Human colon cancer extracellular matrix in-vitro model and preparation method thereof

A colon cancer cell and extracellular matrix technology, applied in the field of basic medical research, can solve the problems of inability to get rid of, cannot truly reflect the characteristics of ECM changes, and cannot accurately reflect the role of ECM cells, so as to reduce the time-consuming process and simplify the process flow Effect

Inactive Publication Date: 2014-12-10
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, all ECM-related studies have been conducted through studies on the expression levels of related genes or proteins in tumor tissues, or in vitro experimental studies using tumor cells in a three-dimensional culture environment with ECM components added, which is very one-sided: 1. The ECM is not separated and studied separately, and it cannot get rid of the interference of other factors such as cells or peptide factors, so it cannot truly reflect the change characteristics of the ECM; 2. Since the ECM in vivo cannot be truly simulated, the research based on the in vitro culture environment cannot Exactly reflect the effect of ECM on cells in the tissue
Therefore, the current research results on tumor ECM are still far from revealing the characteristics and process of tumor ECM remodeling and its effect on cells.
There are similar deficiencies in the study of EMT: in tumor tissue, EMT occurs temporarily and occasionally in time and space, and conventional pathological analysis cannot detect EMT.
However, so far, no study has been able to construct an in vitro model that truly reflects the ECM

Method used

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  • Human colon cancer extracellular matrix in-vitro model and preparation method thereof
  • Human colon cancer extracellular matrix in-vitro model and preparation method thereof
  • Human colon cancer extracellular matrix in-vitro model and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of an extracellular matrix model of human colon cancer cells

[0036] 1. Pretreatment of colon cancer tissue samples

[0037] A total of 30 cases of colorectal cancer specimens and corresponding normal tissues of stages I to IV were collected. The specific case information is shown in Table 1. Fresh colon cancer tissue with a size of 1 cm×1 cm×1 cm was taken and washed 3 times with pure water. The fresh tissue refers to surgically removed tumor tissue within 4 hours of in vitro.

[0038] 2. Sterilization

[0039] Sterilization by low-concentration peracetic acid-ethanol solution method, this step is carried out in a constant temperature shaker, wherein the volume percentage of peracetic acid is 0.1%, the sterilization time is 1h, the shaking frequency is 200rpm, and the temperature range at 4-40°C, then wash in phosphate buffer for 2-5 times, each time for 15 minutes, check the pH value of the washed phosphate buffer, when the pH reaches 6.5-7....

Embodiment 2

[0049] Example 2: Detection of porosity of the extracellular matrix of acellular colon cancer cells prepared in Example 1

[0050] The porosity of the material was determined by mercury intrusion porosimetry. Result: The porosity is not less than 85%.

Embodiment 3

[0051] Example 3: Histological detection of the extracellular matrix of acellular colon cancer cells prepared in Example 1

[0052] 1) Observation with optical microscope: Method: The paraffin-coated material was stained with hematoxylin-eosin, and observed with an inverted phase-contrast microscope. Results: No cells and cell debris remained, collagen fibers were well preserved, and with the increase of colon cancer stage, the content, density and distribution of collagen fibers in the extramatrix of colon cancer cells increased, as shown in figure 1 shown.

[0053] 2) Observation of ultrastructure. Results: The material has a porous structure with uniform pore size, the average pore size is 200um, and the porosity is greater than 85%. With the increase of colon cancer stage, the porosity and surface roughness of colon cancer cell extramatrix increase, as shown in figure 2 , image 3 shown.

[0054] 3) Masson staining was used to observe the configuration characteristics...

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Abstract

The invention provides a human colon cancer extracellular matrix in-vitro model and a preparation method thereof. The model is a freeze-dried sterile porous membrane without cytotoxic substance residues; and in the model, the cell components are completely removed, and the components and structure of the extracellular matrix are completely maintained. The preparation method comprises the following steps: taking a human colon cancer or rectum cancer tissue which is exsomatized within 4 hours, and carrying out pretreatment; sterilizing in a constant-temperature oscillating table by using a low-concentration peroxyacetic acid-ethanol solution; cleaning with a 5-100 mmol / L sodium hydroxide solution in a constant-temperature ultrasonic cleaner with an oscillatory cleaning tank for 30-90 minutes to perform decellularization; putting the material in a freeze drier, and carrying out freeze-drying forming according to a predesigned freeze-drying process; and packaging in a sterile state. According to the method, the colon or rectum cancer extracellular matrix is separated for independent research to construct the model outside the ideal body and inside the animal body for colon or rectum cancer microenvironment and EMT research.

Description

technical field [0001] The invention relates to the technical field of basic medical research, in particular to a method for preparing an extracellular matrix model of human colon cancer cells, and an extracellular matrix model of human colon cancer cells obtained by the preparation method. Background technique [0002] Malignant tumors are not just the autonomous proliferation or cloning of tumor cells caused by gene mutations, but the complex development of the interaction and mutual influence between tumor cells, stromal cells and other types of cell components and special microenvironment The study of tumor cells itself cannot fully reveal the mechanism of tumor occurrence and development. The tumor microenvironment is a complex comprehensive system, which is composed of extracellular matrix (ECM), interstitial cells, various peptide factors (growth factors, chemokines, cytokines, antibodies) and their metabolites. It is a complex interaction between tumor cells and str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 赵博王振军
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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