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Human normal vaginal epithelial cells and application thereof

A technology of vaginal epithelial cells and normal cells, which is applied in the field of cell biology, can solve problems such as inflammation, and achieve the effects of normal response ability, strong three-dimensional sense, and normal differentiation function

Inactive Publication Date: 2017-03-15
SHENZHEN RES INST OF WUHAN UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the normal vaginal flora, antimicrobial factors can inhibit or kill other bacteria, however, once the ecological balance in the vagina is broken or invaded by exogenous pathogens, it can lead to inflammation

Method used

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  • Human normal vaginal epithelial cells and application thereof
  • Human normal vaginal epithelial cells and application thereof
  • Human normal vaginal epithelial cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] [Example 1] Primary isolation and culture of primary human normal vaginal epithelial cells

[0051] (1) With the informed consent of the patient or the patient guardian, collect the paracancerous normal tissue samples from surgically resected patients with vaginal cancer.

[0052] (2) Preparation of digestive solution: HL medium containing 0.2 mg / mL of collagenase and dispase; among them, HL medium is: complete DMEM (GIBCO#11965-092) and Ham's F-12 (GIBCO#GNM21700 ) culture medium was mixed at a volume ratio of 3:1, and 5% (v / v) fetal bovine serum was added at the same time, as well as 0.4 μg / mL cortisol (hydrocortisone), 5 μg / mL insulin (insulin), and 8.4 ng / mL cholera toxin (cholera toxin), 10ng / mL epidermal growth factor (epithelial growth factor (EGF)), 24μg / mL adenine (adenine), 100U / mL penicillin (penicillin), 100μg / mL streptomycin (streptomycin), 0.25μg / mL Amphotericin B (Fungizone), 30 μM Fasudil (Fasudil), the above culture medium needs to be filtered through ...

Embodiment 2

[0062] [Example 2] subculture of human normal vaginal epithelial cells

[0063] (1) When human normal vaginal epithelial 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), and then wash with 0.05% ( Mass volume ratio) trypsin-EDTA digest monolayer cells for 2-5 minutes.

[0064] (2) Add 10 mL of complete DMEM to neutralize the digestion reaction for 1-2 minutes.

[0065] (3) Centrifuge at 1000rmp for 5 minutes, discard the supernatant, resuspend the cell pellet and inoculate in 10mL HL medium.

[0066] (4) If necessary, 1×10 6 Epithelial cells were resuspended in 1-2 mL of cell freezing medium (90% fetal bovine serum and 10% DMSO, v / v), and stored in liquid nitrogen for future use.

[0067] Subculture human normal vaginal epithelial cells according to the above method, and the cell growth curve of the culture establishment line is as follows: figure 2 A, continuous subculturing for 140 days, the no...

Embodiment 3

[0068] [Example 3] Genotyping analysis and identification of human normal vaginal epithelial cells

[0069] (1) Human normal vaginal epithelial cells (1×10 6 ), wash the cells twice with 1×PBS, digest the monolayer cells with 0.05% trypsin-EDTA for 2-5 minutes, and neutralize the digestion reaction with 10 mL of complete DMEM.

[0070] (2) Centrifuge at 10,000 rpm for 1 minute, pour off the supernatant, add 200 μL buffer GA (Cell / Tissue Genomic DNA Extraction Kit DP304, Tiangen Company), and shake until thoroughly suspended.

[0071] (3) Add 20 μL of Proteinase K solution and mix well.

[0072] (4) Add 200 μL buffer solution GB (cell / tissue genomic DNA extraction kit DP304, Tiangen Company), fully invert and mix well, place at 70°C for 10 min, and briefly centrifuge.

[0073] (5) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, and briefly centrifuge.

[0074] (6) Add the obtained solution and the flocculent precipitate into an adsorption column (cell / tiss...

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Abstract

The invention discloses human normal vaginal epithelial cells and application thereof. The cells are named human normal vaginal epithelial cells HNVEC / HL-016, and the collection number is CCTCC NO:C2015113. The primary isolated culture method comprises the following steps: removing fat in a para-carcinoma tissue sample excised from a vaginal carcinoma patient, digesting, adding dispase and DNase I to act, filtering, centrifugating to collect the cells, resuspending the cells by using an HL culture medium, inoculating, and culturing. The subculture method comprises the following steps: carrying out cell proliferation until the abundance is 70-90%, carrying out pancreatin-EDTA (ethylene diamine tetraacetic acid) digestion, and neutralizing with a DMEM (dulbecco's modified eagle medium); and centrifugating to collect the cells, resuspending the cells by using an HL culture medium, inoculating, and culturing. The cells can be used for physiological research of human normal cells, drug toxicity research and detection of in-vitro normal cells, and research of pathogenesis of vagina / cervix-related diseases (including vaginal carcinoma, cervical carcinoma and infectious diseases).

Description

technical field [0001] The invention belongs to the field of cell biology and relates to a normal human vaginal epithelial cell and its application. Background technique [0002] The female uterus is an important place to conceive the fetus and reproduce the next generation. Therefore, the uterus is very important to every woman. Once the uterus becomes lesioned (usually caused by cervical cancer), it will seriously endanger the reproductive health of women and even be critical. life. The cervix is ​​one of the important tissues and organs in the female reproductive system. Its upper end is connected with the uterus, and its lower end penetrates into the vagina. This kind of specifically differentiated epithelial cells is directly related to the specific functions of this organ. For example, the epithelial cells of the ectocervix (vaginal epithelial cells) can protect the cervix from the damage of viruses and bacteria in the external environment, thereby ensuring the integr...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02C12R1/91
CPCC12N5/0625G01N33/5014G01N33/5044
Inventor 李晖叶立娜丘建斌吴小婷朱雅琪吴绪峰
Owner SHENZHEN RES INST OF WUHAN UNIVERISTY
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