CN114891722A - Avian intestinal epithelial cells and preparation method and application thereof - Google Patents

Avian intestinal epithelial cells and preparation method and application thereof Download PDF

Info

Publication number
CN114891722A
CN114891722A CN202210521390.1A CN202210521390A CN114891722A CN 114891722 A CN114891722 A CN 114891722A CN 202210521390 A CN202210521390 A CN 202210521390A CN 114891722 A CN114891722 A CN 114891722A
Authority
CN
China
Prior art keywords
avian
cells
small intestine
intestinal epithelial
embryo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210521390.1A
Other languages
Chinese (zh)
Inventor
孙铭飞
陈慧芳
李娟�
白银山
戚南山
潘晓婷
廖申权
吕敏娜
林栩慧
蔡海明
胡俊菁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Original Assignee
Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Animal Health of Guangdong Academy of Agricultural Sciences filed Critical Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority to CN202210521390.1A priority Critical patent/CN114891722A/en
Publication of CN114891722A publication Critical patent/CN114891722A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention relates to an avian intestinal epithelial cell and a preparation method and application thereof. An avian intestinal epithelial cell is derived from avian embryo small intestine segment cells, and the genome contains a large T antigen gene of SV 40. The researchers of the invention find that the integration of the large T antigen gene of SV40 in the genome of avian embryo small intestine segment cells can lead the avian intestinal epithelial cells to be cultured for a long time in vitro, can be continuously cultured for more than 6 months and can be passaged for more than 30 generations, and the gene expression is stable, presents the specific characteristics of the intestinal epithelial cells, expresses the intestinal epithelial cell marker molecules, and is suitable for the research related to the health of avian intestinal tracts and the development of biological products.

Description

Avian intestinal epithelial cells and preparation method and application thereof
Technical Field
The invention relates to the field of cell biology, in particular to an avian intestinal epithelial cell and a preparation method and application thereof.
Background
The intestinal organs can be divided into small intestine (duodenum, jejunum and ileum) and large intestine (rectum, cecum and colon), the former can resist the invasion of external pathogenic bacteria and maintain the steady state of digestive tract, and is responsible for the digestion and absorption of nutrient substances; the latter absorbs mainly excess moisture. During the development of the intestinal tract, the small intestine dominates. The small intestine is an important immune and endocrine organ and is also an important site for digestion and absorption of nutrients. The small intestinal epithelium is the fastest-renewing tissue, plays an important role in maintaining intestinal health, repairing injuries, preventing invasion of viruses, bacteria and parasites, and the like, and is particularly important for poultry. The health of the intestinal tract directly concerns the immunity of the birds, and once the intestinal tract of the birds is destroyed, the health of the birds is difficult to guarantee. Therefore, the method has important significance for exploring the epithelial function of the small intestine and is also an important aspect for promoting the healthy breeding of poultry industry.
In vitro cell models are important research tools that enable the study of cellular mechanisms by increasing the reproducibility of the experiment and the availability of samples and by reducing the use of experimental animals. However, at present, it is still difficult to have available avian intestinal tract cells, and only by isolated culture of intestinal tract primary cells, primary cells can be maintained under the condition of adding a large amount of growth factors, and the culture time is generally maintained within 2 weeks, and long-term culture cannot be performed, so that the basic requirements of drug screening and development of some biological products cannot be met in time, which severely limits the current research on the intestinal tract health of avian and the development of biological products.
Disclosure of Invention
Based on this, there is a need to provide avian intestinal epithelial cells that can be cultured in vitro for a long period of time.
In addition, a preparation method and application of the avian intestinal epithelial cells are also provided.
An avian intestinal epithelial cell is derived from avian embryo small intestine segment cells, and the genome contains a large T antigen gene of SV 40.
The researchers of the invention find that the integration of the large T antigen gene of SV40 in the genome of avian embryo small intestine segment cells can lead the avian intestinal epithelial cells to be cultured for a long time in vitro, can be continuously cultured for more than 6 months and can be passaged for more than 30 generations, and the gene expression is stable, presents the specific characteristics of the intestinal epithelial cells, expresses the intestinal epithelial cell marker molecules, and is suitable for the research related to the health of avian intestinal tracts and the development of biological products.
In one embodiment, the avian embryo small intestine segment cells comprise 9 embryo-old avian embryo small intestine segment cells.
In one embodiment, the avian embryo small intestine segment cells comprise avian embryo small intestine segment cells.
In one embodiment, the avian intestinal epithelial cells express an intestinal epithelial cell marker molecule comprising TJP 1.
