Co-culture method and application of benzene-contaminated mouse adipose tissue and K562 cell

Abstract

The invention discloses a co-culture method of benzene-contaminated mouse adipose tissue and K562 cells, which comprises the following steps: (1) collection of benzene-contaminated mouse adipose tissue: performing benzene contamination on a mouse for 5-7 days per week, and after continuous contamination for 5-7 weeks, collecting the benzene-contaminated mouse adipose tissue under an aseptic condition; (2) inoculating K562 cells, namely inoculating the K562 cells to a 12-pore plate one day before collecting the adipose tissues of the benzene-contaminated mice, and culturing in a CO2 incubator; and (3) co-culturing the benzene-contaminated mouse adipose tissue and the K562 cell: putting the collected benzene-contaminated mouse adipose tissue into a transwell chamber, putting the transwell chamber into a 12-pore plate inoculated with the K562 cell, adding a culture medium, and co-culturing the benzene-contaminated mouse adipose tissue and the K562 cell. The invention belongs to the technical field of preventive medicine, can intuitively reflect the influence of adipose tissue on benzene-induced K562 cytotoxicity, and is expected to play an important role in regulation and control of benzene-induced blood toxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of preventive medicine, and particularly relates to a co-culture method and application of benzene-exposed mouse adipose tissue and K562 cells. Background Art

[0002] Benzene is an important industrial raw material and organic solvent, widely used in petrochemical industry, synthetic rubber and other fields, and is confirmed as a Class I carcinogen by the International Agency for Research on Cancer (IARC). Long-term low-concentration benzene exposure can lead to hematopoietic dysfunction and cause blood system damage such as aplastic anemia and leukemia.

[0003] As an organic whole, when the human body responds to exogenous chemical exposure, there may be interactions between different tissues and organs, which can affect the toxic effects of the target organ. Benzene has strong liposolubility and is mainly distributed in tissues and organs rich in lipids after entering the human body. Among them, adipose tissue is one of the main enrichment sites of benzene. Therefore, the amount of body fat will directly affect the distribution of benzene in the body. In 1975, Sato et al. reported that the elimination rate of benzene was significantly slowed down in mice and humans with higher body fat content, suggesting that body fat content may affect the toxicokinetics of benzene in the body. Some adipose tissues have important endocrine functions, which can affect the functions of other tissues and organs or the occurrence and development of diseases. In addition, adipose tissue can also express metabolic enzymes closely related to benzene metabolism such as CYP2E1 and NQO1. It is speculated that adipose tissue may affect the hematotoxicity of benzene. In recent years, population studies and animal experiments have shown that adipose tissue in the body may participate in the regulation of benzene toxic effects, suggesting that people with different body fat contents have different susceptibilities to benzene. Therefore, in-depth exploration of the specific regulatory role and molecular mechanism of adipose tissue in benzene-induced hematotoxicity will help to screen high-risk populations exposed to benzene.

[0004] Although population studies and whole-animal experiments have, to some extent, revealed that adipose tissue may affect the toxicity of benzene, due to the high complexity of organisms and the intricate interactions that may exist between different tissues and organs in the body, it is difficult to distinguish the effects that other tissues and organs, apart from adipose tissue, may have, making it hard to intuitively reflect the specific role of adipose tissue therein. During in vitro culture, K562 cells can be induced to differentiate into erythroid blood cells, and treatment with benzene or its metabolites such as hydroquinone and benzoquinone can affect their differentiation. Therefore, K562 cells are widely used in the study of benzene-induced erythroid blood cell toxicity. Chinese Patent Application CN 113652401 A discloses a co-culture method of multi-cells and human liver microsomes, a co-culture medium thereof, and their applications, providing a reference for the study of re-injury after exposure and metabolism jointly participated by the lungs, blood circulation, liver, and immune response. However, there is currently a lack of research methods that directly reflect the regulatory role of adipose tissue in benzene-induced blood toxicity, and there are no relevant reports on the co-culture of adipose tissue from benzene-exposed mice and K562 cells.

