CN114958959A - Processing method of human mesenchymal stem cell IDO1 activity data - Google Patents
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Abstract
The invention discloses a method for processing activity data of human mesenchymal stem cells IDO1, which comprises the following steps: (1) thawing the cell culture supernatant in the EP tube, and heating for pretreatment to obtain a pretreatment solution; (2) after the pre-treatment liquid is treated by the kynurenine standard liquid, standing for 10-15 minutes, and detecting by an enzyme-linked immunosorbent assay (ELISA) instrument A 492 A value; (3) with A 492 Drawing a standard curve by using the value and the concentration of the kynurenine standard solution, and calculating a regression equation; (4) calculating A of active medium culture group and basal medium culture group 492 And (4) carrying out value difference and substituting the value difference into a regression equation to obtain the required data. The active culture medium and the culture process in the method have good high-density stem cell culture effect, can effectively prevent the cell reduction phenomenon during high-density stem cell culture, promote cell proliferation and reduce cell aging, so that the data processing result is more accurate, and the method is suitable for popularization in the field of stem cells and has wide development prospect.
Description
Technical Field
The invention relates to the field of C12Q1/00, in particular to a method for processing activity data of human mesenchymal stem cells IDO 1.
Background
Mesenchymal Stem Cells (MSCs) are widely present in most tissues of the body, can be isolated from bone marrow, fat, muscle, and other tissues, have a property of being differentiated across germ layers, can be differentiated into adipocytes, osteoblasts, chondroblasts, nerve cells, and the like, and are involved in tissue regeneration and repair and maintenance of homeostasis in the body. The expression of indoleamine 2, 3-dioxygenase 1(IDO1) is a precondition for the regeneration of nerve cells and the regulation of an immune system of mesenchymal stem cells, so that the research on the activity expression of indoleamine 2, 3-dioxygenase 1 of the mesenchymal stem cells in recent years also shows a trend of increasing year by year, and the detection of the activity expression of indoleamine 2, 3-dioxygenase 1 also becomes a hot spot of the recent research.
The prior art (CN202011561297.0) discloses an immunoregulation stem cell IDO1 activity detection method meeting the product release requirements, and claims to establish a relative biological activity detection method and effectively eliminate incorrect results in detection results. However, the mesenchymal stem cell complete culture medium used in the method cannot effectively reduce the negative oxidation influence of free radicals in the culture medium on lipid, protein and DNA of the stem cells, cannot promote the cell membrane permeability of the stem cells, further reduces the damage condition of the stem cells, is simple and convenient in analysis steps of detection results, and cannot further improve the accuracy of activity detection results.
Therefore, in order to solve the above problems, there is a need for a detection method and a detection data processing method that can effectively culture a large amount of mesenchymal stem cells and greatly reduce the loss of stem cells during the culture process, so that the accuracy of the detection result of IDO1 activity is significantly improved, and both of them have the same effect to improve the accuracy of the detection result of IDO1 activity in mesenchymal stem cells.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for processing activity data of human mesenchymal stem cell IDO1, comprising the following steps: (1) thawing the cell culture supernatant in the EP tube, and heating for pretreatment to obtain a pretreatment solution; (2) after the pre-treatment liquid is treated by the kynurenine standard liquid, standing for 10-15 minutes, and detecting by an enzyme-linked immunosorbent assay (ELISA) instrument A 492 A value; (3) with A 492 Drawing a standard curve by using the value and the concentration of the kynurenine standard solution, and calculating a regression equation; (4) calculating A of active medium culture group and basal medium culture group 492 And (4) carrying out value difference and substituting the value difference into a regression equation to obtain the required data.
In some preferred embodiments, the specific method of step (1) is: (1) thawing the cell culture supernatant in the EP tube, centrifuging, and removing suspended cells; (2) after centrifugation, taking 120-180 mu L of supernatant from each EP tube, adding the supernatant into a new EP tube, adding 30-50 mu L of 30 wt% trichloroacetic acid solution respectively, and uniformly mixing by vortex; (3) after mixing, heating at 50 ℃ for reaction for 30 minutes, and after reaction, centrifuging for 10 minutes to obtain a pretreatment solution.
