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Tumor perivascular cell as well as separation method and application thereof

A technology of tumor blood vessels and separation methods, which is applied in the direction of cell dissociation methods, vascular endothelial cells, tumor/cancer cells, etc., can solve the problems that the biological functions of tumor pericytes cannot be reflected, and achieve low cost, comprehensive types, and simple operation Effect

Pending Publication Date: 2021-11-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of tumor cells to educate HBVPs cannot reflect the biological functions of real tumor pericytes because the physiological functions and characteristics of cerebral blood vessels are very different from those of tumor blood vessels.

Method used

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  • Tumor perivascular cell as well as separation method and application thereof
  • Tumor perivascular cell as well as separation method and application thereof
  • Tumor perivascular cell as well as separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Dissection of human colorectal cancer blood vessels and isolation and culture of human colorectal cancer perivascular cells

[0073] Experimental method: refer to figure 1The flow shown is to take fresh human colorectal cancer samples (collected by the Department of Gastrointestinal Surgery of Guangzhou Overseas Chinese Hospital, and the collected samples were informed of the research purpose and signed the informed consent. According to clinical imaging, serum oncogenic protein test, For malignant colorectal cancer diagnosed preoperatively by biopsy and other technical means, fresh tumor specimens including cancer focus and paracancerous tissue are obtained for surgical resection, and washed with DMEM medium containing 1% (v / v) green chain double antibody to remove residual Pollutants such as feces and blood stains are stored in DMEM medium containing 1% (v / v) penicillin-streptomycin), and in a clean bench, use 1% (v / v) PS (penicillin-streptomycin, penicilli...

Embodiment 2

[0075] Example 2 Determination of expression of molecular markers of human colorectal cancer pericytes by flow cytometry

[0076] Experimental method: the human colorectal cancer vascular cells obtained in Example 1 were resuspended with 100 μL of cell staining buffer (staining buffer) and then transferred to a 1.5 mL EP tube, and then 1 μL of anti-CD32-PEblocking (Miltenyi Cat.No.130-097-521) prototype control solution was blocked on ice for 10 minutes; then added anti-FAPα-PE (R&D Cat.No.FAB3715P), anti-NG2-PE (Miltenyi Cat.No.130-097-458), anti-PDGFRβ-PE (Miltenyi Cat.No.130-105-323) and anti-CD146-PE (Miltenyi Cat.No.130-097-939) four pericyte positive molecular markers and anti-CD31-PE (Miltenyi Cat.No.130-110-807), MYH11( Cat.No.PA5-82526) two negative molecular marker flow antibodies, incubate on ice in the dark for 30-60 minutes, wash twice with PBS; add 1 μg / mL DAPI for nuclear staining, keep in the dark on ice Incubate for 10 minutes, wash twice with PBS;...

Embodiment 3

[0078] Example 3 Immunofluorescence assay for the expression of molecular markers of human colorectal cancer perivascular cells

[0079] Experimental method: After the human colorectal cancer perivascular cells obtained in Example 1 were resuspended, 1×10 5 Cells were seeded on laser confocal small dishes at a density of 24 hours, discarded the culture medium and washed with PBS, fixed with 4% (w / v) paraformaldehyde (PBS with a solvent of 0.1M, pH=7.4) at room temperature for 30 minutes, and 0.1 Permeabilize the membrane with %Triton-X100 for 3 minutes, block with 5% BSA at room temperature for 1 hour, add anti-FAPα (R&D Cat.No.AF3715), anti-NG2 (R&D Cat.No.MAB2585), anti-PDGFRβ (R&D Cat.No.AF385) and anti-CD146 (R&D Cat.No.AF932) four pericyte positive molecular markers and anti-CD31 (R&D Cat.No.BBA7), anti-MYH11 ( Cat.No.PA5-82526) the primary antibodies of two negative molecular markers were incubated overnight at 4°C. Wash 3 times with PBS every other day, ea...

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Abstract

The invention discloses a tumor perivascular cell as well as a separation method and application thereof, and belongs to the technical field of biology. The method provided by the invention comprises the steps: stripping a fresh solid tumor tissue sample to obtain tumor blood vessels, cutting the tumor blood vessels into annular segments, then putting the tumor blood vessels of the annular segments into a pore plate pre-paved with matrigel, culturing by using a perivascular cell culture medium, and finally recovering the tumor perivascular cells from the matrigel. The tumor perivascular cells provided by the invention highly express four positive molecular markers of FAP[alpha], NG2, PDGFR[beta] and CD146, and do not express two negative molecular markers of CD31 and MYH11. By utilizing the tumor perivascular cells, a breakthrough of zero construction of an immortalized human colorectal cancer perivascular cell model is realized. The invention can provide an original material with reliable source, sufficient quantity and stable quality for researching biological functions and drug targets of solid tumor perivascular cells, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a tumor vascular pericyte and its separation method and application. Background technique [0002] Tumor pericytes (tumor pericytes, TPCs) are a kind of wall cells located in the periphery of tumor blood vessels and embedded in the basement membrane. aspects play an important role (1-3). The regulatory role of tumor pericytes on tumor progression has become a research hotspot in recent years. At present, one of the biggest obstacles in the field of tumor pericytes research is the lack of specific molecular markers for tumor pericytes, which not only affects the identification of tumor pericytes, but also limits the isolation and sorting of tumor pericytes. First, unlike other types of cells in tumor tissues, tumor pericytes still lack specific molecular markers, and most studies generally use markers such as NG2, CD146, PDGFRβ, CD13 and αSMA to indicate tumor pericytes ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/10C12N15/37C12N15/867
CPCC12N5/069C12N15/86C07K14/005C12N2509/00C12N2509/10C12N2501/135C12N2710/22022C12N2740/15043C12N2510/04C12N2533/90C12N5/0679C12N5/0693C12N2502/28C12N2510/00C12N2740/16043A61K35/44A61P35/00
Inventor 张冬梅陈敏锋叶文才潘京华李勇刘同征齐琦
Owner JINAN UNIVERSITY
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