CN113388581A

CN113388581A - Primary cell culture method for sleeve gastrectomy mouse vagus nerve

Info

Publication number: CN113388581A
Application number: CN202110757009.7A
Authority: CN
Inventors: 申晓军; 张新; 刘兆瑞; 郑瑞; 张隆江; 印慨
Original assignee: First Affiliated Hospital of Naval Military Medical University of PLA
Current assignee: First Affiliated Hospital of Naval Military Medical University of PLA
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-09-14
Anticipated expiration: 2041-07-05
Also published as: CN113388581B

Abstract

The invention provides a primary cell culture method of vagus nerve of a sleeve gastrectomized mouse, which is characterized by comprising the following steps: step 1, ganglion digestion and separation: step 1-1, cutting the back of an SD mouse and stripping ganglion cells, and step 1-2, washing and cutting the ganglion; step 1-3, adding a digestive juice, performing low-speed rotation in a shaking table at 37 ℃ for digestion, centrifuging, removing the digestive juice, adding an adherence culture medium, and uniformly blowing to obtain a suspension; step 2, culturing the vagus nerve cells: and 2-1, filtering the suspension by using a 70-micrometer filter, 2-2, diluting the filtrate by using an adherent culture medium, inoculating the diluted filtrate into a 6-well plate, culturing 10 ten thousand cells/well, 2-3, putting the culture box into an incubator for culture, removing non-nerve cells by using a differential adherence method, changing the culture medium into a growth culture medium after 8 hours, and continuously culturing the cells for 4-6 days to obtain the cultured vagal neuron cells.

Description

Primary cell culture method for sleeve gastrectomy mouse vagus nerve

Technical Field

The invention relates to the field of cell culture, in particular to a primary cell culture method of vagus nerve of a sleeve gastrectomized mouse.

Background

The vagus nerve provides bidirectional communication of information between the brain and peripheral organs and plays an important role in the regulation of energy metabolism by mediating gut-brain communication. Among them, 70% of vagus nerve fibers are from the corpus and located in Vagal Afferent Neurons (VANs) of the Nodose Ganglia (NG), and their nerve endings form the following three connections with the intestinal tract: smooth intramuscular intraganglionic plate terminals and intramuscular arrays, endosomal process terminals, and the formation of rapid excitatory synapses with gut endocrine cells. VANs can directly sense mechanical stimulation such as intestinal tract traction and contraction, respond to chemical signals such as GM (XX) products, intestinal tract hormones and neurotransmitters through receptors, and are main neural mechanisms for regulating energy metabolism and glycolipid metabolism.

The neurochemical phenotype of vagal afferent neurons in different vegetative states is plastic, participates in important mechanisms for maintaining the energy balance of the body, and plays an important role in the regulation of metabolic homeostasis and the development of obesity. Therefore, the culture of vagal neuronal cells in obese mice is the basis for the study of VANs function. However, in the prior art, few reports on primary culture of vagus neurons exist, and no suitable method is available for primary culture of vagus neurons, which greatly restricts further research on the functions of VANs.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for culturing primary cells of vagal nerve in sleeve gastrectomized mice.

