CN115323034A - Rapid evaluation method for activity of tryptophan metabolism rate-limiting enzyme - Google Patents
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Abstract
The invention relates to a rapid evaluation method for tryptophan metabolism rate-limiting enzyme activity and application thereof. The method comprises the following specific steps: s1, incubating target cells; s2, performing contrast culture; s3, preparing gradient standard solution by kynurenine; and S4, developing the target cells and the gradient standard solution, and detecting. The invention overcomes the defect of large data deviation commonly existing in the IDO1 detection method by controlling the specific culture medium and the culture process conditions; the activity of the indoleamine2,3-dioxygenase1 (IDO 1) of the mesenchymal stem cell can be rapidly detected in a short time, stable and credible data can be obtained, and the detection efficiency of the IDO1 activity of the mesenchymal stem cell is greatly improved; meanwhile, the method is convenient to operate and has wide application prospects.
Description
Technical Field
The invention relates to a rapid evaluation method for activity of tryptophan metabolism rate-limiting enzyme and application thereof.
Background
Indoleamine2,3-dioxygenase1 (IDO 1) is a rate-limiting enzyme for mediating tryptophan along the kynurenine pathway, regulates tumor immunity by degrading tryptophan-tryptophan starvation environment and producing metabolites, and is a potential tumor immunotherapy drug target. The detection of IDO1 activity is currently carried out mainly by detecting tryptophan or its metabolites (e.g.formylkynurenine, kynurenine). The mature measuring means in the prior art mainly comprise the following steps: 1. high Performance Liquid Chromatography (HPLC): detection of IDO1 Activity by high performance liquid chromatography was first proposed in 1990 by Beal MF et al to measure IDO1 enzyme activity by measuring the content of kynurenine produced by tryptophan and degradation. The method has the advantages of high accuracy, small sample amount, good specificity and no interference of other substances, and has the disadvantages of setting different experimental conditions aiming at different samples to reduce tailing peaks and improve peak shapes, along with poor universality; 2. and (3) measuring absorbance: tamantha et al proposed an absorbance assay based on Takikawa's theory. The method needs to temporarily prepare a mixed IDO1 enzyme reaction system, and determines the activity of the IDO1 enzyme by detecting the absorbance of a complex generated by the reaction of kynurenine and p-dimethylamino benzene, namely p-DMAB, by using an enzyme label plate; the method is simple and convenient, but has high requirement on the freshness of the reaction solution, needs to be prepared at present (particularly ascorbic acid and catalase), and brings inconvenience to practical application; 3. fluorescence detection: the fluorescence detection object can directly select formyl kynurenine or hydrolyzed kynurenine. Formyl kynurenine is a direct product of tryptophan catalyzed by IDO1 enzyme, has high selectivity, but is easy to quench due to short excitation wavelength and needs to be derivatized by piperidine; the fluorescence method has high sensitivity and small dosage, can be used for enzyme labeling to carry out high-throughput screening of IDO1 enzyme inhibitors, but the method needs strong acid with pH =1 and strong base with pH =11, piperidine has pungent smell and high risk coefficient, and limits wide application of the method in the clinical field.
Chinese patent CN202011561297.0 discloses a method for detecting the activity of immunoregulatory stem cell IDO1 meeting the product release requirements, which uses a culture medium containing/not containing IFN-gamma to act on a test sample to be detected to detect the relative biological activity of the test sample, and cn201680053185.X discloses a screening assay for identifying IDO1 and/or TDO modulators, which uses 520-560nm fluorescence to measure the expression of cellular IDO1 after contacting with kynurenine receptors. In the prior art, the detection precision of the activity of the human mesenchymal stem cell IDO1 is low, the RSD standard of the prior industry is not more than 20 percent, namely the product is qualified, and the improvement of the data quality is in a bottleneck breakthrough stage. The invention explores an IDO1 detection method which is high in accuracy, simple, convenient, safe and practical, reduces the data RSD to be below 10%, greatly improves the data quality, and effectively promotes the accuracy of the IDO1 activity assessment method.
Disclosure of Invention
The invention provides a rapid evaluation method for the activity of tryptophan metabolism rate-limiting enzyme, which overcomes the defects of low detection efficiency and inaccurate detection result of the prior art for the activity of human mesenchymal stem cells IDO1 and realizes the rapid evaluation method for the activity of tryptophan metabolism rate-limiting enzyme, which has high accuracy, is simple, convenient, safe and practical.
