WO2011054290A1 - Methods and reagent kits for determining the activity of thioredoxin reductase and the uses thereof - Google Patents

Abstract

Methods and reagent kits for determining the activity of thioredoxin reductase in a sample and the uses thereof are disclosed. The methods comprise determining the sulphydryl content of selen-compounds before and after treating the sample individually, consequently counting the activity of thioredoxin reductase in the sample by the difference of sulphydryl content before and after treating the sample. Additionally, the present invention also discloses the methods for evaluating the level of disease resistance in vivo, which comprise the step of determining the activity of thioredoxin reductase in a sample, and the step of counting the level of oxidative stress and the activity of thioredoxin reductase.

Description

 Method and kit for determining the activity of stearoxygen reductase in a sample. The present application relies on and claims the priority of Chinese Patent Application No. 200910207455.X filed on Nov. 3, 2009, the entire content of Incorporated herein.

Technical field:

 The invention belongs to the field of enzyme activity detection, and particularly relates to a method, reagent and application for measuring thioredoxin reductase activity in a sample. Background technique:

About 2 million people suffer from cancer every year in China, and 1.5 million people die of cancer, and they are still increasing, and they are gradually becoming younger. The direct economic losses caused by cancer to China each year exceed 100 billion yuan (from Beijing 4th, 2007/03/31). According to the Ministry of Health in 2007, the number of deaths due to cancer in China has reached 1.8 million in 2006, which is 300,000 higher than in 2005. Early human tumors have no sensations and symptoms, so only early physical examination can be detected early. However, at present, more than 80% of cancer patients are treated with symptoms after they have symptoms. Once diagnosed, they are not early. Most patients have metastasis and have lost the chance of surgery. Many patients with surgical tumors have to repeat Radiotherapy, chemotherapy to prevent recurrence and metastasis. Therefore, early diagnosis and treatment is one of the effective means to fight cancer, and early diagnosis of tumor is an important factor in determining its prognosis. As an important indicator, Tumor marker (TM) plays an increasingly important role in the early diagnosis of tumors. TM is a type of substance produced by tumor tissue, present in the tumor tissue itself, or secreted into blood or other body fluids, or produced by host cells by tumor tissue stimulation, which is significantly higher than the normal reference value. TM has certain application value in the treatment monitoring and prognosis of tumors. At present, there are more than one hundred kinds of tumor markers, but these tumor markers are mainly antibodies or proteins formed after tumor formation. By detecting these tumor markers, the occurrence of tumors and related abnormal proliferation can be detected to some extent. Related diseases Symptoms, but these markers cannot be used to predict the anti-tumor ability of the body before the onset of tumor or hyperplasia. Therefore, there is an urgent need in the art to develop new methods for evaluating the disease resistance in vivo before tumor onset, thereby predicting the body's ability to resist tumor or abnormal proliferation.

 The spirulina reductase system includes Thioredoxin reductase (TrxR), thioredoxin (Trx) and nicotinamide adenine dinucleotide phosphate (NADPH), which is a wide range. A distributed NADPH-dependent disulfide reductase system. Among them, Trx is a growth factor that can be produced by many cells and secreted by lymphocytes, hepatocytes and fibroblasts, as well as many cancer cells. The thiol redox activity of Trx has a general and important role in the maintenance of cellular physiological activity.

 Studies have shown that under oxygen stress, Trx and TrxR in lymphocytes and tumor cells, as well as some normal cells, can rapidly regulate and slow down the stress of oxygen stress. Under normal conditions, TrxR reduces the oxidation state of Trx. In the presence of NADPH, the reduced form of Trx acts as an electron donor for PRDX to reduce hydrogen peroxide to water. Hydrogen peroxide generated by ultraviolet light or the like induces the production of TrxR. However, persistent hydrogen peroxide induces a high level of tumor suppressor p53, which in turn negatively regulates TrxR, so that TrxR activity is better under the control of hydrogen peroxide. On the other hand, the lower the activity of TrxR, the higher the level of tumor suppressor p53 in the body, the higher the level of p53, the easier the body can make tumor cells tend to apoptosis, so the ability to fight tumors is stronger. Therefore, the level of TrxR activity reflects the anti-tumor level in vivo to some extent.

At present, TrxR activity is determined by the inhibition method of Aurothioglucose (see formula a). The working principle is: using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) Total reduction ability in the sample; After inhibiting the activity of TrxR in the sample with thioglucose gold, the reducing ability was measured by DTNB; the difference between the two reducing powers before and after the sample was calculated to determine the activity of TrxR in the sample. However, glucosinolate gold lacks specificity and can also block other sulfhydryl substances in the sample, and is not suitable for direct determination of TrxR activity in blood or tissue samples. 1" Column, KE, etc. (Determination of thioredoxin reductase activity in rat liver Anal Biochem. , 1997 , Nov l ;253(l):123-5) When glucosinolate is used to measure TrxR activity in rat liver pulp, dialysis should be performed on liver pulp to remove After the small molecule sulfhydryl-based substance (such as glutathione), the TrxR activity was determined by adding thioglucose gold. It can be seen that other small molecule sulfhydryl substances (such as glutathione) can interfere with the thioglucose gold pair.