A preparation method of avian intestinal epithelial cells takes avian embryo small intestine segment cells as host cells, and integrates large T antigen genes of SV40 into genomes of the host cells.
In one embodiment, the preparation method comprises the following steps: separating the small intestine section cells of the avian embryo; transducing the avian embryo small intestine segment cells with a retroviral vector containing the large T antigen gene of SV 40; cell passage, screening and identification.
In one embodiment, the transduction time is 36-60 h.
In one embodiment, the avian embryo small intestine segment cells comprise 9 embryo-old avian embryo small intestine segment cells.
In one embodiment, the avian embryo small intestine segment cells comprise avian embryo small intestine segment cells.
The use of the avian intestinal epithelial cells of any one of the embodiments described above in the preparation of bioengineered avian intestinal-related products or in vitro screening of avian intestinal-related therapeutic drugs.
Drawings
FIG. 1 is a schematic flow chart of the experimental design of example 1;
FIG. 2 is a schematic diagram of embryos, whole intestine sections and small intestine sections of the Zhongling south yellow chickens of example 1 at 9, 11, 13, 15 and 18 embryo ages;
FIG. 3 is a schematic representation of the tissue structure of the cross-section of the small intestine section of the Nanhuang chicken of example 1 at 9, 11, 13, 15 and 18 embryo ages under microscope;
FIG. 4 is a schematic diagram showing the cell morphology and virus expression of avian embryo intestinal epithelial cells transduced with the lentiviral vector carrying the large T antigen gene in example 1 for 2 days;
FIG. 5 is a schematic diagram showing the cell morphology and virus expression of avian embryo intestinal epithelial cells successfully transduced by the lentiviral vector with a large T antigen gene in example 1 after monoclonal amplification culture;
FIG. 6 is a schematic diagram showing the cell morphology and virus expression of subculture after successful transduction of avian embryo intestinal epithelial cells with a lentiviral vector carrying a large T antigen gene in example 1;
FIG. 7 is a graph showing the results of electrophoretic detection of marker genes of small intestinal epithelial cells of F10 and F30 generations after successful transduction of avian embryo intestinal epithelial cells with a lentiviral vector carrying a large T antigen gene in example 1;
FIG. 8 is an immunofluorescence assay of the expression of TJP1 protein in the avian intestinal epithelial cell passage F30 in example 1;
FIG. 9 is a FPKM hierarchical cluster analysis chart of the differential gene expression of avian intestinal epithelial cells of generations F10, F20 and F30 in example 1, in which the horizontal axis represents the cell groups of different generations, the vertical axis represents the gene, the dark color represents the up-regulation of gene expression, and the light color represents the down-regulation of gene expression;
FIG. 10 is a KEGG pathway analysis graph of differentially expressed genes of avian intestinal epithelial cells at generations F10 and F30 in example 1;
FIG. 11 is a diagram showing analysis of cell cycle signaling pathway of differentially expressed genes of avian intestinal epithelial cells of generations F10 and F30 in example 1;
FIG. 12 is a statistical chart showing the results of real-time fluorescent quantitative PCR of the differentially expressed genes of avian intestinal epithelial cells of generations F10 and F30 in example 1;
fig. 13 is a schematic view of the development of e.tenella sporozoites in avian intestinal epithelial cells under microscope for bright field observation in example 1;
fig. 14 is a graph of HE staining of e.tenella sporozoites in the development of avian intestinal epithelial cells in example 1;
FIG. 15 is a schematic diagram showing cell morphology of 9-embryo-old southern yellow chick embryo small intestine epithelial cell primary cells isolated in comparative example 1 after subculture.
Detailed Description
The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
"SV 40" as used herein is an abbreviation for "Simian vacuolating virus 40" or "Simian virus 40", monkey vacuolar virus 40. The SV40 virus genome early genes encode two tumor antigens (T antigens) with molecular weights of 94000 (large T antigen) and 17000 (small T antigen), respectively.
The term "avian embryo" refers to avian embryo, and is mainly used for developmental biology research.
The term "age of embryo" refers to the age of the embryo in days, for example, "age of 9 embryo" refers to the age of 9 days.
The term "TJP 1" is an abbreviation for light Junction Protein 1, also known as ZO-1.
By "transduction" is meant the transfer of DNA or RNA from one cell to another cell upon infection with a viral vector.
The term "FPKM" is an abbreviation of "Fragments Per Kilobase Million", and is the number of Reads Per one Million Reads Per Kilobase length from a certain gene, which is a commonly used method for normalizing gene expression level, and this method considers the influence of both sequencing depth and gene length on the gene expression level count.
