Method for establishing hypoxia induction model by using cobalt chloride

Abstract

The invention relates to the technical field of biological medicines, in particular to a method for establishing an anoxia induction model by using cobalt chloride. The method comprises the following steps: culturing cells; determining whether oxygen distribution in the incubator is uniform or not; when the oxygen in the incubator is not uniformly distributed, analyzing the reaction of the cells in the hypoxia induction model according to the oxygen concentration deviation degree and the cell state so as to carry out corresponding regulation and control; and when the oxygen in the incubator is uniformly distributed, monitoring the impedance through a cell analyzer, and selecting the cobalt chloride concentration and the adding time according to the monitoring result. The oxygen concentration in the incubator is monitored in real time, so that the oxygen deficit degree is accurately monitored, the stability of an oxygen control system of the incubator is ensured, confusion variables are avoided, and the optimal concentration and the treatment time of cobalt chloride are more accurately determined by analyzing the influence of the oxygen deficit degree on the cell survival rate, the solubility of cobalt chloride and the biological activity. And a more accurate hypoxia induction model is established.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for establishing an oxygen induction model by using cobalt chloride. Background Art

[0002] Hypoxia refers to a pathological process in which abnormal changes in tissue metabolism, function, and morphological structure occur due to insufficient oxygen supply to tissues or impaired oxygen utilization.

[0003] Cobalt chloride (CoCl2) is currently the most common chemical used to induce hypoxia in vitro and can simulate hypoxia-ischemia responses in a variety of ways, such as 2+ Activation, accumulation of hypoxia-inducible factor-1α (HIF-1α) protein and accumulation of reactive oxygen species. CoCl2 simulated hypoxia will increase the expression level of HIF-1α protein in cells and has been widely used to explore various hypoxia experimental models.

[0004] The Chinese patent document with the publication number CN113943696A discloses a method for establishing a hypoxia-induced three-dimensional angiogenesis model using porcine vascular endothelial cells, comprising: under in vitro culture conditions, using porcine vascular endothelial cells as a porcine placental vascular endothelial cell model, adding cell microcarrier beads, inducing hypoxia and incubating so that the cell microcarrier beads wrap the porcine vascular endothelial cells to form coated carrier beads; then mixing the coated carrier beads with matrix glue, inducing hypoxia and incubating so that the matrix glue embeds the coated carrier beads to form a hypoxia-induced three-dimensional angiogenesis model of porcine vascular endothelial cells. The three-dimensional model of the present invention simulates the in vivo growth environment of porcine placental vascular endothelial cells, and studies the migration and tube formation of porcine endothelial cells under hypoxic conditions; the existing hypoxia-induced model does not consider the influence of fluctuations in oxygen concentration on the monitoring results, and it is difficult to ensure the stability of the experimental conditions and the reliability of the experimental results. Summary of the invention

[0005] To this end, the present invention provides a method for establishing an oxygen induction model using cobalt chloride, so as to overcome the problem in the prior art that the accuracy of the experimental results is low due to the lack of real-time monitoring of the oxygen concentration fluctuation in the incubator.

[0006] To achieve the above object, the present invention provides a method for establishing an hypoxia induction model using cobalt chloride, comprising:

[0007] Step S1, cell culture, inoculating cells into a certain amount of complete culture medium containing cobalt chloride, and culturing in a 37° C., 5% CO 2 incubator;

[0008] Among them, each complete medium contains different concentrations of cobalt chloride;

[0009] Step S2, monitoring the maximum oxygen concentration difference in real time by each concentration sensor arranged in the incubator, and determining whether the oxygen distribution in the incubator is uniform according to the maximum oxygen concentration difference;

[0010] Step S3, when the oxygen distribution in the incubator is uneven, analyzing the response of the cells in the hypoxia-induced model according to the degree of oxygen concentration deviation and the cell state, so as to perform corresponding regulation;

[0011] The corresponding regulation includes reducing the maximum concentration of cobalt chloride, starting the self-regulation mode and adjusting the preset processing time;

[0012] Step S4, when the oxygen in the incubator is evenly distributed, the impedance is monitored by a cell analyzer, and the concentration and addition time of cobalt chloride are selected according to the monitoring results.

[0013] Further, determining whether the oxygen distribution in the incubator is uniform according to the maximum oxygen concentration difference includes,

[0014] Obtaining the corresponding unit oxygen concentration detected by each concentration sensor in the incubator;

[0015] Calculating the maximum oxygen concentration difference according to the oxygen concentration of each unit;

[0016] The maximum oxygen concentration difference is compared with the allowable oxygen concentration difference, and whether the oxygen distribution in the incubator is uniform is determined according to the comparison result;

[0017] When the maximum oxygen concentration difference is greater than the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uneven, and when the maximum oxygen concentration difference is less than or equal to the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uniform.

[0018] Further, calculating the maximum oxygen concentration difference according to the oxygen concentration of each unit includes:

[0019] Obtain the real-time oxygen concentration detected by each concentration sensor in each unit detection area to obtain the unit oxygen concentration;

[0020] Add the oxygen concentration of each unit and divide it by the number of unit detection areas to get the average oxygen concentration;

[0021] Subtracting the average oxygen concentration from the oxygen concentration of each unit to obtain a number of real-time oxygen concentration fluctuation differences;

[0022] Take the absolute value of each real-time oxygen concentration fluctuation difference, obtain the value with the largest absolute value, and obtain the maximum oxygen concentration difference.

