CN107686830A - A kind of grass carp hypothalamus cells extracorporeal culturing method - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and specifically discloses a method for culturing grass carp hypothalamic cells in vitro. The method for purifying and culturing primary grass carp hypothalamic cells of the present invention can obtain primary hypothalamic cells with a large number and high purity, and The NM culture medium prepared by our applicant can significantly improve the viability of the obtained grass carp hypothalamic cells. This primary cell culture method can lay a solid foundation for in-depth research on the neuroendocrine system of fish.

Description

A kind of in vitro culture method of grass carp hypothalamic cells

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a method for culturing grass carp hypothalamic cells in vitro.

Background technique

Grass carp is the freshwater fish with the largest aquaculture output in my country (5.68 million tons in 2015). One of the main problems it faces in the aquaculture process is the serious degradation of germplasm resources. Therefore, the breeding of new grass carp species is particularly urgent. However, due to the long sexual maturity time of grass carp, female fish generally need 4-5 years to reach sexual maturity, which seriously restricts the process of its improved species breeding. Therefore, it is of great significance for grass carp breeding to reveal the molecular mechanism of grass carp sexual maturation initiation regulation, find out the key factors that regulate its gonad development and sexual maturation initiation, and then edit the key factors to shorten the sexual maturity time of grass carp through targeted editing of genetic engineering technology.

The initiation of sexual maturation in fish is influenced by the environment. Sensory organs transmit the stimuli of the external environment (such as temperature, light and water stimulation, etc.) Secretion of sex steroid hormones to promote gonad maturation and ovulation and sperm production. Among them, the GnRH secreted by the hypothalamus plays an important role in the initiation of sexual maturation in fish, but how external factors regulate the expression of GnRH in the hypothalamus is still unclear. The structure of fish hypothalamus is complex, and in vivo research is affected by the interaction between cells and other neuroendocrine transmitters. The isolation and culture of grass carp hypothalamus cells in vitro has become an important way to study the function of hypothalamus and the expression regulation mode of GnRH in hypothalamus. By isolating and culturing grass carp hypothalamic cells, establishing the in vitro isolation and culture methods of grass carp hypothalamic cells, understanding the morphological characteristics and vitality characteristics of hypothalamic cells under in vitro culture conditions, and laying the foundation for further in vitro studies to reveal the initiation mechanism of grass carp sexual maturation.

Fish cell culture is the same as mammalian cell culture, including two stages of primary culture and subculture. Primary culture refers to the first culture of cells obtained directly from animal tissues and organs; subculture cell culture refers to the expansion and subculture of cells grown in vitro. Generally speaking, as the number of passages increases, the subcultured cells will gradually lose the physiological and biochemical functional characteristics of the primary cultured cells, while the primary cultured cells still retain some or all of the same physiological functional characteristics as the somatic cells, and the cells still maintain a high degree of Therefore, primary cultured cells are considered to be able to replace living animals for some life science research. As far as the current application status of cell culture is concerned, the research fields applied by primary cell culture are broader, and the role of subculture in certain research fields such as environmental toxicology research, fish physiology research and genetics research is even more important. irreplaceable cells.

Due to the different physiological structures of fish species, ages, and different tissues and organs, the internal environment of different cells is also very different. different. In the primary culture of fish cells, the nerve cells of brain tissue are relatively difficult to culture. In recent years, significant progress has been made in the culture of seawater fish brain cells. Cobia brain cell lines, grouper brain cell lines, flounder brain cell lines, and turbot brain cell lines have been established successively. However, the primary culture of mesencephalic cells and the establishment of cell lines in freshwater fish are relatively rare; the primary culture of grass carp hypothalamic cells and the establishment of cell lines have not been reported yet. The invention establishes methods for the isolation, primary culture and subculture of brain cell lines of grass carp hypothalamic cells.

Contents of the invention

The object of the present invention is to provide a method for culturing grass carp hypothalamic cells in vitro, the method is simple and easy to implement, the obtained primary cells have stable properties, the hypothalamic cells maintain good vitality, and the cells grow relatively uniformly.

