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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 difficulty in obtaining precursor cells

Inactive Publication Date: 2013-03-20
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

Experimental program
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Effect test

preparation example Construction

[0043] Preferably, the preparation method of the cochlear large epithelial crest cell line provided by the present invention further includes the step of gene detection.

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

[0045] Preferably, after the cells of the cochlear large epithelial crest cell line are massively expanded, total RNA is extracted, and RT-PCR is used to detect whether the Math1 gene exists.

[0046] The present invention also provides an application of a cochlear large epithelial crest cell line containing the Math1 gene and having a preservation number of CGMCC No. 4719 in the research on the differentiation and regeneration of hair cells. The cell line grows fast and is easy to culture. After being introduced into the noise-induced cochlear model, it can express the characteristic marker protein myosin VIIa of hair cells, and can develop into stereocilia clusters with characteristics ...

Embodiment 1

[0049] Example 1 Isolation of large cochlear epithelial crest cells

[0050] Take P 0 -P 3 Rats were soaked in 75% alcohol for 5 minutes, decapitated along the junction of the head and neck, ophthalmic scissors removed most of the parietal bone and all brain tissue from the foramen magnum, exposed the middle cranial fossa and posterior cranial fossa, and completely removed the auditory bulb; In the PBS solution of pH=7.4, open the auditory bulb under a dissecting microscope (Olympus Company), peel off the volute, remove the bony cochlea, and gradually peel off the basement membrane from the bottom (tear off the spiral ligament and spiral ganglion); The basement membrane was transferred into 0.5 mg / mL thermolysin (Sigma company) solution (prepared from 0.01 mol / L PBS solution with pH=7.4, and 5 U / μL DNase (Promage company) was added at the same time, and incubated at 37 ° C for 27 min ;Under a high-power microscope, the epithelial cell sheet on the basement membrane was torn ...

Embodiment 2

[0051] Example 2 Establishment of cochlear large epithelial crest cell line

[0052] The large epithelial crest cells were co-cultured with retrovirus containing SV40 large T antigen, and 20ng / mL EGF and 8μg / mL polybrene were added at the same time. After two infections (6 hours each), the culture medium was replaced with 10% DMEM (20 ng / mL EGF was added). After culturing for 10 days, the culture was transferred to a 24-well culture plate by digestion with 0.125% trypsin. Through daily observation with an inverted microscope, clones of infected cells can be seen after a few days. While digesting with 0.125% trypsin, single clones are extracted with a pipette to obtain cell purification. The extracted cells were monoclonal inoculated into 96-well culture plates for proliferation and passage.

[0053] Transfection: Transfection was carried out when the cells in the 24-well culture plate grew to 80% confluence. 2 hours before transfection, 400 μL serum-free medium was changed t...

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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 application thereof. Background technique [0002] Deafness is the sensory or functional defect disease with the highest incidence rate in humans. It not only seriously affects the quality of life, study, social interaction 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. According to the survey results released by the Ministry of Health of my country in 2000, sensorineural deafness accounts for about 63% of deaf patients, and its absolute number exceeds 80 million. At present, the treatment of sensorineural hearing loss mostly requires the help of cochlear implants. The clinical application of cochlear implantation and hearing aid technology has partially improved...

Claims

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Application Information

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Patent Type & Authority Patents(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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