[0023] Further, the response of cells in the hypoxia-induced model was analyzed based on the degree of oxygen concentration deviation and cell status, including:

[0024] When the oxygen distribution in the incubator is uneven, obtain the real-time oxygen concentration corresponding to the maximum oxygen concentration difference, obtain the oxygen analysis concentration value, and compare the oxygen analysis concentration value with the standard oxygen concentration.

[0025] If the oxygen analysis concentration value is greater than the standard oxygen concentration, the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate;

[0026] If the oxygen analysis concentration value is less than or equal to the standard oxygen concentration, a curve of the oxygen analysis concentration value changing with time is plotted to obtain an oxygen concentration change curve, and whether to adjust the preset processing time is determined based on the oxygen concentration change curve and the difference in stress protein content.

[0027] Further, determining whether to adjust the preset processing time based on the oxygen concentration change curve and the stress protein content difference includes:

[0028] Obtain the actual slope of the oxygen concentration change curve and the difference in stress protein content,

[0029] If the actual slope is less than or equal to the standard slope, the actual cell survival rate is obtained, and the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate;

[0030] If the actual slope is greater than the standard slope, obtain several stress protein content differences, and compare each stress protein content difference with the standard protein content difference.

[0031] If the stress protein content difference is less than or equal to the standard protein content difference, the preset processing time is not adjusted, and the maximum concentration of cobalt chloride is increased;

[0032] If the real-time protein content difference is greater than the standard protein content difference, the current time is updated to the preset processing time.

[0033] Further, reducing the maximum concentration of cobalt chloride or starting the self-regulation mode based on the actual cell survival rate includes:

[0034] Obtain the actual cell survival rate and compare it with the standard cell survival rate.

[0035] If the actual cell survival rate is greater than or equal to the standard cell survival rate, the self-regulation mode is turned on. The self-regulation mode is to adjust the oxygen concentration of the corresponding area through the gas flow controller. After the adjustment, after a preset waiting time, the maximum oxygen concentration difference is obtained again for judgment. If it is still determined that the oxygen analysis concentration value is greater than the standard oxygen concentration, a calibration alarm is issued;

[0036] If the actual cell viability is lower than the standard cell viability, adjust the maximum concentration of cobalt chloride to a lower level.

[0037] Further, obtaining the difference in the content of several stress proteins includes:

[0038] The content of stress proteins in each complete culture medium is obtained at a preset collection interval, and the change curve of the content of stress proteins over time is plotted to generate the change curve of the content of each stress protein;

[0039] The peak values ​​corresponding to the stress protein content change curves were obtained, and the differences in the corresponding stress protein contents in the two complete culture media were calculated in the order of the amount of cobalt chloride added from low to high, which were recorded as the differences in the stress protein contents.

[0040] Furthermore, the cells are cardiomyocytes.

[0041] Further, culturing in a 37°C, 5% CO2 incubator also includes:

[0042] When the cells grew to 80% confluence, they were digested with 0.25% trypsin-EDTA and passaged every 2-3 days.

[0043] Furthermore, the complete culture medium contains 10% FBS and 1% penicillin-streptomycin dual antibody.

[0044] Compared with the prior art, the beneficial effect of the present invention is that by monitoring the oxygen concentration in the incubator, the cobalt chloride concentration and the treatment time are optimized to ensure the stability of the experimental conditions and the reliability of the experimental results, that is, the oxygen concentration in the incubator is monitored to consider whether the preset oxygen concentration reaches the preset hypoxia degree. Since high concentrations of cobalt chloride will lead to increased cell death and reduced metabolic activity, thereby reducing oxygen consumption and increasing the oxygen concentration in the incubator, or if the stress response of the cells is enhanced under cobalt chloride treatment, and oxygen consumption is increased, resulting in changes in local oxygen concentration, oxygen fluctuations in the incubator occur, therefore, the oxygen concentration in the incubator must be precisely regulated, and the oxygen concentration of each area in the incubator is monitored in real time, and then the oxygen concentration of the incubator is systematically evaluated, so as to accurately monitor the degree of hypoxia, ensure the stability of the incubator oxygen control system, avoid confounding variables, and analyze the effects of the degree of hypoxia on cell survival rate, solubility of cobalt chloride and biological activity, so as to more accurately determine the optimal concentration and treatment time of cobalt chloride, and then establish a more accurate hypoxia induction model.

[0045] Furthermore, when it is determined that the actual slope is less than or equal to the standard slope, it indicates that oxygen consumption is reduced, which may be related to cell damage or metabolic inhibition. The cell state is determined by obtaining the actual cell survival rate to accurately analyze the cause. When it is determined that the actual slope is greater than the standard slope, it indicates that oxygen consumption is increased, which may be related to the enhanced stress response. Therefore, the difference in the content of each stress protein is compared with the difference in the standard protein content. If it is determined that the real-time protein content difference is greater than the standard protein content difference, indicating that the enhanced stress response leads to increased oxygen consumption, the current addition time is the optimal value. If it is determined that the stress protein content difference is less than the standard protein content difference, it means that the concentration of cobalt chloride does not significantly promote the stress response, that is, the set initial concentration of cobalt chloride is low, and the concentration of added cobalt chloride needs to be optimized to more comprehensively analyze the response of cells in the hypoxia-induced model.

[0046] Furthermore, when it is detected that the gas distribution in the incubator is uneven and the local oxygen concentration increases, or the oxygen consumption decreases, it may be caused by weakened cell respiration, poor gas flow in the incubator itself, or errors in oxygen concentration measurement. In this case, the actual cell survival rate can be monitored to accurately analyze the cause.