The invention provides a primary cell culture method of vagus nerve of a sleeve gastrectomized mouse, which is characterized by comprising the following steps: step 1, a ganglion digestion and separation step, which comprises the following substeps: step 1-1, cutting off the back of an obesity-cultured SPF-grade Sprague-Dawlcy mouse, peeling off ganglion cells by using forceps with the help of a dissecting microscope, step 1-2, washing the peeled ganglion by using Hanks salt balanced solution, and cutting the cleaned ganglion into pieces to obtain cut-off tissues; step 1-3, adding a digestive juice into the sheared tissue, carrying out low-speed rotation in a shaking table at 37 ℃ for digestion, then centrifuging, removing the digestive juice, adding an adherence culture medium, and uniformly blowing to obtain a suspension; and step 2, a step of culturing vagal neurons, which comprises the following substeps of 2-1, filtering the suspension by using a 70-micron filter to obtain a filtrate, 2-2, diluting the filtrate by using an adherent culture medium, inoculating the diluted filtrate into a 6-well plate, mixing 10 ten thousand cells/well, gently and uniformly mixing the cells to uniformly spread the cells in a culture dish, 2-3, putting the culture dish into an incubator for culturing, removing the non-neural cells by using a differential adherence method in the culture process, after 8 hours, discarding the adherent culture medium and replacing the adherent culture medium with a growth culture medium, continuously culturing the cells for 4-6 days, and replacing the fresh growth culture medium every two days to obtain the cultured vagal neurons.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: in step 1-1, Sprague-Dawlcy mice are obtained from 30-40 days of birth and weigh 30-40g, and are male and female.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein in the step 1-3, the mass volume of the sheared tissue and the digestive juice is (0.2 g-1 g): 2 ml.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein in the step 1-3, the digestive juice is trypsin with the mass volume concentration of 0.25%.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: in the step 1-3, the digestive juice comprises trypsin and collagenase type I in a volume ratio of 1:1, wherein the mass volume concentration of the trypsin is 0.25%, and the mass volume concentration of the collagenase type I is 0.2%.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein in the step 1-3, the digestion is carried out for 40-80 min, and the blowing beating process comprises the following steps: respectively using a large-caliber Pasteur pipe, a medium-caliber Pasteur pipe and a small-caliber Pasteur pipe, lightly blowing and beating the tissues by using the large-caliber Pasteur pipe for 4-5 times when the tissues become soft, replacing the middle-caliber Pasteur pipe, lightly blowing and beating the tissues into small tissues, replacing the small-caliber Pasteur pipe, and continuously blowing and beating to obtain suspension.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein, in the step 2-3, the temperature in the incubator is 37 ℃, and CO is added₂The volume content was 5%.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein, the adherent culture medium is a DMEM culture medium added with fetal calf serum, and the growth culture medium is a Neurobasal culture medium added with B27 additive, N2 additive, growth factor EGF and growth factor FGF.

In the primary cell culture method for the vagus nerve of the sleeve gastrectomized mouse provided by the invention, the method can also have the following characteristics: wherein, the volume fraction of fetal calf serum in the adherent culture medium is 10%, the mass ratio of the B27 additive, the N2 additive, the growth factor EGF and the growth factor FGF in the growth culture medium is 2:1:1:1, and the concentration of the N2 additive in the growth culture medium is 1ug/100 ml.

Action and Effect of the invention

According to the primary cell culture method for the vagus nerve of the sleeve gastrectomy mouse, which is disclosed by the invention, the method comprises a digestion and separation step of a transganglion and a culture step of a vagus neuron, wherein the digestion and separation step comprises the steps of firstly cutting the back of the mouse and stripping off the ganglion, then cleaning and cutting up the ganglion, adding a digestive juice, placing the mixture in a 37 ℃ shaking table for low-speed rotation for digestion, centrifuging, adding a culture medium, and uniformly blowing to obtain a suspension; the culture step of the vagus nerve cells comprises the steps of filtering at the level of 70 microns, diluting filtrate, inoculating the filtrate into a 6-hole plate, culturing 10 thousands of cells/hole in an incubator, removing non-nerve cells by using a differential adherence method in the culture process, removing an adherence culture medium and replacing the adherence culture medium with a growth culture medium after 8 hours, continuously culturing the cells for 4-6 days, and replacing fresh growth culture medium every two days to obtain the cultured vagus nerve cells. Therefore, the method provided by the invention can be used for simply culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse with strong operability and good repeatability, and lays a foundation for further researching the action of VANs-mediated signal channels in intestinal-brain communication.

Drawings

FIG. 1 is an inverted photomicrograph of two groups of digestive juices treated during the culture of vagal neurons in an example of the present invention;

FIG. 2 is a graph showing the change in the number of cells during the culture of vagal neurons after two groups of digestive juices treatment according to the example of the present invention as a function of the number of days of culture;

FIG. 3 is a photograph of fluorescent staining of MAP2 from primary cells cultured in an example of the invention;

FIG. 4 is a graph of the proportion of vagal MAP2 positive cells obtained from primary cell culture methods in examples of the invention.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the efficacy of the invention easy to understand, the following examples are combined with the accompanying drawings to specifically describe the culture method of the primary cells of the vagus nerve of the sleeve gastrectomized mouse.