In order to solve the above problems, the first aspect of the present invention provides a method for rapidly evaluating the activity of a tryptophan metabolism rate-limiting enzyme, comprising the steps of:
s1, incubating target cells;
s2, performing contrast culture on the incubated target cells by adopting a first culture medium and a second culture medium;
s3, preparing kynurenine gradient standard solution;
s4, performing color development and detection on the target cells and the kynurenine gradient standard solution after contrast culture in the step S2;
the first medium and the second medium are distinguished in that: the first medium does not contain gamma interferon, and the second medium contains gamma interferon.
In some preferred embodiments, the raw materials of the first medium are MM3 medium, and the raw materials of the second medium are MM3 medium and gamma interferon; the MM3 culture solution is a mixture of a serum substitute and a basic serum-free culture solution, and the volume ratio of the serum substitute to the basic serum-free culture solution is 1:19.
in some preferred embodiments, the incubating the target cells in the S1 step is specifically: the target cells were inoculated into the first medium using a 24-well plate in which 0.3-0.5mL of the first medium was added per well in 12 wells and incubated for 22-26h.
In some preferred embodiments, the seeded target cell density is 4-6X 10 4 One for each well.
In some preferred embodiments, the control culture in the S2 step is specifically to aspirate the culture medium from 12 wells and add the first culture medium to 6 wells; adding a second culture medium into another 6 holes, and performing contrast culture for 22-26h to obtain a culture solution A and a culture solution B; wherein culture solution A is a control culture product of the first culture medium, and culture solution B is a control culture product of the second culture medium.
In some preferred embodiments, the feed stock for the second medium comprises MM3 broth and gamma interferon.
Further preferably, the second medium has a concentration of 5 to 15ng/mL by mass of gamma interferon, and the balance is supplemented with MM3 medium.
Still more preferably, the concentration by mass of gamma interferon in the second medium is 10 ng/mL.
In some preferred embodiments, the concentration of kynurenine in the kynurenine gradient standard solution is in the range of 0-600 μ M, and the solvent is ultrapure water.
In some preferred embodiments, the step S4 is to take the gradient standard solutions prepared in the steps S2, S3 and S3, respectively, to react with an organic acid, then react with a color-developing agent, and after the reaction, stand still and detect.
In some preferred embodiments, the organic acid is TCA (trichloroacetic acid) aqueous solution with concentration of 25-35g/100mL, and the color developer is PDAB (p-dimethylaminobenzaldehyde) glacial acetic acid solution with mass concentration of 1-3%.
In some preferred embodiments, the aqueous TCA solution is prepared by the steps of: dissolving TCA in ultrapure water, vortexing at more than 3000r/min for 20-150s, and standing in dark for 20-30 min. If the storage time is too long, the data deviation is too high, and the data quality is negatively affected.
In some preferred embodiments, the organic acid has an expiration date of 24 hours; the effective period of the color developing agent is 6h.
Further preferably, an antioxidant is added during the reaction with the color developing agent; the antioxidant is 5-15mM Vc (ascorbic acid) aqueous solution; preferably 10mM.
The invention discovers that when the chromogenic agent is used for developing the color of the target cell, the repeatability of the color developing result of human mesenchymal cell IDO1 with higher activity is poor, and the reason may be that when the IDO1 activity is higher, the concentration fluctuation of kynurenine in a reaction system is larger, the requirement on the stability of the chromogenic agent is higher, and when the freshness of the chromogenic agent is lower, the capture capability of kynurenine with low concentration is poorer, the repeatability of the color developing result is lower, and the reproducibility of the detection result is poorer. The antioxidant is added in the color development process, so that the stability of the color developing agent in a kynurenine system is improved, the activity of the human mesenchymal cell IDO1 can be quickly detected in a short time, the possibility of re-detection when the detection result has large deviation is effectively reduced, and the universality degree of the method on the human mesenchymal cell IDO1 body is improved.
In some preferred embodiments, the detection is in particular: and detecting the absorbance value of 440-495nm by using a microplate reader.