The "GFP" is an abbreviation of "Green fluorescent protein", namely Green fluorescent protein, which can be stably inherited in the descendants and can be specifically expressed according to a promoter, and the GFP becomes a better research tool for replacing the traditional chemical dye in some experiments.
The "DEGs" is an abbreviation for "Differentially expressed genes", i.e., Differentially expressed genes, which refers to genes that are significantly Differentially expressed at mRNA levels under different conditions, such as different environmental pressures, times, or spaces.
The "retrovirus" also called retrovirus belongs to a class of RNA viruses, and its genetic information is stored not on deoxyribonucleic acid (DNA) but on ribonucleic acid (RNA). The retrovirus genome is diploid, two identical single-stranded positive-strand RNAs (ribonucleic acids) are provided with Long Terminal Repeats (LTRs) at two ends, contain stronger promoters and enhancers and play an important role in the transcriptional control of virus DNA. The virus core contains reverse transcriptase and integrase, and is different from other RNA viruses in that the RNA of the reverse transcriptase does not replicate autonomously, after entering a host cell, the RNA synthesizes double-stranded DNA through the reverse transcriptase, the double-stranded DNA is integrated on the chromosomal DNA of the host cell by the integrase to form provirus, and lifetime infection is established and can be transmitted to progeny cells along with the division of the host cell.
The described "lentivirus vector" is a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency virus I), said vector can effectively integrate exogenous gene into chromosome of host so as to attain the goal of persistent expression of exogenous gene. In addition, this vector is distinguished from a typical retroviral vector in that a foreign gene can be integrated into the genome of dividing and non-dividing cells.
One embodiment of the application provides an avian intestinal epithelial cell which is derived from avian embryo small intestine segment cells, and the genome of the avian intestinal epithelial cell contains a large T antigen gene of SV 40.
The researchers of the invention find that the integration of the large T antigen Gene (Gene ID: 29031019) of SV40 in the genome of avian embryo small intestine segment cells can lead the avian intestinal epithelial cells to be cultured in vitro for a long time, can be continuously cultured for more than 6 months and passaged for more than 30 generations, and has good cell morphology, stable Gene expression, specific characteristics of the intestinal epithelial cells, expression of intestinal epithelial cell marker molecules and suitability for the related research on the health of avian intestinal tracts and the development of biological products.
Further, the avian embryo intestinal segment cells include 9-embryo-aged avian embryo intestinal segment cells.
Specifically, researchers of the invention find that the avian embryo small intestine cells have strong activity and strong division capacity in the early embryonic development process, and are more suitable for in vitro cell preparation, and preferably 9-embryo-aged avian intestine segments are used for cell preparation and culture.
In one embodiment, the avian embryo small intestine segment cells comprise avian embryo small intestine segment cells.
In an alternative embodiment, the avian embryo small intestine segment cell is a green south yellow chicken small intestine segment cell.
In one embodiment, the avian intestinal epithelial cells express an intestinal epithelial cell marker molecule comprising TJP 1.
Further, the above intestinal epithelial cell marker molecule further comprises at least one of KRT18(Keratin 18), CDH1(Cadherin 1), CLDN1(Claudin 1), ocln (occludin), and VILL (Villin-like protein).
In one example, a retroviral vector containing the large T antigen gene of SV40 is used to transduce the large T antigen gene into avian embryo small intestine segment cells.
Further, the above-mentioned retroviral vector includes a lentiviral vector.
A preparation method of avian intestinal epithelial cells takes avian embryo small intestine segment cells as host cells, and integrates large T antigen genes of SV40 into genomes of the host cells.
Specifically, by adopting the preparation method of the avian intestinal epithelial cells, the prepared avian intestinal epithelial cells have high purity, are suitable for research of drug screening and biological product development, and can clearly show the types of cells.
In one embodiment, the preparation method comprises step a1, step a2 and step a3, specifically:
step a 1: and (4) separating the small intestine section cells of the avian embryo.
Preferably, the avian embryo small intestine segment cells comprise 9 embryo-aged avian embryo small intestine segment cells.
In one embodiment, the avian embryo small intestine segment cells comprise avian embryo small intestine segment cells.
In an alternative specific example, the avian embryo small intestine section cell is a green south yellow chicken small intestine section cell.
Step a 2: the avian embryo small intestine segment cells were transduced using a retroviral vector containing the large T antigen gene of SV 40.
Specifically, the above-mentioned retroviral vector includes a lentiviral vector.
In one embodiment, the transduction time is 36-60 h. Further, the transduction time is 40-55 h. Furthermore, the transduction time is 45-50 h.
Step a 3: cell passage, screening and identification.
