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Human tumor-associated fibroblast cell lines and application thereof

A fibroblast, tumor-related technology, applied in the direction of tumor/cancer cells, animal cells, vertebrate cells, etc., can solve problems such as direct research difficulties

Pending Publication Date: 2022-01-25
SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, existing studies have shown that the appearance of tumor-associated fibroblasts may precede tumorigenesis
Due to the heterogeneity of tumor-associated fibroblasts, and the same cell shape, there are also different functions, so it is difficult to conduct direct research at the tissue level

Method used

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  • Human tumor-associated fibroblast cell lines and application thereof
  • Human tumor-associated fibroblast cell lines and application thereof
  • Human tumor-associated fibroblast cell lines and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Detection of protein expression in human tumor-associated fibroblasts by Western blotting

[0033] 1) Take the cells in the logarithmic growth phase, suck off the medium and wash with PBS three times, add an appropriate amount of protein lysate, and lyse on ice for 15 minutes;

[0034] 2) Scrape off the cells with a clean cell scraper, and then transfer them to a 1.5ml EP tube;

[0035] 3) Place the EP tube in a 4-degree centrifuge at 12,000 rpm for 10 minutes, take the supernatant and place it in a 1.5ml EP tube;

[0036] 4) For protein quantification by BCA method, add 5×SDS-PAGE protein loading buffer according to the volume of the sample, mix it well, cook for 10-15min in a metal bath at 95°C. Centrifuge at 1000rpm for 5 minutes, wait for the sample to cool down, then you can choose to load the sample or - store it in a refrigerator at -20°C;

[0037] 5) Glue dispensing: Place the glass plate vertically and align it, put it on the sponge pad of the shel...

Embodiment 2

[0045] Example 2: Tumor-associated fibroblasts promote tumor metastasis

[0046] 1. Method

[0047] 2. Ⅰ. Scratch test:

[0048] 1) Digest the cells with trypsin containing 0.25% EDTA, resuspend the cells with basal medium and adjust the cell suspension to 5×10^5 cells / ml.

[0049] 2) Evenly spread the counted cell suspension into a 12-well plate at an amount of 1ml per well.

[0050] 3) After the cells adhere to the wall for 24 hours, scrape with a sterile wound replicator. After scraping, add PBS to the well, shake the well plate lightly to remove the scratched cells, and repeat 3 times.

[0051] 4) Add 95CAF-CM to the experimental group, place it under a microscope to take pictures and take samples, and mark the sampling position. Continue to cultivate after taking pictures.

[0052] 5) After culturing in the incubator for 48 hours, take pictures again at the same sampling point.

[0053] 6) The scratch healing rate is calculated as follows: healing rate (%)=(scratch di...

Embodiment 3

[0065] Example 3: Tumor-associated fibroblasts promote HUVEC tube formation

[0066] 1. Method

[0067] 1. Take 95-CAF in the logarithmic growth phase, culture with DMEM medium plus 1% double antibody for 48 hours, filter the obtained medium and add 10% serum;

[0068] 2. Stimulate HUVEC cells with 95-CAF conditioned medium for half an hour;

[0069] 3. Spread matrigel in 96-well plate. The process of laying matrigel should be low temperature, fast, uniform, spread on the ground, uniform in thickness, and the tip of the pipette should be pre-cooled, 50 μl per well, and then placed in a 37° incubator for 30 minutes polymerization;

[0070] 4. After the stimulation, remove the CM, wash with PBS, then digest it, count, control the same number of cells in each well, and plant them in the matrigel of a 96-well plate;

[0071] 5. The laid cells were cultured in a 37° incubator, and observed under a microscope at 4h and 6h.

[0072] 2. Results

[0073] In the 96-well plate, comp...

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Abstract

The invention belongs to the field of tumor biology, and relates to human tumor-associated fibroblast cell lines and an application thereof. The human tumor-related fibroblast cell lines have the following biological characteristics: long fusiform or astral shape, rich cytoplasm and eosinophilic property, cell nucleus positioned in the center of the cell body in circular or oblong shape, radial or beam arrangement of cells , part of the cells disordered in arrangement, polarity disappearance, obvious crossing and overlapping phenomena, and stable passage in ten generations. The proteins of alpha-SMA, FAP, E-cadherin and Vimentin expressed by the cells are positive. The human tumor-associated fibroblast cell lines have the advantages of achieving long-term in-vitro culture while keeping cell characteristics unchanged. The human tumor-associated fibroblast cell lines are a powerful tool for related researches of tumor-related fibroblast cells, and a powerful tool for researching generation and development of tumor-related fibroblast mediated tumors and related molecular action mechanisms, developing drug targets for treating tumors, novel tumor biological treatment technologies and detecting related bioengineering product researches.

Description

technical field [0001] The invention belongs to the field of tumor biology and relates to a human tumor-associated fibroblast and its application. Background technique [0002] The tumor microenvironment provides strong support for the tumor during the occurrence and development of the tumor, and tumor-associated fibroblasts are one of the most important components in the tumor microenvironment. It can be differentiated from various types of cells under the action of the inside of the cell, such as normal fibroblasts, adipocytes, pericytes, etc., which can be used as a source of human tumor-associated fibroblasts. Human tumor-associated fibroblasts can build and remodel the structure of extracellular matrix, and also have a certain secretion function, which can secrete cytokines and inflammatory factors such as interleukin 6 (IL-6), chemokine (CXCL12), etc. While maintaining tumor tissue morphology, it can mediate multidrug resistance, invasion and proliferation of tumor ce...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02A01K67/027C12R1/91
CPCC12N5/0693G01N33/5011A01K67/0271A01K2207/12A01K2267/0331G01N2500/10
Inventor 徐可殷佩浩朱锐秋张祎稀王海静詹月萍袁泽婷李炜王杰方凯汪玉倩许健
Owner SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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