Activity assay of TrxR. Another study showed that glucosinolate gold increased the rate of reaction of thiol-based substances with DTNB in blood (Hu ML, Dillard CJ, Tappel AL In vivo effects of aurothioglucose and sodium thioglucose on rat tissue sulfhydryl levels and plasma sulfhydryl reactivity. Agents Actions 1988, Aug; 25(l-2): 132-8).

 Therefore, there is an urgent need to solve the problem of low sensitivity and poor specificity in the detection of TrxR activity in a sample.

Summary of the invention:

 As mentioned above, overexpression of TrxR is closely related to the development of diseases, especially tumors. Therefore, accurate and reliable detection of TrxR levels or activity levels in tissues is extremely important for predicting the ability of the body to resist diseases such as tumors, however, There are many shortcomings in the detection of TrxR activity, such as pre-treatment of the sample, complicated operation, unreliable measurement results, and the like. It is an object of the present invention to provide a reagent for determining thioredoxin reductase activity in a sample, the reagents comprising separately present separately:

 An agent for determining the total thiol content in the sample; and

 Thioredoxin reductase specifically inhibits compounds.

Another object of the present invention is to provide a kit for determining thioredoxin reductase activity in a sample, the kit comprising the above reagent for determining thioredoxin reductase activity in a sample. Another object of the present invention is to provide a method for determining thioredoxin reductase activity in a sample, the method comprising:

 1) determining the total sulfhydryl content in the sample using the reagent for determining the total sulfhydryl content in the sample;

 2) using the thioredoxin reductase specific inhibitory compound to react with the sample, and then determining the content of the sulfhydryl group other than the sulfhydryl group in the thioredoxin reductase in the sample by the method in 1);

 3) Calculate the activity of thioredoxin reductase in the sample by subtracting the total thiol content in the sample obtained in 2) from the total thiol content in the sample obtained in 1).

 Another object of the invention is a method for determining thioredoxin reductase activity in a sample, the method comprising:

 1) reacting with a sample containing a solution containing 5,5-dithiobis(2-nitrobenzoic acid), and then measuring the absorbance value in the sample by ultraviolet light;

 2) adding the gram-reductase-reducing enzyme-specific inhibitory compound to the sample, and then measuring the absorbance value in the sample under the same conditions using the method in 1);

 3) Calculate the activity of the gram-reductive reductase in the sample by subtracting the absorbance value in the sample obtained in 2) from the absorbance value in the sample obtained in 1).

 Another object of the present invention is to provide a method for assessing the level of disease resistance in vivo, the method comprising:

 1) 测量 measuring the thioredoxin reductase activity in the sample by the above method;

 2) obtaining the level of oxidative stress in the sample;

 3) obtaining a ratio of the oxidative stress level to the thioredoxin reductase activity;

4) when the activity of the spirulina reductase is about 1 or less, the anti-disease level is high in the body; when the activity of the spirulina reductase is from about 1 or more to about 10 or less, and The ratio of the oxidative stress level to the oxidoreductin reductase activity is 5 to 10 or more, such as 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and preferably 5 or more, the in vivo anti-disease level is high; When the activity of the thioredoxin reductase is from about 1 or more to about 10 or less, and the ratio of the oxidative stress level to the thioredoxin reductase activity is 5 or less, the in vivo anti-disease level is low; The activity of the reoxygenase reductase is 10-20 or more, such as 9 or more, 10 or more, 11 or more, 12 or more, or 15 or more, but when it is preferably 10 or more, the anti-disease level in the body is reduced.

 Further, the present invention provides the use of the above agent for the preparation of a medicament for evaluating an anti-disease level in vivo.

 With the reagent or kit of the present invention, the TrxR activity in the sample can be effectively determined without any pretreatment of the sample, and the measurement result is more accurate and reliable than the measurement using thioglucose gold.

 Studies have shown that using the method of the present invention, a comprehensive evaluation system based on the evaluation of the function of the thioredoxin system by measuring the TrxR or OS/TR in the sample, and the antioxidant capacity/oxidative stress level is supplemented, The characteristics of the occurrence of diseases in the body indicate that the results evaluated by the method of the present invention are more than 80% consistent with the results of the clinical examination, and therefore, the method of the present invention can more accurately and sensitively predict the level of disease resistance in the body. DRAWINGS

 Figure 1. Inhibition of glutathione reductase by ethaneselen.

 Figure 2 is the effect of glutathione on the inhibition of ethaneselenase. Detailed ways:

 In the present invention, TrxR activity is inhibited by a specific inhibitor of TrxR, and the activity or content of TrxR in the sample is determined by measuring the TrxR activity in the biological sample by background subtraction.

The inventors have further found that by measuring the TrxR activity or content in a sample, and combining the ratio of oxidative stress (OS) level in the sample to the measured activity (TR) of TrxR, ie, OS/TR can be evaluated in vivo. Anti-disease, especially anti-tumor levels. The specific measurement principle is: 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB) can be reduced by the -SH group to produce 2-nitro-5-mercaptobenzoic acid (TNB), while TNB is at 405-412 nm. There is a strong UV absorption, so the amount of TNB can be detected by reflecting the amount of TNB produced in the sample.