The use of the avian intestinal epithelial cells of any one of the embodiments above in the preparation of an avian intestinal-related bioengineered product or in vitro screening of an avian intestinal-related therapeutic drug.
Specifically, the avian intestinal epithelial cell model prepared by using the avian intestinal epithelial cells has the specific characteristics of the avian intestinal epithelial cells, is stable in gene expression, is suitable for long-term research, and is suitable for preparing biological engineering products related to avian intestinal tracts or screening therapeutic drugs related to avian intestinal tracts in vitro.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.
The avian embryo small intestine epithelial cells in the examples and comparative examples were derived from Lingnan yellow chicken.
Example 1
The experimental scheme of example 1 is shown in figure 1.
1. Isolated culture of avian embryo small intestine cells
The small intestine tissues of different embryo ages have different degrees of development, as shown in fig. 2-3, fig. 2 is a schematic diagram of the embryo, the whole intestine section and the small intestine section of Lingnan yellow chicken at 9, 11, 13, 15 and 18 embryo ages; FIG. 3 is a schematic representation of the tissue structure of the small intestine section of Lingnan yellow chicken at 9, 11, 13, 15 and 18 embryo ages under low and high power microscope in cross section.
By separating intestinal section tissues of different embryo ages, researchers of the invention observe that mucous layer epithelial cells are distributed on the surface of villus of chicken intestinal tracts of 9 embryo ages, which probably can meet the requirement that small intestinal epithelial cells have dry primordial cells, and although the number of the cells is small, the proliferation capacity is strong; the crypt depth of the small intestine with the age of 11 embryos is changed, the crypts are obvious and are arranged orderly; with the increase of embryo age and the continuous development of intestinal tissues, the inherent layer structure of intestinal villi is widened and thickened, and the number and the types of cells are increased. Preferably, 9-embryo-aged chicken small intestine segments are subjected to cell isolation culture.
The specific steps of the isolated culture of the avian embryo small intestine cells are as follows:
separating small intestine tissue of 9 embryo-aged chicken, transferring into EP tube, shearing, and making the volume less than 1mm 3 Washing for 2-3 times, centrifuging for 3min at 1000RPM, and discarding the supernatant; adopting a tissue block primary culture method for culture, inoculating the tissue suspension into a culture flask, adding a small amount of complete culture medium (DMEM (11960) containing 10% serum), covering the tissue, and culturing at 37 ℃; observing at different time periods, washing the cells for 1-2 times by PBS after the cells hatch out of the adherent wall, and continuing culturing; and changing a new culture medium once at intervals of 1-2 days, growing the cells in the logarithmic phase, ensuring the confluence degree of the cells to be about 80%, and carrying out passage and cryopreservation.
SV40 Large T antigen lentivirus vector transduction
1) Lentiviral packaging and concentration
(a) Recovering 293FT cells in advance, and when the cells are in a growth logarithmic phase and the density is 80-90%, carrying out passage and plating; when the number of cells is 10 5 Discarding the old culture medium when the fusion degree is 70% -80%, washing with PBS for 1-2 times, and adding 3mL of new culture medium containing 10% FBS for continuous culture;
(b) two 1.5mL sterile EP tubes are respectively taken, 300 mu L basal medium DMEM is added, 3.5 mu g packaging plasmid pMD2.G, 10.4 mu g psPAX2, 10 mu g SV40 large T antigen plasmid connected with GFP and transfection reagent with relative dose are added, and the mixture is gently mixed and acts for 15 min; uniformly dripping the mixed solution into a culture bottle containing cells, uniformly mixing and culturing;
(c) after 5-6 h, carrying out first liquid change, and adding a new culture medium for continuous culture; carrying out second liquid change the next day; collecting supernatant after 48h and 72h, centrifuging at 3000RPM for 3min, filtering with 0.45 μm filter, and concentrating virus; sequentially transferring the virus liquid into an ultrafiltration tube respectively, and centrifuging at 4 ℃ and 4000RPM for 0.5 h; the filtrate was discarded, 100. mu.L of the basic culture medium was added to the filtrate to resuspend the precipitate, and the precipitate was stored at-80 ℃ in 20. mu.L/tube.
2) Lentiviral infection
Calculating the concentrated virus solution according to the MOI value of 20, and finally adding 50 mu L of virus solution into 1mL of complete culture medium to infect the chick embryo small intestine epithelial cells in a good growth state; adding Polybrene (Polybrene) with the final concentration of 6 mug/mL for assisting infection, and culturing in an incubator at 37 ℃; and observing the green fluorescence expression condition after 24h, judging the cell transduction efficiency, and if the efficiency is lower, carrying out multiple infection.
