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Free-type HPV18-containing cervical epithelia interior tumor-like lesion cell line and application thereof

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

Active Publication Date: 2019-04-23
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a new Chinese female, long-term in vitro cultured and stable human cell line for the sustainable passage of patient-derived cell lines containing free HPV and unable to reflect the characteristics of the Chinese population. Cervical intraepithelial neoplasia cell line

Method used

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  • Free-type HPV18-containing cervical epithelia interior tumor-like lesion cell line and application thereof
  • Free-type HPV18-containing cervical epithelia interior tumor-like lesion cell line and application thereof
  • Free-type HPV18-containing cervical epithelia interior tumor-like lesion cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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 patient guardian, collect clinical tissue samples of cervical intraepithelial neoplasia 1–2 cm from patients with HPV18 infection 3 (cubic centimeters), without other treatment.

[0045] (2) Preparation of digestive solution: HCIN medium containing 0.2 mg / mL of collagenase and dispase; wherein, HCIN medium is a medium mixed with DMEM and Ham's F 12 NUTRIENT MIX at a volume ratio of 4:1, and at the same time Add 4-6% fetal bovine serum (FBS), 1-3nM triiodothyronine (triiodothyronine), 0.4-0.65% insulin (insulin) reagent, 9-11ng / mL epidermal growth factor (epithelial growth factor ), 0.3-0.5 μg / mL hydrocortisone (hydro-cortisone), 35-45 μg / mL gentamicin (gentamicin), 45-55 nM calpeptin, 35-45 ng / ml recombinant human IL-1RA, and 3 μg / mL ml recombinant human R-Spondin-1.

[0046] (3) Wash the isolated tissue sample once wit...

Embodiment 2

[0053] [Example 2] Subculture of human cervical intraepithelial neoplasia cells

[0054] (1) When the human cervical intraepithelial neoplasia cells cultured in T25 or T75 culture flask 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% in mL was gently shaken to make it fully contact with the cells on the culture flask, and after 20 seconds, tap the outer wall of the culture flask to make the mouse fibroblasts fall off the wall of the culture flask. After all the mouse fibroblasts were detached from the flask wall, EDTA was quickly removed, and the cells were washed 3 times with PBS.

[0055] (2) Digest the monolayer cells with 0.05% trypsin-EDTA for 5 min.

[0056] (3) Add 10 mL DMEM to neutralize the digestion reaction for 1 min, centrifuge at 1000 rpm for 5 min, and remove the supernatant.

[0057] (4) Resuspend the cell pellet in HCIN medium at a ratio of about 1:3, inoculate it in a culture flask...

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 at a final concentration of 0.2 μg / mL, and continue culturing for 3.5 hours.

[0062] (2) Beat the cells repeatedly to make them fall off, and centrifuge at 2000rpm 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 blow and beat the cell mass to mix, and place 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 pipette, mix, and centrifuge at 2000 rpm for 5 minutes.

[0065] (5) Discard the supernatant, add 8 mL of fixative, pipette to form a cell suspension, and fix at room temperature for 20 minutes.

[0066] (6) Centrifuge at 2000rpm for 5 minutes, discard the...

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Abstract

The invention discloses a free-type HPV18-containing cervical epithelia interior tumor-like lesion cell line and application thereof. A clinic sample from the cell line is 1-2 cm<3> of a cervical epithelia interior tumor-like lesion clinic tissue sample of a HPV18 infected patient, no other treatment is adopted, after in-vitro direct culture, in-vitro stable passage is performed, on the basis of PCR and rolling circle amplification experiments, it is authenticated as a free-type HPV18-containing Chinese woman cervical epithelia interior tumor-like lesion cell named as a cervical epithelia interior tumor-like lesion cell HCINC / HL-017 preserved at CCTCC, and the preservation number is CCTCC NO:C2015124. The cell line can have the application prospect at the aspect of HPV18 virus biology, HPV18 infected cervical cancer tumor biology, and anti-HPV and anti-cervical-cancer drug and vaccine development.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, in particular to a cervical intraepithelial neoplasia cell line containing free human papillomavirus 18 subtype and its application. Background technique [0002] Human papillomavirus (Human papillomavirus, HPV) is a minimal, spherical double-stranded DNA virus, HPV infection is known to cause cervical cancer. There is a special cervical epithelial transition zone or transformation zone in the endocervix, which is the junction of the single layer of columnar epithelium and squamous epithelium (external cervix) of the cervical canal. The basal cells of the endocervix in the transformation zone have the potential to proliferate and differentiate into columnar epithelial cells and squamous epithelial cells. If the cells in the transformation zone are continuously exposed to high-risk human papillomavirus (High-risk human papillomavirus, HR-HPV ), it is easy to produce abnormal hyperplasia...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02
CPCG01N33/5014C12N5/0693C12N2500/32C12N2500/84C12N2501/11C12N2501/2301C12N2501/33
Inventor 李晖叶立娜朱雅琪陈雨曾志宏
Owner WUHAN UNIV
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