Construction method of cadmium ion mediated deciduation damaged cell model

Abstract

The invention discloses a construction method of a cadmium ion mediated deciduous damage cell model, and belongs to the technical field of cell model construction. The specific model construction method comprises the following steps: 1) extracting primary endometrial stromal cells from endometrial tissues; 2) culturing primary endometrial stromal cells in vitro; and 3) inducing endometrial stromal cell decidualization in vitro under the cadmium ion exposure condition. The invention finds that the expression of a deciduation related marker can be obviously reduced by cadmium ion treatment, so that the endometrial stromal cell deciduation injury is caused, and the cadmium ion mediated deciduation injury cell model is successfully constructed.

Description

Technical Field

[0001] The invention belongs to the technical field of cell model construction, and in particular relates to a method for constructing a cadmium ion-mediated decidualization damaged cell model. Background Art

[0002] Cadmium (Cd) is a ubiquitous harmful metal. Currently, about 10% of the world's population is at risk of chronic low-level Cd exposure. Cd has a long biological half-life and a low excretion rate, which leads to its long-term accumulation in the body. Cd has toxic effects on multiple organs such as the liver, kidneys, uterus, ovaries, and testicles. Population surveys have found that the cadmium concentration in the blood or serum of patients with spontaneous abortion (SA) is significantly higher than that of patients with normal pregnancy. In addition, the Cd accumulation concentration in the decidual tissue of patients with spontaneous abortion is significantly higher than that of the control group.

[0003] Decidualization is a special differentiation process in which endometrial stromal cells (ESCs) transform from fibroblasts to large polygonal decidual stromal cells (DSCs) with rich glycogen and lipid droplets in the cytoplasm under the stimulation of ovarian steroid hormones and embryo implantation in vivo or in vitro. In early pregnancy, DSCs are the most abundant cell type at the maternal-fetal interface, with unique biosynthetic and secretory functions, which are essential for the establishment and maintenance of pregnancy. Impaired decidualization is widely considered to be an important cause of spontaneous abortion.

[0004] Therefore, we speculate that the accumulation of cadmium ions in endometrial tissue damages the decidualization process, but there is currently no relevant research on cadmium damage to decidualization. Summary of the invention

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides the use of cadmium ions in constructing a decidualization damaged cell model.

[0007] On the other hand, the present invention also provides a method for constructing a cadmium ion-mediated decidualization injury cell model, wherein the construction method comprises: culturing endometrial stromal cells in a culture medium containing cadmium ions to obtain a decidualization injury cell model.

[0008] Furthermore, the endometrial stromal cells are cultured in a culture medium containing cadmium ions for 2-4 days, and the final concentration of cadmium ions in the culture medium is 5-10 μM.

[0009] Furthermore, the culture medium is a 2% complete culture medium; the 2% complete culture medium is a DMEM / F-12 culture medium containing FBS and PSA.

[0010] Furthermore, the FBS concentration in the culture medium is 2% and the PSA concentration is 1%.

[0011] Furthermore, the culture medium also contains a decidualization inducer.

[0012] Furthermore, the decidualization inducer is MPA or cAMP.

[0013] Furthermore, the MPA concentration is 1 μM and the cAMP concentration is 0.5 mM.

[0014] In addition, the present invention also provides the use of the decidualization injury cell model constructed by the above method in screening products for preventing and / or treating decidualization injury.

[0015] Furthermore, the product is a drug for preventing and / or treating decidualization damage.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention induces decidualization of endometrial stromal cells in vitro under cadmium ion exposure conditions, and detects through PCR and Western blot that cadmium accumulation impairs the decidualization of ESCs by downregulating the expression of PRL, IGFBP1, PGR, and FoxO1.

[0018] 1. To provide a basis for further study of the damage of decidualization caused by cadmium;

[0019] 2. To provide a theoretical basis for the treatment and prevention of cadmium-induced decidualization damage;

[0020] 3. This decidualization injury model can be used for drug research related to the treatment of decidualization impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The Western blot test results in Example 1 (Figures B and C are the corresponding test results after 4 days of cultivation);

[0022] Figure 2 This is a graph showing the results of detecting the mRNA expression of PRL and IGFBP1 in Example 1;

[0023] Figure 3 The results of the detection of the expression of the upstream regulatory factors of PRL and IGFBP1 in Example 1 are shown (wherein, Figures A, B, C, and D correspond to the mRNA expression results of PGR, ESR1, ESR2, and FOXO1 after 4 days of culture, respectively); DETAILED DESCRIPTION

[0024] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0025] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0026] Example 1

[0027] 1.1 Extraction of primary endometrial stromal cells from human endometrial tissue