[0005] Therefore, it is of great significance to provide a co-culture method and application of adipose tissue from benzene-exposed mice and K562 cells. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a co-culture method of adipose tissue from benzene-exposed mice and K562 cells, which can intuitively reflect the effect of adipose tissue on the toxicity of benzene to K562 cells and is expected to play an important role in the research of benzene-induced blood toxicity regulation and screening of key biomarkers.

[0007] The object of the present invention will be further illustrated by the following detailed description.

[0008] The present invention provides a co-culture method of adipose tissue from benzene-exposed mice and K562 cells, comprising the following steps:

[0009] (1) Collection of adipose tissue from benzene-exposed mice: The mice are exposed to benzene, 5 - 7 days per week, for 5 - 7 consecutive weeks. Then, under sterile conditions, the adipose tissue from benzene-exposed mice is collected.

[0010] (2) Seeding of K562 cells: One day before collecting the adipose tissue from benzene-exposed mice, K562 cells are seeded in a 12-well plate and cultured in a CO2 incubator.

[0011] (3) Co-culture of adipose tissue from benzene-exposed mice and K562 cells: The collected adipose tissue from benzene-exposed mice is placed in a transwell chamber and then put into the 12-well plate seeded with K562 cells. Medium is added for co-culture with K562 cells.

[0012] Preferably, the benzene poisoning is achieved by gavage, static inhalation or dynamic inhalation.

[0013] Preferably, in the step (1), the mice are poisoned for 6 days per week for 6 consecutive weeks. It is found that continuous poisoning for 6 weeks has a significant effect on the differentiation rate of K562 cells after co-culture, while continuous poisoning for 4 weeks has no significant effect on the differentiation rate of K562 cells after co-culture.

[0014] Preferably, the mice used are C57 mice, and the dose of benzene poisoning is 190 - 210 mg / kg body weight.

[0015] Preferably, the medium used is 1640 basal medium.

[0016] Preferably, the CO2 incubator is a CO2 constant temperature incubator with a CO2 content of 5% v / v.

[0017] Preferably, the co-culture time is 22 - 26 hours. More preferably, the co-culture time is 24 hours.

[0018] In addition, the present invention also provides the application of the mouse adipose tissue obtained by the co-culture method of the benzene-poisoned mouse adipose tissue and K562 cells in the study of the regulation of benzene-induced hematotoxicity.

[0019] In addition, the present invention also provides the application of the K562 cells and the culture solution obtained by the co-culture method of the benzene-poisoned mouse adipose tissue and K562 cells in the study of benzene toxic effects and the screening of key biomarkers.

[0020] Compared with the prior art, the beneficial effects of the present invention include:

[0021] (1) The present invention first provides a co-culture method of benzene-poisoned mouse adipose tissue and K562 cells, which not only realizes the integrity of the benzene-poisoned mouse adipose tissue but also excludes the possible interference from other tissues and organs such as the liver, and can intuitively reflect the role of adipose tissue in benzene-induced erythroid hematotoxicity, avoiding / reducing the inherent complexity of whole animal experiments and population studies.

[0022] (2) Adipose tissue can be collected before and after the co-culture, as well as K562 cells and the culture solution after the co-culture. These biological samples can be used for subsequent mechanism research and key biomarker screening, and are conducive to carrying out the following research fields: 1) exploring the regulatory role and molecular mechanism of adipose tissue in benzene-induced hematotoxicity; 2) conducting health risk assessment on benzene-exposed populations with different body fat contents; 3) screening for susceptible populations to benzene poisoning based on different body fat contents; 4) formulating effective prevention or intervention measures for benzene poisoning. Description of the Drawings

[0023] Figure 1 This is the flow chart of co - culturing adipose tissue of benzene - poisoned mice and K562 cells provided by the embodiments of the present invention.