In some preferred embodiments, the specific method of step (2) is: taking 100 mu L of pretreatment solution from each EP tube, respectively adding the pretreatment solution into a 96-well plate, respectively taking 100 mu L of kynurenine standard solution with each concentration, adding 100 mu L of 2 wt% p-dimethylaminobenzaldehyde solution into each well, uniformly mixing, standing at room temperature for 10 minutes, and detecting A by using an enzyme-linked immunosorbent assay (ELISA) instrument 492 The value is obtained.
In some preferred embodiments, the step of preparing the kynurenine standard solution at each concentration comprises the following steps: (1) preparing a kynurenine standard solution with the concentration of 500 mu mol/L by using ultrapure water and the kynurenine; (2) mixing half volume of kynurenine standard solution with ultrapure water of the same volume, and sequentially preparing kynurenine standard solutions with the concentrations of 250, 125, 62.5, 31.25, 15.6, 7.8 and 3.9 mu mol/L; (3) ultrapure water was taken as a kynurenine standard solution of 0. mu. mol/L.
In some preferred embodiments, the specific method of step (3) is: absorbance A of ultrapure water 492 The value is taken as a background value, and the other kynurenine standard liquid A with different concentrations 492 Value minus background value is A 492 Correction values, respectively A 492 And (5) drawing a standard curve by using the correction value and the concentration of the kynurenine standard solution, and calculating a regression equation.
In some preferred embodiments, the specific method of step (4) is: calculating A of active medium culture group and basal medium culture group 492 Value difference value according to regression equation and A 492 And calculating the difference to obtain the kynurenine concentration of the mesenchymal stem cells after the treatment of the medicament and the SD and RSD values of the data, thereby evaluating the activity and the immunoregulatory capacity of the mesenchymal stem cells IDO 1.
In some preferred embodiments, the step of preparing the cell culture supernatant comprises the steps of: (1) inoculating and culturing cells by using a 24-hole plate, and adding 0.3-0 part of the culture medium into each hole6mL of basal medium, 12 wells in total, and 24-well plates at 37 ℃ with 5.0% CO 2 Culturing for the first time in a cell culture box for 22-26 hours; (2) after the first culture is finished, the basic culture medium is sucked and discarded, 0.3-0.6 mL of active culture medium is added into 6 holes of the basic culture medium, 0.3-0.6 mL of basic culture medium is added into the other 6 holes of the basic culture medium, and a 24-hole plate is placed at 37 ℃ and 5.0% CO 2 Carrying out secondary culture in a cell culture box for 23-25 hours; (3) after the culture is finished, completely sucking the culture supernatant, adding the culture supernatant into an EP tube, dividing the sample into an active culture medium culture group and a basal culture medium culture group, and storing all the samples in a refrigerator below 20 ℃ below zero.
In some preferred embodiments, the basal medium is a serum-free medium and a serum replacement in a volume ratio of 1: 16-19.
In some preferred embodiments, the active medium is a basal medium containing gamma interferon; the concentration of gamma interferon in the active culture medium is 7-11 ng/mL.
In some preferred embodiments, afzelechin and human lipocalin 2 are also added to the active medium; the concentration ratio of the African catechin (CAS:2545-00-8) to the human lipocalin 2 is 1-3: 5 to 7.
In some preferred embodiments, the concentration of African catechin is 4-12 μ g/mL; the concentration of the human lipocalin 2 is 20-28 mug/mL.
