Cochlear greater epithelial ridge (GER) cell line and its application

A cell line and epithelial technology, applied in the field of cell line construction, can solve problems such as difficulties in obtaining precursor cells

Inactive Publication Date: 2011-11-02
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very difficult to obtain a large number of such cochlear hair cell precursors due to the small tissue in the inner ear, the complex bone structure in vivo, and the lack of a culture system composed of hair cell progenitor cells

Method used

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  • Cochlear greater epithelial ridge (GER) cell line and its application
  • Cochlear greater epithelial ridge (GER) cell line and its application
  • Cochlear greater epithelial ridge (GER) cell line and its application

Examples

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Effect test

preparation example Construction

[0043] Preferably, the method for preparing the cochlear large epithelial ridge cell line provided by the present invention further includes a step of gene detection.

[0044] The above gene detection is the RT-PCR detection of the Math1 gene in the cochlear large epithelial crest cell line.

[0045] Preferably, after a large number of cells of the cochlear large epithelial ridge cell line are amplified, total RNA is extracted, and RT-PCR detects the presence of the Math1 gene.

[0046] The present invention also provides an application of the cochlear large epithelial ridge cell line containing Math1 gene and depositing number CGMCC No. 4719 in the study of hair cell differentiation and regeneration. The cell line grows fast and is easy to cultivate. After being introduced into the noise-induced deafness cochlea model, it can express the characteristic marker protein myosin VIIa of hair cells, and can develop into static cilia clusters with the characteristics of hair cells. The ce...

Embodiment 1

[0049] Example 1 Isolation of large epithelial crest cells of the cochlea

[0050] Take P 0 -P 3 The rat was immersed in 75% alcohol for 5 minutes, and the head was decapitated along the head-neck joint. Ophthalmological scissors were cut from the foramen magnum to remove most of the parietal bone and all the brain tissue, and the middle and posterior fossa were exposed. In pH=7.4 PBS solution, open the auditory vesicle under a dissecting microscope (Olympus), peel off the volute, remove the bony cochlea, and gradually peel off the basement membrane (removing the spiral ligament and spiral ganglion) by turning the bottom; Transfer the basement membrane into 0.5mg / mL thermolysin (Sigma) solution (prepared from 0.01mol / L PBS solution with pH=7.4, and 5U / μL DNase (Promage) at the same time, incubate at 37°C for 27 minutes ; Under a high-power microscope, the epithelial cell sheet on the basement membrane was torn off from the connected connective tissue with a tungsten needle; the s...

Embodiment 2

[0051] Example 2 Establishment of Cochlear Greater Epithelial Crest Cell Line

[0052] Co-culture with large epithelial crest cells with retrovirus containing SV40 large T antigen, while adding 20ng / mL EGF and 8μg / mL polybrene. After two infections (6h each), the medium was changed to 10% DMEM medium (20ng / mL EGF was added). After culturing for 10 days, the culture was transferred to a 24-well culture plate using 0.125% trypsin digestion. Through daily inverted microscope observation, a few days later, the clones of infected cells can be seen. While 0.125% trypsinization is performed, single clones are extracted using a pipette to obtain cell purification. The extracted cells were seeded into 96-well culture plates for proliferation and passage.

[0053] Transfection: Transfection is carried out when the cells in the 24-well culture plate have grown to 80% confluence. 2 hours before transfection, 400μL serum-free medium per well; 1μg prv5-EGFP plasmid and 2μL Lipoofectamine TM 20...

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Abstract

The invention discloses a cochlear GER cell line, the preservation number of which is CGMCC No.4719. The cochlear GER cell line is obtained through the steps of transfection of SV40 large tumor antigen into a cochlear GER cell through a viral vector, introduction of the eukaryotic expression plasmid prv5-EGFP of the encoding gene Math1 (mammalian atonal homolog1), screening with G418 to obtain ananti-G418 positive cell, amplification culture of the anti-G418 positive cell and screening. The cell line has a fast growing speed, is easy to culture, can express characteristic marking protein myosin VIIa of hair cells when introduced to a cochlea model for deafening caused by noise, and can grow to be a stereocilia bundle with the characteristics of hair cells. The cell line is convenient forpopularization and is applicable to research on differentiation and regeneration of hair cells.

Description

Technical field [0001] The invention relates to the field of cell line construction, in particular to a cochlear large epithelial crest cell line and its application. Background technique [0002] Deafness is a sensory or functional defect disease with the highest incidence in humans. It not only severely affects the quality of life, study, social communication and family life of the affected population, but also brings physical and psychological pain to patients and their families. Deafness is divided into conductive deafness, sensorineural deafness and mixed deafness. The survey results published by the Ministry of Health of my country in 2000 show that sensorineural hearing loss accounts for about 63% of deaf patients, and its absolute number exceeds 80 million. At present, the treatment of sensorineural hearing loss mostly requires cochlear implants. The clinical application of cochlear implants and hearing aid technology has partially improved the hearing impairment of pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02C12R1/91
Inventor 翟所强杨仕明郭维
Owner GENERAL HOSPITAL OF PLA
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