Chronic heat stress model construction method based on chick embryo primary skeletal muscle cells and application

Abstract

The invention relates to the technical field of cell engineering, and discloses a chronic heat stress model construction method based on chick embryo primary skeletal muscle cells, which comprises the following steps: step 1, separating and purifying cells, and recording as P0 generation; step 2, differentiating after the cells are subcultured to a P2 generation; step 3, constructing a chronic heat stress model: continuously treating the differentiated cells in an incubator at 43 DEG C for 24 hours; 4, model verification, wherein the effectiveness of the model is confirmed by detecting at least one index of lactic dehydrogenase activity, cell apoptosis rate, ROS level, oxidation reduction state and HSP70 / Caspase-3 gene expression quantity. According to the method, a chronic heat stress model based on chick embryo primary skeletal muscle cells is established, and the in-vitro simulation of the chronic heat stress state of the broiler chicken is realized through standardized cell separation, passage differentiation and continuous 24-hour treatment at 43 DEG C.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell engineering, and in particular to a method for constructing a chronic heat stress model based on chicken embryo primary skeletal muscle cells and application thereof. BACKGROUND

[0002] In recent decades, the continuous directional selection, the progress of feed nutrition technology and the promotion of intensive high-density breeding mode have made the growth performance of broilers improve rapidly. However, the rapid growth rate of broilers means more vigorous metabolism and more metabolic heat production. Since broilers are covered with feathers and have no sweat glands, they are more sensitive to high temperature, mainly through respiratory and surface heat dissipation. When the environmental temperature exceeds the upper critical temperature limit (28℃), broilers (in the middle and later stages) will produce heat stress. Generally, heat stress can be divided into acute heat stress and chronic heat stress according to the duration of stress. Acute heat stress refers to the heat stress caused by sudden exposure to extreme high temperature environment for a short time (several hours), which will cause changes in animal physiology and metabolism to maintain animal survival. Chronic heat stress is the heat stress caused by long-term (several days to several weeks) exposure to high temperature environment, and the animal body will adapt to the environment to a certain extent.

[0003] Heat stress not only reduces the feed intake of broilers, damages the intestinal health, increases the risk of disease infection and mortality, but also causes muscle tissue damage, significantly reduces the growth performance and muscle quality, thereby seriously restricting the sustainable development of the broiler industry and causing huge economic losses. Previous studies have shown that chronic heat stress can affect muscle quality by affecting muscle metabolic homeostasis, redox state, cell apoptosis and other pathways, but the specific mechanism is still not very clear.

[0004] In order to further study the effects of chronic heat stress on the function, physiological state and molecular mechanism of chicken skeletal muscle cells, it is of great significance to establish a stable and repeatable chicken embryo primary skeletal muscle cell isolation and culture and chronic heat stress in vitro model. As the cell model closest to the physiological state in vivo, primary cells can more truly reflect the response process of muscle cells under complex environments such as heat stress, and are an important basis for revealing the regulation mechanism of muscle development, screening anti-heat stress intervention means and promoting the research of broiler genetics and breeding.

[0005] Therefore, it is of great theoretical value and application prospect to develop a scientific, efficient and standardized chicken embryo skeletal muscle primary cell isolation and culture method, and on this basis, to establish a chronic heat stress cell model, which can promote the research of heat stress related mechanism and the technical improvement of livestock and poultry breeding industry. SUMMARY

[0006] In order to solve the technical problems proposed in the background art, the present application provides a method for constructing a chronic heat stress model based on chicken embryo primary skeletal muscle cells and application thereof.

[0007] The application adopts the following technical scheme to achieve the purpose: one of the purposes of the application is to propose a chronic heat stress model construction method based on chicken embryo primary skeletal muscle cells, which comprises the following steps: Step 1, cell separation and purification, denoted as P0 generation; Step 2, cell passage to P2 generation for differentiation; Step 3, chronic heat stress model construction: the differentiated cells are placed in a 43℃ incubator for continuous treatment for 24 h; Step 4, model verification: the model effectiveness is confirmed by detecting at least one of the following indexes: lactate dehydrogenase (LDH) activity, cell apoptosis rate (TUNEL method), ROS level, oxidation-reduction state and HSP70 / Caspase-3 gene expression amount.

[0008] Preferably, in step 1, specifically, the 12-day-old chicken embryo pectoral muscle tissue is taken, disinfected with 75% alcohol, rinsed with PBS containing double antibodies for 3 times, and the muscle membrane, blood vessels and skeleton are removed; after being cut into small pieces, 0.25% trypsin is added for digestion until the tissue disappears, and 3 times the volume of complete culture medium containing 15% FBS is added to terminate the digestion; after filtration, centrifugation is performed, the cells are resuspended and inoculated and cultured.

[0009] Preferably, in step 1, a 200-mesh (70 μm) cell sieve is used for filtration, and the centrifugation condition is 1000 rpm for 5 min.

[0010] Preferably, in step 2, when the cell density reaches 70%, the cells are passaged to P2 generation by using 0.25% trypsin for digestion; when the density reaches 90%, the differentiation culture medium containing 2% horse serum (HS) and 1% double antibodies is replaced, and the differentiation is induced for 48 h Preferably, in step 2, the differentiation culture medium is DMEM / F12 basic culture medium, and 2% HS and 1% penicillin-streptomycin double antibodies are added.

[0011] Preferably, the heat stress treatment time in step 3 is 24 h, which is determined based on the LDH activity peak value, glycogen depletion and significant increase in apoptosis rate (P<0.05).

[0012] Preferably, the model verification in step 4 comprises: LDH activity is increased by more than 2.5 times of the control group; Glycogen content is decreased by more than 50%; The proportion of TUNEL positive cells is more than 25%.

[0013] One of the purposes of the application is to propose the application of the broiler skeletal muscle cell chronic heat stress model constructed by the above method in heat stress drug screening, molecular mechanism research and broiler genetic breeding.

[0014] Compared with the prior art, the present application has the beneficial effects that: The present application establishes a chronic heat stress model based on chicken embryo primary skeletal muscle cells, realizes the simulation of chronic heat stress state of broilers in vitro by standardizing cell isolation, passage differentiation and 43℃ continuous 24-hour treatment; the model verification index is clear (LDH activity ≥2.5 times, glycogen decrease ≥50%, TUNEL positive ≥25%), and the repeatability and stability are high; the system can truly reflect the metabolic disorder, oxidative stress, cell apoptosis and HSP70 / Caspase-3 signal change of skeletal muscle under high temperature, provides a reliable tool for revealing the chronic heat stress damage mechanism, can be applied to high-throughput screening of anti-heat stress drugs, molecular target identification and heat-resistant strain breeding, significantly improves the research efficiency and reduces the animal experiment cost, and has important theoretical value and industrial popularization prospect for promoting the research and development and genetic improvement of the anti-heat stress technology of broilers. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The flow chart of the construction method proposed in the present application is shown in the figure; Figure 2 The influence of different heat stress times on cell morphology in the embodiment of the present application is shown in the figure; Figure 3 The influence of different heat stress times on the LDH activity of the culture medium in the embodiment of the present application is shown in the figure; Figure 4 The influence of different heat stress times on the redox state in the embodiment of the present application is shown in the figure; Figure 5 The influence of different heat stress times on the cell apoptosis level in the embodiment of the present application is shown in the figure; Figure 6 The influence of different heat stress times on the HSP70 and Caspase-3 gene expression in the embodiment of the present application is shown in the figure. DETAILED DESCRIPTION

[0016] In the following, the present application will be further described in combination with the drawings and specific embodiments, and it should be noted that the following described embodiments or technical features can be combined in any manner to form new embodiments without conflict.

[0017] EMBODIMENT The present application will be further described in combination with specific embodiments: 1. Isolation, culture differentiation and identification of skeletal muscle cells Select 12-day-old chicken embryos, 75% alcohol disinfection, separation of the chest muscles, rinsed with PBS containing double-antibody 3 times, during which the fascia, blood vessels, bones and other tissues were removed, then transferred to the surface dish and cut into meat paste with ophthalmic scissors. After adding 0.25% trypsin to digest most of the muscle, add 3 times the volume of complete medium containing 15% FBS to terminate digestion. Use a 200-mesh (70 μm) cell screen, and centrifuge the filtrate at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells with 2 mL of complete medium, and evenly add them to the cell culture dish, and place it in the cell culture incubator for culture, which is recorded as P0 generation. When the cell density reaches 80%, subculture, use P2 generation cell density to reach 70% for Desmin and PAX7 immunofluorescence staining identification, and when the P2 generation cell density reaches 90%, replace it with 2% HS differentiation medium to induce differentiation for 48 h, and collect the cells for MyHC immunofluorescence staining identification.