[0047] In particular, since high concentrations of cobalt chloride may induce cell death or metabolic inhibition, resulting in reduced oxygen consumption, thereby indirectly affecting the dynamic balance of oxygen in the incubator, when it is determined that the oxygen analysis concentration value is greater than the standard oxygen concentration and the actual cell survival rate is less than the standard cell survival rate, it is analyzed that this situation is caused by weakened cell respiration, and the maximum concentration of cobalt chloride is reduced to ensure that the concentration used is within the range that the cells can withstand. When the actual cell survival rate is greater than or equal to the standard cell survival rate, it means that there is no cell death causing reduced oxygen consumption. In this way, the gas flow controller of the corresponding area is turned on to adjust the oxygen concentration of the corresponding area, that is, the fan is added to promote full mixing of the gas to adjust the oxygen concentration of the corresponding area. After the adjustment, the gas distribution in the incubator is analyzed again. If there is no improvement, a calibration alarm is issued to remind the staff to calibrate the incubator oxygen sensor to ensure data accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a flow chart of a method for establishing an oxygen induction model using cobalt chloride according to an embodiment of the present invention;

[0049] Figure 2 A schematic diagram of a process for determining whether oxygen distribution in an incubator is uniform according to an embodiment of the present invention;

[0050] Figure 3 A schematic diagram of a process for calculating the maximum oxygen concentration difference according to an embodiment of the present invention;

[0051] Figure 4A schematic diagram of a process for analyzing the response of cells in a hypoxia-induced model according to an embodiment of the present invention;

[0052] Figure 5 Schematic diagram of the stress protein content change curve of the embodiment of the present invention;

[0053] Figure 6 Schematic diagram of the proliferation changes of AC16 cells under hypoxic conditions in an embodiment of the present invention;

[0054] Figure 7 This is a schematic diagram of the proliferation changes of KD cells under hypoxic conditions in an embodiment of the present invention;

[0055] Figure 8 This is a schematic diagram of the proliferation changes of KDNC cells under hypoxic conditions in an embodiment of the present invention;

[0056] Fig. 9 Schematic diagram of the proliferation changes of OE cells under hypoxic conditions in an embodiment of the present invention;

[0057] Fig.10 Schematic diagram of the proliferation changes of OENC cells under hypoxic conditions in an example of the present invention. DETAILED DESCRIPTION

[0058] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0060] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is merely for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0061] In addition, it should be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0062] See also Figure 1 As shown, it is a flow chart of a method for establishing an oxygen induction model using cobalt chloride according to an embodiment of the present invention. The present invention provides a method for establishing an oxygen induction model using cobalt chloride, comprising:

[0063] Step S1, cell culture, inoculating cells into a certain amount of complete culture medium containing cobalt chloride, and culturing in a 37° C., 5% CO 2 incubator;

[0064] Among them, each complete medium contains different concentrations of cobalt chloride;

[0065] Step S2, monitoring the maximum oxygen concentration difference in real time by each concentration sensor arranged in the incubator, and determining whether the oxygen distribution in the incubator is uniform according to the maximum oxygen concentration difference;

[0066] Step S3, when the oxygen distribution in the incubator is uneven, analyzing the response of the cells in the hypoxia-induced model according to the degree of oxygen concentration deviation and the cell state, so as to perform corresponding regulation;

[0067] The corresponding regulation includes reducing the maximum concentration of cobalt chloride, starting the self-regulation mode and adjusting the preset processing time;

[0068] Step S4, when the oxygen in the incubator is evenly distributed, the impedance is monitored by a cell analyzer, and the concentration and addition time of cobalt chloride are selected according to the monitoring results.

[0069] The cells are cardiomyocytes; the complete culture medium contains 10% FBS and 1% penicillin-streptomycin; in step S1, culturing in a 37° C., 5% CO2 incubator also includes:

[0070] When the cells grew to 80% confluence, they were digested with 0.25% trypsin-EDTA and passaged every 2-3 days.

[0071] HIF-1α is the main regulator of the human body under hypoxic conditions. Under normal oxygen conditions, the oxygen-dependent degradation domain of HIF-1α binds to prolyl hydroxylase, HIF-1α is hydroxylated, then recognized and ubiquitinated, and finally degraded by the proteasome. However, in a hypoxic environment, HIF-1α is not hydroxylated by PHD and therefore not degraded. The addition of cobalt chloride can stabilize the HIF-1α protein, thereby simulating a hypoxic environment.