In order to achieve the above object, the present invention takes the following technical measures:

A method for culturing grass carp hypothalamic cells in vitro, comprising the steps of:

(1) Cut the cleaned healthy fresh grass carp hypothalamus into pieces with a diameter of 0.5-1.0mm;

(2) Collect the cut hypothalamus fragments in a centrifuge tube, add WM medium to wash;

(3) Transfer the cleaned hypothalamic fragments to a new centrifuge tube, add WM medium (containing 3-8mg/ml trypsin), and digest in a water bath at 20-37°C;

(4) Aspirate the WM medium containing Trypsin, and then add the WM medium containing 2-5mg/ml Trypsin inhibitor;

(5) Aspirate the WM medium containing trypsin inhibitor, add WM medium containing 0.1-0.5 mg/ml DNaseII, and let it stand at room temperature to dissolve the DNA connection between cells;

(6) Aspirate the WM medium containing DNase II, add BM medium containing 1.5-3.0mM EDTA to shake or stand still, and then aspirate the medium;

(7) Transfer the hypothalamic fragments to a new centrifuge tube, add BM medium into the tube, gently blow the hypothalamic fragments with a pipette, and the cells are blown away. At this time, use a cell sieve with a pore size of 40-100 μm to remove the upper layer of cells. Collected in a centrifuge tube after filtration;

(8) Transfer the filtered cells to a new centrifuge tube, and collect the cells by centrifugation at 1000-3000rpm for 10-30min;

(9) Remove the upper medium, add PM medium to resuspend the cells;

(10) Take the cell suspension, add trypan blue staining, and count under the microscope;

(11) Add PM medium to dilute the hypothalamic cells, inoculate the cells into a cell culture plate, inoculate 0.5-3.0×10 ⁶ cells per well, add PM medium containing FBS, so that the final content of FBS in each well is 5-10%;

(12) After the hypothalamic cells are cultured and adhered in PM medium containing 5-10% FBS, the medium is sucked off and added:

A. Subculture in NM medium containing 5-15ng/ml basic fibroblast growth factor; or B. Subsequent dosing experiment after adding NM medium for culture.

In the method described above, preferably, the specific method of step (1) comprises: healthy fresh grass carp hypothalamus is to place the fresh brain tissue of healthy grass carp in WM medium; Separate it from the brain tissue, use tweezers to peel off the capsule on the surface of the hypothalamus; put the separated hypothalamus into a centrifuge tube and add WM medium to clean it; put the cleaned healthy fresh grass carp hypothalamus Put it on a tissue chopper and cut it into pieces with a diameter of 0.5-1.0mm;

In the method described above, preferably, in step (12), when the NM medium containing 5-15ng/ml basic fibroblast growth factor is added for subculture, the medium is replaced every three days;

In the method described above, preferably, in step (12), when adding the NM medium for cultivation and then continuing the drug addition experiment, the drug is dissolved in the TM medium and then incubated with the cells.

The formula of described WM medium comprises:

MEM: 10.0-12.0mg/ml, HEPES: 6.0-8.0mg/ml, NaHCO ₃ : 2.0-3.0mg/ml, BSA: 3.0-5.0mg/ml, penicillin: 50-100U/ml, streptomycin: 50 -100μg/ml;

The formula of described BM medium comprises:

S-MEM: 10.0-12.0mg/ml, HEPES: 6.0-8.0mg/ml, NaHCO ₃ : 2.0-3.0mg/ml, BSA: 3.0-5.0mg/ml, penicillin: 50-100U/ml, streptomycin : 50-100 μg/ml, the S-MEM is Ca ²⁺ freeMEM;

The formula of described PM medium comprises:

MEM: 10.0-12.0mg/ml, NaHCO ₃ : 2.0-3.0mg/ml, HEPES: 6.0-8.0mg/ml, penicillin: 100-200 U/ml, streptomycin: 100-200μg/ml;

