Hertwig epithelial root sheath cell lines hers-h1 and hers-c2 of sd rats and their establishment method and application
A technology for establishing methods and cell lines, which is applied in botany equipment and methods, biochemical equipment and methods, epidermal cells/skin cells, etc. It can solve the problems of large cell damage, poor passage effect, and difficult induction, etc., and achieve a proliferation rate Improved, repeatable, and easy-to-operate results
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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] Cell line identification:
[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 confocal ...
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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