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Method for establishing human nasopharyngeal carcinoma tumor stem cell line

A tumor stem cell line, human nasopharyngeal carcinoma technology, applied in the biological and medical fields, can solve the problem of high incidence of nasopharyngeal carcinoma, and achieve the effects of simple operation, strong clone formation ability, and strong drug tolerance

Inactive Publication Date: 2015-04-22
GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the current high incidence of nasopharyngeal carcinoma, the present invention provides a method for establishing a human nasopharyngeal carcinoma tumor stem cell line, so as to promote and promote the diagnosis and treatment of nasopharyngeal carcinoma

Method used

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  • Method for establishing human nasopharyngeal carcinoma tumor stem cell line
  • Method for establishing human nasopharyngeal carcinoma tumor stem cell line
  • Method for establishing human nasopharyngeal carcinoma tumor stem cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Obtaining nasopharyngeal carcinoma sphere-forming cells

[0036] 1. Place nasopharyngeal carcinoma cells CNE-2, SUNE-1, and HONE-1 in DMEM / F12 (Gibco) medium containing 10% FBS (fetal bovine serum, Gibco) plus double antibodies at 37°C, 5% CO 2 Culture in the incubator;

[0037] 2. Select nasopharyngeal carcinoma cells CNE-2, SUNE-1, and HONE-1 in the logarithmic growth phase, digest them with trypsin at a mass volume percentage concentration of 0.25% to form a single-cell suspension, and add an appropriate amount of serum-containing medium to terminate Trypsinize, centrifuge at 1000rpm for 5min, discard the supernatant, add 1ml of serum-free stem cell medium containing epidermal growth factor EGF and basic fibroblast growth factor bFGF, and mix well. The medium is DMEM / F12, where The final concentration of EGF and bFGF is 20ng / ml;

[0038] 3. Cell counting: take 20ul and 20ul trypan blue to mix, take a cell counting plate and flush the pool, and count t...

Embodiment 3

[0052] Example 3 Drug resistance of nasopharyngeal carcinoma sphere-forming cells to the antineoplastic drug gemcitabine

[0053] 1. Prepare a 100 μmol / mL gemcitabine solution:

[0054] The molecular weight of gemcitabine is 299.66, that is, 29.966 mg / mL = 0.1mol / L, so weigh 0.2997g of gemcitabine and dissolve it in 10ml of DMEM / F12 medium, filter and sterilize, and store at 4°C for later use;

[0055] 2. Collect nasopharyngeal carcinoma sphere-forming cells CNE-2s, HONE-1s, and SUNE-1s in the logarithmic growth phase, and nasopharyngeal carcinoma cells CNE-2, HONE-1, and SUNE-1 used as reference, and digest them as Single cell suspension, count the cells, the counting steps are the same as step 3 in Example 1, adjust the cell concentration to 3 × 10 4 / mL;

[0056] 3. MTT detects the sensitivity of two types of cells to gemcitabine:

[0057] (1) Take nasopharyngeal carcinoma sphere-forming cells CNE-2s, HONE-1s and SUNE-1s in the logarithmic growth phase, and nasopharyn...

Embodiment 4

[0060] Example 4 RT-PCR method to detect the expression of stemness factor

[0061] 1. Design primers according to Table 1

[0062] Table 1

[0063]

[0064] 2. Total RNA extraction (TRIZOL, Invitrogen)

[0065] (1) Treatment of adherent cells CNE-2, HONE-1 and SUNE-1: Rinse the treated cells once with ice-cold PBS, directly add Trizol for lysis, the volume of Trizol is 10cm 2 / mL ratio added;

[0066] Treatment of suspension culture cells CNE-2s, HONE-1s and SUNE-1s: collect the cell spheroid suspension, centrifuge to remove the cell culture medium, then wash once with ice-cold PBS, and directly add Trizol to lyse;

[0067] (2) After adding Trizol, place at room temperature for 5 minutes to fully lyse;

[0068] (3) Centrifuge at 12000rpm at 4°C for 15 minutes, and discard the precipitate. This step is used to remove some insoluble substances such as cell outer membrane, high molecular weight DNA, polysaccharides, etc.;

[0069] (4) Add chloroform to 200μl chloroform...

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Abstract

The invention provides a method for establishing a human nasopharyngeal carcinoma tumor stem cell line. The method comprises the following steps: (1) obtaining human nasopharyngeal carcinoma cells in the single cell state; (2) adding the human nasopharyngeal carcinoma cells in the single cell state, obtained in the step (1), to a serum-free stem cell culture medium containing epidermal growth factors and basic fibroblast growth factors to obtain single cell suspension; (3) adjusting the concentration of the single cell suspension in the step (2) to 900-1100 single cells / ml; (4) inoculating 1ml of the single cell suspension obtained in the step (3) into single pores on a cell culture six-pore plate and then adding the equivoluminal stem cell culture medium for culture for 72-96 hours; and (5) collecting the cells obtained in the step (4), carrying out trypsinization till the single cell and carrying out subculture. The method provided by the invention is simple and convenient to operate; and the obtained nasopharyngeal carcinoma stem cell line has obvious stem cell characteristics.

Description

technical field [0001] The invention relates to the fields of biology and medicine, in particular, the invention relates to a method for establishing a human nasopharyngeal carcinoma tumor stem cell line. Background technique [0002] Nasopharyngeal carcinoma (NPC) refers to epithelial malignant tumors that occur on the epithelium of the surface of the nasopharyngeal cavity or the epithelium of the nasopharyngeal crypts. The incidence of nasopharyngeal carcinoma has local characteristics. my country is the country with the highest incidence of nasopharyngeal carcinoma in the world. South China, especially Guangdong and its adjacent areas such as Guangxi and Hong Kong are high-incidence areas, and the incidence rate is higher than that of most other countries. The national area is more than 100 times higher, so it is also called "Guangdong tumor". At present, the pathogenesis of nasopharyngeal carcinoma is still unclear and needs further study. [0003] Stem cells (stem cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/095
Inventor 周克元李涛李彩虹冀天星黄颖林碧华张鑫
Owner GUANGDONG MEDICAL UNIV
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