[0072] In this embodiment, the placement of items in the incubator should be reasonably planned, leaving enough space to ensure gas circulation, avoiding placing too many culture dishes, bottles or other items in the incubator to hinder gas flow and cause uneven local oxygen concentration. Due to frequent opening and closing of the incubator door, external air enters and destroys the internal gas environment. Therefore, the number of opening and closing of the door should be reduced as much as possible, or it should be completed quickly during operation. When adjusting the oxygen concentration each time, it is necessary to wait for 30 minutes to 1 hour to allow the gas to be fully mixed to avoid causing oxygen fluctuations in the incubator, which affects the degree of hypoxia and causes inaccurate experimental results and low repeatability. A control group, a hypoxic group and a cobalt chloride treatment group were set. The control group was cultured under normoxic conditions (21% O2) without adding cobalt chloride; the hypoxic group was cultured under hypoxic conditions without adding cobalt chloride. The hypoxic condition 1% O2 is to control the oxygen concentration in the incubator at 1% (volume fraction), and the remaining gases are nitrogen and carbon dioxide (5%); the cobalt chloride treatment group was cultured under normoxic conditions with different concentrations (such as 125 μM, 250 μM, 500 μM, etc.) The cells were treated with cobalt chloride (20 μM) and different treatment times (such as 6 h, 12 h, 24 h, and 48 h) were set. The oxygen concentration in the incubator was monitored to optimize the cobalt chloride concentration and treatment time to ensure the stability of the experimental conditions and the reliability of the experimental results. That is, the oxygen concentration in the incubator was monitored to consider whether the preset oxygen concentration reached the preset hypoxia level. Since high concentrations of cobalt chloride will lead to increased cell death and reduced metabolic activity, oxygen consumption will be reduced, causing the oxygen concentration in the incubator to increase. Alternatively, if the stress response of cells is enhanced under cobalt chloride treatment, oxygen consumption will increase, resulting in changes in local oxygen concentration, causing oxygen fluctuations in the incubator. Therefore, the oxygen concentration in the incubator must be precisely regulated. By real-time monitoring of the oxygen concentration in each area of ​​the incubator, the oxygen concentration of the incubator can be systematically evaluated, thereby accurately monitoring the degree of hypoxia, ensuring the stability of the incubator oxygen control system, and avoiding confounding variables. By analyzing the effects of the degree of hypoxia on cell survival rate, the solubility of cobalt chloride, and biological activity, the optimal concentration and treatment time of cobalt chloride can be more accurately determined, thereby establishing a more accurate hypoxia induction model.

[0073] See also Figure 2 As shown, it is a schematic diagram of a process for determining whether the oxygen distribution in the incubator is uniform according to an embodiment of the present invention;

[0074] Specifically, determining whether the oxygen distribution in the incubator is uniform based on the maximum oxygen concentration difference includes:

[0075] Step S2001, obtaining the corresponding unit oxygen concentration detected by each concentration sensor in the incubator;

[0076] Step S2002, calculating the maximum oxygen concentration difference according to the oxygen concentration of each unit;

[0077] Step S2003, comparing the maximum oxygen concentration difference with the allowable oxygen concentration difference, and determining whether the oxygen distribution in the incubator is uniform according to the comparison result;

[0078] When the maximum oxygen concentration difference is greater than the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uneven, and when the maximum oxygen concentration difference is less than or equal to the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uniform.

[0079] The allowable oxygen concentration difference in this embodiment represents the set concentration sensor indication error. The set allowable indication error shall not exceed 5% of the standard oxygen concentration. When the maximum oxygen concentration difference is less than or equal to the allowable oxygen concentration difference, it indicates that the oxygen distribution in the incubator is uniform.

[0080] By dividing the incubator into grids, either 3×3 or 4×4, the oxygen concentration in the corresponding area can be monitored to accurately analyze the oxygen distribution inside the incubator, and then analyze the impact of oxygen concentration on cell status to avoid unreliable experimental results caused by uneven oxygen distribution.

[0081] See also Figure 3 As shown, it is a schematic diagram of the process of calculating the maximum oxygen concentration difference according to an embodiment of the present invention;

[0082] Specifically, the maximum oxygen concentration difference is calculated based on the oxygen concentration of each unit, including:

[0083] Step S2012, obtaining the real-time oxygen concentration detected by each concentration sensor in each unit detection area to obtain the unit oxygen concentration;

[0084] Step S2022, adding the oxygen concentrations of each unit and dividing by the number of unit detection areas to obtain an average oxygen concentration;

[0085] Step S2032, subtracting the average oxygen concentration from the oxygen concentration of each unit to obtain a number of real-time oxygen concentration fluctuation differences;

[0086] Step S2042, taking the absolute value of each real-time oxygen concentration fluctuation difference, obtaining the value with the largest absolute value, and obtaining the maximum oxygen concentration difference.

[0087] By real-time monitoring of the oxygen concentration in each area of ​​the incubator, the degree of hypoxia can be precisely controlled to ensure the stability of the incubator's oxygen control system.

[0088] See also Figure 4 As shown, it is a schematic diagram of the process of analyzing the response of cells in a hypoxia-induced model according to an embodiment of the present invention;

[0089] Specifically, the analysis of cell responses in hypoxia-induced models based on the degree of oxygen concentration deviation and cell status includes:

[0090] Step S301, when the oxygen distribution in the incubator is uneven, obtaining the real-time oxygen concentration corresponding to the maximum oxygen concentration difference to obtain the oxygen analysis concentration value;

[0091] Step S302, comparing the oxygen analysis concentration value with the standard oxygen concentration, and analyzing the response of the cells in the hypoxia-induced model based on the comparison result to perform corresponding regulation, wherein:

[0092] If the oxygen analysis concentration value is greater than the standard oxygen concentration, the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate;

[0093] If the oxygen analysis concentration value is less than or equal to the standard oxygen concentration, a curve of the oxygen analysis concentration value changing with time is plotted to obtain an oxygen concentration change curve, and whether to adjust the preset processing time is determined based on the oxygen concentration change curve and the difference in stress protein content.

[0094] When it is determined that the oxygen concentration distribution is uneven, the real-time oxygen concentration of the corresponding area is obtained as the oxygen analysis concentration value for accurate analysis. If the oxygen analysis concentration value is greater than the standard oxygen concentration, it means that the local oxygen concentration is too high, which may be due to cell damage that weakens cell respiration. The actual cell survival rate is analyzed to accurately determine the cause. If the oxygen analysis concentration value is less than the standard oxygen concentration, it means that the local oxygen concentration is too low. In this case, the oxygen concentration changes will continue to be monitored, and the cause will be further analyzed based on the change trend.

