CN112961830A - Oral squamous carcinoma tissue acellular matrix and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of medical materials, and discloses an oral squamous carcinoma tissue acellular matrix and a preparation method and application thereof. The preparation method mainly comprises the following steps: cleaning oral squamous carcinoma tissue and cutting into blocks; treating with 1% Triton-X100 on a shaking table for 12h to obtain a primary acellular tissue; treating with 1% SDS for 12h on a shaker; shaking the mixture on a shaking table for 24 hours by using DNAse I with the concentration of 100U/ml; washing with PBS for 12h on a shaking table to obtain a acellular matrix; the acellular matrix is placed in a 0.1U/L streptomycin solution for closed preservation. The preparation method has low requirement on required equipment, and the cell-removing reagent is common and cheap, so the preparation method has low construction cost and strong practicability and is suitable for popularization.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to an oral squamous carcinoma tissue acellular matrix and a preparation method and application thereof.
Background
Worldwide, the incidence of Oral cancer is the sixth place of systemic malignancy, at least 80% of which are Squamous Cell carcinomas, and in recent decades, despite advances in therapeutic approaches, 5-year survival rates of patients with Oral Squamous Cell Carcinoma (OSCC) have not been significantly improved, and the mechanisms of development of OSCC have not been elucidated, so that there is an urgent need for further research on tumor biological behavior. The current common tumor research methods include: two-dimensional (2D) culture, tumor xenograft animal models, and synthetic bioscaffolds. Three-dimensional (3D) structures in which 2D culture lacks appropriate cell-cell and cell-Extracellular matrix (ECM) interactions; animal models can mimic the human tumor microenvironment, but animals introduce a number of uncontrollable variables, including the production of endogenous growth factors and lack of the immune system; the artificial scaffold material can establish a 3D structure for cell growth, but lacks various biological components required by cells.
The extracellular matrix (ECM) is composed of a three-dimensional network, including capillaries, lymphatic and nerve endings, defense cells and basement membranes, in addition to proteoglycans and glycosaminoglycans, collagen, elastin, and basal fibers. The ECM function is important, and is responsible for not only transport of nutrients and elimination of metabolic end products, but also immune defense functions. The scaffold material prepared by the acellular method removes cell components in tissues, completely retains extracellular matrix (ECM) components and a three-dimensional structure to the maximum extent, and can simulate a three-dimensional microenvironment for tumor growth through a decellularized oral squamous cell carcinoma tissue scaffold (dECM) based on the characteristics, so that a new thought is provided for the research of tumor biology.
Disclosure of Invention
The present invention aims to solve the above technical problem at least to some extent. Therefore, the first purpose of the invention is to provide a preparation method of the oral squamous carcinoma tissue acellular matrix.
The technical scheme adopted by the invention for realizing the first purpose is as follows:
a preparation method of an oral squamous carcinoma tissue acellular matrix comprises the following steps:
(1) cleaning fresh or freeze-thawed squamous cancer tissue of the oral cavity and cutting into blocks;
(2) treating with 1% Triton-X100 on a shaking table for 12h to obtain a primary acellular tissue;
(3) treating with 1% SDS for 12h on a shaker;
(4) shaking the mixture on a shaking table for 24 hours by using DNAse I with the concentration of 100U/ml;
(5) washing with PBS for 12h on a shaking table to obtain a acellular matrix;
(6) the acellular matrix is placed in a 0.1U/L streptomycin solution for closed preservation.
Preferably, in the step (1), the oral squamous carcinoma tissue is washed 3 times with PBS, and the oral squamous carcinoma tissue is cut into pieces at 4 ℃.
Preferably, the steps (2) to (5) are all carried out at room temperature.
Preferably, after the step (2), the step (3) and the step (4) are finished, the acellular tissues are washed by PBS.
Preferably, the preservation temperature in the step (6) is 3-5 ℃ or-90 ℃ to-70 ℃.
Preferably, the rotation speed of the shaking table in the steps (2) to (5) is 225 rpm.
The second purpose of the invention is to provide the oral squamous carcinoma tissue acellular matrix prepared by the method.
The third purpose of the invention is to provide the application of the oral squamous carcinoma tissue acellular matrix as a culture scaffold.
The fourth purpose of the invention is to provide an application of the oral squamous carcinoma tissue acellular matrix in the construction of oral squamous carcinoma organoids.
The fifth purpose of the invention is to provide an application of the oral squamous carcinoma tissue acellular matrix in tumor drug screening and related detection.