Drawing a standard curve according to the determination result of the kynurenine standard solution, calculating a regression equation, and calculating A after gamma interferon culture and gamma interferon-free culture 492 Substituting the difference value into a regression equation to calculate a determination result, namely the kynurenine concentration of the cells after the drug treatment; according to the kynurenine concentration of the cells after drug treatment and the SD and RSD values of the data, the activity and the immunoregulatory capacity of the mesenchymal stem cells IDO1 are evaluated.
The invention provides an application of a rapid evaluation method of tryptophan metabolism rate-limiting enzyme activity, and the method is applied to IDO1 activity detection of human bone marrow mesenchymal stem cells, human amniotic mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells and human placenta mesenchymal stem cells.
Has the advantages that:
the invention solves the defect of large data deviation commonly existing in the IDO1 detection method by controlling the specific culture medium and the culture process condition; the activity of the indoleamine2,3-dioxygenase1 (IDO 1) of the mesenchymal stem cell can be rapidly detected in a short time, stable and credible data can be obtained, and the detection efficiency of the IDO1 activity of the mesenchymal stem cell is greatly improved; the method can stably detect the activity of IDO1 in the range of 440-495nm, the RSD value of the data is lower than 10%, the cell shape is uniform and stable in the culture process, no aggregation and clustering exists, no fracture and depression exist, the culture process is stable and controllable, and the accurate acquisition of the data is ensured; meanwhile, the method is convenient to operate and has wide application prospect.
Description of the drawings:
FIG. 1 shows the growth pattern of the target cells in example 1;
FIG. 2 shows the growth pattern of the target cells in example 4;
FIG. 3 shows the growth pattern of the target cells in example 5;
wherein, a represents the cell morphology after the target cells are incubated in the step S1, b represents the cell morphology after the control culture of the stimulation group, and c represents the cell morphology after the control culture of the control group.
Detailed Description
Example 1.
The embodiment provides a method for rapidly evaluating activity of tryptophan metabolism rate-limiting enzyme, which comprises the following specific steps:
s1, incubating target cells;
s2, performing contrast culture on the incubated target cells by adopting a first culture medium and a second culture medium;
s3, preparing a kynurenine gradient standard solution;
s4, developing and detecting the target cells and the kynurenine gradient standard solution after contrast culture in the step S2;
the first medium and the second medium are distinguished in that: the first medium does not contain gamma interferon, and the second medium does contain gamma interferon.
The raw materials of the first culture medium are MM3 culture solution, and the raw materials of the second culture medium are MM3 culture solution and gamma interferon.
The MM3 culture solution is a mixture of a serum substitute and a basic serum-free culture solution, and the volume ratio of the serum substitute to the basic serum-free culture solution is 1:19; the serum replacement was purchased from the EliteCell biomedical group, inc. under the product number EliteGroTM; basal serum-free media was purchased from Biological Industries, inc. under the catalog number 05-200-1B.
Raw materials of the second culture medium comprise MM3 culture solution and gamma interferon; the mass concentration of gamma interferon in the second culture medium is 10 ng/mL, and the balance is supplemented by MM3 culture solution; gamma interferon was purchased from petoltek biotechnology (suzhou) limited (PeproTech china).
The incubation of the target cells in the step S1 specifically comprises the following steps: using 24-well plates, in 12 wells of which 0.4mL of the first medium was added per well, inoculating the target cells into the first medium, placing the 24-well plates at 36.8 ℃,5.0% CO 2 And (5) incubating for 24h in a cell incubator.
The target cell is human umbilical cord mesenchymal stem cell, is derived from ELPIS hUC-MSC, and has the cell batch number of 2004D200814.
The target cell density of the inoculation is 5 x 10 4 Per well.
The control culture in the step S2 is specifically that culture medium in 12 holes is discarded, and 0.4mL of first culture medium is added into 6 holes; adding 0.4mL of the second medium into another 6 wells, placing the 24-well plate at 36.8 deg.C, 5.0% 2 Performing contrast culture in a cell culture box for 24h, centrifuging, collecting supernatant to obtain culture solution A and culture solution B, and storing in a refrigerator at-20 ℃ in a dark place; wherein culture solution A corresponds to the supernatant of the first culture medium and culture solution B corresponds to the supernatant of the second culture medium. Wherein 6 wells added with the first culture medium are control group parallel samples, and 6 wells added with the second culture medium are stimulation group parallel samples.
