal Root Ganglion (DRG) in sequence from high to low; wherein FIG. 2B shows the semi-quantitative PCR analysis of CXCR3 expression in Spleen (Spleen), Lymph node (Lymph node), Spinal cord (Spinal cord), Brain (Brain) and Dorsal Root Ganglion (DRG), CXCR3 expression in these tissues is consistent with the results shown in FIG. 2B, and β -actin is an internal reference gene.

FIG. 3 is a graph comparing CXCL10 and CXCR3 expression in human spinal cord and lymph node tissue in accordance with the present invention;

wherein, fig. 3A is an RT-PCR result showing that CXC10 and CXCR3mRNA can be expressed in human spinal cord, DRG and lymph node in the order of lymph node, spinal cord and drg.; FIG. 3B is a Western Blot result further demonstrating that CXCR3 protein can be expressed in human spinal cord and lymph nodes; FIG. 3C is an ELISA result showing that CXCL10 protein can be expressed in human spinal cord and lymph node at a level higher than that of spinal cord,^***P<0.001，Student's t test。

FIG. 4 is a graph comparing the change in the amount of CXCL10 in cerebrospinal fluid of patients of various groups of patients in the present invention, NP indicates neuropathic pain,^*P<0.05，^**P<0.01，one way ANOVA。

FIG. 5 is a graph comparing the changes in the serum levels of CXCL10 in the patients of each group of patients according to the invention, NP indicates neuropathic pain,^*P<0.05，^**P<0.01，one way ANOVA。

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example one

A biological diagnostic marker for chronic pain disorders comprising at least chemokine 10.

Example two

Use of chemokine 10 in the manufacture of a medicament for chronic pain.

Wherein the medicament comprises chemokine 10.

Verification I, RT-PCR detection of CXCL10 and CXCR3 expression profiles in different tissues of mice

CXCL9, CXCL10 and CXCL1 can be used as ligands of CXCR3, and gene expression profiling analysis results show that CXCL9, CXCL10 and CXCL11 can be expressed in mouse spinal cord, but the expression level of CXCL10 in spinal cord is significantly higher than that of CXCL9 and CXCL10 (as shown in fig. 1A). Tissue expression profiling analysis shows that CXC10 can be expressed in immune organs and nerve tissues of normal mice, and the expression content of the CXC10 is spleen, lymph node, spinal cord, brain and DRG from high to low; in the nerve group, CXC10 was expressed in much higher amounts in the spinal cord than in the brain and spinal cord (see fig. 1B). Tissue expression profiling analysis also showed that CXCR3 can also be expressed in normal mouse immune organs and neural tissues with expression levels ranging from high to low in spleen, lymph node, spinal cord, brain and DRG, and its expression in neural tissues is not significantly different in spinal cord, brain and DRG (see fig. 2).

1.1 tissue total RNA extraction:

(1) mice were anesthetized by isoflurane inhalation, and 1ml Trizol was added to L5 spinal cords after perfusion with normal saline. Then, the mixture was homogenized by an electric homogenizer and allowed to stand on ice for 5 minutes after homogenization.

(2) 200 microliter of chloroform was added and shaken vigorously for 15s, and after standing for 2 minutes, centrifuged at 12000rpm at 4 ℃ for 10 minutes, and the supernatant was collected.

(3) Adding isopropanol with the same volume into the supernatant, gently mixing uniformly, and standing for 10 minutes. Centrifuge at 12000rpm for 10 minutes at 4 ℃ and discard the supernatant.

(4) The precipitate was washed with 1ml of absolute ethanol, centrifuged at 12000rpm at 4 ℃ for 10 minutes, and the supernatant was discarded.

(5) Air-drying at room temperature, adding 10 microliters of Free water, and promoting dissolution in a water bath at 60 ℃. The RNA concentration and purity were then determined using a nucleic acid protein analyzer.

