CN112577803B - Method for preparing turtle chromosome specimen by using peripheral blood cells - Google Patents
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150015—Source of blood
- A61B5/15003—Source of blood for venous or arterial blood
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/153—Devices specially adapted for taking samples of venous or arterial blood, e.g. with syringes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2503/00—Evaluating a particular growth phase or type of persons or animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2503/00—Evaluating a particular growth phase or type of persons or animals
- A61B2503/42—Evaluating a particular growth phase or type of persons or animals for laboratory research
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Abstract
The invention discloses a method for preparing a turtle chromosome specimen by using peripheral blood cells, belonging to the field of cytogenetics. According to the invention, the metaphase specimen of the soft-shelled turtle chromosome, which is convenient to observe and clear in image, is prepared by collecting peripheral blood of the soft-shelled turtle, and performing the steps of blood cell culture, colchicine treatment, dyeing and flaking and the like. The invention overcomes the problem of great damage to animals in the existing method for preparing chromosome specimens of aquatic animals, realizes non-destructive sampling, only needs to collect a small amount of peripheral blood of the samples, and has little influence on the survival state of the animals. The method for preparing the soft-shelled turtle chromosome is simple and rapid, the metaphase mitotic phase of the soft-shelled turtle chromosome can be obtained 72 hours after blood collection, observation is facilitated, and a favorable tool is provided for further developing relevant researches on the genome and evolution of the soft-shelled turtle and realizing more sufficient protection of the soft-shelled turtle.
Description
Technical Field
The invention relates to the field of cytogenetics, in particular to a method for preparing a turtle chromosome specimen by using peripheral blood cells.
Background
Chromosomes are important components of cell nuclei and are main substance carriers of biological genetic information, and the karyotype of an organism not only has the characteristics of species, but also can reflect the evolution history of the organism. The chromosome karyotype analysis is an important research content in the work of genetic breeding and germplasm identification of aquatic products.
At present, three conventional chromosome preparation methods exist in aquatic animals, and 1) an in vivo cell culture method is adopted, namely, phytohemagglutinin (PHA-P) is injected into an abdominal cavity or a thoracic cavity to stimulate B lymphocytes in hematopoietic organs such as head, kidney or spleen of an experimental animal to multiply in large quantity, and then colchicine is used for treatment to enable cell division to stay in metaphase, so that a metaphase chromosome division phase can be obtained. 2) The embryo cell direct method is to blow and disperse the cells by utilizing normally developed blastocyst or early gastrula embryo, and then add colchicine for treatment, thereby obtaining the metaphase chromosome split phase of the cells. 3) Somatic cell direct method. The method is mainly applied to fishes by soaking the larva of the animals in colchicine with a certain concentration to inhibit the actively dividing gill silk epithelial cells in the metaphase.
However, the above method has a problem in chromosome preparation in that it requires a living body from the experimental animal, and the experimental animal hardly survives after taking a sample. Therefore, it is not applicable to precious experimental materials or protected animals requiring non-invasive sampling.
Soft-shelled turtle (pelocheys cantonii Gray, 1864), belonging to the family trionyhidae (pelochelonys) of the suborder tortoiseshes (Cryptodira), pelargoni, asian giant soft-shelled turtle, is a large-scale animal of the family trionyx naturally distributed in southern regions of China, southeast asia and south asia continental lands, with adult individual carapace reaching 30-80 cm and weight reaching 15-35 kg. Due to the rare number of wild population individuals, the turtle is classified as a first-class protection animal in the last 80 th century in China. In the beginning of the 21 st century, the endangered state of soft-shelled turtle has received attention from the relevant international organizations. In 2000, Yuan-Yuan was classified as endangered species (EN) by the International Union of Nature protection (IUCN) under the red name of endangered species. In 2003, turtle is listed in appendix II of International trade Convention (CITES) of endangered species of wild animals and plants. Because of the rare number, only the existing living body samples are extremely precious, and the application of the traditional chromosome specimen preparation method in soft-shelled turtle is quite difficult. At present, the chromosome number and karyotype of Yuanzao are still unknown for animal protectors.
Disclosure of Invention
The invention aims to provide a method for preparing a turtle chromosome specimen by using peripheral blood cells, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for preparing a turtle chromosome specimen, which is characterized in that turtle peripheral blood cells are utilized to prepare the turtle chromosome specimen.