 There are many components in the biological sample containing the -SH group, and the direct measurement of thioredoxin reductase in the sample with DTNB produces a very large error. However, the inventors have found that the compound of Formula 1 or Formula 2, especially the organic selenium compound I-XI in Table 1, can specifically react with the sulfhydryl group in TrxR as a specific inhibitor of TrxR, and it is difficult Reacts with sulfhydryl groups other than TrxR. The use of the compounds of the invention enables the determination of TrxR activity in complex systems, including blood and tissue extract samples.

 In addition, considering the complex changes in the body, such as dysplasia, when developing a variety of biological and physiological changes in the body, the accuracy of evaluating the level of disease resistance by measuring a single test index is very limited, in order to further improve the prediction of anti-disease, such as anti-tumor levels. The reliability of the present invention was first proposed by calculating the ratio of oxidative stress level to thioredoxin reductase activity in the sample, that is, OS/TR to the disease resistance in the body.

通式 1 其中： The reagent for determining the activity of streptohol reductase in the sample of the present invention contains at least an agent for determining the total thiol content in the sample and the above-mentioned thioredoxin reductase specific inhibitory compound, wherein the 'oxygen reductase specific enzyme "Suppressor compound" (hereinafter sometimes referred to as "inhibitor") refers to a compound highly selective for thioredoxin reductase, which is capable of specifically binding to thioredoxin reductase and inhibiting sulfhydryl groups therein. The preferred thioredoxin reductase specific inhibitory compound is a compound having the structure represented by the following Formula 1 or Formula 2, and it has been found that the compound of Formula 1 or Formula 2 used in the present invention is particularly It is a strong inhibitory effect of selenoline compound I-XI on TrxR. Up to now, this kind of compound is the only inhibitor with specific targeting of TrxR, which can effectively bind to SeCys/Cys activity in TrxR. The site acts as a suppressor to suppress TrxR with a high degree of selectivity, see Figure 1.

Formula 1 where:

 Xo is C or N;

X^ Se; is hydrogen, dC ₆ alkyl, C ₃ -C ₇ cycloalkyl, R ^{; 1} -

NCNR^eR^f、 CNR^eR^f或 R^g— OH; 其中 R^a、 R^b、 R^e、 R^f、 R^g 各自独立地为： d-C₆烷基； R^c , R^d各自独立地为： 氢、 卤素、 腈基、 C Ce烷基、 d-C₆烷氧基、 羟基、 S0₃R、 COOR、 聚乙二 醇、 0-S0₃R或 0-P0₃RR, 其中 R可以为氢、 d-C₆烷基或芳基； R₂和 R₃分别独立的选自： 氢、 卤素、 腈基、 d-C₆烷基、 d-C₆ 烷氧基、 羟基、 S0₃R、 COOR、 聚乙二醇、 0-S0₃R或 0-P0₃RR, 其中 R可以为氢、 d-C₆烷基或芳基， 其中芳基可以为苯基或苯 并基。

NCNR ^e R ^f , CNR ^e R ^f or R ^g — OH; wherein R ^a , R ^b , R ^e , R ^f , R ^g are each independently: dC ₆ alkyl; R ^c , R ^d are each independently: Hydrogen, halogen, nitrile group, C Ce alkyl group, dC ₆ alkoxy group, hydroxyl group, S0 ₃ R, COOR, polyethylene glycol, 0-S0 ₃ R or 0-P0 ₃ RR, wherein R can be hydrogen, dC ₆ alkyl or aryl; R ₂ and R ₃ are each independently selected from the group consisting of: hydrogen, halogen, nitrile group, dC ₆ alkyl group, dC ₆ alkoxy group, hydroxyl group, S0 ₃ R, COOR, polyethylene glycol, 0 -S0 ₃ R or 0-P0 ₃ RR, wherein R may be hydrogen, dC ₆ alkyl or aryl, wherein the aryl group may be phenyl or benzo.

通式 2 ,

Formula 2

among them:

Xo is C or N; X^ Se;

! ^ is alkylene, phenylene, biphenylene, cyclohexane, cyclopentane, -(CH ₂ ) _m SS(CH ₂ ) _m - , -((CH ₂ ) _m O) _n (CH ₂ ) _m - ,

-(CH ₂ )((CH ₂ ) _m O) _n (CH ₂ ) _m - , -(CH ₂ ) _m N(CH ₃ ) ₂ (CH ₂ ) _m - , -(CH ₂ ) _m NH(CH _{2 m} ) _m is an integer from 1 to 6, and n is an integer greater than one;

R' ₂ , R' ₃ , R'4 and R' 5 are each independently: hydrogen, [3⁄4], nitrile group, dC ₆ alkyl group, dC ₆ alkoxy group, hydroxyl group, S0 ₃ R, COOR, polyethyl b A diol, 0-S0 ₃ R or 0-P0 ₃ RR, wherein R can be hydrogen, dC ₆ alkyl or aryl, wherein the aryl group can be phenyl or benzo.