1.2 reverse transcription (20 microliter system)

(1) Place 200. mu.l RNase-free centrifuge tube on ice, add the reaction:

table 1: DNA digestion system

(2) Centrifuging for a short time;

(3) placing in a gradient PCR instrument at 42 deg.C for 2 min;

(4) the following reaction solution was prepared on ice, added to the above mixture, and centrifuged for a short time.

Table 2: reverse transcription system

(5) In a gradient PCR instrument, the temperature is 37 ℃ and the time is 15 min; 85 ℃ for 5 s.

(6) Diluted 8 times for use and stored at 4 ℃.

1.3 primer sequence information

Designing a primer, selecting an mRNA sequence of a specific gene of a mouse in an NCBI database, designing the primer on an NCBI website, carrying out BLAST specificity verification, prejudging the size of an amplification product, and specifically verifying the specificity of the primer according to a melting curve and an agarose gel running fruit, wherein the primer sequence is synthesized by an industrial process or Invitrogen (Shanghai).

Table 3: primer sequence information of mouse CXCL9, CXCL11, CXCL10, CXCR3 and internal reference beta-actin

1.4 Real-time PCR

(1) The Real-time PCR reaction (10. mu.l) was configured as follows:

table 4: real-time PCR reaction system

(2) The reaction was performed using a Roche LightCycle 96 under the following conditions:

table 5: real-time PCR reaction conditions

(3) And analyzing the Ct value of each sample to determine the expression of CXCL9, CXCL10, CXCL11 and CXCR3 in different tissues according to the amplification curve.

1.5 RT-PCR and agarose gel electrophoresis

1.5.1 semi-quantitative RT-PCR

The samples, reaction systems and primers adopted by the semi-quantitative RT-PCR experiment are consistent with those of Real-time PCR, but the reaction cycle numbers are different: ct values of 28 were used to compare the expression levels of CXCL9, CXCL10, and CXCL11 in spinal cords, as shown in fig. 1A; ct values taken for comparing CXCL10 expression levels in spleen, lymph nodes, spinal cord, brain and DRG were 30, as shown in fig. 1B; ct values taken for comparison of CXCR3 expression levels in spleen, lymph nodes, spinal cord, brain and DRG were 35, as shown in fig. 2. The PCR product was subjected to agarose electrophoresis.

1.5.2 agarose gel electrophoresis

(1) Agarose gel with 3% concentration is prepared according to molecular weight, 3g of agar powder is mixed with 100ml of 1 XTBE buffer solution, the mixture is heated by a microwave oven, 5 microliter EB is added after agar is melted, and the mixture is poured into a gel tank after even mixing to prepare gel.

(2) 10.78g of Tris (hydroxymethyl) aminomethane, 5.51g of Borate, and 0.75g of EDTA were dissolved in 1L of an aqueous solution to prepare an electrophoretic solution.

(3) Selecting DL500DNA Marker of TAKARA company;

(4) constant voltage 70-100V electrophoresis, ultraviolet gel imaging.

Verification II, detecting the expression conditions of CXCL10 and CXCR3 in normal human immune tissues and nerve groups

Gene expression profiling analysis results show that CXCL10 and CXCR3 mrnas can be expressed in lymph nodes, spinal cord and DRG of normal humans, CXCL10mRNA expression is lymph nodes, spinal cord and DRG in sequence from high to low, and CXCR3mRNA expression is lymph nodes, DRG and spinal cord in sequence from high to low (fig. 3A). The WesternBlot results indicate that CXCR3 protein is expressed in human lymph nodes and spinal cord, with lower expression levels in spinal cord than in lymph nodes (fig. 3B). The results of WesternBlot showed that CXCL10 protein was expressed in normal human spinal cord and lymph nodes, with higher expression in lymph nodes than in spinal cord (fig. 3C).

