CN111849873A - Method for inducing autophagy of embryonic stem cells of chicken - Google Patents
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Abstract
A method for inducing chicken embryonic stem cell autophagy belongs to the technical field of biology. The method comprises the steps of firstly detecting the proliferation capacity of cells added with rapamycin with different concentrations through EdU, secondly detecting the expression quantity of autophagy related genes through fluorescent quantitative PCR, and finally detecting the expression quantity of LC3 protein through Western blotting, and comprehensively evaluating the effects of the rapamycin on the cell proliferation efficiency and the autophagy induction effect so as to determine the optimal induction concentration and time. Discussing the induction conditions of rapamycin to the poultry germ cells lays a foundation for the research on the effect of autophagy in the induction and differentiation of embryonic stem cells to primordial germ cells.
Description
Technical Field
The invention relates to a method for inducing the optimum concentration and time of chicken embryonic stem cell autophagy by rapamycin, in particular to a method for inducing chicken embryonic stem cell autophagy, and belongs to the technical field of biology.
Background
Rapamycin (mammalian target of rapamycin, mTOR) is a negative regulatory molecule of autophagy, is extracted from streptomyces metabolites at the earliest, is applied to clinic as an antifungal agent and an immunosuppressant, and is discovered to have the effects of starting autophagy and resisting tumors. The basic principle of initiating autophagy is that rapamycin can enter mammalian cells to form a complex with FKBP12 receptor, and the complex is particularly sensitive to the receptor in mTORC1, can bind to mTORC1 and inhibit its activity, thereby activating ULK1 and initiating the autophagy pathway.
Autophagy is a life phenomenon that is widely present in eukaryotes. Numerous studies have shown that autophagy plays a very important role in both the development and differentiation of cells and organisms. If rapamycin is added into human leukemia HL-60 cells, the results show that 1, 2, 3 and 5 mu mol/L of rapamycin has inhibition effect on HL-60 cell proliferation, and the inhibition efficiency is increased along with the increase of the concentration. After 0, 12 and 24h of treatment of HL-60 cells with 2 mu mol/L of rapamycin, the number of autophagosome in cytoplasm is increased, the morphology is typically specific, and large vacuoles appear in the cytoplasm, which indicates that the rapamycin can induce the autophagy of the HL-60 cells.
Disclosure of Invention
The induction concentration and induction time of rapamycin in various species such as mice and humans have been reported, but the conditions for induction of germ cells in poultry are still unclear. There are studies showing that rapamycin acts differently in different cells. For example, in human leukemia HL-60 cells, 1, 2, 3 and 5 umol/L rapamycin all have inhibitory effect on HL-60 cell proliferation, and the inhibitory efficiency is increased along with the increase of the concentration. After the mesenchymal stem cells are added with 1, 5, 10, 20, 50 and 100nM rapamycin and treated for 12, 24, 36 and 48 hours respectively, the proliferation rate of the BMSCs of the mice shows a trend of increasing firstly and then decreasing, and the cell proliferation rate reaches a peak value in 24 hours. At present, the condition of inducing autophagy by poultry germ cells by rapamycin and the influence of rapamycin on the cell proliferation efficiency are unknown.
The technical scheme of the invention is as follows:
the method comprises the steps of firstly detecting the proliferation capacity of cells added with rapamycin with different concentrations through EdU, secondly detecting the expression quantity of autophagy related genes through fluorescent quantitative PCR, and finally detecting the expression quantity of LC3 protein through Western blotting, and comprehensively evaluating the effects of the rapamycin on the cell proliferation efficiency and the autophagy induction effect so as to determine the optimal induction concentration and time.
The invention has the following beneficial effects:
the invention has simple operation and is practical. The inventor conducts sequencing in the process of inducing embryonic stem cells of chicken to primordial germ cells at the early stage to find that an autophagy signal pathway is activated, the expression of autophagy-related genes INS, VAC8, ATG7 and ATG8 is up-regulated, and the expression of AMPK and ATG4 is down-regulated. This elicits a discussion of the role of autophagy in the induction of differentiation of primordial germ cells. And the research on the induction conditions of rapamycin to the poultry germ cells lays a foundation for the research on the effect of autophagy in the induction and differentiation from embryonic stem cells to primordial germ cells. The inventor has a mature technology for separating germ cells, and has the equipment and research foundation required by the invention. The invention lays a foundation for the subsequent research of the functional mechanism of autophagy in the PGC formation process, and simultaneously provides more experimental materials for the research of poultry cell and embryo engineering.