< example >

This example provides a method for culturing primary cells of vagal nerve in sleeve gastrectomized mice and a method for identifying vagal neurons.

The materials used in this implementation include:

(a) laboratory animal

6 obesity cultured SPF-grade Sprague-Dawlcy (SD) mice were selected, born 30-40 days, weighed 30-40g, male and female unlimited, and provided by the animal testing center of the university of naval medicine.

(b) Reagent

Digestion culture: hanks salt balanced solution (HBSS), trypsin, collagenase type I (Worthington, usa), Neurobasal medium, B27 additive, N2 additive, growth factor EGF, growth factor FGF, DMEM medium, Fetal Bovine Serum (FBS), penicillin-streptomycin, GlutaMAX medium.

Dyeing and identifying: 4% paraformaldehyde, rabbit anti-mouse MAP2 monoclonal antibody (CST antibody, USA), Alexa Fluor488 fluorescent secondary antibody, PBS, a fixative (5% Normal coat Serum, 0.25% TritonX-100in PBS), a mounting medium (Vector labs, USA), nail polish, a glass slide and a cover glass.

(c) Instrumentation and equipment

Dissecting microscope (purchased from Calcaisi corporation), forceps, scissors, glass bottle, cell culture incubator (37 ℃, 5% CO)₂) A biosafety cabinet, a Milli-Q Biocel type ultrapure water system (Millipore, USA), a DK-600 type constant temperature water tank (Shanghai sperm macroexperimental facilities, Inc.), an inverted microscope (Olympus, Japan), and a confocal fluorescence microscope (Lycra, China, Inc.).

The primary cell culture method for the sleeve gastrectomized mouse vagus nerve in the present example comprises the following steps:

step 1, the digestion and separation step of ganglia.

Wherein, step 1 comprises the following substeps:

step 1-1, the backs of adiposity cultured SPF grade SD mice were dissected and ganglion cells were dissected cautiously with forceps with the aid of a dissecting microscope.

Step 1-2, the stripped ganglia is firstly placed in a preservation solution (the preservation solution contains penicillin-streptomycin and GlutaMAX culture medium) and transferred into a biological safety cabinet, the ganglia is washed twice by Hanks salt balanced solution, then the ganglia are cut into pieces by sterilized scissors, and the cut-up tissues are transferred into a 15ml centrifuge tube.

Step 1-3, adding digestive juice into the sheared tissue, and dividing the tissue into two groups during digestion: one group used trypsin at a mass volume concentration of 0.25% as a digestive fluid and added 2ml of 0.25% trypsin to 0.5g of tissue; another group used a digest of 0.25% by volume trypsin and 0.2% by volume collagenase type I (1: 1 ratio by volume of the two enzymes), and added to 0.5g of tissue 1ml of 0.25% trypsin and 1ml of 0.2% collagenase type I. Then rotating at low speed in a shaker at 37 ℃ for 60 minutes for digestion, then centrifuging at 2000r/min for 15 minutes, removing digestion solution, adding an adherence culture medium, and blowing and beating uniformly to obtain a suspension.

Wherein, DMEM culture medium of Fetal Bovine Serum (FBS) is added into the adherent culture medium, and the volume fraction of the fetal bovine serum in the adherent culture medium is 10%. The composition of the adherent medium may be DMEM medium + 10% FBS.

The blowing process comprises the following steps: the tissue is lightly blown and beaten by the large-caliber Pasteur tube for 4-5 times after the tissue becomes soft, so that the tissue is divided into small tissues, and the small-caliber Pasteur tube is replaced to continuously blow and beat to obtain suspension. Note that: in the operation process, the action must be gentle and gentle again, so that bubbles are avoided.

And 2, culturing the vagus nerve cells.

Wherein, step 2 comprises the following substeps:

step 2-1, the above suspension was filtered using a 70 μm filter, and the tissue cake was filtered off to obtain a filtrate.

And 2-2, firstly, diluting 10 mu L of filtrate by 10 times, measuring the concentration of cells by using a cell counter, then diluting the rest filtrate by using an adherent culture medium (DMEM culture medium + 10% FBS), inoculating the diluted filtrate into a 6-well plate (10 ten thousand cells/well), and gently mixing the cells uniformly to ensure that the cells are uniformly spread in a culture dish.