 Further, the selenium-based compound used in the present invention, in particular, the inhibitory effect of the organic selenium compound I-XI on TrxR is not interfered by other sulfhydryl substances in the system, and has high selectivity. Studies have shown that even in the presence of high concentrations of other sulfhydryl species in the sample system, such as 0.5 mM reduced glutathione, the selenium compounds used in the present invention, especially the organic selenium compound I-XI, still exhibit high selectivity for TrxR. For strong inhibition, see Figure 2.

 The most preferred thioredoxin reductase specific inhibitory compound in the present invention is the following compound I-XI.

 XI

 The "reagent for determining the total thiol content in the sample" in the present invention is not particularly limited as long as it can react with the thiol group and indicates the content in the sample. Preferred

The "reagent for determining the total thiol content in the sample" is 5,5-dithiobis(2-nitrobenzoic acid) (DTNB).

 The reagent for determining the total thiol content in the sample and the thioredoxin reductase specific inhibitory compound may be provided in any form such as a solid, a powder, a solution or the like. These two components may be provided in the form of a composition, but are preferably provided in a form which is independently present. The reagent for thioredoxin reductase activity or the thioredoxin reductase specific inhibitory compound in solution form is not particularly limited in concentration, and is preferably formulated into a concentration of 1 X working solution, more preferably formulated. The concentration of the 5 X working solution is most preferably formulated to a concentration of 10 Torr of working fluid.

In addition to the above two components, the reagent for determining the activity of thioredoxin reductase in the sample may further contain other components, for example, may contain NADPH, and may also contain other components required for measuring thioredoxin reductase activity, such as maintenance. activity stabilizing buffer components, including K ₂ HP0 ₄ and the like KH ₂ P0 _4, EDTA and / or the like BSA.

 The "sample" in the present invention means any tissue derived from a living organism or a portion separated therefrom, and the "sample" is preferably blood, body fluid, tissue tumbling liquid, and most preferably blood, wherein the blood may be serum, plasma, or the like. Component.

The thioredoxin reductase activity (TR) described in the present invention refers to an activity expressed by a thiol content in a thioredoxin reductase, wherein a thiol content in a sample is obtained by reacting a thiol group with a D TNB to produce a TNB at 405 nm. The absorbance at the place The calculation method of thioredoxin reductase activity (TR) is as follows:

^{ΔΑΑηίη χ 2} χ sample dilution factor _xf

 TR = 6.35x 0.02 χ Ι ΟΟΟ , where △ A/min = ( \ A sample - △ A sample inhibitor) ] / 7 min; Δ A sample = eight sample 420 s - A sample 0 s; Δ A sample inhibition Agent = Eight Sample Inhibitor 420s-A Sample Inhibitor 0s, resulting thioredoxin reductase activity, ie nanomolar amount of DTNB catalyzed per minute per mL of sample. The kit for measuring thioredoxin reductase activity in a sample of the present invention contains at least the reagent for determining the total thiol content in the sample and the thioredoxin reductase inhibiting compound, and may further contain other reagents. A component necessary for measuring the activity of the enzyme, such as a buffer or the like. The above various components may be packaged in a kit in any form, such as a bottle-shaped container or the like each containing the above-described different reagents or compounds. In addition, the kit may be provided in any form including, but not limited to, in the form of a square package.

 In the method for determining the activity of thioredoxin reductase in a sample according to the present invention, the total thiol content in the sample can be determined by any known method capable of indicating the total thiol content, including direct measurement of the thiol content. The method, or an indirect method capable of indicating an index of thiol content, the indicator includes an absorbance value or the like. When the sample is measured, different amounts of the above reagents or compounds are selected depending on the sample, and this is not particularly limited.

 In the method for evaluating the anti-disease level in vivo according to the present invention, the oxidative stress level can be determined by any known measurement method, such as the barbituric acid method, and the measurement TrxR of the present invention can also be utilized. The total Δ A in the sample measured in the method without the addition of the gram-reducing enzyme-specific inhibitor was calculated by Δ A sample X 224 . The OS/TR described in the present invention can be calculated by (ΔΑ sample x224)/TR.

In addition, when the activity of the spirulina reductase is 1 or less, The level of anti-disease in vivo is high; when the activity of the thioredoxin reductase is near 1 or above 1 to 10 or below 10, and the ratio of the oxidative stress level to the oxydoxin reductase activity is around 5 Or 5 or more, the anti-disease level in the body is high; when the activity of the strepto-reoxygenase reductase is 10 or more, the anti-disease level in the body is low.

 The "disease" as used in the present invention includes, but is not limited to, diseases such as abnormal hyperplasia, wherein the abnormal proliferation described herein includes a tumor, and includes diseases associated with abnormal hyperplasia, such as benign hyperplasia of medicine, atypical hyperplasia, and malignant hyperplasia. In addition, abnormal proliferation may also include combined abnormal proliferation.

 The present invention will be specifically described with reference to the embodiments, and the embodiments of the present invention are only intended to illustrate the technical solutions of the present invention, and do not limit the essence of the present invention.

Determination of strepto-reoxygenase reductase activity in blood:

Example 1

 1. Microplate reader measurement conditions

In the mode controlled by Ascent Software Version 2.6,

Oscillation: time 5s, speed 1020rpm;

 Measurement mode: continuous;

 Measurement type: dynamic;

 Time interval: 30s;

 Number of measurements: 15 ;

 Wavelength: 405 certificate.