[0018] 1.2 Heat stress treatment Transfer the cells differentiated for 48 h to a 43 ℃ incubator for culture, and collect the cell supernatant after heat stress treatment for 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h to measure the culture medium LDH activity and LA content, and collect the cells to measure the glycogen content, mRNA and protein expression.

[0019] 1.3 Cell morphology observation Use to cause microscopic observation and take pictures of the morphology of cells at different heat stress times.

[0020] 1.4 Culture medium LDH determination Use the lactate dehydrogenase kit from Nanjing Jiancheng Biological Engineering Institute to determine the lactate dehydrogenase activity of the cell culture medium, and refer to the reagent manual for specific operation steps.

[0021] 1.5 Oxidative stress indicators Use commercial kits to measure the ROS level, SOD activity, GSH-Px activity, T-AOC activity, CAT activity, MDA level and carbonyl content of the cells. Use the BCA kit from Shenguo Biological Engineering Co., Ltd. to determine the protein concentration of the sample.

[0022] 1.6 TUNEL detection of cell apoptosis level When the P1 generation cell density reaches 80%, digest and subculture and plate into a 12-well plate containing a fear sheet for culture and induction of differentiation, then collect cells at different times, and fix them. Use the TUNEL detection kit (A112-03, Vazyme, Nanjing, China) to perform staining according to the kit's operation steps. After staining, observe the sections under the LSM900 laser confocal microscope and collect images. The cell nuclei are blue under the microscope, and the apoptotic nuclei are green.

[0023] 1.7 HSP70 and Caspase3 gene expression level detection Cell samples were collected at different times of heat stress, RNA was extracted, and reverse transcription was performed using the HiScript III RT Super Mix reverse transcription kit (Novogene Bioinformatics Co., Ltd., Nanjing) using RNA reverse transcription. Primer3 software was used to design HSP70 and Caspase3 specific primers. ChamQ Universal SYBR qPCR Master Mix kit (Novogene Bioinformatics Co., Ltd., Nanjing) was used for quantitative detection on ABI QuantStuRT-PCRdio™5 (Applied Biosystems, Waltham, MA, USA). The specific operation steps and quantitative PCR reaction program are described in the reagent instruction manual. The expression of related genes was calculated by 2-ΔΔCt method, and β-actin was used as the internal reference gene for normalization: 2. Refer to Figures 2-6 Results analysis: Combined Figure 2 - The effect of different heat stress times on cell morphology; it shows the microscopic morphological changes of differentiated skeletal muscle cells at different time points (0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h) of 43°C heat stress treatment.

[0024] The key features include: Untreated (0 h) cells were typically long spindle-shaped or multinucleated myotube-like, with intact structure.

[0025] With the extension of treatment time, the cells gradually shrank, rounded, and some cells detached and suspended (typical heat injury morphology).

[0026] At 24 h, the intercellular space increased and the cell body shrank, indicating that the heat stress injury reached the modeling threshold at this time Combined Figure 3 - LDH assay of culture medium at different heat stress times; the line chart or column chart shows the dynamic changes of lactate dehydrogenase (LDH) activity at different time points of heat stress treatment; The key data include: LDH activity increased continuously with the extension of treatment time, reaching a peak at 24 h (about 2.5 times higher than the control group).

[0027] After 24 h, the activity tended to be stable or slightly decreased (possibly due to massive cell death).

[0028] 24 h group was significantly different from the control group (P<0.05), which supported the choice of 24 h as the key time point of the model.

[0029] Combining Figure 4 The influence of different heat stress times on the redox state; the multi-index composite chart (may contain sub-charts) shows the change trend of reactive oxygen species (ROS) level and antioxidant enzyme (SOD, GSH-Px, CAT) activity, total antioxidant capacity (T-AOC); The key indicators include: ROS level: 24 h significantly increased to the highest (oxidative stress marker).

[0030] Antioxidant indicators: SOD, GSH-Px enzyme activity significantly decreased at 24 h (P<0.05), indicating that the antioxidant system was imbalanced.

[0031] With Figure 3 LDH peak, Figure 5 apoptosis peak formation, to verify the effectiveness of the model.

