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Mucosal melanoma cell and application thereof

A technology for melanoma cells and melanoma, applied in the field of mucosal melanoma cells, can solve the problems of difficult to obtain samples, cell aging, growth stagnation, etc.

Active Publication Date: 2021-08-03
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common problem of isolating mucosal melanoma cells from primary tumor foci to construct stable cell lines is that the samples are difficult to obtain and the samples are relatively small. Half of the samples cannot isolate tumor cells, even if tumor cells are isolated, but Cell aging and growth stagnation appear in most of the subcultures until about P5~P10 generation
To a certain extent, this reflects the complex in vitro culture conditions and high technical requirements for the construction of mucosal melanoma cell lines.

Method used

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  • Mucosal melanoma cell and application thereof
  • Mucosal melanoma cell and application thereof
  • Mucosal melanoma cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Establishment of cell line

[0071] The sources of specimens were the oral and maxillofacial head and neck of the Ninth People's Hospital of Shanghai Jiaotong University, an example of a 67-year-old female mucosal melanoma, primary stove is located in the lower teeth mucosa. Before the freezing activity, the patient and the guardian agreed to sign the informed consent, and the postoperative pathological diagnosis was: mucosa melanoma. Organizational dyeing is shown in Figure (9A).

[0072] Specific culture method:

[0073] The biopsy sample quickly loaded into the centrifuge tube containing the commercial tissue storage liquid (MACS, Germany). The centrifuge tube is loaded into the ice cartridge to the laboratory.

[0074] In the biosafety cabinet, the tissue was transferred to the culture dish, and the PBS immersion tissue containing amphotericin B (500 ng / ml) and 5% pyroxin double anti-anti-double anti-antigen was about 10 minutes. The mucosal tissue was remov...

Embodiment 2

[0080] Example 2 Cell scratches experiment

[0081] Cell scratches showed that MM9H-1 migration capacity is stronger than classic human skin melanoma cells A375 ( image 3 ).

[0082] Cell scratches experiments use wound healing plugs (IBIDI, Germany, # 81176) experiment, which can generate an neat scale area. The specific steps are as follows: Put the plug in 12-well plates, and the MM9H-1 cell digestion is prepared as a suspension of about 5 x 106 / ml cell suspension, inoculated in the plug-in, 80 microliter cytosin suspensions per well; 24 After the hour, under the mirror, the cells were observed, about 90 ~ 100% aggregation, remove the plug, add 500 microlitrated serum culture based on 12-well plate; microscope, as 0 baseline; observe once every 24 hours, take pictures, Determination of cell migration distance between scratches.

Embodiment 3

[0083] Example 3 Cell line melanoma-related marker staining

[0084] MM9H-1 of human mucosa tumor cells MM9H-1 was identified by immunofluorescence technology MM9H-1, MMB45, MELAN-A, S100A6, S100B for identification. Such as Figure 4 As shown, human mucosa melanoma cells MM9H-1 expression marker HMB45, MELAN-A, S100A6. MM9H-1 was detected by MM9H-1 with melanoma hybrid label antibody in P50 generation, HMB45, Melan-A and other markers were stable ( Figure 5 ).

[0085]MM9H-1 cells were inoculated into a cop-focus peani having a diameter of 12 mm. After 24 h, the culture solution, PBS was washed twice, and the pre-cooling 4% polymethyledehyde was fixed for 30 min, and 5 min × 3 times at room temperature PBS. 0.1% Triton X-100 treatment 10min, room temperature PBS wash 5 min × 3 times, 5% goat blood cleaning closed 30 min, add dilution imperative (HMB45, Melan-a, S100A6) placed in a wet cartridge, 4 ° C overnight, room temperature PBS wash To the anti-anti-affordable, 10 min × 3 tim...

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Abstract

The invention relates to the field of cell engineering, in particular to a mucosal melanoma cell and application thereof, and the preservation number of the human mucosal melanoma cell MM9H-1 is CCTCC (China Center For Type Culture Collection) NO: C 202174. Themucosal melanoma cell can be used for constructing a melanoma animal model. The mucosal melanoma cells and the melanoma animal model can be used for preparing products for diagnosing or treating melanoma, screening melanoma biomarkers or researching the occurrence and development mechanism of melanoma. The human mucosal melanoma cell MM9H-1 has immortality, does not age after continuous passage for one hundred generations, can express HMB45, Melan-A and S100A6 markers, and has a triploid karyotype.

Description

Technical field [0001] The present invention relates to the field of cellular engineering, and more particularly to a mucosa melanoma cell and its use thereof. Background technique [0002] Mucosa malignant melanoma [0003] Malignant melanoma is a highly malignant tumor derived from melanin cells. Tumoroma can occur in the skin or mucosa, which is mostly developed by mucosal black spots or skin melanin. The onset of melanoma has a certain race species: Caucasians are more likely to occur skin malignant melanoma, and mucosa black tumor is relatively small; and the yellow human mucosa and limb melanoma are more common. Mucosal Melanoma, MM) includes oral, oral, nasal / sinus, esophagus, rectal anal tube, reproductive tract, urinary tract, melanoma, is the second larger subtype of Asian black tumor (accounting for 22.6 %), Especially in China, mucosa malignant melanoma accounts for 22.6% -25% of the general malignant melanoma. It is much higher than the European and American popula...

Claims

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Application Information

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IPC IPC(8): C12N5/09A01K67/027
CPCC12N5/0693A01K67/0271A01K2227/105A01K2267/0331Y02A50/30
Inventor 石超吉顾子悦周榕徐声铭韩永任国欣张志愿
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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