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Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats as well as establishing method and applications of Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2

A technology for establishing methods and cell lines, applied in botany equipment and methods, epidermal cells/skin cells, biochemical equipment and methods, etc., can solve the problems of poor passaging effect, large cell damage, difficult induction, etc., and achieve repeatability High, increased proliferation rate, easy operation

Active Publication Date: 2018-05-18
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because HERS is a temporary structure in tooth root development and consists of only two layers of epithelial cells surrounded by ectodermal mesenchymal cells, it is very difficult to isolate HERS cells from tooth germs alone and the number of cells is small
The isolated HERS cells have relatively tight intercellular connections. Digestion with commonly used trypsin takes a long time to separate the cells. However, trypsin acts on the cells for a long time, causing great damage to the cells and making it difficult to culture HERS cells. Therefore, in order to facilitate the exploration of the molecular mechanism of tooth root development and the use of HERS cells to construct biological tooth roots, it is necessary to obtain HERS cells that can be stably passaged and expanded

Method used

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  • Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats as well as establishing method and applications of Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2
  • Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats as well as establishing method and applications of Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2
  • Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats as well as establishing method and applications of Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction of rat Hertwig epithelial root sheath cell lines HERS-H1 and HERS-C2, the preservation number of Hertwig epithelial root sheath cell line HERS-H1 in the present invention is CCTCC NO.C2017249, said Hertwig The epithelial root sheath cell line HERS-C2 has a preservation number of CCTCC NO.C2017250:

[0056] (1) Culture of primary cells

[0057] Select 10 SD rat suckling mice 6-10 days after birth. After anesthesia, they were disinfected with 75% ethanol twice, separated the maxilla and mandible, stripped the complete tooth germ of the mandibular first molar, and cut about 1mm dental neck tissue, rinsed with PBS and shredded until chylus;

[0058] Use a mixed enzyme digestion solution containing 1-2.5U / mL dispase and 300U-800U / mL type I collagenase to digest the tissue together, digest in a water bath at 37°C for 30-100 minutes, and shake it every 10 minutes; 10% FBS DMEM / F12 culture medium to stop digestion, wash with PBS containing double antibody, centri...

Embodiment 2

[0067] Identification of cell lines:

[0068] Step 1 Immunofluorescence detection

[0069] The HERS-H1 and HERS-C2 cells were inoculated in special confocal microscope dishes. When the cells grew to a cell density of about 50%, the cells were fixed with 4% paraformaldehyde, and the two cell lines were identified by immunofluorescence staining.

[0070] Immunofluorescence staining steps are:

[0071] a. Cells were fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS;

[0072] b. Add 0.1% TritionX-100 at room temperature for 15 minutes, wash with PBS 3 times;

[0073] c. Add goat serum for 30 minutes at 37°C;

[0074] d. Add the primary antibody, overnight at 4°C;

[0075] e. Rewarm for 10 minutes, wash with PBS 3 times, act for 5 minutes each time, add fluorescent secondary antibody, and act for 2 hours at 37°C;

[0076] f. Wash with PBS for 3 times, and stain the nucleus with DAPI for 5 minutes;

[0077] g. Wash with PBS 3 times and observe under a confo...

Embodiment 3

[0080] Biological properties of cells:

[0081] Step 1 Cell growth curves of HERS-H1 and HERS-C2 cells

[0082] The P1 HERS primary cells in good growth state and the P5 generation cells of HERS-H1 and HERS-C2 were seeded in a 96-well plate at a cell density of 1×10 5 cells / well, with 3 replicates in each group, cultured in a cell culture incubator, and changed the medium every 2 days. Take three wells for each group of cells every day, discard the culture medium, add 110 μl of fresh culture medium containing 10 μl of CCK-8, and incubate for 2 hours in the dark. Take the supernatant, and measure the absorbance (optical density, OD value) of each well at 450 nm with a microplate reader. Continuous detection for 7 days, draw growth curve, such as image 3 .

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Abstract

The invention belongs to the field of biotechnology, and relates to an establishing method of Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats and applications of the Hertwig's epithelial root sheath cell lines HERS-H1 and HERS-C2 of SD rats in tooth regeneration. The establishing method and the applications are characterized in that the Hertwig's epithelial root sheath cell line HERS-H1 is assigned with the accession number of CCTCC NO.C2017249, and the Hertwig's epithelial root sheath cell line HERS-C2 is assigned with the accession number of CCTCC NO.C2017250. According to the method and the applications, for the cell lines, the features of the primary Hertwig's epithelial root sheath cells in the tooth development process are reserved, passage to 50 generations is available at least, and the multiplication rate is greatly improved compared with that of the primary cells. The establishing method is easy to operate, and high in stability and repeatability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the establishment method and application of SD rat Hertwig epithelial root sheath cell lines HERS-H1 and HERS-C2. Background technique [0002] Teeth are important human organs, responsible for chewing, pronunciation, and maintaining the shape of the maxillofacial region. However, tooth loss has become a major factor affecting human life. Adults aged 20-64 only had 24.92 teeth remaining on average, and an average of 3.08 teeth were missing per person (excluding terminal teeth). Among them, 3.75% of the people were completely missing teeth. At present, the methods of repairing missing teeth mainly include bridge restoration, dental implant restoration and removable denture restoration. Bridge restoration and removable denture restoration mainly rely on the remaining teeth to restore the missing teeth, which often cause irreversible damage to the remaining teeth, and the comfort of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61L27/38C12R1/89
CPCA61L27/3813A61L27/3865A61L2430/12C12N5/0625C12N15/86C12N2510/00C12N2740/15043
Inventor 陈国庆田卫东李雪冰
Owner SICHUAN UNIV
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