Method for constructing HK-2 cell mitochondrial damage model based on inorganic phosphate

Abstract

The invention discloses a method for constructing an HK-2 cell mitochondrial damage model based on inorganic phosphate, which is characterized in that HK-2 cells are selected as objects for establishing the model, and the HK-2 cell mitochondrial damage model of inorganic phosphate food additives with different concentrations is constructed. A more excellent cell model is provided for the effective and safe use dosage of the food additive, a safer range reference is provided in the current safe use dosage range, and a very important experimental material and a technical method are provided for the use of the food additive. Compared with the prior art which focuses on an endocrine regulation signal transduction mechanism of FGF-23 and alpha-kloth axis and rarely researches directly aiming at kidney tubular mitochondria, the HK-2 cell mitochondrial injury model is constructed, a reasonable research thought is provided for establishing and knowing the action change of an inorganic phosphate food additive on the kidney, and the application of the HK-2 cell mitochondrial injury model is developed. The method has positive influence on how to prevent and treat CKD, and has guiding significance on healthy diet of common people.

Description

Technical Field

[0001] The present invention relates to the technical field of cell injury model construction, and in particular to a method for constructing a HK-2 cell mitochondrial injury model based on inorganic phosphate. Background Art

[0002] Prior art research has found that high-sodium and high-lead diets are risk factors for chronic kidney disease quantified by the global burden of disease. In addition, ultra-processed foods are becoming more and more popular among consumers due to their easy availability and convenience, but ultra-processed foods often require the addition of more food additives, including phosphate food additives. At present, the most prominent ultra-processed foods are pre-prepared dishes. In order to maintain the better taste of pre-prepared dishes, food additives such as the phosphate food additives mentioned above are often required. There are about 18 types of phosphoric acid and phosphate additive products allowed for use in my country, including multiple functions such as water retention and preservation, and a wide range of applications. As people consume too much of these ultra-processed foods, the risk of chronic kidney disease (CKD) will also increase. Therefore, studying the impact of phosphate food additives on health has guiding significance for the safe use of food additives in the pre-prepared food industry.

[0003] As we all know, the kidney is the second highest oxygen consumption organ in the human body, second only to the heart. Compared with other cells in the kidney, the proximal tubule requires more energy for reabsorption, so the mitochondria in the proximal tubule are the most abundant in all kidney structures. However, there are few studies directly targeting the mitochondria in the renal tubules in the existing technology. Therefore, it is particularly important to study the effects of food additives on the mitochondria in the renal tubules and then intervene in the diet. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a method for constructing a mitochondrial damage model of HK-2 cells based on inorganic phosphate. The specific technical scheme is as follows:

[0005] A method for constructing a mitochondrial damage model of HK-2 cells based on inorganic phosphate, the method comprising the following steps:

[0006] S1. HK-2 cells were resuscitated;

[0007] S2. Count the stably cultured HK-2 cells and set different cell densities for plating, with 6 replicates per group. Use the CCK-8 kit to obtain the optimal plating density of HK-2 cells. 4 / mL density was plated in a 96-well plate. After 24 hours, culture medium containing different concentrations of inorganic phosphate was added to the 96-well plate for co-culture. After 72 hours of culture, the cells were taken out of the cell culture incubator, the culture medium was discarded, and the complete culture medium containing CCK-8 solution was added to the 96-well plate in the form of replacement medium. After incubation for 1 hour, the cells were detected at a wavelength of 450nm using an automatic microplate reader to obtain the active oxygen data of HK-2 cells under different concentrations of inorganic phosphate.

[0008] S3. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene HK-2 cells. Four replicate wells were prepared for each concentration. After 48 hours, the culture medium was removed and replaced with a new medium containing different concentrations of drugs that had been taken out of a 4°C refrigerator and re-warmed. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded. The cell morphology of HK-2 cells in the 12-well plate was analyzed using an inverted microscope at 0 hours and 96 hours of culture. Then, the DCFH-DA staining working solution was added. After incubation for 30 minutes, the supernatant was discarded. The sealing medium was added and the data of cell mitochondrial reactive oxygen content were obtained by detection on a microscope.

[0009] S4. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene HK-2 cells. Each concentration was repeated for 4 wells. After 48 hours, the medium was changed and the medium containing different concentrations of inorganic phosphate was taken out of the 4°C refrigerator and re-warmed. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded, and then JC-1 staining working solution was added. The cells were incubated for 30 minutes, the staining solution was discarded, and the cells were washed. The sealing medium was added. After the detection on the machine, the data were analyzed using image J software to obtain the data related to the changes in mitochondrial membrane potential of HK-2 cells.