3) Single cell clonal screening
Screening out positive cells for digestion, washing and centrifuging, and counting the cells; inoculating 100 mu L of suspension containing 1 cell into a 96-well plate, wherein each well contains one cell, observing after culturing for 1 week, marking a monoclonal well for expressing GFP, continuously culturing for 2-3 weeks, and digesting, passaging and freezing after the positive monoclonal cells grow full.
FIG. 4 is a schematic diagram showing the cell morphology and virus expression of chick embryo intestinal epithelial cells transduced with lentivirus vectors carrying large T antigen genes for 2 days. As can be seen from FIG. 4, after passage, the chicken embryo intestine segment cells are infected by SV40 large T antigen lentivirus, and after 2 days of culture, part of the cells express green fluorescent protein GFP, which proves that the large T antigen is expressed in the chicken embryo small intestine epithelial cells.
Counting cells by a monoclonal cell screening method, paving the cells in a 96-well plate, wherein each well contains one cell, and observing after culturing for one week, the cell fusion reaches about 80%. FIG. 5 is a schematic diagram showing the cell morphology and virus expression after monoclonal amplification culture of chick embryo intestinal epithelial cells successfully transduced by lentiviral vectors with large T antigen genes. As can be seen from FIG. 5, the cells all expressed GFP, and were cloned like epithelial cells according to preliminary judgment of morphology, and were selected for expanded culture, and were found to have a high proliferation rate.
3. Cell passage and expanded culture
Passage: when the cell density is more than 80-90%, discarding the old culture medium, washing with PBS for 1-2 times, digesting at 37 ℃ for 30s, adding the complete culture medium to terminate the digestion, blowing the cells, transferring all the exfoliated cells into a centrifuge tube, and centrifuging; cell pellet was measured according to 1: 3, inoculating the cell suspension into a culture flask for culture.
The chick embryo small intestine epithelial cells successfully expressing GFP are screened out by a single cell screening method, the continuous culture is carried out for 6 months, the cells still keep a rapidly growing state after passage for 30 generations, and the fusion rate reaches 80-90%.
FIG. 6 is a schematic diagram showing the cell morphology and virus expression of subculture after the successful transduction of chick embryo intestinal epithelial cells by a lentiviral vector with a large T antigen gene. As can be seen from FIG. 6, the cells of the F5 generation are in short spindle shape and uniformly distributed by subculture; f10 generation cell confluent chip; f20 generation minority cells are polygonal, other cells are collected by short fusiform; the F30 generation is the same as the F10 generation, most cells are gradually gathered, the cells are uniformly distributed, the activity is better, and the morphological structure is complete.
The above results confirm that the cell morphology is well maintained after passage, and the cell can be continuously proliferated and passed for many times.
4. Detection of transcriptional level of marker gene of small intestine epithelial cell
Extracting RNA of chick embryo small intestine epithelial cells of F10 generation and F30 generation, carrying out reverse transcription, and carrying out agarose gel electrophoresis. FIG. 7 is a diagram showing the results of electrophoretic detection of small intestine epithelial cell marker gene cDNAs of F10 generation and F30 generation after successful transduction of chick embryo intestinal epithelial cells by a lentiviral vector carrying a large T antigen gene. As can be seen from FIG. 7, the chick embryo small intestine epithelial cell marker genes Krt-18, Cdh1, Cldn1, Tjp1, Ocln and Vill were expressed in the F10 and F30 generations.
5. Immunofluorescence detection of small intestine epithelial cell marker protein
The immunofluorescence detection of TJP1 protein was carried out on F30 intestinal epithelial cells, the cell nucleus was labeled with Hochest33342 antibody (Solambio C0031), and the TJP1 protein was labeled with TJP1 antibody (Biyuntian Biotechnology company, Cat. No.: AF 8394). Fig. 8 is an immunofluorescence test chart of the expression of the TJP1 protein of chicken intestinal epithelial cells of generation F30, and it can be seen from fig. 8 that the TJP1 protein of intestinal epithelial cells of generation F30 is positive, i.e. shows fluorescence, and the negative control result without addition of the TJP1 antibody is negative. This result demonstrates that the cells prepared highly express the intestinal epithelial cell marker TJP 1.