Preferably, the method comprises the following steps:
1) sampling peripheral blood: collecting 1ml of peripheral blood from the neck blood sinuses of live soft-shelled turtles by using a negative pressure blood collection tube and a sterilization needle; after blood collection, temporarily storing the blood at 4 ℃;
2) culturing peripheral blood cells, namely performing cell culture operation under the aseptic condition; in CO2Culturing at 28 deg.C for 72h with concentration of 5%;
3) colchicine treatment: adding colchicine with the final concentration of 100-150 ng/ml into the cultured blood cells for treating for 3-4 hours;
4) chromosome flaking: hypotonic for 50min after colchicine treatment; collecting cells at the rotating speed of 1500r/min after hypotonic, fixing for 1-2 times by using Carnot fixing solution, and then performing slide preparation by adopting a cold slide drying method; staining with Giemsa dye liquor for 20 min.
Preferably, the negative pressure blood collection tube contains heparin sodium.
Preferably, a modified commercial Eagle basal medium is used in step 2), and the medium modification formula is that: 20mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acids, 1mM sodium pyruvate, 1mM penicillin and streptomycin double antibody, 55 μ M β -mercaptoethanol, 15% fetal bovine serum, 5ng/mL recombinant human basic fibroblast growth factor; each 3ml of the medium was supplemented with 200. mu.L of whole blood and 750. mu.g of PHA-P was added thereto.
Preferably, the volume ratio of methanol to glacial acetic acid in the carnot stationary liquid used in step 4) is 3: 1.
preferably, the volume ratio of the stock giemsa solution to the phosphate buffer solution in the giemsa staining solution used in the step 4) is 1: 6, the medicine is ready for use.
The invention also provides a soft-shelled turtle chromosome specimen prepared by the preparation method of any one of the soft-shelled turtle chromosome specimens.
The invention discloses the following technical effects:
1. and the sampling is not damaged, only a small amount of peripheral blood of the sample needs to be collected, and the influence on the survival state of the animal is small.
2. The method for preparing the soft-shelled turtle chromosome is simple and rapid, and the metaphase mitosis phase of the soft-shelled turtle chromosome can be obtained 72 hours after blood collection, so that the observation is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a specimen of a turtle chromosome prepared in example 1;
FIG. 2 is a specimen of a turtle chromosome prepared in example 2;
FIG. 3 is a specimen of turtle chromosome prepared in example 3.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Unless otherwise indicated, the examples were run under conventional experimental conditions or in accordance with the manufacturer's instructions. Reagents and instruments used in the examples were purchased from conventional bioproduct agencies unless otherwise specified.
Example 1
1. Peripheral blood sampling
Peripheral blood 1ml was collected from the neck antrum of live turtle using a commercially available negative pressure blood collection tube (containing heparin sodium) and a sterile needle. After blood collection, the blood was stored at 4 ℃.
2. Peripheral blood cell culture
Cell culture procedures were performed under sterile conditions using a modified commercial Eagle basal medium. The improved culture medium formula is that 20mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acid, 1mM sodium pyruvate, 1mM penicillin and streptomycin double antibody, 55 MuM beta-mercaptoethanol, 15% fetal bovine serum and 5ng/mL recombinant human basic fibroblast growth factor are added on a basic culture medium. Each 3ml of the culture medium was supplemented with 200. mu.L of whole blood and 750. mu.g of PHA-P.
3. Colchicine treatment
Blood cell culture at 28 deg.C (CO)25%) for 72h, and adding colchicine with final concentration of 100ng/ml, 125ng/ml and 150ng/ml respectively for treatment.
4. Chromosome slide preparation
And (3) performing hypotonic treatment on the colchicine for 3-4 hours, wherein the hypotonic treatment time is preferably 50 min. After hypotonic, cells were collected at 1500r/min, fixed with Carnot fixative (methanol: glacial acetic acid: 3: 1, V/V) for 1-2 times, and then slide-dried by cold slide drying. Staining with Giemsa staining solution (Giemsa stock solution: phosphate buffer solution: 1: 6, V/V, ready for use) for 20min, and observing under an objective lens of 40-100 × to obtain soft-shelled turtle chromosome specimen as shown in FIG. 1.
Example 2
1. Peripheral blood sampling
Peripheral blood 1ml was collected from the neck antrum of live turtle using a commercially available negative pressure blood collection tube (containing heparin sodium) and a sterile needle. After blood collection, the blood was stored at 4 ℃.
2. Peripheral blood cell culture
Cell culture procedures were performed under sterile conditions using a modified commercial Eagle basal medium. The improved culture medium formula is that 20mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acid, 1mM sodium pyruvate, 1mM penicillin and streptomycin double antibody, 55 MuM beta-mercaptoethanol, 15% fetal bovine serum and 5ng/mL recombinant human basic fibroblast growth factor are added on a basic culture medium. Each 3ml of the culture medium was supplemented with 200. mu.L of whole blood and 750. mu.g of PHA-P.