[0028] (1) Proliferative endometrial tissue from women of childbearing age was collected and placed in a 15 mL centrifuge tube. The tissue was immersed in sterile phenol red-free DMEM / F12 medium to complete the transfer. The tissue was rinsed with PBS under sterile conditions, and then the tissue was placed at the mouth of a 15 mL centrifuge tube and chopped into a paste;

[0029] (2) Add 5 mL of DMEM / F12 digestion solution containing 2 mg / ml Cls IV (collagenase IV) + 0.1 mg / ml DNase I + 1% PSA (triple antibody) to the centrifuge tube, wrap the centrifuge tube with tin foil to avoid light, and place it in a shaker at 37°C, 150 rpm, and digest for 1.5 h;

[0030] (3) Add 5 mL of 10% complete culture medium (10% FBS fetal bovine serum + 1% PSA phenol red-free DMEM / F12 medium) to terminate digestion;

[0031] (4) Place a sterile 100 μm nylon mesh on a 50 mL centrifuge tube and grind with the piston handle of a 1 mL syringe while filtering;

[0032] (5) The filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 10% complete medium;

[0033] (6) The resuspension was filtered through a 40 μm nylon mesh and centrifuged at 1500 rpm for 5 min. The supernatant was removed to obtain the ESCs pellet;

[0034] (7) The obtained ESCs pellet was resuspended again in 10% complete medium, and the resuspended liquid was transferred to a 6 cm adherent cell culture dish and cultured in a standard cell culture incubator at 37°C and 5% CO2;

[0035] (8) After 24 hours, the culture medium was replaced with fresh 10% complete culture medium, and non-attached red blood cells and white blood cells were further removed to obtain ESCs.

[0036] 1.2 In vitro culture of primary endometrial stromal cells to verify their purity

[0037] At 37°C and 5% CO2, the obtained ESCs were grown in 10% complete medium to the fourth generation, and the purity of ESCs was verified by Vimentin immunofluorescence staining. ESCs with positive Vimentin fluorescence staining and purity exceeding 98% were used for subsequent experiments.

[0038] 1.3 Induction of decidualization of endometrial stromal cells in vitro under cadmium ion exposure conditions

[0039] ESCs were grown in 10% complete medium until they were 80-90% confluent and then cultured according to Cd 2+ Intervention concentration: ESCs that were fused to 80-90% were divided into 4 groups. The 4 groups of ESCs were replaced with 2% complete culture medium containing different components and cultured at 37°C, 5% CO2 for 4 days. The 2% complete culture medium was DMEM / F-12 culture medium containing 2% FBS+1% PSA.

[0040] The 4 groups of culture media with different components are:

[0041] ①2% complete culture medium;

[0042] ②2% complete medium supplemented with 1μM MPA+0.5mM cAMP;

[0043] ③2% complete medium supplemented with 1μM MPA+0.5mM cAMP+5μM CdCl2;

[0044] ④ 2% complete medium with 1μM MPA+0.5mM cAMP+10μM CdCl2;

[0045] Among them, MPA and cAMP in the culture medium are decidualization inducers. In addition, estrogen E2 can also be used as the decidualization inducer described in the present application.

[0046] Related reagent configuration:

[0047] CdCl2 solution: Weigh 36.7 mg of CdCl2 powder and dissolve it in 20 mL of pure water to obtain a 10 mM CdCl2 stock solution.

[0048] cAMP solution: Take 100 mg of cAMP (Bucladesine sodium, CAS No: 16980-89-5) powder, add 2.0351 mL of DMEM / F12 culture medium to obtain 100 mM cAMP stock solution, and freeze it at -80°C for later use.

[0049] MPA solution: Take 100 mg of MPA (Medroxyprogesterone acetate, CAS No: 71-58-9) powder, dissolve it in 2.5872 mL of DMSO solution to obtain a 100 mM super concentrated stock solution, then take 100 μL of the 100 mM super concentrated stock solution, add 9.9 mL of DMSO solution, vortex and mix well to obtain a 1 mM MPA stock solution, and store it in aliquots at -80°C for later use.

[0050] 1.4 Detection of the expression of decidualization-related markers and regulatory factors

[0051] Total RNA and protein of the cells were extracted to verify the expression of decidualization markers: prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1), as well as the mRNA and protein expressions of the upstream regulatory factors of PRL and IGFBP1, progesterone receptor (PGR), ESR1, ESR2 and Forkhead Box O1 (FoxO1).

[0052] 1.4.1 Western blotting

[0053] (1) Glue making:

[0054] A. Wash the plate and 15-hole comb with ultrapure water, and prepare a 10% lower glue solution and a 4% upper glue solution while drying;

[0055] B. Install the board on the glue rack, add the coagulant to the lower glue solution and mix well, pour 7mL of the lower glue solution into the glue board, add 2mL of isopropyl alcohol to flatten the lower glue, and let it stand at room temperature for about 30 minutes to wait for the lower glue to solidify;

[0056] C. Pour out the isopropyl alcohol, invert the glue rack for 2 minutes to allow the isopropyl alcohol to flow out as completely as possible, and then place the glue rack;

[0057] D. Add the coagulant to the upper glue solution and mix well. Pour 2.5 mL of the lower glue solution into the glue plate, quickly insert the comb, and let it stand at room temperature for about 30 minutes to wait for the upper glue to solidify.

[0058] (2) Electrophoresis:

[0059] A. Leak detection: Install the rubber sheet on the electrophoresis tank, fill the inner tank with ultrapure water, and observe whether there is leakage. If there is leakage, reinstall the rubber sheet. After checking for leaks, fill the inner tank with electrophoresis liquid;

[0060] B. Pull out the comb: Gently pull out the comb and use a syringe to clean the impurities in the glue hole;

[0061] C. Sample loading: 3-5μL per well, add 3μL of Maker to the left of the sample and 1.5μL of Maker to the right

[0062] D. Electrophoresis: After about 30 minutes at a constant voltage of 80V, the markers are well separated, and the voltage is adjusted to 120V and electrophoresis is continued for about 60 minutes;

[0063] (3) Transfer:

[0064] A. PVDF membrane with a pore size of 0.2 μm was placed in methanol for activation for 3 minutes;

[0065] B. Pour the pre-cooled transfer solution into the tray, place the sandwich with the black side facing down, open the clamp, put a layer of transfer sponge and 2 pieces of transfer filter paper, and use a roller to gently drive away the bubbles;

[0066] C. Place the PVDF membrane in the tray and equilibrate for 1 minute;

[0067] D. Cutting glue: pry open the glue board and cut off the remaining glue except the marker;

[0068] E. Gently place the gel on the filter paper, cover the gel with the PVDF membrane, use a roller to remove bubbles, cover the PVDF membrane with 2 sheets of filter paper and 1 layer of sponge, and close the clamp;

[0069] F. Insert the black sandwich clip into the transfer tank with the black side facing the black side. Fill the device with transfer solution, place it in a pre-prepared foam box filled with ice, and connect the power supply.

[0070] G. Set the transfer conditions: constant current 250mA, 60 minutes.

[0071] (4) Blocking: The PVDF membrane was washed twice with TBST on a fast shaker at 120 rpm for 10 min each time. TBST was prepared with 5% skim milk, the membrane was immersed in the milk, and incubated on a slow shaker at 80 rpm at room temperature for 1.5 h.

[0072] (5) Primary antibody incubation: Wash three times with TBST, 10 min each time, dilute the antibody with primary antibody diluent according to the instructions, immerse the strips in the primary antibody, and place on a slow shaker at 40 rpm at 4°C overnight;

[0073] (6) Secondary antibody incubation: Wash with TBST three times, 10 min each time, dilute the secondary antibody with TBST at 1:10,000, and incubate at room temperature for 1.5 h on a slow shaker at 80 rpm;

[0074] (7) Development: The ECL-added membrane was imaged using a Tanon 5200 luminescence imaging system.

[0075] (8) Analysis: ImageJ software (National Institutes of Health, MD, USA) was used to analyze the grayscale values ​​of the stripes.

[0076] 1.4.2 Total RNA extraction and real-time fluorescence reverse transcription PCR (qRT-PCR)

[0077] 1.4.2.1 Total RNA extraction

[0078] (1) Cracking:

[0079] Cells: Wash twice with PBS, add 1 mL TRIzol reagent (Cat: 15596026CN, Ambion, Austin, US), shake the culture dish gently up and down, left and right, observe the lysis effect under the microscope after 1 minute, transfer to a 1 mL enzyme-free EP tube without cell residue at the bottom of the dish, and let stand on ice for 10 minutes;

[0080] Tissue: Weigh about 10 mg of tissue into an EP tube, add 500 μL TRIzol, use an ultrasonic probe cleaned with DEPC water, sonicate for 5 seconds, add 500 μL TRIzol, let stand on ice for 10 minutes; centrifuge at 12000 rpm, 4 degrees Celsius for 10 minutes, and take the supernatant;

[0081] (2) Chloroform extraction: Add 200 μL of chloroform, shake vigorously up and down 30 times, let stand at room temperature for 3 minutes, centrifuge at 2000 rpm, 4 degrees Celsius for 10 minutes, and observe that the liquid is divided into three layers: a transparent upper RNA phase, a white middle DNA and protein phase, and a pink lower organic phase;