[0024] Figure 2 This is the representative picture of co - culturing adipose tissue of mice and K562 cells provided by the embodiments of the present invention; among them, 2 - A is the illustration example of collecting adipose tissue of mice; 2 - B is the schematic diagram of co - culturing adipose tissue of mice and K562 cells.

[0025] Figure 3 This is the influence of co - culturing adipose tissue of mice provided by the embodiments of the present invention on the survival rate of K562 cells; among them, 3 - A is the influence of co - culturing adipose tissue poisoned by benzene for 4 weeks and K562 cells for 24 hours on the survival rate of K562 cells; 3 - B is the influence of co - culturing adipose tissue poisoned by benzene for 6 weeks and K562 cells for 24 hours on the survival rate of K562 cells.

[0026] Figure 4 This is the influence of co - culturing adipose tissue of benzene - poisoned mice for 4 weeks provided by the embodiments of the present invention on the differentiation of K562 cells; among them, 4 - A is the differentiation rate of K562 cells poisoned by different doses of hydroquinone; 4 - B, 4 - C, and 4 - D are the differentiation rates of K562 cells after co - culturing with epididymal adipose tissue, perirenal adipose tissue, and mesenteric adipose tissue for 24 hours respectively; *P < 0.05, **P < 0.001.

[0027] Figure 5 This is the influence of co - culturing adipose tissue of benzene - poisoned mice for 6 weeks provided by the embodiments of the present invention on the differentiation of K562 cells; among them, 5 - A is the differentiation rate of K562 cells poisoned by different doses of hydroquinone; 5 - B, 5 - C, and 5 - D are the differentiation rates of K562 cells after co - culturing with epididymal adipose tissue, perirenal adipose tissue, and mesenteric adipose tissue for 24 hours respectively; *P < 0.05, **P < 0.001. Detailed implementation manners

[0028] The following further elaborates on the present invention in detail with reference to the drawings and embodiments.

[0029] Unless otherwise specified, the raw materials or reagents used in the embodiments of the present invention are all commercially available or can be obtained by conventional technical means in the art. Unless otherwise specified in the text, the percentages used are mass percentages.

[0030] Some of the raw materials used in the examples are described as follows: benzene (analytical grade, ≧99%, purchased from Sigma), K562 cell line purchased from ATCC, hydroquinone and hemin purchased from Sigma Aldrich, corn oil purchased from Yuanye Biotechnology Co., Ltd., 1640 basal culture medium and fetal bovine serum were purchased from GIBCO, gentamicin and amphotericin B were purchased from Guangzhou Jiakaijie Biological Products Co., Ltd., and benzidine was purchased from Maclean.

[0031] Some of the instruments used in the examples are described as follows: electronic balance (Shuangjie Company, USA), CO2 constant temperature incubator (ESCO, Singapore), desktop low-temperature centrifuge (Eppendorf, Germany), liquid nitrogen tank (STATEBOURNE, UK), cell counter (BECKMAN, USA), inverted microscope (Leica, Germany), and biological safety cabinet (ESCO, Singapore).

[0032] Example 1 Benzene poisoning in mice

[0033] 1.1 Acquisition and daily feeding of C57BL / 6 male mice

[0034] Fifteen 8-week-old SPF C57BL / 6 male mice were purchased from the Guangdong Animal Experiment Center. After 1 week of adaptive feeding in the quarantine room, they were placed in the SPF animal room. The temperature in the animal room was maintained at 20±3°C, the light duration was 12h / day, and the relative humidity was maintained at 55%±5%RH. After the SPF animal room for mice was inspected, 3 mice were placed in each cage, and ordinary maintenance feed and drinking water were provided by the SPF animal room of the School of Public Health of Sun Yat-sen University. During the feeding period, they were free to drink water and eat.

[0035] All animal experiments involved in this study were approved by the Animal Ethics Committee of Sun Yat-sen University and complied with the relevant regulations for the management and use of laboratory animals. Discarded samples were handled strictly in accordance with the principles and procedures for handling biological contaminants.

