Human prostate cancer cell and its primary isolation culture and subculture method and application

A prostate cancer, prostate technology, applied in the field of cell biology, can solve the problems of tumor research and new anti-cancer drug research going astray

Active Publication Date: 2015-10-21
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, genetic manipulation will change the genetic background and phenotype of these cells. For example, the p53 and pRB signaling pathways are often inhibited. Therefore, using this gene-transferred cell as a model will lead to fundamental changes in tumor research and anti-cancer drug research. astray

Method used

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  • Human prostate cancer cell and its primary isolation culture and subculture method and application
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  • Human prostate cancer cell and its primary isolation culture and subculture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] [Example 1] Primary isolation and culture of human prostate cancer cells

[0051] (1) With the informed consent of the patient or the patient's guardian, 1–2 cm prostate cancer tissue was collected from a 57-year-old prostate cancer patient 3 (cubic centimeters), the patient's clinical stage was T3b poorly differentiated adenocarcinoma, and no other treatment was given.

[0052] (2) Preparation of digestive solution: HL medium containing 0.2 mg / mL of collagenase and dispase; among them, HL medium is a mixed medium of DMEM and Ham's F-12 NUTRIENT MIX, and the volume ratio is 3: 1. At the same time add 5% fetal bovine serum, 0.4μg / mL cortisol (hydrocortisone), 5μg / mL insulin (insulin), 8.4ng / mL cholera toxin (cholera toxin), 10ng / mL epidermal growth factor (epithelial growth factor) factor (EGF)), 24 μg / mL adenine, 100 U / mL penicillin and 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B (Fungizone,) 30 μM fasudil (Fasudil).

[0053] (3) Wash the isolated tissue sample...

Embodiment 2

[0062] [Example 2] Subculture of human prostate cancer cells

[0063] (1) When human prostate cancer cells cultured in T25 or T75 culture flasks proliferate to 70-90% abundance, wash the cells twice with 1×PBS (0.01M, pH7.4), and then wash with 0.05% ( Mass volume ratio) trypsin-EDTA digest monolayer cells for 2-5 minutes.

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

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

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

[0067] Subculture human prostate cancer cells according to the above method, and the cell proliferation doubling curve of the culture line is as follows: figure 2 , continuously subcultured for 90 days, and the number of culture ...

Embodiment 3

[0068] [Example 3] Karyotype analysis and identification of human prostate cancer cells

[0069] (1) When human prostate cancer 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.

[0070] (2) Repeatedly blow the cells to make them fall off, and centrifuge at 2000rpm for 5 minutes to harvest the cells.

[0071] (3) Discard the supernatant, add 8 mL of 0.075mol / 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.

[0072] (4) Add 1 mL of freshly prepared fixative (methanol : Glacial acetic acid=3 : 1, v / v), carefully pipette, mix, and centrifuge at 2000rpm for 5 minutes.

[0073] (5) Discard the supernatant, add 8 mL of fixative, pipette to make a cell suspension, and fix at room temperature for 20 minutes.

[0074] (6) Centrifuge at 2000rpm for 5 minutes, discard the supernatant, and repeat the fixati...

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Abstract

The invention discloses a cell strain from human prostate cancer. The cell is named as human prostate cancer cell HPCC / HL-002, and the preservation number is CCTCCNO:C2013102. The cell strain is prepared from an exairesis cancer tissue sample of a prostate cancer patient with T3b poorly differentiated adenocarcinoma according to clinical stages, has typical tumor cellular biology characteristics, can be continuously passed for a long time in vitro, and is kept stable in character. In addition, the invention further discloses a preparation method of the cell strain. The human prostate cancer cell disclosed by the invention has the biology characters of clinical essential prostate, and can be applied to molecular mechanism researches on pathogenesis, invasion and metastasis of prostate, pharmacodynamics study and detection on anti-cancer medicines selected in vitro.

Description

technical field [0001] The invention belongs to the field of cell biology, and relates to a human prostate cancer cell and its primary separation culture and subculture method and application. Background technique [0002] The establishment of tumor cell lines in vitro is a basic method for screening tumor-targeted drugs. However, in basic research and clinical applications at home and abroad, almost all tumor cell lines are not primary site cancer cell lines, and most of them are highly differentiated and metastatic tumor sources, which cannot reflect and represent the true primary site tumor biology. characteristic. At present, nearly a thousand cancer cell lines used in the world have been cultured and passaged in vitro for several years or even decades, and they can no longer reflect the characteristics of cancer cells in cancer patients. Some cell lines have more than 50% of their genes. Variation, and cross-contamination of a large number of cancer cell lines in some...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12Q1/02
Inventor 李晖张百芳付振明彭晓叶琳
Owner WUHAN UNIV
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