A cervical intraepithelial neoplasia cell line containing free HPV18 and its application
A diseased cell, free-type technology, applied in the field of microbial animal cell lines, can solve the problem that HPV cell lines cannot reflect the characteristics of the population, and achieve the effect of stable properties
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Embodiment 1
[0043] [Example 1] Primary isolation and culture of human cervical intraepithelial neoplasia cells
[0044] (1) With the informed consent of the patient or the patient’s guardian, collect 1–2 cm clinical tissue samples of cervical intraepithelial neoplasia from patients with HPV18 infection 3 (cubic centimeters), no other treatment.
[0045] (2) Preparation of digestion solution: HCIN medium containing both 0.2 mg / mL of collagenase and dispase; wherein, the HCIN medium is: a medium in which DMEM and Ham's F 12 NUTRIENT MIX are mixed in a volume ratio of 4:1, and at the same time Add 4~6% fetal bovine serum (FBS), 1~3nM triiodothyronine, 0.4~0.65% insulin reagent, 9~11ng / mL epidermal growth factor (EGF) ), 0.3~0.5μg / mL hydro-cortisone, 35~45μg / mL gentamicin, 45~55nM calpeptin, 35~45ng / ml recombinant human IL-lRA, and 3μg / ml ml recombinant human R-Spondin-1.
[0046] (3) Wash the separated tissue samples once with 95-100% (v / v) ethanol, and then twice with PBS (0.01M, pH 7.4)...
Embodiment 2
[0053] [Example 2] Subculture of human cervical intraepithelial neoplasia cells
[0054] (1) When human cervical intraepithelial neoplasia cells cultured in T25 or T75 culture flasks proliferate to 70-90% abundance, wash the cells twice with 1×PBS (0.01M, pH 7.4), add 0.6 EDTA with a concentration of 0.02% was shaken gently to make it fully contact with the cells on the culture flask. After 20s, the outer wall of the culture flask was tapped to make the mouse fibroblasts fall off the wall of the culture flask. After the murine fibroblasts were all detached from the flask wall, the EDTA was quickly removed, and the cells were washed three times with PBS.
[0055] (2) The monolayer cells were digested with 0.05% trypsin-EDTA for 5 min.
[0056] (3) 10 mL of DMEM was added to neutralize the digestion reaction for 1 min, centrifuged at 1000 rmp for 5 min, and the supernatant was removed.
[0057] (4) Resuspend the cell pellet in HCIN medium at a ratio of about 1:3, inoculate it ...
Embodiment 3
[0060] [Example 3] Karyotype analysis and identification of human cervical intraepithelial neoplasia cells
[0061] (1) When human cervical intraepithelial neoplasia cells (1×10 6 ) in the exponential growth phase, add colchicine to a final concentration of 0.2 μg / mL, and continue to culture for 3.5 hours.
[0062] (2) The cells were detached by repeated pipetting and centrifugation at 2000 rpm for 5 minutes to harvest the cells.
[0063] (3) Discard the supernatant, add 8 mL of 0.075 mol / L KCl solution pre-warmed at 37°C, gently pipette the cell mass to mix, and place it at 37°C for hypotonic treatment for 25 minutes.
[0064] (4) Add 1 mL of freshly prepared fixative (methanol:glacial acetic acid=3:1, v / v), carefully pipet, mix, and centrifuge at 2000 rpm for 5 minutes.
[0065] (5) Discard the supernatant, add 8 mL of fixative, pipette to make a cell suspension, and fix at room temperature for 20 minutes.
[0066] (6) Centrifuge at 2000 rpm for 5 minutes, discard the sup...
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