Detailed Description
Effect of rapamycin on embryonic Stem cell proliferation at varying concentrations and Induction times
Embryonic stem cells of chickens were first isolated and seeded at 2X 10 per well in 24-well plates4And (4) cells. 8 concentration gradients of 0, 20nM, 50nM, 100nM, 200nM, 300nM, 400nM, 500nM were set up, 6h, 12h, 24h, 36h, 48h, 72h6 time gradients per concentration. Dissolving rapamycin in DMSO, and inducing according to the required concentration configurationAnd (4) a culture medium. Adding 500 mul of induction culture medium into each hole, and after inducing for a certain time, using a cell complete culture medium according to a ratio of 1000: 1, preparing a proper amount of 50 mu M EdU culture medium, adding 100 mu l of 50 mu M EdU culture medium into each hole, incubating for 2h, collecting cell suspension, centrifuging, and washing for 1-2 times with PBS (phosphate buffer solution) for 5 minutes each time. Adding 50 mul of cell fixing solution into each tube, incubating for 30 minutes at room temperature, centrifuging to discard the fixing solution, adding 50 mul 2mg/ml glycine into each tube, incubating for 5 minutes by using a decoloring shaking table, centrifuging to discard the glycine solution, adding 100 mul PBS into each tube, washing for 5 minutes by using the decoloring shaking table, centrifuging to discard the PBS, adding 100 mul of penetrant decoloring shaking table into each tube, incubating for 10 minutes, washing for 1 time by using the PBS, washing for 5 minutes, and centrifuging to discard the supernatant. Adding 100 mul 1 XApollo dyeing reaction liquid into each tube, incubating for 30 minutes in a dark place at room temperature and a decoloring shaker, centrifuging to discard the reaction liquid, adding 100 mul of penetrating agent decoloring shaker to clean for 2-3 times, 10 minutes each time, centrifuging to discard the penetrating agent, finally adding 100 mul methanol into each tube to clean for 1-2 times, 5 minutes each time, and cleaning for 1 time by PBS (phosphate buffer solution) and 5 minutes each time.
Detecting the expression level of autophagy genes in each group of cells
Primer5 software design ATG4, ATG5, ATG7, ATG13, ATG14, Beclin-1 and ULK 17 quantitative primers of autophagy related genes, collect samples of each group, add 1ml Trizol to each tube to crack cells, add 200 mul chloroform to each tube, stand at room temperature for 3 minutes after violent shaking for 15 seconds, centrifuge at 4 ℃, 12000rpm for 15 minutes, transfer the upper colorless aqueous phase to a new enzyme-free tube, add isopropanol with equal volume, stand at room temperature for 10 minutes after uniform mixing, centrifuge at 4 ℃ 12000rpm for 10 minutes, discard the supernatant and leave white colloidal precipitate. The precipitate was washed by adding 1ml of 75% ethanol to each tube. Centrifuging at 4 deg.C and 10000rpm for 15 min, discarding liquid, air drying at room temperature, and dissolving in appropriate amount of non-enzyme water. After RNA extraction is successful, reverse transcription is carried out by a Vazyme kit, and the reaction steps comprise the first step of removing genome DNA, the temperature of 42 ℃ is 2 minutes, and the second step of preparing a reverse transcription reaction system, the temperature of 37 ℃ is 15 minutes, and the temperature of 85 ℃ is 5 seconds. After reverse transcription of RNA into cDNA, expression of 7 autophagy genes was quantitatively determined using the Vazyme kit.
Detection of the LC3 II/I ratio in each group of cells
After extracting each histone and measuring the concentration, starting to prepare a sample, and carrying out membrane transfer after 2h of electrophoresis. And immersing the PVDF membrane into 100% methanol for 10 seconds and deionized water for 5min in sequence, and then immersing the glue, the filter paper and the membrane into a transfer buffer for balancing for 5-10 min. The gel was covered on filter paper, manually adjusted to align with the filter paper, and gently rolled with a glass rod to remove air bubbles. The membrane was covered on the glue and air bubbles were removed. The membrane was covered with 3 sheets of filter paper and the air bubbles were removed. Finally, another spongy cushion is covered. After the transfer of the membrane, the membrane was soaked with TBS from bottom to top, transferred to a dish containing blocking solution (TBST containing 5% skimmed milk powder), and then blocked by shaking on a decolourization shaker at room temperature for 2 h. The LC3 antibody was reacted with TBST at 1: after 1000 dilution, the primary antibody was incubated overnight at 4 ℃ and washed three times with TBST on a decolorizing shaker at room temperature for 5min each. Secondary antibodies were mixed with TBST at 1: 1000 dilution, incubation for 2h at room temperature, washing three times with TBST on a destaining shaker at room temperature, 5min each time. And transferring the film into a freshly prepared developing solution, and developing and photographing.
Claims (2)
1. A method for inducing autophagy of embryonic stem cells of chickens, which is characterized by comprising the following steps:
1) the effect of rapamycin at various concentrations and induction times on embryonic stem cell proliferation;
2) qRT-PCR detects the expression quantity of autophagy genes in each group of cells;
3) western Blot was used to determine the ratio of LC3 II/I in each cell group.
2. The method for inducing autophagy of embryonic stem cells of chicken according to claim 1, which is characterized in that the method comprises the following steps: the method comprises the steps of firstly detecting the proliferation capacity of cells added with rapamycin with different concentrations through EdU, secondly detecting the expression quantity of autophagy related genes through fluorescent quantitative PCR, and finally detecting the expression quantity of LC3 protein through Western blotting, and comprehensively evaluating the effects of the rapamycin on the cell proliferation efficiency and the autophagy induction effect so as to determine the optimal induction concentration and time.
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| 袁霞: "CEF转分化为iPGCs诱导体系的研究", 《中国优秀硕士论文全文数据库》 * |
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