6. Clustering analysis and homology comparison of differentially expressed genes
Hierarchical clustering analysis is performed on the FPKM values of the differential gene expression of the small intestine epithelial cells of different generations as the expression levels, and FIG. 9 is an FPKM hierarchical clustering analysis graph of the differential gene expression of chicken intestine epithelial cells of F10 generation, F20 generation and F30 generation, wherein the horizontal axis represents cell groups of different generations, the vertical axis represents genes, the dark color represents the up-regulation of the gene expression, and the light color represents the down-regulation of the gene expression. As can be seen in FIG. 9, the gene expression patterns within the same group are similar and may have similar functions or participate in common biological processes. The results show that the differential expression genes of different groups of cells of the F10 generation, the F20 generation and the F30 generation are respectively gathered together, and the expression modes of the genes involved in regulation are gradually changed along with the increase of the generations. Meanwhile, from the clustering graphs of the F20 generation and the F30 generation, the gene expression of the two generations is stable, the difference between the same generation is not obvious, and the gene expression is more and more stable along with the continuous change of the generations.
7. KEGG pathway analysis of intercellular differential expression gene
According to the sequencing result of the transcriptome, the high-expression differential genes between the epithelial cells of the small intestine of the F10 generation and the F30 generation are selected for KEGG enrichment analysis, and the genes are found to be significantly enriched in 204 paths. FIG. 10 is a KEGG pathway analysis diagram of differentially expressed genes of chicken intestinal epithelial cells of generations F10 and F30, and the results show that the differential genes mainly relate to 9 pathways, namely a cell cycle pathway, a MAPK signal pathway, a Wnt signal pathway, cell senescence, a TGF-beta signal pathway, a FoxO signal pathway, a p53 signal pathway, apoptosis and an ErbB signal pathway, and the signal pathways are mainly pathways related to material metabolism and cell cycle regulation, so that the intestinal epithelial cells with the established main functions of material transport and metabolism are laterally illustrated. Among them, 51 Differentially Expressed Genes (DEGs) were found to be significantly enriched in the CELL CYCLE (cell cycle) pathway, in which Cyclin and Cyclin-dependent kinase CDK complexes play an important role.
8. Cell cycle differential analysis of intercellular differentially expressed genes
Functional network interaction relation of differential expression genes among cells in CELL CYCLE signal channels is analyzed by using a KEGG database (http:// www.genome.jp/KEGG /). Fig. 11 is a diagram of analyzing cell cycle signal pathways of differentially expressed genes of chicken intestinal epithelial cells of generations F10 and F30 (light gray represents down-regulated genes, dark gray represents up-regulated genes, and black represents no-differential genes in the diagram), and the results show that after cell culture and passage times are increased, the cell proliferation capacity is remarkably increased compared with that of an early generation, the cell cycle is stable, and the cell is favorable for carrying out a repeatability experiment after the passage times are increased.
Subsequently, by using the sequencing result of the transcriptome, genes with significant differential expression are screened out, and the expression levels of related mRNA of the F10 generation and F30 generation cells are verified by using a real-time fluorescent quantitative PCR technology. FIG. 12 is a statistical chart of the real-time fluorescence quantitative PCR verification results of the differentially expressed genes of the chicken intestinal epithelial cells F10 and F30, and the results show that the mRNA expression of Cdk1, Cdc25a, Perp2, Ctase 1 and Ccne2 promoting cell proliferation is up-regulated; and the results of inhibiting the down-regulation of the mRNA expression of the periodically operated Tp53i3, Cdknla, Ccnb1, Ccng1 and Tgfb3 were consistent with the transcriptome sequencing results.
9. Inoculation of chicken intestinal epithelial cell model with Eimeria tenella (E.tenella)
The purified e.tenella sporozoites were inoculated into the chicken intestinal epithelial cell model, and fig. 13 is a schematic view of the e.tenella sporozoites under a microscope of the development conditions of chicken intestinal epithelial cells for bright field observation. The results show that 0h sporozoite has an isolated cell surface, is similar to a crescent, and has better activity (shown as A in figure 13); after culturing for 6h, the sporozoites begin to invade the cells in pairs, the middle parts of the sporozoites are sunken, the two ends are sharper, and vacuoles with insects are formed (shown as B in figure 13); after 12h, a plurality of sporozoites are planted in the cells for development, the morphology is changed into fertilizer, the centers are straightened, and the sporozoites are arranged in order (as shown in C in figure 13); after 48h, coccidian sporozoites were found to be round, tightly connected, and rotationally moved (as shown in D in FIG. 13); an increase in the number of merozoites, free inside the cell, was found after 72h (as shown in fig. 13E); after 140h, schizonts developed, with round follicles surrounding the merozoites, and slow peristalsis in situ observed under the mirror (as shown by F in FIG. 13).