2.1 semi-quantitative RT-PCR detection of CXCL10 and CXCR3 expression in human lymph nodes, spinal cord and DRG

The samples, reaction systems and primers adopted by the semi-quantitative RT-PCR experiment are consistent with those of Real-time PCR, but the reaction cycle numbers are different: ct value of 28 was used to compare the expression levels of CXCL9, CXCL10 and CXCL11 in spinal cords, as shown in FIGS. 1A-B, CXCL9, CXC10 and CXCL11 mRNAs can be expressed in the spinal cordIn the spinal cord of the mouse, the expression of beta-actin as an internal reference in each sample is kept unchanged; the smaller the Ct value is, the higher the gene expression content is, the Ct values of CXCL9, CXC10 and CXCL11 expressed in spinal cords are counted, and the result shows that the expression level of CXCL10 is obviously higher than that of CXCL9 and CXCL 11; comparing the expression level of CXCL10 in spleen, lymph node, spinal cord, brain and DRG with a Ct value of 30, as shown in FIGS. 1C-D, wherein CXCL10 is expressed in spleen and lymph node higher than DRG, spinal cord and brain, and β -actin is maintained as an internal reference in each tissue; comparing the expression level of CXCR3 in spleen, lymph nodes, spinal cord, brain and DRG using a Ct value of 35, as shown in fig. 2, CXCR3 can be expressed in spleen, popliteal lymph nodes, DRG, spinal cord and brain, where CXCR3 is expressed in spleen and lymph nodesThe amount is higher than in the neural group. Expression of CXCR3 was higher in spinal cord than in brain and drg. The PCR product was subjected to agarose electrophoresis.

Table 6: human CXCL10, CXCR3, and internal reference GAPDH primer sequence information

2.2 WesternBlot detection of CXCR3 expression in human spinal cord and lymph nodes

2.2.1 extraction of human tissue sample proteins

(1) Putting human lymph nodes, spinal cords and DRG tissues into prepared RIPA protein lysate, and adding 100 mul lysate/sample into the spinal cords;

(2) homogenizing on ice sufficiently by using a homogenizer, and placing the homogenized tissue on ice for 30 min;

(3) centrifuging at 4 deg.C and 15000RPM for 18min, collecting supernatant to obtain protein solution, and collecting precipitate;

(4) a part of the protein was taken and diluted with ultrapure water (spinal cord diluted 5-fold, DRG diluted 8-fold) to give a protein solution for concentration measurement.

2.2.2 BCA assay for protein concentration

(1) Using a 96-well plate, add bovine serum total protein standards (gradient concentration) in the first column, 10 μ l/well;

(2) adding protein diluent in the second row, wherein each hole is made with 10 mu l;

(3) preparing working solution, solution A: mixing solution B at 50:1 in dark, sucking with a discharge gun, adding 100 μ l/well, and incubating at 37 deg.C in dark for 30 min;

(4) the absorbance (OD value) at 562nm was measured using a microplate reader, and the protein concentration was converted from the standard curve.

2.2.3 protein denaturation

Calculating the protein loading amount according to the experiment requirement, calculating the protein loading volume of each hole according to the required protein amount/protein concentration, taking the protein loading amount of 30 mu g of total protein loading amount of each hole sample as an example, judging the total protein loading volume according to the sizes of different SDS gel lanes, adding SDS protein loading buffer solution (6X), not supplementing the protein volume with dd H2O, heating in boiling water for 5min after vortex oscillation, taking out, centrifuging for a short time, and storing at-20 ℃ for later use.