[0082] (3) Isopropanol precipitation: transfer the transparent upper RNA phase to a new EP tube, add 3 volumes of isopropanol, mix well, let stand at 4°C for 20 minutes, centrifuge at 12000 rpm, 4°C for 10 minutes, discard the supernatant, and obtain RNA precipitate;

[0083] (4) Ethanol wash × 2: Prepare 75% ethanol with DEPC water, add 1 mL of 75% ethanol into the EP tube, turn it upside down, and centrifuge at 12000 rpm, 4 degrees Celsius for 10 minutes;

[0084] (5) Drying: Absorb as much 75% ethanol as possible and dry in a fume hood for 30 minutes;

[0085] (6) Dissolution: Add 30-50 μL DEPC water according to the amount of precipitate;

[0086] (7) Use a nucleic acid protein analyzer to detect RNA concentration and purity.

[0087] 1.4.2.2 Reverse transcription

[0088] According to PrimeScriptTM The RT kit (Cat:RR037A, TaKara, Osaka, Japan) was configured into a 20 μL reverse transcription system as shown in Table 1; the silver fast gradient PCR instrument was set to the following program: 37 degrees Celsius for 15 minutes, 85 degrees Celsius for 5 seconds, and 4 degrees Celsius for ∝; and the reverse transcription was completed.

[0089] Table 1 Reverse transcription system

[0090]

[0091] 1.4.2.3 Real-time fluorescence quantitative PCR

[0092] According to Table 2, the real-time fluorescence quantitative PCR reaction system was configured. The reaction program was set on the LC480 instrument according to Table 3 to obtain the ct value of the target gene. GAPDH was used as the housekeeping gene. 2 -ΔΔCt The primer sequences are shown in Table 4.

[0093] Table 2 Real-time fluorescence quantitative PCR system

[0094]

[0095]

[0096] Table 3 Real-time fluorescence quantitative PCR reaction conditions

[0097]

[0098] Table 4 qRT-PCR primer sequences

[0099]

[0100] according to Figure 1 The results showed that Western blot confirmed that on the 4th day of decidualization, 5 μM Cd 2+ The protein expressions of PRL and IGFBP1 in the two groups were significantly downregulated.

[0101] qRT-PCR showed that the levels of PRL and IGFBP1 mRNA in the control group continued to increase with the extension of decidualization time. Compared with the control group, PRL mRNA in cadmium-treated cells was significantly reduced on the 2nd and 4th days of decidualization ( Figure 2 A) However, on the second day of decidualization, 10 μM Cd 2+ The IGFBP1 mRNA in the group was reduced, and 5 μM Cd 2+ The IGFBP1 mRNA expression in the group was not significantly decreased. On the 4th day of decidualization, 5 μM Cd 2+ The IGFBP1 mRNA in the group was significantly decreased ( Figure 2B). In addition, the mRNA expression of PRL, IGFBP1 upstream regulators PGR, ESR1, ESR2, and FOXO1 was measured and it was found that 5 μM Cd 2+ In the intervention group, the decidualization was damaged for 4 days, and the mRNA expressions of PRL, IGFBP1 upstream regulators PGR, ESR1, ESR2, and FOXO1 all showed a downward trend.

[0102] Therefore, we propose that Cd accumulation impairs decidualization of ESCs by downregulating the expression of genes upstream of PRL and IGFBP1.

[0103] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of cadmium ions in constructing a decidualization damaged cell model.

2. A method for constructing a cadmium ion-mediated decidualization injury cell model, characterized in that: The construction method is: placing endometrial stromal cells in a culture medium containing cadmium ions for culture, thereby obtaining a decidualization damaged cell model.

3. The construction method according to claim 2, characterized in that: The endometrial stromal cells are cultured in a culture medium containing cadmium ions for 2-4 days, wherein the final concentration of the cadmium ions in the culture medium is 5-10 μM.

4. The construction method according to claim 2, characterized in that: The culture medium is a 2% complete culture medium; the 2% complete culture medium is a DMEM / F-12 culture medium containing FBS and PSA.

5. The construction method according to claim 4, characterized in that: The FBS concentration in the culture medium is 2%, and the PSA concentration is 1%.

6. The construction method according to claim 5, characterized in that: The culture medium also contains a decidualization inducing agent.

7. The construction method according to claim 6, characterized in that: The decidualization inducing agents are MPA and cAMP.

8. The construction method according to claim 7, characterized in that: The MPA concentration was 1 μM, and the cAMP concentration was 0.5 mM.

9. Use of the decidualization injury cell model constructed by the method according to any one of claims 2 to 8 in screening products for preventing and / or treating decidualization injuries.

10. The use according to claim 9, characterized in that: The product is a drug for preventing and / or treating decidualization damage.

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