[0095] Specifically, determining whether to adjust the preset processing time based on the oxygen concentration change curve and the stress protein content difference includes:

[0096] Obtain the actual slope of the oxygen concentration change curve and the difference in stress protein content,

[0097] If the actual slope is less than or equal to the standard slope, the actual cell survival rate is obtained, and the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate;

[0098] If the actual slope is greater than the standard slope, obtain several stress protein content differences, and compare each stress protein content difference with the standard protein content difference.

[0099] If the stress protein content differences are all less than or equal to the standard protein content differences, the preset processing time is not adjusted, and the maximum concentration of cobalt chloride is increased to a first correction concentration;

[0100] If the real-time protein content difference is greater than the standard protein content difference, the current time is updated to the preset processing time;

[0101] Among them, the first correction concentration is the product of the initially set concentration and the first correction step, the first correction step is the sum of 1 and the first correction degree, and the first correction degree is the ratio of the part of the standard protein content difference that exceeds the minimum value of each stress protein content difference to the standard protein content difference.

[0102] In this embodiment, the standard slope is set to 0.01% O2 / hour, indicating that under hypoxic conditions, the standard reduction rate of oxygen consumption of cardiomyocytes per hour is 0.01% O2; the standard oxygen concentration is set to 1% O2 to simulate an oxygen-deficient environment; the standard protein content difference is detected by impedance using an RTCA real-time cell analyzer, and the impedance value is characterized to express the content range of stress-related proteins produced by the stress response of cells in the culture medium. The set value is related to the cell density, type, specifications of the culture medium, etc., and can be set to 105 mg / L, and is selected based on the characteristics of the actual established induced model.

[0103] When it is determined that the actual slope is less than or equal to the standard slope, it indicates that oxygen consumption is reduced, which may be related to cell damage or metabolic inhibition. The cell state is determined by obtaining the actual cell survival rate to accurately analyze the cause. When it is determined that the actual slope is greater than the standard slope, it indicates that oxygen consumption is increased, which may be related to the enhanced stress response. Therefore, the difference in the content of each stress protein is compared with the difference in the standard protein content. If it is determined that the real-time protein content difference is greater than the standard protein content difference, indicating that the enhanced stress response leads to increased oxygen consumption, the current addition time is the optimal value. If it is determined that the stress protein content difference is less than the standard protein content difference, it means that the concentration of cobalt chloride does not significantly promote the stress response, that is, the set initial concentration of cobalt chloride is low, and the concentration of added cobalt chloride needs to be optimized to more comprehensively analyze the response of cells in the hypoxia-induced model.

[0104] Specifically, reducing the maximum concentration of cobalt chloride or starting the self-regulation mode based on the actual cell survival rate includes:

[0105] Obtain the actual cell survival rate and compare it with the standard cell survival rate.

[0106] If the actual cell survival rate is greater than or equal to the standard cell survival rate, the self-regulation mode is turned on. The self-regulation mode is to adjust the oxygen concentration of the corresponding area through the gas flow controller. After the adjustment, after a preset waiting time, the maximum oxygen concentration difference is obtained again for judgment. If it is still determined that the oxygen analysis concentration value is greater than the standard oxygen concentration, a calibration alarm is issued;

[0107] If the actual cell survival rate is less than the standard cell survival rate, the maximum concentration of cobalt chloride is adjusted to the second correction concentration;

[0108] The second correction concentration is the product of the initially set concentration and the second correction step length, the second correction step length is the difference between 1 and the second correction degree, and the second correction degree is the ratio of the portion of the standard cell survival rate that exceeds the actual cell survival rate to the standard cell survival rate.

[0109] In this embodiment, the standard cell survival rate is the expected cell survival ratio, which is set to 85%; the preset waiting time is 30 minutes to 1 hour;

[0110] For example, when it is determined that the oxygen distribution in the incubator is uneven and the maximum oxygen concentration difference is 0.12%, it exceeds the allowed 0.05%. At this time, the oxygen distribution is determined to be uneven, and the self-regulation mode is started. The oxygen concentration is adjusted to the target value (for example, from 1.12% to 1.05%) through the gas flow controller to get as close as possible to the standard oxygen concentration of 1% O2. After 60 minutes, the maximum oxygen concentration difference is obtained again for determination. If the maximum oxygen concentration difference drops to 0.04% at this time, which is less than or equal to the allowed 0.05%, the oxygen distribution is determined to be uniform and the self-regulation mode is stopped; if the maximum oxygen concentration difference drops to 0.08% at this time, but is still greater than the allowed 0.05%, a calibration alarm is issued to remind the staff to check and calibrate the oxygen sensor or gas flow controller to ensure data accuracy.

[0111] When uneven gas distribution and local oxygen concentration increases or reduced oxygen consumption are detected in the incubator, it may be caused by weakened cell respiration, poor gas flow in the incubator itself, or errors in oxygen concentration measurement. The actual cell survival rate can be monitored to accurately analyze the cause.

[0112] Since high concentrations of cobalt chloride may induce cell death or metabolic inhibition, resulting in reduced oxygen consumption, thereby indirectly affecting the dynamic balance of oxygen in the incubator, when it is determined that the oxygen analysis concentration value is greater than the standard oxygen concentration and the actual cell survival rate is less than the standard cell survival rate, it is analyzed that this situation is caused by weakened cell respiration. The maximum concentration of cobalt chloride is reduced to ensure that the concentration used is within the range that the cells can withstand. When the actual cell survival rate is greater than or equal to the standard cell survival rate, it means that there is no cell death causing reduced oxygen consumption. The gas flow controller in the corresponding area is turned on to adjust the oxygen concentration in the corresponding area, that is, the fan is added to promote full mixing of the gas to adjust the oxygen concentration in the corresponding area. After the adjustment, the gas distribution in the incubator is analyzed again. If there is no improvement, a calibration alarm is issued to remind the staff to calibrate the incubator oxygen sensor to ensure data accuracy.