 In the analysis of the actual blood sample, each analysis batch (Run) establishes a standard curve for calculating the concentration of the analyte in the analysis batch.

 2. Determination process

2.1 Preparation of working fluid: Mix 1M K ₂ HP0 ₄ 615ml, 1M KH ₂ P0 ₄ 385ml, adjust pH to 7.0, internal pressure EDTA 2924mg, B SA 200mg, dissolve, 4 °C save.

 2.2 Preparation of inhibitor working solution (25μ1): Dilute the stock solution of 20mM compound I to 25μΜ with working solution and store at 4°C in the dark.

 2.3 Pre-heat the microplate reader and set its measurement parameters: first shake and mix at 1020rpm for 5s, then measure the absorbance continuously at wavelength 405nm, interval 30s, measure 15 times, total time 7min. The blood sample stored at -20 °C was thawed into liquid at 4 °C, and the working solution and inhibitor working solution stored at 4 °C were placed on a shaker and mixed.

 2.4 Take a 96-well plate, set the sample well and inhibitor hole, and set up 3 sets of parallel experiments for each hole.

 2.5 The sample well and inhibitor well were added to the working solution 60μ1, 40μ1.

2.6 ? Preparation of each person 20 _μ 1 ρ preparation Wang Zuo.

 2.7 Samples L, p preparations Each human blood sample 20μ1.

 2.896 well plate immediately wrapped in tin foil, protected by light, mixed on a shaker for 5s, immediately incubated at 37 ° C for 30min.

 2.9 Preparation of DTNB working solution and NADPH working solution: According to the ratio of 600μ1 working solution + lmgNADPH, 4ml working solution + 15.9mg DTNB, avoid light, dissolve, shake at room temperature on a shaker.

 After the incubation at 2.1037 °C, add NADPH working solution 20μ1 to the sample well and suppression well at room temperature, avoiding light and mixing on a shaker for 5 s.

 2.11 Place the 96-well plate on the microplate reader, and quickly add ΙΟΟμΙ DTNB working solution to each well with a lance. After ensuring that there are no air bubbles, start the measurement according to the measurement conditions of the above-mentioned microplate reader.

Example 2-10

The same sample was measured in the same manner as in Example 1 except that the compound I in Example 1 was changed to the compound Π-ΧΙ shown in Table 1, and the results are shown in Table 1. Table 1 Results of TrxR activity in blood samples determined using various inhibitors

* : Because the blood samples of the subjects are limited, it is not possible to measure TrxR activity in the same blood sample with 11 compounds. Therefore, blood samples of subjects whose experience results are healthy are selected, and TrxR activity is measured under the same conditions. TrxR activity is less than 0, indicating that there is almost no TrxR activity in the body.

 Comparison of the effects of the reagent of the invention with the glucosinolate reagent:

 Three clinical examination subjects were selected, and TrxR activity in the blood of the above subjects was measured under the same conditions using the gram-deoxyglucose inhibition method and the inhibitory compound I of the present invention, respectively, and the results are shown in Table 2.

 Table 2 Comparison of the effects of the reagent of the present invention and glucosinolate reagent

 It can be seen from the above results that the TrxR activity measured by the present invention is significantly lower than that of TrxR activity measured by thioglucose gold, and the TrxR activity is related to the level of abnormal proliferation in vivo. The higher the value, the higher the degree of abnormal hyperplasia and the deviation of body health. The higher the degree of normality, the inhibitor of the present invention is more realistic and more reliable because it can specifically inhibit TrxR.

 Kit for the determination of thioredoxin reductase activity in blood:

 The kit of the present invention (100 test) may contain:

 1. DTNB powder 49.8mg, protected from light, stored at room temperature;

2. NADPH powder 4.2mg, protected from light, stored at -20 °C; 3. 10 χ working solution (1M potassium citrate buffer, PH = 7.4, containing 73.1mg EDTA, 5mg BSA.) 2mL, stored at 4 ° C;

 4. TrxR inhibitor solution (containing 2.5 mM inhibitor selected from one of compounds I-XI), lOOuL, stored at -20 ° C;

 5. Positive control: rat liver TrxR, 300U/mg Unit, stored at -20 °C;

6. TNB standard products;

 The method of using the kit of the invention:

 1. Take 2 mL of 10 χ working solution (0.5 M potassium citrate buffer, pH 7.4) and dilute to 20 mL with deionized water (containing 73.1 mg of ethylenediaminetetraacetic acid (EDTA), 5 mg of bovine serum albumin ( BSA)), get lx working solution, use at room temperature, can be used for 100-well detection;

2. Weigh 39.6 mg DTNB dissolved in 10mL lx working solution, store at room temperature in the dark, and use it now.

 3. Weigh 4.2 mg of NADPH dissolved in 2ml of lx working solution, avoid it at 4 °C, and return to room temperature before use.

 4. TrxR inhibitor working solution (25 μΜ): Take 10 μΐ inhibitor stock solution (2.5 mM), add 1 χ working solution, dilute to 1 mL, and test for 50 wells. Use at room temperature.