The formula of described TM medium comprises:

MEM: 10.0-12.0mg/ml, NaHCO ₃ : 2.0-3.0mg/ml, HEPES: 6.0-8.0mg/ml, BSA: 1.0-3.0mg/ml, penicillin: 100-200U/ml, streptomycin: 100 -200μg/ml;

The formula of described NM medium comprises:

MEM: 10.0-12.0mg/ml, NaHCO ₃ : 2.0-3.0mg/ml, HEPES: 6.0-8.0mg/ml, Penicillin: 100-200U/ml, Streptomycin: 100-200μg/ml, GlutaMax Supplement: 25 -50 μM, B27 Supplement (50×): 2-5%, FBS: 5-10%.

The culture medium described above, preferably:

The formula of described WM medium comprises:

MEM: 9.6 mg/ml, HEPES: 6.0 mg/ml, NaHCO ₃ : 2.2 mg/ml, BSA: 3.0 mg/ml, penicillin: 50 U/ml, streptomycin: 50 μg/ml; PH=7.7.

The formula of described BM medium comprises:

S-MEM: 10.4 mg/ml, HEPES: 6.0 mg/ml, NaHCO ₃ : 2.2 mg/ml, BSA: 3.0 mg/ml, penicillin: 50 U/ml, streptomycin: 50 μg/ml; PH=7.7.

The formula of described PM medium comprises:

MEM: 9.6 mg/ml, NaHCO ₃ : 2.2 mg/ml, HEPES: 6.0 mg/ml, penicillin: 100 U/ml, streptomycin: 100 μg/ml; PH=7.7.

The formula of described TM medium comprises:

MEM: 9.6 mg/ml, NaHCO ₃ : 2.2 mg/ml, HEPES: 6.0 mg/ml, BSA: 1.0 mg/ml, penicillin: 100 U/ml, streptomycin: 100 μg/ml; PH=7.7.

The formula of described NM medium comprises

MEM: 9.6mg/ml, NaHCO ₃ : 2.2mg/ml, HEPES: 6.0mg/ml, Penicillin: 100U/ml, Streptomycin: 100μg/ml, GlutaMax Supplement: 25μM, 2% B27Supplement (50×), 5 %FBS; pH=7.7.

Compared with the prior art, the present invention has the following advantages:

(1) The present invention establishes the method for the isolation, primary culture and subculture of grass carp hypothalamus cells for the first time, and a large amount of primary culture cells of grass carp hypothalamus with stable vitality can be quickly obtained by this method, which lays the foundation for further hypothalamus in vitro experiments Base;

(2) the separation medium (WM medium, BM medium, PM medium) and in vitro medium (NM medium and TM medium) used in the present invention are all configured according to the characteristics of grass carp nerve cells, which can be very good It meets the needs of grass carp hypothalamic cells, and the cost is much cheaper than buying ready-made media.

Description of drawings

Figure 1 is a schematic diagram of grass carp hypothalamus cells in different culture periods.

Fig. 2 is a schematic diagram showing the effect of estradiol on the expression of β-actin, TAC3a and TACR3 mRNA in primary cultured cells of grass carp hypothalamus.

Figure 3 is a schematic diagram of the subculture results of grass carp hypothalamic cells.

detailed description

The technical solutions described in the present invention, if not specified, are conventional solutions in the art, and the reagents or materials, if not specified, are all derived from commercial channels.