[0113] See also Figure 5 As shown, it is a schematic diagram of the stress protein content change curve of the embodiment of the present invention;

[0114] Specifically, obtaining the difference in the content of several stress proteins includes:

[0115] The content of stress proteins in each complete culture medium is obtained at a preset collection interval, and the change curve of the content of stress proteins over time is plotted to generate the change curve of the content of each stress protein;

[0116] The peak values ​​corresponding to the stress protein content change curves were obtained, and the differences in the corresponding stress protein contents in the two complete culture media were calculated in the order of the amount of cobalt chloride added from low to high, which were recorded as the differences in the stress protein contents.

[0117] According to the monitoring results, the concentration of cobalt chloride and the time of addition are selected, including:

[0118] Use a cell analyzer to monitor cell impedance changes in real time and determine the cell status based on the impedance change curve;

[0119] If cell impedance decreases, reduce the cobalt chloride concentration or shorten the addition time;

[0120] If the cell impedance is stable, maintain the current cobalt chloride concentration and addition time, select the cobalt chloride concentration and treatment time with a cell survival rate higher than 80%, a HIF-1α expression level greater than 2 times the control, and no significant damage to cell activity as the optimal conditions, and construct a hypoxia-induced model based on the selected cobalt chloride concentration and addition time.

[0121] Specifically, the hypoxia-induced model is used in the detection and screening of antioxidant drugs.

[0122] In this example, the optimized cobalt chloride concentration is 250 μmol / L and the treatment time is 60 h;

[0123] Example 1: The optimized cobalt chloride concentration and addition time were used to simulate the hypoxic environment and the iCELLigence RTCA real-time cell analysis system was used to detect the proliferation of cardiomyocytes under hypoxic conditions.

[0124] Specifically, 1. Activate the E-Plate 16-well plate, preheat the culture medium 30 minutes in advance, use a pipette to carefully add 100 μL of preheated culture medium to the E-Plate 16-well plate, place the E-Plate 16-well plate in the RTCA instrument, open the RTCA software, and the instrument automatically detects that the electrode values ​​of the horizontal parallel duplicate holes of the click plate are similar, and the electrode values ​​of the vertical symmetrical holes are similar. Click New Experience to create a new experiment: add the experiment name; Layout the experiment layout, click Add Program to start Step 1 to run the baseline; 2. Target cell treatment, collect Collect 1 bottle (T75 culture bottle) of AC16, gene overexpression group (OE), overexpression empty control group (OENC), gene knockdown group (KD), knockdown empty control group (KDNC) cells (fusion degree 60%-80%), discard the supernatant, wash once with 10mL PBS, digest with 1mL 0.25% trypsin at 37℃ for 1min, add 7mL 10% FBS DMEM / F12 medium, collect cells after pipetting evenly, centrifuge at 1000rpm / min for 5min, discard the supernatant, add 10% FBS complete medium to resuspend the cells, count and dilute the cells to 5×10 4 cells / mL (100μL / well); 3. Sample addition, take out the E-Plate 16-well plate from the RTCA, carefully discard the culture medium (suck it off along the border, do not touch the bottom electrode part), add the above cell dilution into the E-Plate 16-well plate at a volume of 100μL / well (quickly inject along the wall), so that the number of target cells in each well is 5000; let the E-Plate 16-well plate stand at room temperature for 30 minutes to allow the cells to settle to the bottom, set the program, Step 2 is 240h, read the reading every 30min, then move the E-Plate 16-well plate into the RTCA instrument in the constant temperature cell culture incubator (37℃, 5% CO2), after balancing for 15min, click Step 2 to start the RTCA program, monitor the impedance for 240h, read the reading every 30min, and observe the proliferation curve of the target cells; 4. Add medicine, group setting: Blank control group, different concentrations of cobalt chloride groups;

[0125] The drug configuration is shown in Table 1.

[0126]

[0127] Table 1 Drug configuration table

[0128] Where: “*” indicates AssayBuffer;

[0129] The next day, pause the program, take out the E-Plate 16-well plate, add 100 μL / well of cobalt chloride solution according to the group settings, move the E-Plate 16-well plate into the RTCA instrument in the constant temperature cell culture incubator (37°C, 5% CO2), equilibrate for 20 minutes, click Step 2 to start the RTCA program, monitor the impedance, read it every 30 minutes, and observe the proliferation of cells under hypoxic conditions.

[0130] See also Figure 6 As shown, it is a schematic diagram of the proliferation changes of AC16 cells under hypoxic conditions in an embodiment of the present invention. There are some problems with the proliferation of AC16 cells. The CI value reaches about 4.3 2 hours after cell plating, and the drug addition result is unreliable.