[0010] S5. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene HK-2 cells. Each concentration was repeated for 4 wells. After 48 hours, the medium was changed and the medium containing different concentrations of inorganic phosphate taken out of the 4°C refrigerator and re-warmed was added. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded and Rhod-2 AM staining working solution was added. The cells were incubated for 30 minutes. The cells were washed with complete medium 2-3 times, each time for 5 minutes. The sealing agent was added. The cells were detected and photographed using an automatic microplate reader and an inverted fluorescence microscope, respectively, to obtain the change data of mitochondrial calcium ion concentration in HK-2 cells.

[0011] The reactive oxygen data of HK-2 cells under different concentrations of inorganic phosphate, the reactive oxygen content data of mitochondria of cells, the mitochondrial membrane potential data of HK-2 cells, and the mitochondrial calcium ion concentration data of HK-2 cells were statistically analyzed, and charts were drawn to construct a model of inorganic phosphate damage to HK-2 cell mitochondria.

[0012] Furthermore, in step S1, the resuscitation treatment is: adding HK-2 cells to a complete culture medium containing 10% FBS, stably subculturing at 37°C and 5% CO2, and using HK-2 cells within 4 to 10 generations for the experiment.

[0013] Furthermore, in step S2, the different cell densities were: 1.2×10 4 / mL, 2.4×10 4 / mL, 4.8×10 4 / mL, 9.6×10 4 / mL.

[0014] Furthermore, the different concentrations of inorganic phosphate are 0.375 mM, 0.75 mM, 1 mM, 1.25 mM and 1.5 mM, respectively.

[0015] Furthermore, in step S2, the final concentration of the CCK-8 solution is 10%.

[0016] Furthermore, the incubation conditions in step S2, the incubation conditions in step S3, the incubation conditions in step S4, and the incubation conditions in step S5 are all 37° C. and 5% CO 2 .

[0017] Furthermore, the sealing medium is an anti-fluorescence quenching sealing medium containing DAPI.

[0018] Further, in step S3, DCFH-DA in the DCFH-DA kit was diluted with serum-free DMEM / F12 medium at a ratio of 1:1000, so that the final concentration of the DCFH-DA staining working solution was 10 μM, and HK-2 cells in a 12-well plate were covered when used.

[0019] Furthermore, in step S4, the concentration of the JC-1 staining working solution is 1 μg / μL.

[0020] Furthermore, in step S5, the concentration of the Rhod-2 AM working solution is 5 μg / μL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention uses HK-2 cells as the object of establishing this model, and constructs a model of HK-2 cell mitochondrial damage caused by inorganic phosphate food additives of different concentrations, providing a better cell model for the study of effective and safe dosage of food additives in the food industry, and establishing safe use limits of food additives. In addition, it can also provide a reference for the safety of additives in pre-prepared dishes, and provide very important experimental materials and technical methods for the use of food additives.

[0023] 2. Compared with the existing technology that focuses on the mechanism research of FGF-23 and α-kloth axis, there are few studies directly targeting renal tubular mitochondria. The present invention constructs a HK-2 cell mitochondrial damage model to provide a reasonable research idea for understanding the changes in the effects of inorganic phosphate food additives on the kidneys, which has a positive impact on the prevention and treatment of CKD and has guiding significance for healthy diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention can be further understood from the following description in conjunction with the accompanying drawings. The components in the figures are not necessarily drawn to scale, but the emphasis is placed on illustrating the principles of the embodiments. In different views, the same reference numerals designate corresponding parts.

[0025] Figure 1 is a schematic diagram of observing the mitochondrial morphology under an optical inverted microscope in Example 3 of the present invention;

[0026] Figure 2 is a schematic diagram of the ratio of mitochondrial green fluorescence observed under an optical inverted microscope in Example 4 of the present invention;

[0027] Figure 3 This is a schematic diagram of observing cells and mitochondria using a transmission electron microscope in Example 5 of the present invention;

[0028] Figure 4 This is a schematic diagram of the red and green fluorescence ratio of mitochondria observed under an optical inverted fluorescence microscope in Example 6 of the present invention;

[0029] Figure 5 Schematic diagram of the red and green fluorescence ratio of mitochondria observed under an optical inverted fluorescence microscope in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0032] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0033] Embodiment 1:

[0034] Preparation of inorganic phosphate solutions of different concentrations. The following experiments use disodium hydrogen phosphate-sodium dihydrogen phosphate solution, that is, the inorganic phosphate solution involved in the present invention is sodium phosphate that becomes disodium hydrogen phosphate-sodium dihydrogen phosphate solution after contacting water and air, as shown in the following Table 1:

[0035] Table 1: Inorganic phosphate solutions of different concentrations

[0036]

[0037] Embodiment 2:

[0038] HK-2 cell recovery treatment:

[0039] Take out the pre-prepared complete culture medium containing 10% FBS from the 4°C refrigerator, take out the HK-2 cells from the liquid nitrogen tank for resuscitation, and culture the HK-2 cells in a 37°C, 5% CO2 cell culture incubator. After the adherent growth is stable, subculture is performed every 2-3 days, and cells with a subculture number of 4 to 10 are selected for the experiment.

[0040] Embodiment 3:

[0041] The stable HK-2 cells were counted. In experiment 1, 1.2×10 4 / mL, 2.4×10 4 / mL, 4.8×10 4 / mL, 9.6×10 4 The cells were plated at a density of 1:1 / mL, with 6 replicate wells in each group, and the optimal number of HK-2 cells for plating was determined using the cell counting kit 8 (cck-8) (Biosharp, BS350B).

[0042] Experiment 2 used 5×10 4 / mL density was plated into a 96-well plate. After 24 hours, the culture medium containing different concentrations of phosphate was added to the 96-well plate for co-culture. After 72 hours of culture, the CCK-8 solution was added to the complete culture medium according to the required volume to make the final concentration of CCK-8 10%. The cells were taken out of the cell culture incubator, the culture medium was discarded, and the complete culture medium containing 10% CCK-8 was added to the 96-well plate in the form of liquid replacement using a 12-channel pipette. After incubation at 37°C, 5% CO2 environment for 1 hour, the detection was performed at a wavelength of 450nm using an automatic microplate reader. The results are shown in Figure 2. Figure 1 shown.

[0043] from Figure 1 It can be clearly seen that the fully automatic fluorescent microplate reader detects the OD value at a wavelength of 450nm. The higher the OD, the stronger the cell vitality. The mitochondrial morphology is observed under an optical inverted microscope. Figure 1 Figure a is the control group. Figure 1 Figure b is the experimental group. It can be seen that the mitochondrial morphology of the HK-2 cells in the blank group is approximately elliptical, while the morphology of the HK-2 cells in the experimental group is different and cell deformation occurs.

[0044] In summary, the data were collated to determine 5×10 4 / mL density is the optimal plating density of HK-2 cells in 96-well plates, and the optimal incubation time of CCK-8 is 1h. In experiment 2, after 72h of plating and culture, the inorganic phosphate concentration in the range of 1mM-1.5mM had an effect on the activity of HK-2 cells compared with the control group.

[0045] Embodiment 4:

[0046] Press 2×10 5 / mL density was plated in a 12-well plate, 1mL per well, and cultured at 37℃, 5% CO2 for 24h. After the growth stabilized, different concentrations of inorganic phosphate were added to intervene in HK-2 cells, with 4 replicates for each concentration. After 48h, the culture medium was aspirated and replaced with the medium. The culture medium containing different concentrations of drugs taken out of the 4℃ refrigerator and pre-warmed was added again. The incubation continued for 48h. After 96h of intervention, the supernatant was discarded. The cell morphology of HK-2 cells in the 12-well plate was analyzed using an inverted microscope at 96h of culture. Then the DCFH-DA kit was added and the DCFH-DA kit was diluted with serum-free DMEM / F12 culture medium at a ratio of 1:1000 to a final concentration of 10μM. The cells were incubated at 37℃, 5% CO2 for 30min, and then the sealing agent was added. The level of cell ROS production was observed using an inverted fluorescence microscope. The results are as follows Figure 2 shown.

[0047] Figure 2 Figure c is the control group. Figure 2 Figure d in the figure is the experimental group, and the mitochondrial green fluorescence ratio was observed under an optical inverted fluorescence microscope. The mitochondrial electron transport chain is the main place where cells produce ROS, so detecting the production of cellular ROS helps to show that mitochondria have oxidative stress reactions and produce too much reactive oxygen. The ROS production level in the control group Figure a is significantly lower than that in the experimental group, indicating that inorganic phosphate has an adverse effect on the intervention of HK-2 cells.

[0048] Embodiment 5:

[0049] 5×10 5 HK-2 cells were spread into a 10 cm dish at a density of / mL. After 24 hours of stable culture at 37°C and 5% CO2, the cells were randomly divided into a control group and a treatment group. Different concentrations of inorganic phosphate were added to the experimental groups to intervene in HK-2 cells. The cells were treated with a replacement medium at 48 hours. After 96 hours, the culture medium was discarded and a 2.5% glutaraldehyde solution was added to fix the cells. The cells were then gently scraped off with a cell scraper and collected by centrifugation. Samples were prepared and the cells and mitochondria were observed using a transmission electron microscope. The results are as follows Figure 3 shown.

[0050] Figure 3 Figure e in the figure is the control group. Figure 3 Figure f is the experimental group. Figure 3 It can be seen that the mitochondrial structure of HK-2 cells in the control group is complete, the mitochondria are arranged regularly and clearly (green arrows), while the mitochondrial membrane structure of HK-2 cells in the experimental group is incomplete, the mitochondrial cristae are broken and disappeared (red arrows), and calcium deposition appears in the mitochondria (yellow arrows), Scalebar = 500nm. This indicates that high concentrations of inorganic phosphate intervention stimulated HK-2 cells and HK-2 cells produced oxidative stress.

[0051] Embodiment 6:

[0052] 5×10 4 / mL density was plated in a 96-well plate. After 24 hours, culture medium containing different concentrations of inorganic phosphate was added to the 96-well plate for co-culture. After 72 hours of culture, the cells were taken out of the cell culture incubator, the culture medium was discarded, and then JC-1 working solution with a concentration of 1μg / μL was added. The cells were incubated at 37°C and 5% CO2 for 30 minutes. The staining solution was discarded, and after washing, the sealing medium was added. The red and green fluorescence ratio of mitochondria was observed under an optical inverted fluorescence microscope. After taking pictures, the red and green fluorescence ratio of the pictures was analyzed using Image J. The results are shown in Figure 4 shown.

[0053] Figure 4 Figure g is the control group. Figure 4 Figure h in the figure is the experimental group. Figure 4As can be seen in the figure, the HK-2 cells after intervention were stained with JC-1 staining solution and DAPI solution. The proportion of mitochondrial red fluorescence in the HK-2 cells in the control group was higher, while the mitochondrial red fluorescence in the HK-2 cells in the experimental group was weak and the green fluorescence was obvious, indicating that the mitochondrial membrane potential decreased under the stimulation of high concentrations of phosphate. Under normal conditions, the mitochondrial membrane potential of HK-2 cells is maintained at a constant level. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form a polymer that produces red fluorescence; conversely, when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix as a monomer and produces green fluorescence. After 96 hours of high concentration phosphate stimulation, mitochondrial homeostasis was destroyed, the mitochondrial membrane potential changed, and the green area increased under the microscope. Data analysis showed that the red-green ratio decreased.

[0054] Embodiment 7:

[0055] 2×10 5 / mL density was plated and plated in 12-well plates. After stable culture at 37℃ and 5% CO2 for 24h, the cells were randomly divided into control group and experimental group. Different concentrations of inorganic phosphate were added to the experimental groups to intervene in the cells, and there were 3 replicates in each group. The cells were stained with Rhod-2 AM at a concentration of 5μg / μL and incubated at 37℃ and 5% CO2 for 30min. After washing with PBS, the sealing medium was added and the mitochondrial calcium ion content was observed under an inverted fluorescence microscope. The stronger the red fluorescence intensity, the higher the mitochondrial calcium ion content. The OD value was detected by an automatic fluorescence microplate reader. The higher the OD value, the more it indicates that mitochondrial calcium overload has occurred. The results are as follows Figure 4 shown.

[0056] Figure 5 Figure i is the control group, Figure 4 Figure j in the figure is the experimental group, HK-2 cells after Rhod-2 AM staining intervention. The mitochondrial red fluorescence in the control group was significantly lower than that in the experimental group, indicating that the mitochondria produced calcium overload under the stimulation of high concentrations of phosphate. Under physiological conditions, calcium ions in mitochondria are maintained at a low level. Rhod-2 AM ester can penetrate the cell membrane and is cleaved by its lactase to produce Rhod-2 without membrane permeability, which is retained in the cell. Compared with the control group, as the phosphate concentration increases, the calcium ion level increases. The inverted fluorescence microscope is used to detect and shoot under the RGB channel. It can be seen from the figure that compared with the control group, the red fluorescence is gradually increasing, which is consistent with the results of the microplate reader, indicating that high concentrations of phosphate cause the mitochondrial calcium ion level to increase, resulting in mitochondrial calcium overload.

[0057] Finally, the data of Examples 1 to 7 were statistically analyzed, graphs were drawn, and a model of inorganic phosphate damage to HK-2 cell mitochondria was constructed.

[0058] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a mitochondrial damage model of HK-2 cells based on inorganic phosphate, characterized in that: The construction method comprises the following steps: S1. HK-2 cells were resuscitated; S2. Count the stably cultured HK-2 cells and set different cell densities for plating, with 6 replicates per group. Use the CCK-8 kit to obtain the optimal plating density of HK-2 cells. 4 The cells were plated into a 96-well plate at a density of 100 μg / mL. After 24 hours, culture medium containing different concentrations of inorganic phosphate was added into the 96-well plate for co-culture. After 72 hours of culture, the cells were taken out from the cell culture incubator, the culture medium was discarded, and complete culture medium containing CCK-8 solution was added into the 96-well plate in the form of replacement medium. After incubation for 1 hour, the cells were detected at a wavelength of 450 nm using an automatic microplate reader to obtain the activity data of HK-2 cells under different concentrations of inorganic phosphate. S3. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene in HK-2 cells. Each concentration was repeated for 4 wells. After 48 hours, the culture medium was removed and the medium was replaced. The culture medium containing different concentrations of drugs that had been pre-warmed and taken out of the 4°C refrigerator was added again. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded. The cell morphology of HK-2 cells in the 12-well plate was analyzed using an inverted fluorescence microscope. Then, the DCFH-DA staining working solution was added. After incubation for 30 minutes, the supernatant was discarded. The sealing medium was added and the data of cell mitochondrial reactive oxygen content were obtained by the microscope. S4. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene HK-2 cells. Each concentration was repeated for 4 wells. After 48 hours, the medium was changed and the medium containing different concentrations of inorganic phosphate was taken out of the 4°C refrigerator and re-warmed. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded, and then JC-1 staining working solution was added. The cells were incubated for 30 minutes, the staining solution was discarded, and the cells were washed. The sealing medium was added. After the detection on the machine, the data were analyzed using image J software to obtain the data related to the changes in mitochondrial membrane potential of HK-2 cells. S5. HK-2 cells were inoculated into 12-well plates. After stable culture, different concentrations of inorganic phosphate were added to intervene HK-2 cells. Each concentration was repeated for 4 wells. After 48 hours, the medium was changed and the medium containing different concentrations of inorganic phosphate that had been taken out of the 4°C refrigerator and re-warmed was added. The cells were incubated for another 48 hours. After 96 hours of intervention, the supernatant was discarded and Rhod-2 AM staining working solution was added. The cells were incubated for 30 minutes. The cells were washed with complete medium for 2-3 times, each time for 5 minutes. The sealing agent was added. The cells were detected and photographed using an automatic microplate reader and an inverted fluorescence microscope, respectively, to obtain the change data of mitochondrial calcium ion concentration in HK-2 cells. The activity data of HK-2 cells under different concentrations of inorganic phosphate, the data of cell mitochondrial reactive oxygen content, the data of HK-2 cell mitochondrial membrane potential, and the data of HK-2 cell mitochondrial calcium ion concentration were statistically analyzed, and charts were drawn to construct a model of inorganic phosphate damage to HK-2 cell mitochondria.

2. The construction method according to claim 1, characterized in that: In step S1, the resuscitation treatment is: adding HK-2 cells to a complete culture medium containing 10% FBS, stably subculturing at 37° C. and 5% CO 2 , and using HK-2 cells within 4 to 10 generations for the experiment.

3. The construction method according to claim 1, characterized in that: In step S2, the cell densities were: 1.2×10 4 / mL, 2.4×10 4 / mL, 4.8×10 4 / mL, 9.6×10 4 / mL.

4. The construction method according to claim 1, characterized in that: The different concentrations of inorganic phosphate were 0.375mM, 0.75mM, 1mM, 1.25mM and 1.5mM.

5. The construction method according to claim 1, characterized in that: In step S2, the final concentration of the CCK-8 solution is 10%.

6. The construction method according to claim 1, characterized in that: The incubation conditions in step S2, the incubation conditions in step S3, the incubation conditions in step S4, and the incubation conditions in step S5 are all 37° C. and 5% CO 2 .

7. The construction method according to claim 1, characterized in that: The sealing medium is a sealing medium containing DAPI and is resistant to fluorescence quenching.

8. The construction method according to claim 1, characterized in that: In step S3, DCFH-DA in the DCFH-DA kit was diluted with serum-free DMEM / F12 medium at a ratio of 1:1000, so that the final concentration of the DCFH-DA staining working solution was 10 μM, and the HK-2 cells in a 12-well plate were covered when used.

9. The construction method according to claim 1, characterized in that: In step S4, the concentration of the JC-1 staining working solution was 1 μg / μL.

10. The construction method according to claim 1, characterized in that: In step S5, the concentration of the Rhod-2AM working solution was 5 μg / μL.

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