2.2.4 SDS-PAGE gel electrophoresis

2.2.4.1 glue making

(1) Selecting a corresponding glass plate according to the total volume of the protein sample loading, cleaning the glass plate, and drying in an oven;

(2) selecting corresponding separation gel according to the size of the protein, and preparing a plurality of layers of separation gel if necessary; taking the preparation of 10% polyacrylamide separation gel as an example: 10ml, ddH2O 2.7.7 ml; 3.3ml of 30 percent Acr-Bis; 1M Tris, pH8.83.8ml; 0.1ml of 10% SDS; 0.1ml of 10% AP; TEMED 0.004 ml. Mixing the above reagents, adding into a glass plate clamped in advance to about 2/3 of the interlayer height, adding isopropanol, pressing, and polymerizing at room temperature for 40min-1 h;

(3) preparing concentrated glue, wherein the concentrated glue is generally 5% in concentration, and the amount of 3ml is taken as an example: ddH2O 2.1 ml; 0.5ml of 30 percent Acr-Bis; 1M Tris, pH6.80.38ml; 0.03ml of 10% SDS; 0.03ml of 10% AP; mixing TEMED 0.003ml, adding lower layer gel with isopropanol removed, inserting corresponding comb (no air bubble generation), standing at room temperature for 40min-1h, and wrapping with wet gauze at 4 deg.C.

2.2.4.2 electrophoresis

(1) Fixing the gel plate on an electrophoresis device, and pouring 1X electrophoresis buffer solution without leakage; after the comb is pulled out, broken rubber in the lane is blown off by a gun head;

(2) sucking a sample with a corresponding volume, adding the sample into a lane, adding a protein pre-staining Marker, putting the lane into an electrophoresis tank, adding enough 1X electrophoresis buffer solution into the electrophoresis tank in advance, opening an instrument, adjusting voltage, running glue at constant voltage of 120V, changing the constant voltage of 80V when bromophenol blue runs to a separation glue position, and pre-judging the position of a strip by taking the Marker as a standard according to experimental requirements.

2.2.4.3 transfer film

(1) Cutting off the adhesive tape of the corresponding section according to a Marker, and putting the adhesive tape into a 1X rotating membrane buffer solution;

(2) selecting a corresponding PVDF membrane according to the molecular weight of the protein, wherein the large molecular weight is 0.45 mu m, the small molecular weight is 0.22 mu m, cutting the PVDF membrane according to an adhesive tape, and then putting the PVDF membrane into methanol for activation for 15 s;

(3) placing sponge and filter paper in advance into 1X membrane-rotating buffer solution, balancing for 15min, and carefully paving the negative plate (black), the sponge, the filter paper, the adhesive tape, the PVDF membrane, the filter paper, the sponge and the positive plate (white) in sequence without air bubbles (to avoid short circuit);

(4) placing the clamped device into an electrophoresis tank according to the corresponding sequence of the anode and the cathode, and adding 1X membrane-rotating buffer solution (paying attention to the position of the covering adhesive tape);

(5) placing ice blocks around the electrophoresis tank, preferably placing a biological ice bag, adding water to a proper position, selecting different membrane conversion conditions according to the molecular weight of the protein, generally selecting the protein with 30-100KD, and selecting a constant current of 300 mA for 1.5-2 h; the size of 200-400KD is properly adjusted according to the size of different molecular weights, taking 370KD as an example, 100mA is selected for constant current overnight film transfer for 17-20h (the film transfer device is placed in an environment with 4 ℃).

2.2.4.4 immune response

(1) Placing the transferred membrane in 5% skimmed milk or 5% BSA for sealing at room temperature, selecting sealing time according to different molecular weights, and generally selecting sealing for 2 h;

(2) primary anti-CXCR 3 (rabbitt, 1:100, Boster, BAO759) or GAPDH antibody (mouse,1:20000, Millipore, MAB374) was added. Diluting primary antibody (5% BSA or skimmed milk) according to the instruction, standing at room temperature for 1h, and standing at 4 deg.C overnight;

(3) washing the membrane with TBST for 3 times and 15 min/time after the next day rewarming for 1 h;

(4) secondary antibodies were added. The secondary antibody (5% BSA or skim milk) was diluted as per the instructions and protected from light for 2h at room temperature;

(5) washing the membrane with TBST for 3 times and 15 min/time;

(6) and (5) developing by using an instrument.