 Determination of TrxR by the kit of the invention

 1. Take 96-well plate, the bottom of the plate is clean and transparent, and there is no interference absorption at 405nm;

2. Sample well port: 20μ1 sample +60μ10.1M potassium phosphate buffer;

 3 · Sample + ? Preparation Ligu people: 20μ1 product +20μ1 preparation working fluid +40μ1

0.1M of 1 χ working solution (0.05 Μ potassium phosphate buffer);

 4. Place the 96-well plate after loading at 37 ° C, incubate for 1 h in the dark;

5. Add 20 μl of NADPH working solution to each sample well and apply again. Enter ΙΟΟμΙ DTNB working solution, immediately at room temperature, at 405 nm, record the initial absorbance (AOs) and continuously measure the absorbance of 420s (A420s). See Table 3 for the sample loading method for each hole. Way

 * Depending on the TrxR activity, there is no positive control group.

Alternatively, the kit can be prepared as a 200 Test or 50 Test package. The 10 X buffer in the kit should be a colorless and transparent liquid. After a long period of time, a small amount of visible protein can be precipitated, which is precipitated and precipitated by bovine serum albumin. It can be dissolved after ultrasonication and does not affect the determination. The TrxR activity inhibitor is a pale yellow, yellowish or colorless, clear, clear solution at room temperature, which is solid when stored at a temperature below the melting point of DMSO, and is used after melting. DTNB is a dry, pale yellow powder with no wet agglomerates and uniform powder particles. NADPH Na ₄ is a dry white or off-white amorphous powder.

 Evaluation of anti-disease levels in the body

 Using the kit of the present invention (containing Compound I), the activity of thioredoxin reductase in blood of 40 clinical examination subjects was measured, and the ratio of TR value to OS/TR was calculated according to the above calculation formula. By comparing the measured values with the physical examination results (see Table 4), it is found that:

 A: When the TR is less than 1, the physical examination is in good condition, and the body generally has a high anti-tumor ability;

B: When TR is about 1 to 10, and OS/TR is greater than 5, the physical examination is normal, but no tumor is found in the physical examination, and the body also has high anti-tumor ability; C: When TR is 1 to 10 Left and right, and when the OS/TR is less than about 5, or when TR is greater than about 10, tumors or early symptoms usually appear in the physical examination results, and the body has a low anti-tumor ability. Table 4 Evaluation of anti-tumor levels in vivo using TR and OS/TR

 Anti-tumor

Case number Physical examination status Physical examination results TR OS OS/TR tumor age

 The level of this inspection did not find any difference

 1-(155) Male 28 Health -22.014 27.788 - A Regular

 No swelling found

 1-(147) Force 30 B-ultrasound: Mild fatty liver -18.517 32.380 - A tumor

 Left front branch conduction resistance

 Stagnation, right ventricular hypertrophy, Bp:

 145/95mmHg, ECG

 Figure: The left side of the electric axis is -56°

 No swelling found

 1-(96) Force 45 Asymptomatic, T-CHO: -18.293 23.52 - A tumor

 6.72mmol/L, TG:

 2.32mmol/L,

 LDL-C :

 4.73mmol/L

 Ophthalmology: C/D=0.5-0.6

 Electrocardiogram: left ventricular high electricity, no swelling

 1-(36) Force 24 -15.928 9.259 - A Pressure Glu: 3.6mmol/L tumor

 T-Bil: 35.3umol/L

 This inspection did not find any difference

 1-(94) Force 22 Health -14.348 23.284 - A Regular

 This inspection did not find any difference

 1-(75) Male 31 Health -12.519 20.210 - A Regular

 This inspection did not find any difference

 1-(161) Female 21 Health -9.900 5.624 - A Regular

 This inspection did not find any difference

 1-(113) Male 24 Health -9.806 31.833 - A Regular

 This inspection did not find any difference

 1-(159) Female 22 Health -9.563 7.424 - A Regular

 This inspection did not find any difference

 1-(44) Male 23 Health -8.960 59.584 - A Regular

 This inspection did not find any difference

 1-(160) Female 22 Health -8.775 21.935 - A Regular

 This inspection did not find any difference

 1-(101) Male 27 Health -8.550 22.047 - A Regular

 This inspection did not find any difference

 1-(1) Female 35 Health -7.802 17.248 - A Regular

 This inspection did not find any difference

1-(23) Male 31 Health-5.450 19.588 - A Regular This inspection did not find any difference

