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Establishment and application of porcine respiratory tract epilhelium in-vitro 3D model

A technology of epithelial cells and porcine respiratory tract, applied in the field of biomedicine, can solve the problems of no virus amplification, identification, pathogenic mechanism, and restriction research

Inactive Publication Date: 2016-07-06
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For many emerging and re-emerging respiratory viruses, there is no suitable model in vitro for virus amplification, identification, pathogenic mechanism research and evaluation of antiviral drugs, which seriously restricts related research

Method used

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  • Establishment and application of porcine respiratory tract epilhelium in-vitro 3D model
  • Establishment and application of porcine respiratory tract epilhelium in-vitro 3D model
  • Establishment and application of porcine respiratory tract epilhelium in-vitro 3D model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Establishment of an in vitro differentiation model of porcine airway epithelial cells

[0069] 1.1 Isolation of porcine respiratory epithelial cells A 3.5-month-old white female pig was anesthetized with pentobarbital, fixed on the operating table, and its neck and abdomen were disinfected with iodine and alcohol in turn. Under sterile conditions, the superficial skin of the pig was cut with a scalpel, the trachea and lungs were exposed, the main trachea and lung tissue below the larynx were peeled off, immersed in a sample box containing 4 ℃ pre-cooled PBS, and placed on crushed ice shipped to the laboratory. Use sterilized forceps and scissors to dissect the tracheal tissue, and remove excess tissue and lymph nodes. After rinsing with PBS, it was transferred to another cell culture dish containing fresh PBS, and cut into 1×2 cm tissue pieces with small scissors. Soak twice in Joklik's MEM containing 0.5mg / mL DTT and 10ug / mL DNAse to remove sputum, fungi, b...

Embodiment 3

[0080] Example 3 In vitro differentiation model of porcine respiratory epithelial cells infected with recombinant AAV6-GFP

[0081] 3.1 Preparation and quantitative recovery of AAV6-GFP 293T cells cryopreserved in our laboratory were treated with complete medium (DMEM containing 10% FBS and 1% PS) at 37°C, 5% CO 2 Cultivated and passaged to 60 150mm cell culture dishes. Transfection was performed when 293T cells reached 80% to 90% confluence. During transfection, firstly mix plasmids PSC-GFP (6ug), pXR6 (10ug) and pXX680 (12ug) in a 15mL centrifuge tube, add 500μL of serum-free DMEM medium and mix gently, then add 110μL of PEI transfection reagent , Gently pipetting and mixing, let stand at room temperature for 5-10min, and finally add the prepared transfection complex to the 293T cell dish without changing the medium. After culturing for 48h, discard the medium in the dish and use a cell scraper. The cells were scraped off the culture dish, collected in a centrifuge tube, c...

Embodiment 4

[0083] Example 4. Study on the infection and replication characteristics of EV68 in PAEC model

[0084] 4.1. Virus: EV68, TCID50=10 -6.7

[0085] Cells: PAE cells, density approximately: 1.5X10 6 / hole

[0086] 4.2. Before inoculation: wash the PAE model 3 times with 0.5ml sterile PBS, soaking for 10min each time.

[0087] 4.3. Virus inoculation: Take EV68 virus seed, add 200 μl to each well, incubate at 37°C for 1 h, remove the virus and wash twice with 0.5ml sterile PBS to remove unbound virus, and continue to culture at 37°C.

[0088] 4.4. Collection of infected samples

[0089] After infection, at different time points (6, 24, 48, 72hrs), samples were collected at each time point according to a, b, c, and d. At the same time, a control well without virus incubation was set up, marked as "Con", and the collection at each time point was also carried out.

[0090] a. Collection of top washes (two duplicate wells): add 200ul PBS to the top surface of cells, incubate at 3...

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Abstract

The invention belongs to the field of biomedicine, and relates to establishment and application of a porcine respiratory tract epilhelium in-vitro 3D model. The porcine respiratory tract epilhelium in-vitro 3D model is a highly-differentiated nutrient material in a multilayer epithelium structure, which is obtained by carrying out in vitro culture on porcine respiratory tract epilhelia; and the top end surface of the nutrient material contains ciliated cells, mucous secretory cells and basal cells. The transepithelial electrical resistance is kept within the range of 250-350 ohm cm<2>. The cilia are distributed on the cell surface in clusters; abundant microvilli are visible on the cell surface; the diameter of the cilia is 0.3-0.5 mu m; and the length of the cilia is more than ten micrometers.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the establishment and application of an in vitro 3D (Dimension) model of porcine airway epithelial cells. Background technique [0002] Respiratory epithelial cells are the first line of defense against contact and defense of the respiratory tract with dust and pathogens in the external environment. The interaction between epithelial cells and pathogens leads to the occurrence of respiratory diseases, and the interaction mechanism between the two is crucial to the study of therapeutic methods. At the beginning of the study, conventional subcultured airway epithelial cells lacked extracellular matrix, cell-to-cell contact and communication. Such undifferentiated cells could not simulate the microenvironment of the human airway and could not meet the needs of the experiment. [1] . Afterwards, Whitcutt, Gray et al. studied the improved air-liquid interface to culture airway epithelial cel...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N5/0627C12N2500/30C12N2506/09C12N2509/00C12N2513/00
Inventor 李武平王健伟任丽丽叶磊张少丹相子春肖艳刘红梅
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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