2.2.4.5 image analysis

Image J software is used for Image processing, and the gray value of each strip is counted; the ratio of the gray value of the CXCR3 band to the gray value of the internal reference GAPDH protein (both minus the background) is used as the final statistical data to represent the relative expression of the corresponding protein.

2.3ELISA detection of CXCL10 protein content in human spinal cord and lymph nodes

The expression of CXCL10 in Human spinal cord and lymph node tissues was tested using the Human CXCL10/IP-10 Quantikine ELISA Kit (Catalog #: DIP100) from R & D (FIG. 3C). The specific experimental process is as follows:

2.3.1 preparation work

(1) Dissolving a standard product: the Standard (Human IP-10 Standard) was dissolved with sterile water to a final concentration of 5000pg/mL, referenced to the resuspension volume indicated by the reagent bottle, and diluted with Calibrator dilution RD5K to 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL, and 7.8pg/mL for Standard curve generation.

(2) Sample preparation: human lymph node and spinal cord tissue proteins were extracted using RIPA lysate, the specific method was the same as that used for Western Blot detection, referred to "2.2.1 human tissue sample protein extraction". The extracted protein supernatant was diluted 10-fold with Calibrator dilution RD 5K: 10 μ L of protein supernatant +90 μ L of Calibrator DiluentRD 5K.

(3) Preparing a washing liquid: and adding 480mL of sterile water into 20mL of the Wash Buffer concentrated solution to prepare a Wash Buffer working solution.

2.3.2 working steps

(1) The kit is taken to the room temperature in advance, and the temperature is restored for 30min-60 min.

(2) Add 75. mu.L of Assay dilution RD1-56 per well.

(3) The standard wells were filled with 75. mu.L of standards (500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL, and 7.8pg/mL) at different concentrations, respectively, to prepare a standard curve; blank control and well 75. mu.L

Calibration dilution RD5K sample; the samples were air-diluted with 75. mu.l of Calibrator dilution RD 5K. After mixing gently, shaking and incubating at 500rpm for 2h at room temperature.

(4) The liquid was drained and the wash solution was added to the sample 5 times with a wash bottle, 400 μ L per well/time and then patted dry on paper.

(5) Add 200. mu.L of Human IP-10 Conjugate to each well and incubate at 500rpm with shaking for 2h at room temperature.

(6) The liquid was drained and the wash solution was added to the sample 5 times with a wash bottle, 400 μ L per well/time and then patted dry on paper.

(7) Add 200. mu.L of substrate reaction solution (A, B solution mixed in equal volume, care away from light) into each well, incubate at room temperature for 10-30min, and terminate at appropriate time according to the color change of the reaction.

(8) Adding 50 mu L of stop solution into each hole at a constant speed to stop the reaction, and slightly mixing the solution uniformly and then detecting and reading the solution by using an enzyme-labeling instrument. The OD of the sample at 450nm was read.

2.3.3 fitting a standard curve, calculating the sample concentration according to the OD value

Standards of different concentrations (500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.8pg/mL) and their corresponding OD values were plotted on Excel as scatter plots, to which were added a trend line, the regression analysis type was chosen linear, i.e. Y ═ a × + B, R2 ≧ 0.98. The OD value of each well sample was substituted into the equation to calculate the sample concentration.

Thirdly, ELISA is used for detecting the content of CXCL10 in cerebrospinal fluid and serum of patients in each group

CXCL10 is a secreted protein, and studies conducted in the present invention indicate that CXCL10 expressed by the spinal cord is secreted into cerebrospinal fluid and blood. Under neuropathic pain conditions, including acute neuropathic pain and chronic neuropathic pain, the content of CXCL10 in cerebrospinal fluid was significantly higher than in normal patients and inflammatory pain patients, as shown in fig. 4, the ELISA results showed that CXCL10 could be present in cerebrospinal fluid of normal persons, acute NP, chronic NP and inflammatory pain patients, in which the content of CXCL10 in cerebrospinal fluid of acute NP patients and chronic NP patients was significantly higher than in normal persons and inflammatory pain patients, with the results using t-test (Student's T-test), compared to normal group,^**P<0.01，^*P<0.05. the content of CXCL10 in the blood of the chronic neuropathic pain patients is obviously higher than that of the normal patients and the inflammatory pain patients, as shown in figure 5, the ELISA result shows that CXCL10 can exist in the serum of the normal people, the acute NP, the chronic NP and the inflammatory pain patients, wherein the content of CXCL10 in the serum of the chronic NP patients is obviously higher than that of the normal people and the inflammatory pain patients, and the result adopts t test (Student's T-test) to compare with the normal group,^**P<0.01。