 1-(108) Force 25 Health -5.400 19.348 - A often

 This inspection did not find any difference

 1-(153) Force 36 Health -3.820 31.049 - A Regular

 This inspection did not find any difference

 1-(134) Force 52 Health -3.262 22.047 - A Often

 This inspection did not find any difference

L-(99) Force 27 Health -3.185 28.548 - A often

 This inspection did not find any difference

 1-(110) Male 26 Health -2.475 21.597 - A Regular

 This inspection did not find any difference

L-(83) Female 31 Health 0.945 45.273 47.868 A Often

 T-Bil: 25.8umol/L

 No swelling found

L-(82) Force 28 Asymptomatic, WBC: 1.157 44.091 38.097 B tumor

 3.9*109/L

 This inspection did not find any difference

L-(28) Force 42 Health 1.780 14.672 8.245 B Often

 Bp: 120/90mmHg,

 Mild fat

 1-(141) Force 39 T-Bil: 22.1umol/L, 1.804 26.407 14.634 B Liver

 B-ultrasound: mild fatty liver

 Bp: 145/90mmHg

L-(43) Force 35 B Ultra: Fatty liver TG: Fatty liver 1.891 17.945 9.487 B

 2.61mmol/L

 No swelling found

 1-(14) Male 35 T-Bil: 23.3mmol/L 2.215 27.751 12.528 B tumor

 B ultrasound: liver edema

 2.6*1.6cm ECG

L-(42) Female 39 bloated 2.439 19.724 8.087 B Figure: Incomplete right bundle

 Block, asymptomatic

 Mild breast hyperplasia,

 T-CHO:

L-(2) Female 45 6.34mmol/L, mild hyperplasia 4.240 42.590 10.045 B

 LDL-C :

 4.31mmol/L

 B-ultrasound: hepatic left lobe edema

 2.0*1.8cm right hole

 Upper quadrant may nodule

L-(7) Female 46 bloated 5.699 44.169 7.750 B

 Bun : 7.8mmol/L

 PLT : 443*109/L

 Urine: Protein 3+

 -18.44 -4.099 l-(85) Female 31 double breast hyperplasia 4.500 C

 8 4

 TG: 2.97mmol/L, multiple polyps

 1-(105) Male 28 4.950 8.549 1.727 C

ALT: 52U/L, B-ultrasound: pathological lesions; Mild fatty liver, biliary edema

 Multiple polypoid lesions

 0.3*0.3cm, left renal pelvis

 Swollen 0.8*0.8cm, with sputum

 Wall calcification

 B-ultrasound: hepatic right lobe edema

 L-(89) Female 23 bloated 6.750 2.250 0.333 C

 1.2cm

 ApoB: 1033mg/L

 CK : 192U/L B

 L-(49) Male 34 Ultra: Mild fatty liver, stones 6.932 28.585 4.124 C Multiple stones in the left kidney, most

 Large 0.6cm

 Bp: 125/90mmHg

 ALT: 48U/L B Ultra:

 L-(40) Male 41 bloated 7.199 26.547 3.688 C fatty liver, liver sputum

 Swollen 2.5*2.0cm

 Moderate double breast hyperplasia

 L-(35) Female 23 Moderate hyperplasia 10.155 48.409 4.767 C

 T-Bil: 24.3umol/L

 T-Bil: 31.0umol/L

 L-(30) Male 53 B-moderate: mild fatty liver, bloated 10.349 50.319 4.862 C Liver right lobe edema 1.4cm

 B-ultrasound: fatty liver, gallbladder

 L-(34) Female 54 biliary stones 10.724 39.145 3.650 C 嚢 stones 1.2*1.1cm

 B-ultrasound: timid polyps-like timid polyps

 L-(54) Female 38 14.161 37.358 2.638 C lesion 0.3cm lesion

 0.5*0.5cm; cholestasis

 1-(154) Force 59 shows unsatisfactory, B-ultrasound: small edema 15.505 40.519 2.613 C multiple small edema in the liver

 B-ultrasound: Liver right posterior lobe blood

 1-(51) Male 31 Hemangioma 15.673 5.999 0.383 C Tumor 2.8*2.3cm

 T-CHO:

 6.67mmol/L mammary gland

 L-(46) Female 49 Moderate hyperplasia PLT: Moderate hyperplasia 16.103 42.137 2.617 C

 352*109/L TG:

 7.98mmol/L

In addition, the inventors also compared the consistency of the evaluation method of the present invention with the clinical diagnosis results, specifically: screening out of 300 subjects with absolute clinical normality (44 cases), absolute clinical evaluation of abnormal hyperplasia (17 cases) In the three groups of patients who have been diagnosed as tumors (20 cases), the anti-tumor levels in vivo in the three groups were evaluated by the kit of the present invention. The specific results are shown in Table 5. Table 5 Comparison of the consistency between this evaluation method and clinical diagnosis results

 It is apparent from the above results that the ratio of TR value and 0 S / T R measured by the kit of the present invention is clearly consistent with the physical examination condition, and the consistency thereof is more than 80%. Therefore, the ratio of TR to OS/TR can be used as an indicator of anti-tumor levels in vivo, which can effectively evaluate the anti-tumor level in the body.

 Industrial applicability

 As described above, according to the TR measuring method and kit of the present invention, it is possible to detect a sample, particularly a TR in a complex sample such as blood, with excellent sensitivity, and is very useful for measuring an antitumor level in vivo, and the reagent or method of the present invention. It can also be used for the assessment of the risk of tumorigenesis in vivo, as well as in the medical field such as tumor prognosis.

Claims

Rights request An agent for determining the activity of thioredoxin reductase in a sample, the reagents comprising: each independently present:  An agent for determining the total thiol content in the sample; and  Thioredoxin reductase specifically inhibits compounds.  The reagent according to claim 1, wherein the reagent for determining the total thiol content in the sample comprises 5,5-dithiobis(2-nitrobenzoic acid).  The reagent according to claim 1, wherein the thioredoxin reductase specific inhibitory compound comprises a porphyrin compound.  The reagent according to claim 3, wherein the selenoline compound is a compound represented by Formula 1.