[0036] 1.2 Treatment of benzene contamination

[0037] The mice were numbered and weighed using ear tags, and randomly divided into 2 groups, 7 in each group. The difference in body weight of the animals within each group was less than 10% of the average body weight, and the difference in the average body weight of the animals between groups was less than 5%.

[0038] Prepare benzene dye solution: add 20 ml corn oil to a 50 ml centrifuge tube, add 460 μL benzene in a fume hood, vortex to mix, tighten the lid, and set aside. Prepare benzene dye solution once a week.

[0039] Using the above-mentioned benzene-contaminated solution, mice were gavaged at a dose of 0.1 ml / 10 g body weight to obtain benzene-exposed mice in the 200 mg / kg group. Control mice were gavaged with corn oil at a dose of 0.1 ml / 10 g body weight. There were 7 mice in each group. They were gavaged 6 days a week for 4 weeks and 6 weeks respectively, and then adipose tissue samples were taken for co-culture. The mice were fasted on the night after the last benzene exposure, and samples were collected the next day for in vitro co-culture experiments.

[0040] Example 2 Co-culture of Adipose Tissue of Benzene-Exposed Mice and K562 Cells

[0041] The method for co-culturing adipose tissue of benzene-exposed mice and K562 cells includes the following steps:

[0042] (1) Collection of adipose tissue of benzene-exposed mice: Mice were exposed to benzene, 6 days a week. After 6 weeks of continuous exposure, adipose tissue of benzene-exposed mice was collected under sterile conditions.

[0043] (2) Seeding of K562 cells: One day before collecting the adipose tissue of benzene-exposed mice, exponentially growing K562 cells were seeded in a 12-well plate (2.5×10 5 cells / well, 1.5 ml system) and cultured in a 5% v / v CO2 incubator. K562 cells are suspension-growing cells and were cultured in RPMI 1640 medium (containing 10% fetal bovine serum) at 37°C in a 5% CO2 incubator.

[0044] (3) Co-culture of adipose tissue of benzene-exposed mice and K562 cells: The collected adipose tissue of benzene-exposed mice was placed in a transwell insert and then put into the 12-well plate seeded with K562 cells. Medium was added for co-culture with K562 cells.

[0045] The flow chart of the co-culture of adipose tissue of benzene-exposed mice and K562 cells is as Figure 1 shown.

[0046] Mice to be experimented on were taken out from the SPF animal house and placed in the dissection room. The body weight of the mice was weighed and recorded. Then the mice were euthanized by cervical dislocation and immersed in a beaker containing 75% (v / v) alcohol, and immediately transferred to the animal cell room. The mice were placed on sterile gauze to absorb the alcohol, and then fixed on a foam board. The abdominal skin of the mice was picked up with forceps and the abdominal cortex was cut open, trying to minimize hair contamination of the mice during this process. The peritoneum of the mice was cut in a "T" shape and fixed to the foam board with sterile pins to expose the abdominal organs. The epididymal fat, mesenteric fat, and perirenal fat were taken out in sequence (as Figure 2As shown in Figure -A); Immediately after removing the adipose tissue, place it in a transwell chamber, add 1 ml of 1640 basal medium (containing 50 μg / ml gentamicin and 0.5 μg / ml amphotericin B), and place the chamber into the well of a 12-well plate that has been seeded with K562 cells (as shown in Figure 2 Figure -B).

[0047] In this experiment, cells poisoned with hydroquinone (HQ), a metabolite of benzene, were used as the positive control. Poisoning with 5 and 10 μM HQ had a significant inhibitory effect on the differentiation of K562. Therefore, the HQ treatment concentrations set in this invention were: 0, 5, 10 μM. That is, 1 ml of the poisoning solution was added to each chamber to treat K562 cells simultaneously, and 3 parallels were set for each dose. After the above operations were completed, the 12-well plate was placed back into the incubator and cultured for another 24 hours.