In the application of the invention, a small amount of African catechin and human lipocalin 2 are added into an active culture medium, and when the concentrations of the African catechin and the human lipocalin are respectively 4-12 mu g/mL and 20-28 mu g/mL, and the concentration ratio is 1-3: and 5-7 hours, the proliferation rate and the adherence efficiency of the mesenchymal stem cells can be enhanced in the cell culture process, the overall loss of the stem cells in the culture period is effectively reduced, and the aging of the stem cells is inhibited, so that the final detection accuracy of the culture supernatant is improved, and the SD value and the RSD value of the data obtained by the detection data processing method are finally enhanced. The applicant speculates that: when the concentration ratio of the two is 1-3: 5-7 hours, the human lipocalin 2 can effectively promote the proliferation rate of the mesenchymal stem cells, effectively inhibit the aging of the stem cells and improve the culture effect of the stem cells; at the moment, the existence of the African catechin can effectively stimulate the activity of stem cell antioxidant enzyme, and the negative effects of free radicals on stem cells and ester substances and DNA in a culture medium are effectively avoided by matching with the free radical elimination effect of the African catechin, more importantly, the inactivation of human lipid carrier protein 2 due to the attack of the free radicals is avoided, the synergistic effect of the African catechin and the African catechin can also effectively improve the activity of the African catechin, the cell membrane permeability of mesenchymal stem cells in the culture process is reduced in the culture process of the stem cells, and the damage condition of the cell membrane structure of the cells is reduced. When the concentration of the African catechins is too low, the free radical elimination efficiency is low, the activity of antioxidant active enzyme is low, and proteins such as human lipocalin 2 and the like are easy to inactivate, so that the proliferation and culture of mesenchymal stem cells are influenced; when the concentration of the afzelechin is too high, the permeability of the cell membrane of the stem cell is too low, so that the phenomenon of difficult exchange of substances inside and outside the cell is easy to occur, and the loss of the mesenchymal stem cell is caused.
In some preferred embodiments, the serum replacement is EliteGro sold by EliteCell biomedical group corporation TM A serum replacement product of the type.
In some preferred embodiments, the serum-free Medium is a Nutristem XF Medium model serum-free Medium product sold by Israel BI.
In some preferred embodiments, the interferon gamma is an IFN-interferon gamma product sold by Peprotech, Inc.
In some preferred embodiments, the kynurenine, trichloroacetic acid and p-dimethylaminobenzaldehyde are all the corresponding products sold by Shanghai Michelin reagent company.
The second aspect of the invention also comprises the application of the detection data processing method in the experiment for detecting the activity of the human mesenchymal stem cell IDO 1.
Has the advantages that:
1. according to the mixed culture medium prepared by mixing the basic culture medium and the interferon mother solution without serum and controlling the volume ratio of the serum substitute to the serum-free culture solution in the basic culture medium and the concentration of the interferon in the culture medium, an excellent environment is provided for high-density inoculation of mesenchymal stem cells, and the loss of the stem cells in the culture process is greatly reduced;
2. according to the invention, by adding the African catechin and the human lipocalin 2 into the active culture medium and limiting the concentration ratio of the African catechin and the human lipocalin 2, the synergistic effect is enhanced, the number and activity of free radicals in the culture medium are effectively reduced, the permeability of a stem cell membrane is effectively reduced, and the structural damage of the stem cell membrane is reduced, so that the overall loss of stem cells during the culture period is reduced, the cell adherence rate is enhanced, and the accuracy of culture supernatant detection is finally improved.
3. In the present invention, the A of the active medium culture group and the basal medium culture group is calculated 492 Difference value and according to the regression equation and A obtained by calculation 492 And obtaining the concentration of kynurenine of the mesenchymal stem cells after the treatment of the medicament and the SD and RSD values of the data so as to evaluate the activity and the immunoregulatory capacity of the mesenchymal stem cells IDO 1.
Detailed Description
The present invention will be more readily understood from the following detailed description of preferred embodiments. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and in the event of conflict, the definitions set forth herein shall control.
Examples
The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to all of the examples. The starting materials of the present invention are all commercially available unless otherwise specified.