Example 1:

A method for culturing grass carp hypothalamic cells in vitro, comprising the steps of:

(1) Place the fresh brain tissue of healthy grass carp in WM medium;

(2) Separate the hypothalamus from the brain tissue in a petri dish containing WM medium, and use forceps to peel off the capsule on the surface of the hypothalamus;

(3) Put the separated hypothalamus into a 50ml centrifuge tube and add WM medium to wash it three times;

(4) Put the cleaned hypothalamus into a tissue chopper, and cut it into pieces with a diameter of 0.8mm;

(5) Collect the cut hypothalamus fragments in a 15ml centrifuge tube, add WM medium to wash three times;

(6) Transfer the cleaned hypothalamic fragments to a new 50ml centrifuge tube, add 10ml WM medium (containing 65mg trypsin), and digest in a water bath at 28°C for 30min;

(7) Use a disposable pipette to remove the WM medium containing Trypsin, and then add 10ml of WM medium containing 2.5mg/ml Trypsin inhibitor to neutralize unreacted trypsin;

(8) Aspirate the WM medium containing trypsin inhibitor, add WM medium containing 0.1mg/ml DNase II, and let it stand at room temperature to dissolve the DNA connection between cells;

(9) Aspirate the WM medium containing DNase II, add the BM medium containing 2.0mM EDTA to shake, and then aspirate the medium;

(10) Transfer the hypothalamic fragments to a new centrifuge tube, add BM medium into the tube, gently blow the hypothalamic fragments with a pipette, and the cells are blown away. At this time, the upper layer of cells is filtered through a cell sieve with a pore size of 45 μm collected in a centrifuge tube;

(11) Transfer the filtered cells to a new centrifuge tube, and collect the cells by centrifugation at 1000rpm for 10min;

(12) Remove the upper medium, add PM medium to resuspend the cells;

(13) Take the cell suspension, add trypan blue staining, and count under the microscope;

(14) Add PM medium to dilute the hypothalamic cells, inoculate the cells into a cell culture plate, inoculate 3.0×10 ⁶ cells per well, add PM medium containing FBS, so that the final content of FBS in each well is 5% ; Culture conditions: 28°C, 5% CO ₂ ;

(15) After the hypothalamic cells were cultured in PM medium containing 5% FBS for 24 hours, the medium was aspirated, and NM medium was added to continue culturing for 48 hours.

The hypothalamic cells were isolated and cultured according to the steps described above; as shown in Figure 1, the hypothalamic cells isolated on the first day had a variety of shapes, but did not grow synapses; on the second day, NM medium was added to strengthen the culture around the cells Gradually grow synapses; after three days of culture, the hypothalamic cells on the fourth day have covered the entire culture plate, and the cells are connected to each other through synapses. At this time, the function of nerve cells has been restored, and the next step can be carried out Drug treatment experiments.

The formula of described WM medium comprises:

MEM (Gibico): 9.6 mg/ml, HEPES: 6.0 mg/ml, NaHCO ₃ : 2.2 mg/ml, BSA: 3.0 mg/ml, penicillin: 50 U/ml, streptomycin: 50 μg/ml; PH=7.7.

The formula of described BM medium comprises:

S-MEM (Sigma): 10.4mg/ml, HEPES: 6.0mg/ml, NaHCO ₃ : 2.2mg/ml, BSA: 3.0mg/ml, penicillin: 50U/ml, streptomycin: 50μg/ml, the The S-MEM is Ca ²⁺ freeMEM, pH=7.7.

The formula of described PM medium comprises:

MEM (Gibico): 9.6 mg/ml, NaHCO ₃ : 2.2 mg/ml, HEPES: 6.0 mg/ml, penicillin: 100 U/ml, streptomycin: 100 μg/ml; PH=7.7.

The formula of described TM medium comprises:

MEM (Gibico): 9.6 mg/ml, NaHCO ₃ : 2.2 mg/ml, HEPES: 6.0 mg/ml, BSA: 1.0 mg/ml, penicillin: 100 U/ml, streptomycin: 100 μg/ml; PH=7.7.

The formula of described NM medium comprises

MEM (Gibico): 9.6mg/ml, NaHCO ₃ : 2.2mg/ml, HEPES: 6.0mg/ml, Penicillin: 100U/ml, Streptomycin: 100μg/ml, GlutaMax Supplement: 25μM, 2% B27Supplement (50× ), 5% FBS; PH=7.7.