[0131] See also Figure 7 As shown, it is a schematic diagram of the proliferation changes of KD cells under hypoxic conditions in an embodiment of the present invention. The CI value of KD cells reached about 2.6 at 24 hours after plating. After adding different concentrations of cobalt chloride at 24 hours, compared with the Blank group, 125μmol / L and 250μmol / L cobalt chloride treatment groups, the concentration of 500μmol / L cobalt chloride had the best effect, which caused the CI value of KD cells to decrease at 14 hours after the addition of the drug, and all cells died after 50 hours. The results showed that KD cells simulated hypoxia under 500μmol / L cobalt chloride conditions, leading to KD cell death;

[0132] See also Figure 8 As shown, it is a schematic diagram of the proliferation changes of KDNC cells under hypoxic conditions in an embodiment of the present invention. The CI value of KDNC cells reached about 2.0 at 24 hours after plating. After adding different concentrations of cobalt chloride at 24 hours, compared with the Blank group, 125μmol / L and 250μmol / L cobalt chloride treatment groups, the concentration of 500μmol / L cobalt chloride had the best effect, which would cause the CI value of KDNC cells to decrease at 14 hours after the addition of the drug, and all cells died after 24 hours. The results showed that KDNC cells simulated hypoxia under 500μmol / L cobalt chloride conditions, leading to KDNC cell death;

[0133] See also Fig. 9As shown, it is a schematic diagram of the proliferation changes of OE cells under hypoxic conditions in an embodiment of the present invention. The CI value of OE cells reached about 2.5 at 24 hours after plating. After adding different concentrations of cobalt chloride at 24 hours, compared with the Blank group, 125μmol / L and 250μmol / L cobalt chloride treatment groups, the concentration of 500μmol / L cobalt chloride had the best effect, which would cause the CI value of OE cells to decrease at 30 hours after the addition of the drug, and all cells died after 40 hours. The results showed that the hypoxic conditions of OE cells simulated under 500μmol / L cobalt chloride led to the death of OE cells;

[0134] See also Fig.10 As shown, it is a schematic diagram of the proliferation changes of OENC cells under hypoxic conditions in an embodiment of the present invention. The CI value of OENC cells reached about 1.6 at 24 hours after plating. After adding different concentrations of cobalt chloride at 24 hours, compared with the Blank group, 125 μmol / L and 250 μmol / L cobalt chloride treatment groups, the concentration of 500 μmol / L cobalt chloride had the best effect, which would cause the CI value of OENC cells to decrease at 30 hours after the addition of the drug, and all cells died after 36 hours. The results show that OENC cells simulated hypoxic conditions under 500 μmol / L cobalt chloride conditions, leading to the death of OENC cells.

[0135] Therefore, it can be concluded that the drug treatment can be carried out 24 hours after the cells adhere to the wall; the dosage range that can cause hypoxia without excessively affecting the cell viability was explored, and the results showed that CoCl2 reduced the viability of human myocardial AC16 cells in a dose- and time-dependent manner, and finally 250 μmol / L was selected for incubation for 24 hours to construct a myocardial hypoxia model.

[0136] Among them, 1 represents the control group without adding cobalt chloride, 2 represents the cobalt chloride concentration of 125 μmol / L, 3 represents the cobalt chloride concentration of 250 μmol / L, and 4 represents the cobalt chloride concentration of 500 μmol / L.

[0137] Example 2, in order to establish appropriate hypoxic conditions, cells were plated in a 96-well plate, 5000 cells per well, and cultured overnight. After the cells were completely attached to the wall, the supernatant was discarded, and CoCl2 with different final concentrations (0, 250, 500, 750, 1000 μmol / L) was added and incubated for different times (0, 24, 48, 72h), the supernatant was discarded, 10μl CCK-8 solution CCK-8 reagent and 90μ complete culture medium were added to each well, incubated in the dark for 1.5h, and the absorbance (OD Value) at 450nm was detected by an enzyme marker. Cell survival rate = [(experimental group-blank group) / (control group-blank group)]×100%. Experimental wells: containing cells, culture medium, CCK-8 solution and CoCl2 solution; control wells: containing cells, culture medium, CCK-8 solution, without CoCl2; blank group: containing culture medium, CCK-8 solution, without cells and CoCl2. The most appropriate CoCl2 concentration was screened based on the detected AC16 cell viability to establish a human cardiomyocyte hypoxia injury model.

[0138] Results: The cells were incubated at 37℃ and 5% CO2 for different time periods (24, 48 or 72 h) in DMEM / F12 complete medium with 250, 500, 750 and 1000 μmol / L CoCl2. The results of CCK-8 assay showed that with the increase of CoCl2 concentration (0, 250, 500, 750 and 1000 μmol / L), the viability of human AC16 cardiomyocytes in other CoCl2 concentration groups was significantly reduced compared with the 0 μmol / L CoCl2 group after 24 h of culture (P<0.0001). Compared with the 250 μmol / L CoCl2 concentration group, the 500 and 1000 μmol / L CoCl2 concentration groups had the greatest inhibitory effect on the viability of AC16 cardiomyocytes (P<0.0001), and the cell viability was less than 50%. With the extension of incubation time, the viability of AC16 cardiomyocytes in the 500 μmol / L and above CoCl2 concentration groups reached the lowest level (P<0.0001).

[0139] Therefore, from the perspective of pharmacodynamics and economics, 250 μmol / L was selected for incubation for 24 h to construct the myocardial hypoxia model.

[0140] In this example, a hypoxia-induced model was constructed with the optimized cobalt chloride addition concentration and addition time, and the effects of different concentrations of cobalt chloride on the hypoxia-induced effect of AC16 cardiomyocytes were verified through a series of experiments. The results showed that the method provided by the present invention can effectively simulate the hypoxic environment and provide an important tool for the study of hypoxia-related diseases.