Formula 1 where:  Xo is C or N; X^ Se; R, is hydrogen, alkyl, CVC ₇ cycloalkyl, R;: ^l —

R ^g — OH; wherein R ^a , R ^b R ^e , R ^f , R ^g are each independently: dC ₆ alkyl; R ^c , R ^d are each independently: hydrogen, halogen, nitrile, dC ₆ alkyl , dC ₆ alkoxy, hydroxyl, S0 ₃ R, COOR Polyethylene glycol, 0-S0 ₃ R or 0-P0 ₃ RR , wherein R can be hydrogen, dC ₆ alkyl or aryl; R ₂ and R ₃ are each independently selected from the group consisting of: hydrogen, halogen, nitrile, dC ₆ alkyl, dC ₆ alkoxy, hydroxy, S0 ₃ R, COOR, polyethylene glycol, 0-S0 ₃ R or 0- P0 ₃ RR, wherein R can be hydrogen, d-Ce alkyl or aryl.  The method according to claim 3, wherein the selenoid compound is a compound represented by the passage 2,

Formula 2 among them: X ₀ is C or N; X^ Se;  ! ^ is alkylene, phenylene, biphenylene, cyclohexane, cyclopentane of ^-^ -(CH ₂ ) _m SS(CH ₂ ) _m - ■((CH ₂ ) _m O) _n (CH ₂ ) _m - -(CH ₂ )((CH ₂ ) _m O) _n (CH ₂ ) _m - , -(CH ₂ ) _m N(CH ₃ ) ₂ (CH ₂ ) _m - , -(CH ₂ ) _m NH(CH ₂ ) _m , m is an integer from 1 to 6, and n is an integer greater than one; R' ₂ , R' ₃ , R'4 and R' 5 are each independently: hydrogen, [3⁄4], nitrile group, dC ₆ alkyl group, dC ₆ alkoxy group, hydroxyl group, S0 ₃ R, COOR, polyethyl b A diol, 0-S0 ₃ R or 0-P0 ₃ RR, wherein R can be hydrogen, d-Ce alkyl or aryl. The reagent according to claim 3, wherein the selenoid compound is /o//〇69s/-ososl>d 06ssonoiΜ

X , or

 XI.  A kit for determining thioredoxin reductase activity in a sample, the kit comprising the method for determining the activity of streptohol reductase in a sample according to any one of claims 1 to 6. Reagents.  8. A method for determining thioredoxin reductase activity in a sample, the method comprising:  1) determining the total sulfhydryl content in the sample using the reagent for determining the total sulfhydryl content in the sample in the reagent of claim 1;  2) using the thioredoxin reductase specific inhibitory factor in the reagent of claim 1 to react with the sample, and then using the method in 1) to determine the thiol group in the sample other than the thioredoxin reductase The content of sulfhydryl groups;  3) Calculate the activity of thioredoxin reductase in the sample by subtracting the total thiol content in the sample obtained in 2) from the total thiol content in the sample obtained in 1).  9. A method for determining thioredoxin reductase activity in a sample, the method comprising:  1) reacting with a sample containing a solution containing 5,5-dithiobis(2-nitrobenzoic acid), and then measuring the absorbance value in the sample by ultraviolet light;  2) adding the gram-reductase-reducing enzyme-specific inhibitory compound to the sample, and then measuring the absorbance value in the sample under the same conditions by the method in 1); 3) Subtract the sample obtained in 2) from the absorbance value in the sample obtained in 1) In the absorbance photometric value, the activity of the thioredoxin reductase in the sample is calculated.  10. A method of assessing an anti-disease level in vivo, the method comprising: 1) measuring the thioredoxin reductase activity in the sample by the method according to claim 8 or 9;  2) obtaining the level of oxidative stress in the sample;  3) obtaining a ratio of the oxidative stress level to the thioredoxin reductase activity;  4) when the activity of the spirulina reductase is about 1 or less, the anti-disease level is high in vivo; when the activity of the thioredoxin reductase is about 1 or more to about 10 or less, and the oxidation When the ratio of the stress level to the oxydoxin reductase activity is 5 to 10 or more, the anti-disease level is high in the body; when the activity of the spirulina reductase is from about 1 or more to about 10 or less, and When the ratio of oxidative stress level to oxyredoxin reductase activity is 10 to 5 or less, the anti-disease level is low in vivo; when the activity of spiroxane reductase is 10 to 20 or more, the anti-disease level in vivo is low. .  The method according to any one of claims 1 to 6 wherein the sample is a blood, body fluid or tissue homogenate.  The use of the reagent according to any one of claims 1 to 6 or the kit according to claim 7 for the preparation of a drug or a test agent for evaluating an anti-disease level in vivo.  13. The method of claim 10, wherein the disease comprises an abnormal proliferation or abnormal proliferation related disease in the body.  14. The use according to claim 12, wherein the disease comprises an abnormal proliferation or abnormal proliferation related disease in the body.