Example 2:

Viability assay of cultured cells:

Dissolve estradiol (E2) in TM medium to prepare drug solutions with different concentrations. The concentrations of estradiol are respectively 0.1nM, 1nM, 10nM, 100nM, and 1000nM. For thalamic cells, after incubation for 24 hours, the medium was aspirated, and 0.5ml Trizol was added to each well to collect the cells and extract RNA. After reverse transcription into cDNA, real-time quantitative PCR was used to verify the regulatory effect of estradiol on the expression of related genes in the hypothalamus . Through detection and analysis, it was found that there was no significant difference in the expression of the internal reference gene β-actin among the groups (A in Figure 2), indicating that the cell culture system was relatively stable and could meet the drug treatment experiment. In addition, estradiol can induce TAC3a (B in Fig. 2) and TACR3 (C in Fig. 2) mRNA expression levels in hypothalamic cells, and the standard deviation between each group of four repetitions is controlled within 10%, the above results It shows that the cultured hypothalamic cells maintain good viability, and the cells in each well grow relatively uniformly, which is suitable as a cell model for studying hypothalamus.

Example 3:

Subculture of grass carp hypothalamic cells:

Step (15) of embodiment 1, after hypothalamic cells are cultivated in the PM medium containing 5% FBS for 24 hours, the medium is aspirated, and the NM medium containing 5ng/ml basic fibroblast growth factor is added to continue culturing , the medium was changed every 3 days.

Grass carp hypothalamic cells were cultured in NM medium containing 5ng/ml basic fibroblast growth factor for two weeks, and the cells were digested with 0.25% Trypsin and centrifuged at 1000rpm for 10 minutes to collect the cells; after the cells were resuspended in NM medium, the Transfer one-third of the cells to a new cell culture flask, add NM medium containing 5ng/ml basic fibroblast growth factor to continue culturing, and perform the second passage after the cells are full of the culture flask; follow the above method , when the cells were transferred to the ninth generation, there was no significant difference in their growth and viability compared with the primary generation (Figure 3).

Claims (5)