[0032] Combining Figure 5 The influence of different heat stress times on the level of cell apoptosis; contains two parts: TUNEL staining confocal microscopic image: blue is the normal nucleus (DAPI), green is the apoptotic nucleus (TUNEL positive).

[0033] Apoptosis rate statistical histogram: quantitatively shows the proportion of TUNEL positive cells at different time points.

[0034] The key data include: The proportion of apoptotic cells in the 24 h group exceeded 25% (significantly higher than the control group, P<0.05).

[0035] The 24 h group in the microscopic image showed dense green fluorescence, which directly showed the apoptosis peak Combining Figure 6 The influence of different heat stress times on the expression level of HSP70 and Caspase3 genes; the double-y coordinate line chart shows the relative expression of heat shock protein 70 (HSP70) and apoptosis execution protein (Caspase-3) mRNA (β-actin as internal reference, 2 −ΔΔCt calculated by method); The rules expressed include: HSP70: early (6-12 h) rapid up-regulation (stress protection response), 24 h peak and then down-regulation.

[0036] Caspase-3: continuously rising, 24 h significantly high expression (P<0.05), and the apoptosis rate Figure 5) form molecular mechanisms echo.

[0037] Biological significance: to prove that heat stress induces cell damage by activating HSP70 (early protection) and Caspase-3 (late apoptosis) pathways.

[0038] In summary, the prior art has reported the method of skeletal muscle heat stress model, but the processing time is short for the continuous research of the construction of chronic heat stress model. The present application determines the heat stress temperature on the basis of the previous research, explores the effect of high temperature treatment for different time on cells, and concludes that high temperature treatment for 24h is the best effect as the chronic heat stress model for studying heat stress induced cell damage.

[0039] The present application selects the most appropriate time point as the heat stress model for studying chronic heat stress induced cell damage by high temperature treatment at 43℃ for different time.

[0040] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for constructing a chronic heat stress model based on chicken embryo primary skeletal muscle cells, characterized by, The method comprises the following steps: Step 1, cell separation and purification, recorded as P0 generation; Step 2, cell passage to P2 generation and then differentiation; Step 3, chronic heat stress model construction: placing the differentiated cells in a 43℃ incubator for continuous treatment for 24h; Step 4, model verification: confirming the effectiveness of the model by detecting at least one of the following indexes: lactate dehydrogenase activity, cell apoptosis rate, ROS level, oxidation-reduction state and HSP70 / Caspase-3 gene expression.

2. The method of claim 1, wherein the chronic heat stress model is constructed by, In step 1, specifically, 12-day-old chicken embryo pectoral muscle tissue is taken, disinfected with 75% alcohol, rinsed with PBS containing double antibodies for 3 times, and muscle membrane, blood vessels and bones are removed; after being cut into pieces, 0.25% trypsin is added for digestion until the tissue disappears, and 3 times the volume of complete culture medium containing 15% FBS is added to terminate digestion; after filtration, centrifugation is performed, the cells are resuspended and inoculated and cultured.

3. The method of claim 2, wherein the chronic heat stress model is constructed by, In step 1, a 200-mesh cell sieve is used for filtration, and the centrifugation condition is 1000rpm for 5min.

4. The method of claim 1, wherein the chronic heat stress model is constructed by, In step 2, when the cell density reaches 70%, 0.25% trypsin is used for digestion and passage to P2 generation; when the density reaches 90%, the culture medium is replaced with a differentiation culture medium containing 2% horse serum and 1% double antibodies, and differentiation is induced for 48h.

5. The method of claim 1, wherein the chronic heat stress model is constructed by, In step 2, the differentiation culture medium is DMEM / F12 basic culture medium, and 2% HS and 1% penicillin-streptomycin double antibodies are added.

6. The method of claim 1, wherein the chronic heat stress model is constructed by, The heat stress treatment time in step 3 is 24h, which is determined based on the LDH activity peak value, glycogen depletion and significant increase in apoptosis rate, and P<0.05 represents a significant increase.

7. The method of claim 1, wherein the chronic heat stress model is constructed by, The model verification in step 4 includes: LDH activity is increased by more than 2.5 times of the control group; Glycogen content is decreased by more than 50%; The proportion of TUNEL positive cells is more than 25%.

8. The application of the chronic heat stress model of broiler skeletal muscle cells constructed by the method according to any one of claims 1-7 in heat stress drug screening, molecular mechanism research and broiler genetic breeding.

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