Step 2-3, putting the culture dish into an incubator for culture, wherein the temperature in the incubator is 37 ℃, and CO is adopted₂The volume content is 5%, non-nerve cells are removed by using a differential adherence method in the culture process, after 8 hours, the adherence culture medium is discarded and replaced by a growth culture medium, the cells are continuously cultured for 4-6 days, and the fresh growth culture medium is replaced every two days to obtain the cultured vagal neuron cells.During the culture period, the growth of the cells was observed daily using an inverted microscope, and the results are shown in FIGS. 1 and 2.

The growth medium is a Neurobasal medium added with a B27 additive, an N2 additive, a growth factor EGF and a growth factor FGF, the mass ratio of the B27 additive, the N2 additive, the growth factor EGF and the growth factor FGF in the growth medium is 2:1:1:1, and the concentration of the N2 additive in the growth medium is 1ug/100 ml. The composition of this growth medium can be described as Neurobasal + B27+ N2+ EGF + FGF.

FIG. 1 is an inverted photomicrograph of two groups of digestive juices treated during the culture of vagal neurons in an example of the present invention; FIG. 2 is a graph showing the change in the number of cells during the culture of vagal neurons after two groups of digestive juices treatment according to the example of the present invention, as a function of the number of days of culture.

As shown in fig. 1, primary cells of mouse vagus neuron cells attach slowly under observation of an inverted microscope, the initial density is about 20%, and the cells are mostly circular or oval; after the cells are cultured for 4 days, the number of the cells is obviously increased, and the cells have typical neuron forms; then, partial cells are observed to be attached to the wall, the culture solution is replaced and the cells are continuously cultured, when the cells are cultured for 10 days, the neuron cell bodies are mature and full, and are mostly in a fusiform shape and a cone shape, and a plurality of branches rich in cytoplasm are mutually connected to form a dense staggered nerve fiber network. Meanwhile, two different digestion treatment modes are compared to find that: the combined digestion mode of trypsin and collagenase is superior to the single digestion mode of trypsin. The yield of cells from the former digestion and the subsequent cell viability were significantly higher than those of the latter (P ═ 0.012) (see fig. 1 and 2).

In order to further verify the performance of the vagus neuron cells cultured by the above-mentioned primary cell culture method, the present example also identified them. The method for identifying vagal neurons in this example comprises the following steps:

step one, digesting the cultured vagal neuron cells, spreading the cells on a cover glass in a 24-well plate, and washing the cells on the glass for 2-3 times (keeping gentle and avoiding damaging the nerve cell structure) by using PBS after the cells are attached to the wall and overgrow.

And step two, adding 200 mu L of 4% soluble Polytetrafluoroethylene (PFA) preheated at 37 ℃ into the washed slide cells for fixation, quickly and gently ensuring the integrity of the cells, and fixing for 15 min.

And step three, washing the fixing solution with PBS for 3 times, and washing for 10min each time. 0.25% Triton X-100 was added and incubated for 15min, followed by 3 washes with PBS for 10min each, and the prepared rabbit anti-mouse MAP2 monoclonal antibody (1:500, i.e., 1ul of antibody was added to 500ul of solution) was added and incubated at 4 ℃ for 24 h. The following day, wash 3 times with PBS for 10min each. AlexaFluor488 fluorescent secondary antibody (1: 1000, i.e., 1ul of antibody added to 1000ul of solution) was then added and incubated for 1h at room temperature. PBS wash 3 times for 10min each.

And step four, taking the glass slide, dripping the sealing tablet, taking out the cover glass, inverting the cover glass on the glass slide, and sealing. The results of observation using a confocal laser microscope are shown in fig. 3 and 4.

FIG. 3 is a photograph of fluorescent staining of MAP2 from primary cells cultured in an example of the invention; FIG. 4 is a graph of the proportion of vagal MAP2 positive cells obtained from primary cell culture methods in examples of the invention.

In order to further confirm the purity of the obtained cells as vagus neurons, cultured primary cells are taken for immunofluorescence staining identification, and the expression condition of the neuron-specific protein MAP2 is observed. When images are collected under a laser confocal fluorescence microscope, the positive expression of MAP2 is green, and DAPI-stained cell nuclei are blue. The experimental results show that: most of the cytoplasm and processes were green under fluorescence microscopy, i.e., positive expression of MAP2 (see FIG. 3). The positive rate of MAP2 neurons in the cells isolated in this experiment was calculated using the ratio of the number of positive neurons in 5 random fields to the total cells as the neuron purity. The following are found: the purity of the vagus neurons cultured by trypsin and collagenase type I combined digestion (95.0 + -1.98%) was superior to that of trypsin single digestion (84.8 + -1.3%) (see FIG. 4). In the above, the proportion of the number of positive neurons in 5 random visual fields to the total cells is taken as the neuron purity, and the positive rate of MAP2 neurons in the brain cells isolated by the experiment is calculated to be (97.01+ 0.23%), which indicates that the culture method obtains the vagus neuron cells with higher purity by the combined digestion method of trypsin and collagenase. .

The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.

Claims

1. A primary cell culture method for a myxotomy mouse vagus nerve is characterized by comprising the following steps:

step 1, a ganglion digestion and separation step, which comprises the following substeps:

step 1-1, the backs of obese cultured SPF-grade Sprague-Dawlcy mice were dissected and ganglion cells were dissected off with forceps with the aid of a dissecting microscope,

step 1-2, washing the stripped ganglion with Hanks salt balanced solution, and shearing the cleaned ganglion to obtain sheared tissue;

step 1-3, adding a digestive juice into the sheared tissue, performing low-speed rotation in a shaking table at 37 ℃ for digestion, centrifuging, removing the digestive juice, adding an adherent culture medium, and uniformly blowing to obtain a suspension; and

step 2, a culture step of the vagus nerve cells, which comprises the following substeps:

step 2-1, filtering the suspension by using a 70 μm filter to obtain a filtrate,

step 2-2, diluting the filtrate by using an adherent culture medium, inoculating the diluted filtrate into a 6-hole plate, mixing the cells gently to ensure that the cells are uniformly spread in a culture dish,

and 2-3, putting the culture dish into an incubator for culture, removing the non-nerve cells by using a differential adherence method in the culture process, discarding the adherence culture medium and replacing the adherence culture medium with a growth culture medium after 8 hours, continuously culturing the cells for 4-6 days, and replacing the fresh growth culture medium every two days to obtain the cultured vagus nerve cells.

2. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein, in the step 1-1, the Sprague-Dawlcy mice are obtained from 30-40 days of birth and 30-40g of body weight, and are not limited to males and females.

3. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein in the step 1-3, the mass volume of the sheared tissue and the digestive juice is (0.2 g-1 g): 2 ml.

4. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein in the step 1-3, the digestive juice is trypsin with the mass volume concentration of 0.25%.

5. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

in the steps 1-3, the digestive juice comprises trypsin and collagenase type I in a volume ratio of 1:1, the mass volume concentration of the trypsin is 0.25%, and the mass volume concentration of the collagenase type I is 0.2%.

6. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein in the step 1-3, the digestion is carried out for 40-80 min,

the blowing process comprises the following steps: respectively using a large-caliber Pasteur pipe, a medium-caliber Pasteur pipe and a small-caliber Pasteur pipe, lightly blowing and beating the tissues by using the large-caliber Pasteur pipe, changing the middle-caliber Pasteur pipe for lightly blowing and beating for 4-5 times when the tissues become soft, dividing the tissues into small tissues, changing the small-caliber Pasteur pipe for continuous blowing and beating, and obtaining the suspension.

7. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein in the step 2-3, the temperature in the incubator is 37 ℃, and CO is adopted₂The volume content was 5%.

8. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 1, wherein:

wherein the adherent culture medium is a DMEM culture medium added with fetal bovine serum,

the growth medium is Neurobasal medium added with B27 additive, N2 additive, growth factor EGF and growth factor FGF.

9. The method of culturing primary cells of the vagus nerve of a sleeve gastrectomized mouse of claim 8, wherein:

wherein the volume fraction of the fetal calf serum in the adherent culture medium is 10 percent,

in the growth medium, the mass ratio of the B27 additive, the N2 additive, the growth factor EGF and the growth factor FGF is 2:1:1:1,

the concentration of the N2 additive in the growth medium was 1ug/100 ml.