1. a method for culturing grass carp hypothalamic cells in vitro, comprising the steps of: (1) Cut the cleaned and healthy fresh grass carp hypothalamus into pieces with a diameter of 0.5-1.0 mm; (2) Collect the cut hypothalamus fragments into a centrifuge tube, add WM medium to wash; (3) Transfer the cleaned hypothalamic fragments to a new centrifuge tube, add WM medium containing 3-8mg/ml trypsin, and digest in a water bath at 20-37°C; (4) Aspirate the WM medium containing Trypsin, and then add WM medium containing 2-5 mg/ml Trypsin inhibitor; (5) Aspirate the WM medium containing trypsin inhibitor, add WM medium containing 0.1-0.5mg/ml DNase II, and let it stand at room temperature to dissolve the DNA connection between cells; (6) Aspirate the WM medium containing DNase II, add BM medium containing 1.5~3.0mM EDTA to shake or stand still, and then aspirate the medium; (7) Transfer the hypothalamic fragments to a new centrifuge tube, add BM medium to the tube, gently blow the hypothalamic fragments with a pipette, and the cells are blown away. At this time, use cells with a pore size of 40-100 μm in the upper layer Sieve and collect into centrifuge tubes; (8) Transfer the filtered cells to a new centrifuge tube, and collect the cells by centrifugation at 1000-3000 rpm for 10-30 min; (9) Remove the upper medium and add PM medium to resuspend the cells; (10) Take the cell suspension, stain with trypan blue, and count under the microscope; (11) Add PM medium to dilute the hypothalamic cells, inoculate the cells into a cell culture plate, inoculate 0.5-3.0×10 ⁶ cells per well, add PM medium containing FBS, so that the final content of FBS in each well is 5-10%; (12) After the hypothalamic cells are cultured and attached in PM medium containing 5-10% FBS, the medium is aspirated and added: Subculture in NM medium containing 5-15 ng/ml basic fibroblast growth factor; or add NM medium for subsequent dosing experiments; The formula of described WM medium comprises: MEM: 10.0-12.0 mg/ml, HEPES: 6.0-8.0 mg/ml, NaHCO ₃ : 2.0-3.0 mg/ml, BSA: 3.0-5.0 mg/ml, Penicillin: 50-100 U/ml, Streptomycin: 50-100 μg/ml; The formula of described BM medium comprises: S-MEM: 10.0-12.0 mg/ml, HEPES: 6.0-8.0 mg/ml, NaHCO ₃ : 2.0-3.0 mg/ml, BSA: 3.0-5.0 mg/ml, penicillin: 50-100U/ml, streptomycin : 50-100 μg/ml, the S-MEM is Ca ²⁺ freeMEM; The formula of described PM medium comprises: MEM: 10.0-12.0 mg/ml, NaHCO ₃ : 2.0-3.0 mg/ml, HEPES: 6.0-8.0 mg/ml, penicillin: 100-200 U/ml, streptomycin: 100-200μg/ml; The formula of described NM medium comprises MEM:10.0-12.0 mg/ml, NaHCO ₃ :2.0-3.0 mg/ml, HEPES: 6.0-8.0mg/ml, Penicillin:100-200 U/ml, Streptomycin:100-200 μg/ml, GlutaMax Supplement : 25-50μM, B27 Supplement(50×): 2-5%, FBS: 5-10%. 2. The method according to claim 1, the specific method of step (1) comprising: placing the fresh brain tissue of healthy grass carp in WM medium; separating the hypothalamus from the brain tissue in a petri dish containing WM medium Next, use tweezers to peel off the capsule on the surface of the hypothalamus; put the separated hypothalamus into a centrifuge tube and add WM medium to clean it; put the cleaned and healthy fresh grass carp hypothalamus into the tissue and chop it On-machine, it is cut into pieces with a diameter of 0.5-1.0 mm. 3. The method according to claim 1, in step (11), when the NM medium containing 5-15 ng/ml basic fibroblast growth factor is added for subculture, the medium is replaced every three days . 4. The method according to claim 1, in step (11), when adding the NM medium for cultivation and continuing the dosing experiment, the medicine is dissolved in the TM medium, and then incubated with the cells; The formula of described TM medium comprises: MEM: 10.0-12.0 mg/ml, NaHCO ₃ : 2.0-3.0 mg/ml, HEPES: 6.0-8.0 mg/ml, BSA: 1.0-3.0 mg/ml, Penicillin: 100-200 U/ml, Streptomycin: 100-200 μg/ml. 5. The method of claim 1, The formula of described WM medium comprises: MEM: 9.6mg/ml, HEPES: 6.0mg/ml, NaHCO ₃ : 2.2mg/ml, BSA: 3.0mg/ml, penicillin: 50U/ml, streptomycin: 50μg/ml; PH=7.7; The formula of described BM medium comprises: S-MEM: 10.4mg/ml, HEPES: 6.0mg/ml, NaHCO ₃ : 2.2mg/ml, BSA: 3.0mg/ml, penicillin: 50U/ml, streptomycin: 50μg/ml; pH=7.7; The formula of described PM medium comprises: MEM: 9.6mg/ml, NaHCO ₃ : 2.2mg/ml, HEPES: 6.0mg/ml, penicillin: 100U/ml, streptomycin: 100μg/ml; pH =7.7; The formula of described TM medium comprises: MEM: 9.6mg/ml, NaHCO ₃ : 2.2mg/ml, HEPES: 6.0mg/ml, BSA: 1.0mg/ml, Penicillin: 100U/ml, Streptomycin: 100μg/ml; pH =7.7; The formula of described NM medium comprises MEM: 9.6mg/ml, NaHCO ₃ : 2.2mg/ml, HEPES: 6.0mg/ml, Penicillin: 100U/ml, Streptomycin: 100μg/ml, GlutaMax Supplement: 25 μM, 2% B27 Supplement (50×) , 5% FBS; PH=7.7.