The concentration gradient of kynurenine in the kynurenine gradient standard solution is 0,3.9,7.8, 15.6, 31.25, 62.5, 125, 250 and 500 mu M, and the solvent is ultrapure water.
And the step S4 is specifically to respectively take the culture solution A and the culture solution B which are subjected to contrast culture in the step S2, melt and centrifuge for 5min, absorb 160 mu L of supernatant, add 40 mu L of organic acid, uniformly mix by vortex, react for 30min at 50 ℃, centrifuge for 10min, take 100 mu L of supernatant, add 100 mu L of color developing agent and 10 mu L of antioxidant, uniformly mix, stand for 10min at 25 ℃, and detect the absorbance value at 492nm by using an enzyme labeling instrument.
Taking the kynurenine gradient standard solution prepared in the step S3, repeating the color development operation, drawing a standard curve by taking the kynurenine concentration as an abscissa and the light absorption value at 492nm as an ordinate, and obtaining the R of the standard curve 2 =0.9999。
The organic acid is TCA aqueous solution with the concentration of 30g/100mL, the color developing agent is PDAB glacial acetic acid solution with the mass fraction of 2%, and the antioxidant is Vc aqueous solution with the molar concentration of 10mM.
The preparation method of the TCA aqueous solution comprises the following steps: dissolving TCA in ultrapure water, vortexing at 3500r/min for 60s, and standing in dark for 20-30 min.
The kynurenine, TCA, PDAB are purchased from makelin biochemical technologies, ltd.
The effective period of the organic acid is 24h; the effective period of the color developing agent and the antioxidant is 6h.
Example 2.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the absorbance at 442nm is measured in step S4 using a microplate reader.
Example 3.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the absorbance at 542nm was measured in step S4 using a microplate reader.
Example 4.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the mass concentration of gamma interferon in the second culture medium is 5ng/mL, and the balance of MM3 culture solution is supplemented.
Example 5.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the mass concentration of gamma interferon in the second culture medium is 15ng/mL, and the balance of MM3 culture solution is supplemented.
Example 6.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; except that the addition volume of the color developer was 50. Mu.L.
Example 7.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; except that the color developer was added in a volume of 150. Mu.L.
Example 8.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference lies in that the preparation steps of the TCA aqueous solution are as follows: TCA was dissolved in ultrapure water, vortexed at 2000r/min for 60s, placed in the dark and used within 20-30 min.
Example 9.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the target cell density of the inoculation is 6.4X 10 4 Per well.
Example 10.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the antioxidant is Vc aqueous solution with the molar concentration of 3 mM.
Example 11.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the difference is that the organic acid is TCA aqueous solution with the concentration of 30wt%,
example 12.
The embodiment provides a method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme, and the specific implementation mode is the same as that of the embodiment 1; the different points are that the MM3 culture solution is a mixture of fetal bovine serum and basic serum-free culture solution, and the volume ratio of the fetal bovine serum to the basic serum-free culture solution is 10:90.
performance test method
Precision of data:
according to a standard curve obtained by the kynurenine gradient standard solution, calculating A of target cells in a culture solution A and a culture solution B after contrast culture and culture containing gamma interferon and culture without gamma interferon 492 Substituting the difference value of (2) into regressionEquation calculation is carried out to obtain a measuring result, namely the concentration of kynurenine of the cell after drug treatment, and the RSD value of the detecting result is calculated according to the concentration of kynurenine in each hole; higher RSD indicates poorer reproducibility of the results. The reproducibility is defined as excellent when the RSD is less than 10 percent, good when the RSD is more than or equal to 10 percent and less than 15 percent, and poor when the RSD is more than or equal to 15 percent.
(1) Calculating a regression equation: and (3) taking the absorbance value with the concentration of 0 mu M in the kynurenine gradient standard solution as a background value, subtracting the background value from the absorbance values of the other concentration standard solutions as a correction value, respectively taking the absorbance value as a vertical coordinate and the kynurenine concentration as a horizontal coordinate, drawing a standard curve, and calculating a regression equation.
(2) Calculating the kynurenine concentration and data precision: substituting the absorbance value of the culture solution in each hole after the control culture into a regression equation, and calculating the kynurenine concentration of each hole of the sample; and calculating RSD% of the kynurenine concentration of the parallel samples of the stimulation group according to the kynurenine concentration of the samples, which is shown in Table 1. Lower RSD% indicates higher precision of data.
And (3) recovery rate:
kynurenine aqueous solutions with theoretical concentrations of 101.892, 127.365, 152.838, 84.278, 105.348 and 105.348 μ M are prepared, the absorbance values are measured according to the method of S4 in example 1 and are substituted into the regression curve obtained in example 1 to obtain the actual concentration, and the recovery rate R = (actual concentration-theoretical concentration)/the theoretical concentration × 100% is calculated and shown in table 2.
Cell morphology:
the growth morphology of the cells during the culture was observed using an Olympus microscope (CKX), see FIGS. 1-3. As can be seen from the figure, when the mass concentration of the gamma interferon is 5-15ng/L, the growth state of the cells in the culture process is good, the shapes are uniform, the dispersion is uniform, no obvious fracture and depression exist, and no uneven agglomeration exists.
Performance test data
Table 1.
Table 2.
Claims (7)
1. A method for rapidly evaluating the activity of tryptophan metabolism rate-limiting enzyme is characterized by comprising the following specific steps:
s1, incubating target cells;
s2, performing contrast culture on the incubated target cells by adopting a first culture medium and a second culture medium;
s3, preparing a kynurenine gradient standard solution;
s4, developing and detecting the target cells and the kynurenine gradient standard solution after contrast culture in the step S2;
the first medium and the second medium differ in that: the first culture medium does not contain gamma interferon, and the second culture medium contains gamma interferon;
raw materials of the first culture medium are MM3 culture solution, and raw materials of the second culture medium are MM3 culture solution and gamma interferon; the MM3 culture solution is a mixture of a serum substitute and a basic serum-free culture solution;
the volume ratio of the serum substitute to the basic serum-free culture solution is 1:19;
step 4, specifically, reacting the culture solution A, the culture solution B and the gradient standard solution prepared in the step 3 after the step 2 is compared with the culture solution A, the culture solution B and the gradient standard solution with organic acid, reacting with a color developing agent, standing after the reaction, and detecting; the organic acid is TCA aqueous solution with the concentration of 25-35g/100mL, and the color developing agent is PDAB glacial acetic acid solution with the mass concentration of 1-3%;
the preparation method of the TCA aqueous solution comprises the following steps: dissolving TCA in ultrapure water, vortexing at a speed of more than 3000r/min for 20-150s, and standing in dark for 20-30 min;
adding antioxidant when reacting with color developing agent; the antioxidant is 5-15mM Vc (ascorbic acid) aqueous solution.
2. The method for rapidly evaluating the activity of a tryptophan metabolism rate-limiting enzyme according to claim 1, wherein the incubation of the target cells in the step S1 comprises: the target cells were inoculated into the first medium using a 24-well plate in which 0.3-0.5mL of the first medium was added per well in 12 wells, and incubated for 22-26h.
3. The method for rapidly assessing the activity of a rate-limiting enzyme in tryptophan metabolism according to claim 2, wherein the inoculated target cells have a density of 4 to 6 x 10 4 Per well.
4. The method for rapidly evaluating the activity of a tryptophan metabolism rate-limiting enzyme according to any one of claims 1 to 3, wherein the control culture in the step S2 is specifically that after the step S1 is completed, the culture medium in 12 wells is aspirated and discarded, and the first culture medium is added to 6 wells; adding a second culture medium into another 6 holes, and performing contrast culture for 22-26h to obtain a culture solution A and a culture solution B; wherein, the culture solution A is a control culture product of the first culture medium, and the culture solution B is a control culture product of the second culture medium.
5. The method of claim 2, wherein the concentration of gamma interferon in the second medium is 5-15 ng/mL.
6. The method for rapidly evaluating the activity of a tryptophan metabolism rate-limiting enzyme according to claim 1, wherein the detection is specifically as follows: and detecting the absorbance value of 440-495nm by using a microplate reader.
7. The application of the rapid evaluation method for the activity of the tryptophan metabolism rate-limiting enzyme according to claim 1 is characterized in that the method is applied to the detection of IDO1 activity of human bone marrow mesenchymal stem cells, human amniotic mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells and human placental mesenchymal stem cells.
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| CN115323034B (en) | 2023-02-21 |
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