3. Colchicine treatment
Blood cell culture at 28 deg.C (CO)25 percent) for 72 hours, and colchicine with the final concentration of 125ng/ml is respectively addedAnd (5) treating the element.
4. Chromosome slide preparation
The colchicine can be hypotonic after 3 hr treatment, and the hypotonic time is preferably 50 min. After hypotonic, cells were collected at 1500r/min, fixed with Carnot fixative (methanol: glacial acetic acid 3: 1, V/V)1 time, and then slide-dried by cold slide drying. Staining with Giemsa staining solution (Giemsa stock solution: phosphate buffer solution: 1: 6, V/V, ready for use) for 20min, and observing under an objective lens of 40-100 × to obtain a soft-shelled turtle chromosome specimen as shown in FIG. 2.
Example 3
1. Peripheral blood sampling
Peripheral blood 1ml was collected from the neck antrum of live turtle using a commercially available negative pressure blood collection tube (containing heparin sodium) and a sterile needle. After blood collection, the blood was stored at 4 ℃.
2. Peripheral blood cell culture
Cell culture procedures were performed under sterile conditions using a modified commercial Eagle basal medium. The improved culture medium formula is that 20mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acid, 1mM sodium pyruvate, 1mM penicillin and streptomycin double antibody, 55 MuM beta-mercaptoethanol, 15% fetal bovine serum and 5ng/mL recombinant human basic fibroblast growth factor are added on a basic culture medium. Each 3ml of the culture medium was supplemented with 200. mu.L of whole blood and 750. mu.g of PHA-P.
3. Colchicine treatment
Blood cell culture at 28 deg.C (CO)25%) for 72h, and adding colchicine with final concentration of 150ng/ml for treatment.
4. Chromosome slide preparation
The colchicine can be hypotonic after 4 hr treatment, and the hypotonic time is preferably 50 min. After hypotonic, cells were collected at 1500r/min, fixed 2 times with Carnot fixative (methanol: glacial acetic acid 3: 1, V/V) and then slide-dried by cold slide drying. Staining with Giemsa staining solution (Giemsa stock solution: phosphate buffer solution: 1: 6, V/V, ready for use) for 20min, and observing under an objective lens of 40-100 × to obtain a soft-shelled turtle chromosome specimen as shown in FIG. 3.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (4)
1. A method for preparing a turtle chromosome specimen, which is characterized by comprising the following steps:
1) sampling peripheral blood: collecting 1mL of peripheral blood from the neck blood sinuses of live soft-shelled turtles by using a negative pressure blood collection tube and a sterilization needle; after blood collection, temporarily storing the blood at 4 ℃;
2) peripheral blood cell culture: performing cell culture operation under aseptic conditions; in CO2Culturing at 28 deg.C for 72h with concentration of 5%;
3) colchicine treatment: culturing the blood cells, and adding colchicine with the final concentration of 100-150 ng/mL for treatment for 3-4 hours;
4) chromosome flaking: hypotonic for 50min after colchicine treatment; collecting cells at the rotating speed of 1500r/min after hypotonic, fixing for 1-2 times by using Carnot fixing solution, and then performing slide preparation by adopting a cold slide drying method; dyeing for 20min by using Giemsa dye liquor;
the negative pressure blood collection tube contains heparin sodium;
in the step 2), an improved commercial Eagle basal medium is used, and the culture medium improvement formula is that: 20mM 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acids, 1mM sodium pyruvate, 1mM penicillin and streptomycin double antibody, 55 μ M β -mercaptoethanol, 15% fetal bovine serum, 5ng/mL recombinant human basic fibroblast growth factor; 200. mu.L of whole blood was added to 3mL of the medium, and 750. mu.g of phytohemagglutinin was added thereto.
2. The method for preparing a soft-shelled turtle chromosome specimen according to claim 1, wherein the volume ratio of methanol to glacial acetic acid in the carnot stationary liquid used in step 4) is 3: 1.
3. the method for preparing soft-shelled turtle chromosome specimen according to claim 1, wherein the volume ratio of the stock Giemsa solution to the phosphate buffer solution in the Giemsa staining solution used in step 4) is 1: 6, the medicine is ready for use.
4. A soft-shelled turtle chromosome specimen prepared according to the method for preparing a soft-shelled turtle chromosome specimen according to any one of claims 1 to 3.
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| CN202011453959.2A CN112577803B (en) | 2020-12-10 | 2020-12-10 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
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