[0048] Induction of differentiation of K562 cells: After the co-culture ended, the K562 cells were washed and re-seeded in a 12-well plate, and a medium containing Hemin was added to induce cell differentiation for 48 hours. Detection of the differentiation of K562 cells: After the treatment with Hemin ended, the K562 cells were collected and stained with benzidine. Positive benzidine staining (dyed blue) indicated that the K562 cells had differentiated into erythroid cells. The differentiated and undifferentiated cells were counted, and the cell differentiation rate (%) was calculated.

[0049] Example 3 Effect of Poisoned Mouse Adipose Tissue on the Viability of K562 Cells

[0050] After co-culturing for 24 hours according to the method in Example 2, the viability of K562 cells was detected, and the adipose tissue and the culture medium were collected. The co-cultured adipose tissue was collected into a cryotube, quickly frozen in liquid nitrogen, and then stored at -80°C. The medium in the transwell was collected into another new 1.5 ml EP tube, frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

[0051] The lower-layer K562 cell suspension was collected into a 15 ml centrifuge tube. After mixing, 20 μl was taken out and placed in a 1.5 ml EP tube for subsequent detection of cell viability. The trypan blue staining method was used to determine the cell survival rate: A 4% trypan blue stock solution was diluted with PBS to prepare a 0.4% trypan blue staining solution. The cell suspension was diluted and mixed with the 0.4% trypan blue staining solution in a ratio of 9:1. After mixing, it was stained for 3 minutes, and 10 μl was taken and added to a hemocytometer for counting. The blue-stained dead cells and the unstained live cells were counted, and the cells in each well were counted twice to analyze the survival rate of K562 cells. Co-culturing the adipose tissue of mice poisoned with benzene for 4 weeks and 6 weeks with K562 cells for 24 hours did not have an obvious effect on the survival of K562, as shown in Figure 3 Figure 3-A and 3-B respectively.

[0052] Example 4 Inducing Differentiation of K562 Cells

[0053] Hemin can induce the differentiation of K562 cells, and benzene and its metabolites hydroquinone (HQ) and benzoquinone (BQ) can all inhibit the induction of differentiation process. Therefore, this method is widely used in the toxicity study of benzene and its metabolites on erythroid blood cells. In this invention, K562 cells poisoned with 5 μM and 10 μM HQ were used as positive controls, and cells treated with solvent PBS were used as negative controls (i.e., the hydroquinone 0-dose group) to analyze and compare the effect of co-culture with fat on the differentiation of K562 cells.

[0054] Prepare the Hemin stock solution (20 mM) with DMSO in advance, and then prepare the Hemin-induced differentiation solution with complete 1640 medium, with a final concentration of 40 μM. After co-culturing adipose tissue and K562 cells for 24 hours, collect the cell suspension in a 15 ml centrifuge tube, centrifuge at 300 g / min at room temperature for 5 min, discard the supernatant, add 5 ml of sterile 1×PBS to wash the cells once in the cell pellet, centrifuge at 300 g / min at room temperature for 5 min, discard the supernatant, add 1 ml of Hemin-induced differentiation solution to resuspend the cells respectively, inoculate them into a new 12-well plate, put them back into the incubator, and continue to culture for 48 hours.

[0055] Example 5 Effect of Benzene-Poisoned Mouse Adipose Tissue on the Differentiation of K562 Cells

[0056] Take out the 12-well plate, collect the cell suspension into a 15 ml centrifuge tube, centrifuge at 300 g at 4 °C for 5 minutes, and discard the supernatant. Add 5 ml of pre-cooled PBS to wash the cells, centrifuge at 300 g at 4 °C for 5 minutes, and place the cells on ice after centrifugation.