The development of sporozoites on epithelial cells of chicken intestine was investigated by HE staining of cell slide, and fig. 14 is a HE (hematoxylin-eosin) staining pattern of sporozoites of e. The result shows that the sporozoites invade cells within 6h and are fixedly planted near cell nucleuses, invasion is completed, and nauplius vacuoles are developed, the shapes of the sporozoites are changed into blunt circles from long spindle shapes and protrude towards the side edges to form oval pink trophozoites, and 2 refractile bodies disappear (shown as A in figure 14); the sporozoite takes off the capsule and continues to develop for 24h, a mononuclear trophozoite is formed, the trophozoite almost occupies the whole sporozoite, and the shape of the sporozoite is in an oblong shape and begins to develop towards the first generation schizont (shown as B in figure 14); sporozoite circum plasma membrane vesicles encapsulated sporozoites after 30h (as shown in figure 14, C); the 48h sporozoite outline completely disappears, spherical first generation merozoites are formed, and a large number of crescent I generation merozoites are contained in the merozoites (as shown in D in figure 14); clustered merozoites were formed after 54h (as shown in E in figure 14); after 72h mature merozoites in the second generation appeared and the clumped merozoites began to free around and distribute in the cells (as shown by F in FIG. 14).
The results show that the in vitro culture of E.tenella sporozoites inoculated to the chicken intestinal epithelial cell model can complete the second generation of schizogenesis and release merozoites.
Comparative example 1
9 embryo-aged chicken small intestine epithelial cell separation culture passage
In the experiment, 9 embryo chicken embryo small intestine epithelial cells are isolated and cultured by adopting a tissue block culture method, and fig. 15 is a cell morphology schematic diagram of the 9 embryo chicken embryo small intestine epithelial cell primary cells after subculture. The results showed that after 24h of culture, the cells gradually proliferated and surrounded their tissues; after the culture is carried out for 48h, the cells are rapidly proliferated, the shape is obvious polygonal, and the structure is obvious; observing the cell after 96h, wherein the shape changes to a certain extent, the cell is differentiated into a fusiform from the original oval shape, the growth state of the cell is good, the color of the cell observed under a microscope is bright, the cell is uniformly distributed, and the cell density reaches 90 percent at the moment, and carrying out passage; culturing for 24h after passage, and changing the cell morphology to be in an obvious fusiform shape; culturing for 48h, making partial cells round and polygonal, and brightening the periphery of the cells; after 120 hours of culture, the cells are remarkably changed, the shapes are elongated and tapered, the cells are dark and lackluster when observed by naked eyes, and the cells are vacuolated and finally tend to die.
The results show that the poultry small intestinal epithelial cells are possibly different from mammals, and due to the nature of the cells, the renewal rate is high, the in vitro nutrition requirement conditions are harsh, and the long-term culture is difficult to maintain in vitro.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.

Claims (10)

1. An avian intestinal epithelial cell is characterized in that the avian intestinal epithelial cell is derived from avian embryo small intestine segment cells, and the genome of the avian intestinal epithelial cell contains a large T antigen gene of SV 40.
2. The avian intestinal epithelial cells according to claim 1, wherein said avian embryonic small intestine segment cells comprise 9 embryonic age avian embryonic small intestine segment cells.
3. The avian intestinal epithelial cell according to claim 2, wherein said avian embryo small intestine segment cell comprises an avian embryo small intestine segment cell.
4. The avian intestinal epithelial cell according to claim 3, wherein said avian intestinal epithelial cell expresses an intestinal epithelial marker molecule comprising TJP 1.
5. A preparation method of avian intestinal epithelial cells is characterized in that avian embryo small intestine section cells are taken as host cells, and large T antigen genes of SV40 are integrated into genomes of the host cells.
6. The method of claim 5, comprising the steps of:
separating the small intestine section cells of the avian embryo;
transducing the avian embryo small intestine segment cells with a retroviral vector containing the large T antigen gene of SV 40;
cell passage, screening and identification.
7. The method according to claim 6, wherein the transduction time is 36 to 60 hours.
8. The method of claim 7, wherein the avian embryo small intestine cells comprise 9-embryo-old avian embryo small intestine cells.
9. The method according to any one of claims 6 to 8, wherein the avian embryo small intestine segment cells comprise avian embryo small intestine segment cells.
10. Use of the avian intestinal epithelial cells according to any one of claims 1 to 4 for the preparation of an avian intestinal-related bioengineered product or for in vitro screening of an avian intestinal-related therapeutic drug.