Example 1
Example 1 in a first aspect, there is provided a method for processing data on activity of human mesenchymal stem cell IDO1, comprising the following steps: (1) thawing the cell culture supernatant in the EP tube, centrifuging, and removing suspended cells; after centrifugation, 160. mu.L of supernatant was taken from each EP tube, added to a new EP tube, and 40. mu.L of each 30 wt% trichloroacetic acid solution was addedMixing the solution evenly by vortex; after uniformly mixing, heating and reacting for 30 minutes at 50 ℃, and centrifuging for 10 minutes after reaction to obtain a pretreatment solution; (2) taking 100 mu L of pretreatment solution in each EP tube, respectively adding the pretreatment solution into a 96-well plate, respectively taking 100 mu L of kynurenine standard solution with each concentration, adding 100 mu L of 2 wt% p-dimethylaminobenzaldehyde solution into each well, uniformly mixing, standing for 10 minutes at room temperature, and detecting A by using an enzyme-linked immunosorbent assay (ELIASA) 492 A value; (3) absorbance A of ultrapure water 492 The value is taken as a background value, and the other kynurenine standard liquid A with different concentrations 492 Value minus background value is A 492 Correction values, respectively A 492 Drawing a standard curve by the correction value and the concentration of the kynurenine standard solution, and calculating a regression equation; (4) calculating A of active medium culture group and basal medium culture group 492 Value difference value according to regression equation and A 492 And calculating the difference to obtain the kynurenine concentration of the mesenchymal stem cells after the treatment of the medicament and the SD and RSD values of the data, thereby evaluating the activity and the immunoregulatory capacity of the mesenchymal stem cells IDO 1.
In this example, the preparation of kynurenine standard solutions at various concentrations included the following steps: (1) preparing a kynurenine standard solution with the concentration of 500 mu mol/L by using ultrapure water and the kynurenine; (2) mixing half volume of kynurenine standard solution with ultrapure water of the same volume, and sequentially preparing kynurenine standard solutions with the concentrations of 250, 125, 62.5, 31.25, 15.6, 7.8 and 3.9 mu mol/L; (3) ultrapure water was taken as a kynurenine standard solution of 0. mu. mol/L.
In this example, the preparation of the cell culture supernatant comprises the following steps: (1) the cultured cells were inoculated using a 24-well plate, 0.4mL of the basal medium was added to each well, 12 wells were inoculated in total, and the 24-well plate was placed at 37 ℃ in 5.0% CO 2 Culturing for the first time in a cell culture box for 24 hours; (2) after the first culture, the basal medium was aspirated, 0.4mL of active medium was added to 6 wells, 0.4mL of basal medium was added to the other 6 wells, and the 24-well plate was placed at 37 ℃ and 5.0% CO 2 Culturing for the second time in a cell culture box for 24 hours; (3) after the culture is completed, the culture supernatant is completely sucked and added into an EP tube, and the sample is divided into an active culture medium culture group and a basal culture mediumAnd (4) culturing the group, and storing all samples in a refrigerator below-20 ℃.
In this example, the basal medium was serum-free medium and serum replacement at a volume ratio of 1: and 17, preparing.
In this example, the active medium was a basal medium containing interferon-gamma, African catechin (CAS:2545-00-8) and human lipocalin 2; the concentration of gamma interferon is 10ng/mL, the concentration of African catechin is 8 mug/mL, and the concentration of human lipocalin 2 is 24 mug/mL.
In this example, the serum replacement is EliteGro sold by EliteCell biomedical group TM A serum replacement product of the type.
In this example, the serum-free Medium was a Nutristem XF Medium serum-free Medium product sold by Israel BI.
In this example, the gamma interferon is an IFN-gamma interferon product manufactured and sold by Peprotech corporation.
In this example, kynurenine, trichloroacetic acid and p-dimethylaminobenzaldehyde are all corresponding products sold by Shanghai Mecline reagent company.
Example 2
The embodiment of the present invention is different from embodiment 1 in that: the basic culture medium is a serum-free culture solution and a serum substitute in a volume ratio of 1: 19, preparing.
Example 3
The embodiment of the present invention is different from embodiment 1 in that: the concentration of interferon gamma is 7 ng/mL.
Example 4
The embodiment of the present invention is different from embodiment 1 in that: the concentration of gamma interferon was 11 ng/mL.
Evaluation of Performance
1. And (3) appearance observation: the mesenchymal stem cells were subjected to cell culture and culture supernatant extraction by the methods of all examples and comparative examples, and after completion of the culture, the mesenchymal stem cells were microscopically observed for whether the cell shape was regular, whether there was rupture or other phenomena, and the results of the observation are shown in table 1.
2. Mesenchymal stem cell preservation amount: adopting the methods of all the embodiments and the comparative examples to carry out cell culture and culture supernatant extraction on the mesenchymal stem cells, detecting the residual cell density after the culture is finished, taking the cell density of the embodiment 1 as the reference density, calculating the percentage of the residual cell density relative to the reference density of other embodiments, wherein the larger the percentage is, the better the culture effect of the composite culture medium is, and the smaller the loss amount of the stem cells is, the more accurate the detection result of the final culture supernatant is; each example comparative example tested 5 samples and the average of the values measured is reported in table 1.
3. Cell anchorage amount: mesenchymal stem cells were cultured using the same amount of active medium in all examples and comparative examples, each group of three duplicate wells, each well seeded with 1 × 10 5 The mesenchymal stem cells are placed in a cell culture box for culturing for 2 hours, the active culture medium is discarded, the non-adherent cells are washed by PBS (phosphate buffer solution), digestion, collection and counting are carried out, and the average value of the measured values is recorded in a table 2.
4. Data deviation value: the mesenchymal stem cells were subjected to the activity test of IDO1 by the method of all examples and comparative examples; calculating SD and RSD values of each example and comparative example by using a standard deviation equation and a relative standard deviation equation, and evaluating a deviation value after data processing; each example comparative example tested 5 samples and the average of the values measured is reported in Table 2.
5. TABLE 1
6. TABLE 2
| Examples | Number of cells adherent | SD％ | RSD％ |
| Example 1 | 364.1 | 3.955 | 4.1255 |
| Example 2 | 355.4 | 4.015 | 4.3471 |
| Example 3 | 358.4 | 3.987 | 4.4117 |
| Example 4 | 224.6 | 6.048 | 7.1648 |
As can be seen from examples 1 to 4 and tables 1 and 2, the method for processing activity data of human mesenchymal stem cell IDO1 provided by the present invention has the advantages that the activity culture medium and the culture process in the method have good high-density stem cell culture effect, can effectively prevent the cell reduction phenomenon during high-density stem cell culture, and promote cell proliferation and reduce cell aging, so that the data processing result is more accurate, and the method is suitable for popularization in the stem cell field and has a broad development prospect. Wherein, the example 1 obtains the best performance index under the factors of the best preparation method, the volume ratio of the prepared raw materials, the culture process and the like.
Finally, it should be understood that the above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A processing method of activity data of human mesenchymal stem cells IDO1 is characterized by comprising the following steps: the method comprises the following steps: (1) melting the cell culture supernatant in the EP tube, and heating for pretreatment to obtain a pretreatment solution; (2) after the pre-treatment liquid is treated by the kynurenine standard liquid, standing for 10-15 minutes, and detecting by an enzyme-linked immunosorbent assay (ELISA) instrument A 492 A value; (3) with A 492 Drawing a standard curve by using the value and the concentration of the kynurenine standard solution, and calculating a regression equation; (4) calculating A of active medium culture group and basal medium culture group 492 And (4) carrying out value difference and substituting the value difference into a regression equation to obtain the required data.
2. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 1, wherein the data comprises: the specific method of the step (1) comprises the following steps: (1) thawing the cell culture supernatant in the EP tube, centrifuging, and removing suspended cells; (2) after centrifugation, taking 120-180 mu L of supernatant from each EP tube, adding the supernatant into a new EP tube, adding 30-50 mu L of 30 wt% trichloroacetic acid solution respectively, and uniformly mixing by vortex; (3) after mixing, heating at 50 ℃ for reaction for 30 minutes, and after reaction, centrifuging for 10 minutes to obtain a pretreatment solution.
3. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 1, wherein the data comprises: the specific method of the step (2) is as follows: taking 100 μ L of pretreatment solution in each EP tube, adding into 96-well plate, respectively, taking 100 μ L of kynurenine standard solution with each concentration, adding into 96-well plate, adding into 100 μ L of 2 wt% p-dimethylaminobenzaldehyde solution, mixing, standing at room temperatureStanding for 10 minutes, and detecting A by using an enzyme-linked immunosorbent assay (ELIASA) 492 The value is obtained.
4. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 1, wherein the data comprises: the specific method of the step (3) is as follows: absorbance A of ultrapure water 492 The value is taken as a background value, and the other kynurenine standard liquid A with different concentrations 492 Value minus background value is A 492 Correction values, respectively A 492 And (5) drawing a standard curve by using the correction value and the concentration of the kynurenine standard solution, and calculating a regression equation.
5. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 1, wherein the data comprises: the specific method of the step (4) comprises the following steps: calculating A of active medium culture group and basal medium culture group 492 Value difference value according to regression equation and A 492 And calculating the difference to obtain the kynurenine concentration of the mesenchymal stem cells after the treatment of the medicament and the SD and RSD values of the data, thereby evaluating the activity and the immunoregulatory capacity of the mesenchymal stem cells IDO 1.
6. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 2, wherein the data comprises: the preparation steps of the cell culture supernatant comprise the following steps: (1) inoculating cultured cells by using a 24-pore plate, adding 0.3-0.6 mL of basic culture medium into each pore, inoculating 12 pores in total, placing the 24-pore plate at 37 ℃ and 5.0% CO 2 Culturing for the first time in a cell culture box for 22-26 hours; (2) after the first culture is finished, the basic culture medium is sucked and discarded, 0.3-0.6 mL of active culture medium is added into 6 holes of the basic culture medium, 0.3-0.6 mL of basic culture medium is added into the other 6 holes of the basic culture medium, and a 24-hole plate is placed at 37 ℃ and 5.0% CO 2 Carrying out secondary culture in a cell culture box for 23-25 hours; (3) after the culture is finished, completely sucking the culture supernatant, adding the culture supernatant into an EP tube, dividing the sample into an active culture medium culture group and a basal culture medium culture group, and storing all the samples in a refrigerator below 20 ℃ below zero.
7. The method for processing activity data of human mesenchymal stem cell IDO1, according to claim 6, wherein the data comprises: the basic culture medium is a serum-free culture solution and a serum substitute in a volume ratio of 1: 16-19.
8. The method for processing activity data of human mesenchymal stem cell IDO1 according to claim 6, wherein the data comprises the following steps: the active culture medium is a basic culture medium containing gamma interferon; the concentration of gamma interferon in the active culture medium is 7-11 ng/mL.
9. The method for processing activity data of human mesenchymal stem cell IDO1 according to claim 8, wherein the data comprises: the active culture medium is also added with African catechin and human lipocalin 2; the concentration ratio of the African catechin to the human lipocalin 2 is 1-3: 5 to 7.
10. Use of a method for processing human mesenchymal stem cell IDO1 activity data according to any one of claims 1 to 9, wherein the method comprises the following steps: the application of the detection data processing method in the experiment for detecting the activity of the human mesenchymal stem cell IDO1 is included.
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| CN115323034A (en) * | 2022-10-11 | 2022-11-11 | 华夏源(上海)生命科技有限公司 | Rapid evaluation method for activity of tryptophan metabolism rate-limiting enzyme |
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| CN115323034A (en) * | 2022-10-11 | 2022-11-11 | 华夏源(上海)生命科技有限公司 | Rapid evaluation method for activity of tryptophan metabolism rate-limiting enzyme |
| CN115323034B (en) * | 2022-10-11 | 2023-02-21 | 华夏源(上海)生命科技有限公司 | Rapid evaluation method for activity of tryptophan metabolism rate-limiting enzyme |
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