[0057] Prepare benzidine solution (weigh 0.0020 g + 990 ml of double-distilled water + 10 μL of glacial acetic acid, heat at 85 °C for 10 minutes until completely dissolved), take 50 μl of benzidine solution, add 1.25 μl of 30% H2O2, mix well and let stand at room temperature for 3 min to obtain the benzidine staining application solution. After aspirating and discarding the cell supernatant, add 50 μl of benzidine staining application solution and mix with the cells, let stand at room temperature for 3 minutes, mix well, aspirate 20 μl and drop it on a glass slide, carefully cover it with a coverslip along one side to prevent air bubbles from generating. Then take a photo under a 10× microscope to observe the cell staining situation. Blue cells are differentiated cells. A total of 300 cells are counted, and the counting is repeated 3 times to calculate the differentiation rate of K562 cells. For easy comparison, the cell differentiation results of each treatment group are standardized with the same batch of HQ-poisoned 0-dose group (i.e., the PBS treatment group) to obtain the relative differentiation rate (%) of K562 cells, as shown in Figure 4 -A and 5-A. The differentiation rate of K562 cells in the HQ-poisoned group showed a dose-dependent decrease. Figure 4-B and 4-C showed that, compared with the PBS negative control group without adipose tissue, the periepididymal fat and perirenal fat tissues of mice exposed to benzene for 4 weeks could significantly reduce the differentiation rate of K562 cells (P<0.05); moreover, compared with the control group of mice, the periepididymal fat of benzene-exposed mice could significantly inhibit the differentiation of K562 cells (P<0.05)( Figure 4 -B). With the prolongation of benzene exposure time, significant differences were also observed in the perirenal fat tissue of benzene-exposed mice compared with the control group of mice, and the differentiation of K562 cells could be significantly inhibited (P<0.001)( Figure 5 -C). With the prolongation of benzene exposure time, co-culture with mesenteric fat showed different effects on the differentiation of K562 cells. The mesenteric fat of mice exposed to benzene for 4 weeks did not have an obvious effect on the differentiation of K562 cells( Figure 4 -D), but when the mice were exposed to benzene for 6 weeks, an obvious inhibitory effect on the differentiation of K562 cells was shown (P<0.05)( Figure 5 -D). The above research results indicate that the adipose tissue of benzene-exposed mice can affect the toxic effect of benzene on erythroid blood cells, which provides direct evidence for the toxic regulation of benzene by adipose tissue and also provides a powerful technical means for further studying its action mode and mechanism in the future.

[0058] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A co-culture method of benzene-exposed mouse adipose tissue and K562 cells, characterized in that: It includes the following steps: (1) Collection of benzene-exposed mouse adipose tissue: The mice are exposed to benzene. They are exposed 5 - 7 days a week for 5 - 7 consecutive weeks. Then, under sterile conditions, the adipose tissue of benzene-exposed mice is collected. (2) Seeding of K562 cells: One day before collecting the adipose tissue of benzene-exposed mice, K562 cells are seeded in a 12-well plate and cultured in a CO2 incubator. (3) Co-culture of benzene-exposed mouse adipose tissue and K562 cells: The collected adipose tissue of benzene-exposed mice is placed in a transwell chamber and then put into the 12-well plate seeded with K562 cells. Medium is added for co-culture with K562 cells.

2. The co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to claim 1, characterized in that: The benzene exposure is achieved by gavage, static inhalation or dynamic inhalation.

3. The co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to claim 1, characterized in that: In step (1), the mice are exposed 6 days a week for 6 consecutive weeks.

4. The co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to any one of claims 1 to 3, characterized in that: The mice used are C57 mice, and the benzene exposure dose is 190 - 210 mg / kg body weight.

5. The co - culture method of benzene - poisoned mouse adipose tissue and K562 cells according to any one of claims 1 to 3, characterized in that: The medium used is 1640 basal medium.

6. The co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to any one of claims 1 to 3, characterized in that: The co-culture time is 22 - 26 hours.

9. Application of mouse adipose tissue cultured by the co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to any one of claims 1 to 6 in the study of benzene-induced blood toxicity regulation.

10. Application of K562 cells and culture medium cultured by the co-culture method of benzene-exposed mouse adipose tissue and K562 cells according to any one of claims 1 to 6 in the study of benzene toxic effects and screening of key biomarkers.

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