CN202210521390.1A 2022-05-13 2022-05-13 Avian intestinal epithelial cells and preparation method and application thereof Pending CN114891722A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210521390.1A CN114891722A (en) 2022-05-13 2022-05-13 Avian intestinal epithelial cells and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210521390.1A CN114891722A (en) 2022-05-13 2022-05-13 Avian intestinal epithelial cells and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN114891722A true CN114891722A (en) 2022-08-12

Family

ID=82720744

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210521390.1A Pending CN114891722A (en) 2022-05-13 2022-05-13 Avian intestinal epithelial cells and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114891722A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6280970B1 (en) * 1996-05-23 2001-08-28 Institut Naltional De La Recherche Agronomiqe Immortalized avian cell lines
US6395542B1 (en) * 1996-04-19 2002-05-28 Nestec S.A. Immortalized human colon epithelial cell line
CN105483078A (en) * 2015-12-24 2016-04-13 山西农业大学 Isolation and primary culture methods of chicken small intestinal epithelial cells
CN107002044A (en) * 2014-12-04 2017-08-01 英特维特国际股份有限公司 Immortalize chicken embryo fibroblasts
CN107217040A (en) * 2017-06-16 2017-09-29 中国农业大学 One kind immortalizes rabbit intestinal epithelial cell line and its construction method
US20190144831A1 (en) * 2015-12-11 2019-05-16 Intervet Inc. Immortalised chicken embryonic epithelial kidney cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6395542B1 (en) * 1996-04-19 2002-05-28 Nestec S.A. Immortalized human colon epithelial cell line
US6280970B1 (en) * 1996-05-23 2001-08-28 Institut Naltional De La Recherche Agronomiqe Immortalized avian cell lines
CN107002044A (en) * 2014-12-04 2017-08-01 英特维特国际股份有限公司 Immortalize chicken embryo fibroblasts
US20190144831A1 (en) * 2015-12-11 2019-05-16 Intervet Inc. Immortalised chicken embryonic epithelial kidney cells
CN105483078A (en) * 2015-12-24 2016-04-13 山西农业大学 Isolation and primary culture methods of chicken small intestinal epithelial cells
CN107217040A (en) * 2017-06-16 2017-09-29 中国农业大学 One kind immortalizes rabbit intestinal epithelial cell line and its construction method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
詹康 等: "鸡胚小肠上皮细胞的体外分离培养和鉴定", 《动物营养学报》 *

Similar Documents

Publication Publication Date Title
CN102858951B (en) The pluripotent stem cell that can separate from body tissue
Barretto et al. ASCL1-and DLX2-induced GABAergic neurons from hiPSC-derived NPCs
CN108713059A (en) VCN enhancer combinations object and its application method
Katzenell et al. Isolation, purification, and culture of primary murine sensory neurons
CN106636210B (en) Transcription factor combines the method that induced fibroblast transdifferentiation is class interstitial glands
CN111662907B (en) Method for knocking out induced pluripotent stem cell NANS gene and application thereof
CN110106150A (en) A kind of preparation method and application of synovial sarcoma cells system hSS-005R
CN110121555B (en) Blood brain barrier comprising engineered endothelial cells
CN114891722A (en) Avian intestinal epithelial cells and preparation method and application thereof
WO2020125576A1 (en) Method for delivering gene in cells
CN110029086A (en) The factor of inducing somatic reprogramming and the method reprogrammed using the factor inducing somatic
CN102250841A (en) Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof
CN107858322B (en) Method for establishing primary hippocampal cell culture system
CN106635990B (en) Method for primary culture of dorsal root ganglion satellite glial cells
CN108192925A (en) Applications of the SIRT3 in old human adipose mesenchymal stem cells rejuvenation is promoted
CN114507635A (en) Method for separating animal nervous system endothelial cell single cell
WO2001092482A1 (en) Method of concentrating and separating dopaminergic neurons
CN105073978B (en) Method for inducing customized sub-totipotent stem cells by using plant stem cells or extracts of plant dedifferentiated stem cells and sub-totipotent stem cells prepared by using method
CN112961841A (en) Packaging method for rapidly obtaining high-titer lentiviruses
CN102933702A (en) Methods of inducing tissue regeneration
Shayler et al. Single cell transcriptomics reveals early photoreceptor trajectories and a cancer-predisposed cone precursor state
CN111549033B (en) Lentiviral-infected human epidermal keratinocyte strain and construction method and application thereof
CN110172448A (en) A kind of synovial sarcoma cells system hSS-005R and its progeny cell system
Mueller et al. Effective and steady differentiation of a clonal derivative of P19CL6 embryonal carcinoma cell line into beating cardiomyocytes
CN112126627B (en) Construction method and application of canine corneal epithelial cell immortalized cell line

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination