CN112858679B - Method for dyeing intestinal single cell level NKA protein of Gymnocypris przewalskii - Google Patents
Method for dyeing intestinal single cell level NKA protein of Gymnocypris przewalskii Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention relates to a method for dyeing a single cell level NKA protein in an intestinal canal of a gymnocypris przewalskii, which comprises the steps of tissue taking, enzymolysis, preparation of an intestinal single cell suspension and NKA dyeing. The invention can realize low-cost, rapid and high-efficiency obtaining of the intestinal single-cell suspension of the Gymnocypris przewalskii, ensures that dissociated cells keep good activity, can be applied to dissociation, separation and purification of other fish cells and establishment of cell lines, and provides important reference materials for single-cell and gene function research of the Gymnocypris przewalskii.
Description
Technical Field
The invention belongs to the field of fish cell biology, and particularly relates to a method for dyeing a single-cell level NKA protein in an intestinal tract of a gymnocypris przewalskii.
Background
The naked carp (Gymnocypris przewalskii) in Qinghai lake is commonly called as a rare fish, belongs to the order of Cyprinus carpiodes (Cypriniformes), the family of Cyprinus Carpiod (CYPRINIDAE), the subfamily of schizothorax (Schizothoracinae) and the genus of Gymnocypris (Gymnocypris), is an important economic fish in Qinghai lake, and occupies a central position in the Qinghai lake ecological system. The Gymnocypris przewalskii is a fish spawning by tracing behaviour of river, the sexually mature Gymnocypris przewalskii swims from the Qinghai lake to fresh water tributary spring river, bu Ha He and other river channels every 4-7 months, and parent fish and hatched larvae swim back to the Qinghai lake after spawning and reproduction are completed. The salinity of the Qinghai lake is 13-15, the alkalinity is 26-32 mmol/L, the pH is 9.1-9.5, and the alkalinity of the lake water is increased year by year due to the factors of evaporation, irrigation and the like. The naked carp living in the lake water is paid attention to because of the excellent characteristics of salt and alkali resistance, high and cold resistance and the like. Research shows that in the process of transition from fresh water to high-salt-alkali lake water, physiological and biochemical activities such as respiration, excretion, immunity, energy metabolism and the like of the gymnocypris przewalskii can be obviously changed, and the gymnocypris przewalskii adapts to the salt-alkali environment through in-vivo regulation.
Most aquatic organisms are sensitive to the change of the salt and alkali degree in water environment, the osmotic balance of the aquatic organisms is influenced by high salinity, the tissue function of the aquatic organisms is damaged, the activities of digestive enzymes and immune-related enzymes are reduced due to the high alkalinity, and the salinity, the alkalinity and the pH show a certain synergistic effect on the growth, the propagation, the metabolism and the like of the aquatic organisms. At present, the role of fish gills and kidneys in permeation and acid-base regulation has been studied intensively, and recent studies have found that the intestinal tract plays an important role in maintaining permeation and acid-base balance.
The intestinal epithelial cells of fish are complex in type and function, and besides cells involved in osmotic regulation, there are digestive-related cells, immune-related cells, endocrine-related cells, and respiratory-related cells. If cells related to permeation and acid-base equilibrium are to be resolved and isolated, it is important to study the regulatory mechanisms of such cells. Since Na +-K+ -ATPase (NKA) plays an important role in the osmotic regulation and acid-base balance, most cells involved in osmotic and acid-base regulation appear NKA positive. Screening of NKA positive cells can solve the difficult problem that intestinal epithelial ion regulating cell types are difficult to distinguish, and helps to reveal the permeation regulation and acid-base balance mechanism of naked carp intestinal tracts from the single cell level.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for dyeing the intestinal single cell level NKA protein of the Gymnocypris przewalskii, which is favorable for rapidly and efficiently dissociating the intestinal single cell of the Gymnocypris przewalskii to prepare high-quality single cell suspension, further performs dyeing through NKA antigen-antibody immune reaction, and has important application value for dissociating, separating and purifying fish cells and screening osmotic adjustment positive cells.
The invention provides a method for dyeing a single cell level NKA protein in the intestinal tract of a Gymnocypris przewalskii, which comprises the following steps:
(1) Taking fresh intestinal tissues of Gymnocypris przewalskii, flushing, then putting the fresh intestinal tissues into a centrifuge tube containing a D-PBS buffer solution, centrifuging, and then adding an intestinal tissue digestive enzyme mixed solution for enzymolysis and digestion to obtain a tissue digestive juice;
(2) Filtering, centrifuging and re-suspending the tissue digestion solution obtained in the step (1) to obtain intestinal single-cell suspension;
(3) And (3) dripping the intestinal single-cell suspension obtained in the step (2) onto a glass slide, sucking the cell suspension after sedimentation, fixing and sealing the intestinal single-cell, respectively adding NKA primary antibody and NKA secondary antibody for dyeing, and finally photographing by a fluorescence microscope.
The D-PBS buffer in step (1) does not contain calcium ions, magnesium ions, and phenol red.
The intestinal tissue digestive enzyme mixed solution in the step (1) comprises the following components: 0.1-0.5mg/ml collagenase I, 0.04-0.08mg/ml collagenase II and 0.1-0.5mg/ml collagenase IV, and the solvent is D-PBS buffer solution.
The enzymolysis digestion time in the step (1) is 15-30min.
The re-suspension in step (2) uses pre-chilled D-PBS buffer, which also contains 2% Fetal Bovine Serum (FBS).
The dilution ratio of the NKA primary antibody in the step (3) is 1:100, and the dilution ratio of the NKA secondary antibody is 1:400.
The intestinal tissue digestive enzyme mixed solution adopted by the invention comprises collagenase I, collagenase II and collagenase IV. Pancreatin is commonly added to digestive enzyme cocktails during conventional mammalian tissue single cell digestion. Repeated experiments prove that pancreatin cannot be added into the digestive enzyme mixed solution in the invention, because pancreatin is a virulent enzyme, the intestinal epithelial cells of the gymnocypris are easily broken in the digestion process, DNA is released to be in gel linkage, and the cell activity is not strong and is not easy to pass through a cell sieve. The mixed solution of collagenase I, II and IV does not have the problems in the digestion process, and the cell viability meets the requirements of subsequent experiments.
The cell structure of the intestinal tissue of gymnocypris is composed of complex matrix such as protein (such as collagen), glycoprotein, lipid, glycolipid and mucopolysaccharide. In order to isolate individual cells, such complex matrices must be digested with great care, without irreversible damage to the cell surface or intracellular structures. Collagenase I and II have different specificities and relative activities for natural collagen and synthetic substrates, and collagenase I acts to cleave two of the three long undenatured helical strands of collagen, and collagenase II acts by cleaving short synthetic peptides, and collagenase IV can limit damage to membrane proteins such as NKA. The three collagenases are mixed to digest different protein structures of intestinal tissues, and simultaneously, the integrity of membrane proteins such as NKA and the like is protected.
In the process of intestinal tissue digestion, the number of cell types obtained by different digestion time is quite different, and the purpose of the invention is to obtain cells for osmotic adjustment, wherein the enzymolysis digestion time is 15-30min. NKA is a cell marker with osmotic regulation obtained by long-term experimental screening (fig. 3).
Advantageous effects
The invention can realize low-cost, rapid and high-efficiency obtaining of the intestinal single-cell suspension of the Gymnocypris przewalskii, ensures that dissociated cells keep good activity, can be applied to dissociation, separation and purification of other fish cells and establishment of cell lines, and provides important reference materials for single-cell and gene function research of the Gymnocypris przewalskii.
Drawings
FIG. 1 is the result of staining (20-fold objective) of example 1; wherein, the luminous points are the distribution positions of the NKA protein.
FIG. 2 shows the results of the intestinal single cell viability test of the dissociated Qinghai lake naked carp of example 1.
FIG. 3 is a screen for osmoregulation cell marker proteins.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Example 1
Dissecting gymnocypris przewalskii, cutting midgut tissue, placing the midgut tissue into a sterile culture dish containing precooled D-PBS buffer solution, dissecting the intestinal tract by using dissecting scissors, and cleaning the tissue;
Step two, after the tissue is cleaned, transferring the tissue into a smooth centrifuge tube with an inner wall containing enzyme mixed solution (2 mL), cutting the tissue into small fragments with the size of 1-2mm 2, adding intestinal tissue digestive enzyme mixed solution, and placing the intestinal tissue digestive enzyme mixed solution into a constant-temperature water bath kettle at 37 ℃ for enzymolysis and digestion for 15-30min until the tissue blocks disappear; wherein, the intestinal tissue digestive enzyme mixed solution comprises the following components: 0.2mg/ml collagenase I, 0.06mg/ml collagenase II and 0.2mg/ml collagenase IV, and the solvent is D-PBS buffer solution;
Step three, the mixture is evenly mixed up and down every 5 minutes during digestion until the tissue blocks basically disappear;
Step four, filtering the tissue digestion solution by a 40 mu m cell sieve, and transferring the tissue digestion solution into a new smooth inner wall centrifuge tube;
step five, centrifuging 300g of the solution obtained in the step four for 5min at the temperature of 4 ℃ and removing supernatant;
step six, adding the cell sediment in the step five into a precooled D-PBS buffer solution, lightly blowing and resuspending the cells by using a liquid transfer device, and centrifuging 300g of the cell suspension for 5min at the temperature of 4 ℃;
step seven, repeating the step six for 2-3 times, collecting cell sediment, adding precooled D-PBS buffer solution (containing 2% fetal bovine serum FBS), lightly blowing 3-5 times to resuspend cells, and completing preparation of intestinal single cell suspension;
Step eight, taking a polylysine glass slide, and drawing an experimental area by using a hydrophobic marker;
step nine, a proper amount of single cell suspension is dripped into a glass slide experiment area, living cells are allowed to freely settle, and the cell settlement condition is checked under an optical microscope at any time;
Step ten, when the sedimentation density of the cells is moderate, sucking away the cell suspension, immediately washing the glass slide by using D-PBS, washing for 3 times, and adding/sucking the D-PBS as light as possible;
step eleven, fixing cells on a glass slide by using 4% paraformaldehyde for 10min and washing 3 times by using D-PBS;
Step twelve, blocking the cells for 60min with a 3% bsa blocking solution;
step thirteen, incubating NKA (SC-28800,Santa Cruz Biotechnology) primary antibody (antibody dilution ratio 1:100), overnight at 4 ℃; washing the primary antibody 3 times with D-PBS;
Fourteen, the slide was blocked again for 60min with 3% bsa blocking solution;
fifteen, incubating a secondary antibody (A11036, thermo FISHER SCIENTIFIC) (antibody dilution ratio 1:400), at room temperature for 1h; washing the secondary antibody 3 times by using D-PBS;
sixteenth, incubating Hochest for 30min;
seventeenth, photographing by using a fluorescence microscope, and referring to fig. 1.
The method can realize low-cost, rapid and efficient obtaining of the intestinal single-cell suspension of the gymnocypris przewalskii, and ensure that dissociated cells keep good activity, and the cell activity is about 73 percent, as shown in figure 2.
Claims (4)
1. A method for single cell level NKA protein staining of the gut of a gymnocypris przewalskii, comprising:
(1) Taking fresh intestinal tissues of Gymnocypris przewalskii, flushing, then putting the fresh intestinal tissues into a centrifuge tube containing a D-PBS buffer solution, centrifuging, and then adding an intestinal tissue digestive enzyme mixed solution for enzymolysis and digestion to obtain a tissue digestive juice; wherein, the intestinal tissue digestive enzyme mixed solution comprises the following components: 0.1-0.5mg/ml collagenase I, 0.04-0.08mg/ml collagenase II and 0.1-0.5mg/ml collagenase IV, and the solvent is D-PBS buffer solution;
(2) Filtering, centrifuging and re-suspending the tissue digestion solution obtained in the step (1) to obtain intestinal single-cell suspension;
(3) Dripping the intestinal single-cell suspension obtained in the step (2) onto a glass slide, sucking the cell suspension after sedimentation, fixing and sealing the intestinal single-cell, respectively adding NKA primary antibody and NKA secondary antibody for dyeing, and finally photographing by a fluorescence microscope; wherein, the dilution ratio of the NKA primary antibody is 1:100, and the dilution ratio of the NKA secondary antibody is 1:400.
2. The method according to claim 1, characterized in that: the D-PBS buffer in step (1) does not contain calcium ions, magnesium ions, and phenol red.
3. The method according to claim 1, characterized in that: the enzymolysis digestion time in the step (1) is 15-30min.
4. The method according to claim 1, characterized in that: the re-suspension in the step (2) adopts pre-cooled D-PBS buffer, and the D-PBS buffer also contains 2% FBS.
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