[0141] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for establishing an oxygen induction model using cobalt chloride, characterized in that: include, Step S1, cell culture, inoculating cells into a certain amount of complete culture medium containing cobalt chloride, and culturing in a 37° C., 5% CO 2 incubator; Among them, each complete medium contains different concentrations of cobalt chloride; Step S2, monitoring the maximum oxygen concentration difference in real time by each concentration sensor arranged in the incubator, and determining whether the oxygen distribution in the incubator is uniform according to the maximum oxygen concentration difference; Step S3, when the oxygen distribution in the incubator is uneven, analyzing the response of the cells in the hypoxia-induced model according to the degree of oxygen concentration deviation and the cell state, so as to perform corresponding regulation; The corresponding regulation includes reducing the maximum concentration of cobalt chloride, starting the self-regulation mode and adjusting the preset processing time; Step S4, when the oxygen in the incubator is evenly distributed, the impedance is monitored by a cell analyzer, and the concentration and addition time of cobalt chloride are selected according to the monitoring results.

2. The method for establishing an oxygen induction model using cobalt chloride according to claim 1, characterized in that: Determining whether the oxygen distribution in the incubator is uniform based on the maximum oxygen concentration difference includes: Obtaining the corresponding unit oxygen concentration detected by each concentration sensor in the incubator; Calculating the maximum oxygen concentration difference according to the oxygen concentration of each unit; The maximum oxygen concentration difference is compared with the allowable oxygen concentration difference, and whether the oxygen distribution in the incubator is uniform is determined according to the comparison result; When the maximum oxygen concentration difference is greater than the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uneven, and when the maximum oxygen concentration difference is less than or equal to the allowable oxygen concentration difference, it is determined that the oxygen distribution in the incubator is uniform.

3. The method for establishing an oxygen induction model using cobalt chloride according to claim 2, characterized in that: The maximum oxygen concentration difference calculated based on the oxygen concentration of each unit includes: Obtain the real-time oxygen concentration detected by each concentration sensor in each unit detection area to obtain the unit oxygen concentration; Add the oxygen concentration of each unit and divide it by the number of unit detection areas to get the average oxygen concentration; Subtracting the average oxygen concentration from the oxygen concentration of each unit to obtain a number of real-time oxygen concentration fluctuation differences; Take the absolute value of each real-time oxygen concentration fluctuation difference, obtain the value with the largest absolute value, and obtain the maximum oxygen concentration difference.

4. The method for establishing an oxygen induction model using cobalt chloride according to claim 1, characterized in that: Analysis of cell responses in hypoxia-induced models based on the degree of oxygen concentration deviation and cell status includes: When the oxygen distribution in the incubator is uneven, the real-time oxygen concentration corresponding to the maximum oxygen concentration difference is obtained to obtain the oxygen analysis concentration value, and the oxygen analysis concentration value is compared with the standard oxygen concentration. If the oxygen analysis concentration value is greater than the standard oxygen concentration, the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate; If the oxygen analysis concentration value is less than or equal to the standard oxygen concentration, a curve of the oxygen analysis concentration value changing with time is plotted to obtain an oxygen concentration change curve, and whether to adjust the preset processing time is determined based on the oxygen concentration change curve and the difference in stress protein content.

5. The method for establishing an oxygen induction model using cobalt chloride according to claim 4, characterized in that: Determining whether to adjust the preset processing time based on the oxygen concentration change curve and the difference in stress protein content includes: Obtain the actual slope of the oxygen concentration change curve and the difference in stress protein content, If the actual slope is less than or equal to the standard slope, the actual cell survival rate is obtained, and the maximum concentration of cobalt chloride is reduced or the self-regulation mode is turned on based on the actual cell survival rate; If the actual slope is greater than the standard slope, obtain several stress protein content differences, and compare each stress protein content difference with the standard protein content difference. If the stress protein content difference is less than or equal to the standard protein content difference, the preset processing time is not adjusted, and the maximum concentration of cobalt chloride is increased; If the real-time protein content difference is greater than the standard protein content difference, the current time is updated to the preset processing time.

6. The method for establishing an oxygen induction model using cobalt chloride according to claim 4, characterized in that: Based on the actual cell survival rate, the maximum concentration of cobalt chloride can be reduced or the self-regulation mode can be turned on, including: Obtain the actual cell survival rate and compare it with the standard cell survival rate. If the actual cell survival rate is greater than or equal to the standard cell survival rate, the self-regulation mode is turned on. The self-regulation mode is to adjust the oxygen concentration of the corresponding area through the gas flow controller. After the adjustment, after a preset waiting time, the maximum oxygen concentration difference is obtained again for judgment. If it is still determined that the oxygen analysis concentration value is greater than the standard oxygen concentration, a calibration alarm is issued; If the actual cell viability is lower than the standard cell viability, adjust the maximum concentration of cobalt chloride to a lower level.

7. The method for establishing an oxygen induction model using cobalt chloride according to claim 5, characterized in that: Obtaining the difference in the content of several stress proteins includes: The content of stress proteins in each complete culture medium is obtained at a preset collection interval, and the change curve of the content of stress proteins over time is plotted to generate the change curve of the content of each stress protein; The peak values ​​corresponding to the stress protein content change curves were obtained, and the differences in the corresponding stress protein contents in the two complete culture media were calculated in the order of the amount of cobalt chloride added from low to high, which were recorded as the differences in the stress protein contents.

8. The method for establishing an oxygen induction model using cobalt chloride according to claim 1, characterized in that: The cells are cardiomyocytes.

9. The method for establishing an oxygen induction model using cobalt chloride according to claim 1, characterized in that: Cultivation in a 37°C, 5% CO2 incubator also includes: When the cells grew to 80% confluence, they were digested with 0.25% trypsin-EDTA and passaged every 2-3 days.

10. The method for establishing an oxygen induction model using cobalt chloride according to claim 1, characterized in that: The complete culture medium contained 10% FBS and 1% penicillin-streptomycin double antibody.

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