3.1 cerebrospinal fluid sample Collection and handling

The cerebrospinal fluid is clear and transparent colorless or light yellow water sample liquid. Collecting 3mL cerebrospinal fluid of each group of patients by lumbar puncture method, subpackaging into 1.5 mL EP tubes, and freezing in-80 refrigerator for storage. Providing data relating to the number, sex, age, course of disease and VAS score of patients taking cerebrospinal fluid samples is shown in the following table

Table 7: providing patient-related information on cerebrospinal fluid samples of patients General information of patients (CSF)

3.2 serum sample preparation Collection and handling

5ml of fresh blood was placed in an SST serum separation tube at room temperature for 30 minutes, and after hemagglutination, 1000x g was centrifuged for 15 minutes. Then sucking the serum, subpackaging into 1.5 ml EP tubes, and freezing in a-80 refrigerator for storage. Providing data relating to the number, sex, age, course of disease and VAS score of patients taking cerebrospinal fluid samples is shown in the following table

Table 8: providing patient-related information on Serum samples of patients General information of patients (Serum)

3.3 ELISA procedure and procedure

The specific operation process of detecting cerebrospinal fluid and ELISA is consistent with the method of detecting the content of CXCL10 protein in human spinal cord and lymph node by 2.3 ELISA.

When the content of CXCL10 in cerebrospinal fluid and serum is detected, a sterile water-soluble Standard (Human IP-10 Standard) is subjected to gradient dilution by a calibration dilution RD6Q to obtain concentrations of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.8pg/mL for preparing a Standard curve.

In the invention, in the adopted Spinal nerve ligation induced (SNL) neuropathic pain model, the CXCL10/CXCR3 pathway is blocked or weakened, so that the mechanical hyperalgesia and thermal hyperalgesia of animals can be relieved. The following problems were specifically verified: (1) CXCL10 and its receptor CXCR3 expression was increased in the spinal cord of mice. The mRNA content of CXCL10 is obviously increased at SNL 3d and is continued to 21d, and the ELISA result shows that the protein content is obviously increased at SNL 10 d; (2) immunofluorescence results showed that CXCL10 was distributed primarily in neurons and astrocytes after SNL 10 d; (3) intrathecal injection of CXCL10 can induce hyperalgesia in mice and phosphorylate ERK; (4) behaviorally, both CXCR3 gene knock-out, spinal cord injection of CXCR3 shRNA interfering lentiviruses, and intrathecal injection of CXCR3 antagonists can reduce the mechanical and thermal hyperalgesia phenomena caused by SNL; (5) compared with a sham operation group, the protein of CXCL10 in the serum of the mouse is obviously increased 1 day and 10 days after SNL, and the content of the protein in cerebrospinal fluid is also obviously increased 10 days after SNL; therefore, in experimental animals, CXCL10/CXCR3 can be proved to be involved in the regulation of neuropathic pain, and the content change of CXCL10 in blood and cerebrospinal fluid can reflect the development condition of neuropathic pain, so that the method has diagnostic value.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (3)

1. A biological diagnostic marker for chronic pain disorders, comprising at least CXCL 10.

Use of CXCL10 in the preparation of a medicament for chronic pain.

3. The use of CXCL10 for the preparation of a medicament for chronic pain according to claim 2, wherein the medicament comprises CXCL 10.