The invention has the beneficial effects that:
the invention provides
1. The preparation method of the oral squamous carcinoma tissue acellular matrix has low requirement on equipment, and the acellular reagent is common and cheap, so the construction cost is low, the practicability is high, and the preparation method is suitable for popularization. The prepared oral squamous carcinoma tissue acellular matrix is completely acellular after physical, chemical and enzyme treatment, has little nucleic acid residue, and can basically preserve the biological components (type I collagen, type IV collagen, fibronectin and laminin) of the original matrix, thereby being capable of simulating the extracellular matrix microenvironment for the growth of in vivo tumor cells, leading the biological characteristics of the cultured oral tumor cells and the related cells to be closer to the in vivo growth state, and improving the reliability and repeatability of the experiment.
2. The oral squamous carcinoma tissue acellular matrix is suitable for researching the interaction between tumor cells and related cells and ECM and the interaction mechanism between the tumor cells. The method is used for researching tumor growth microenvironment, and is helpful for further researching tumor biological behaviors, thereby providing possibility for realizing targeted extracellular matrix treatment.
3. The oral squamous carcinoma tissue acellular matrix can be used for researching the influence of a tumor microenvironment on the biological behaviors of tumor cells, macrophages and tumor-related cells, so that the influence of the tumor microenvironment on the aspects of tumor promotion and immunosuppression is researched, the development of oral squamous carcinoma is favorably and deeply known, and the possibility is provided for realizing the accurate treatment of the oral squamous carcinoma.
Drawings
FIG. 1 is a schematic diagram showing a pre-and post-comparison of a decellularized tissue specimen according to the invention.
FIG. 2 is a graph showing the DNA content of the acellular matrix according to the present invention.
FIG. 3 is a schematic representation of the histological staining of the acellular matrix according to the present invention.
FIG. 4 is a schematic representation of the microstructure of the tissue before and after decellularization of the tissue according to the invention.
FIG. 5 is a diagram of the structure of the acellular matrix ECM of the present invention.
FIG. 6 is a graph showing the results of an experiment in accordance with example one of the acellular matrix of the present invention.
FIG. 7 is a graph showing the results of an experiment of example two acellular matrices according to the present invention.
Detailed Description
The advantages and features of the invention will become more apparent from the following further description of the invention given in conjunction with specific embodiments. However, the examples are only for illustrating the present invention and do not set any limit to the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The preparation method of the oral squamous carcinoma tissue acellular matrix comprises the following steps:
(1) the oral squamous carcinoma tissue specimen can be taken from oral and maxillofacial surgery, the specimen is placed in an ice box at 4 ℃ after being separated, the ice box is transferred to a laboratory, the specimen is washed for 3-4 times by PBS (phosphate buffer solution), and the specimen is subjected to subsequent treatment after bloodstain is removed or is placed in a refrigerator at-80 ℃ for preservation.
Taking out the specimen, placing at room temperature, washing the specimen with PBS for 3 times, cutting the tissue into small pieces with a size of about 1cm on ice3。
(2) Treating with 1% Triton-X100 (polyethylene glycol octyl phenyl ether) on a shaker at 225rpm at room temperature for 12 hr to obtain primary acellular tissue;
the primary decellularized tissue was repeatedly washed with PBS several times at room temperature until substantially clean.
(3) Treating with 1% SDS (sodium dodecyl sulfate) at room temperature for 12h on a shaker at 225rpm to obtain decellularized tissue;
the decellularized tissue was washed several times with PBS at room temperature until substantially clean.
In order to ensure the complete cell removing effect, the invention continues to carry out the following treatment on the cell removal.
(4) Shaking the mixture on a shaking table at the rotating speed of 225rpm for 24 hours at room temperature by using DNAse I (deoxyribonuclease I) of 100U/ml to obtain a acellular matrix;
the acellular matrix was washed 3-4 times with PBS at room temperature.
(5) Washing with PBS for 12h at room temperature on a shaker at a rotation speed of 225rpm to obtain the final acellular matrix;
(6) placing the acellular matrix in 0.1U/L streptomycin solution, sealing the opening of an EP tube (centrifugal tube) with sealing glue, and storing in a refrigerator environment at 4 deg.C for use, or storing in a refrigerator at-80 deg.C if long-term storage is required.
The oral squamous carcinoma tissue acellular matrix prepared by the method can be used as a preferred experiment carrier, the acellular matrix material needs to be subjected to aseptic processing before use, and the acellular matrix is washed for 3-4 times by PBS sterilized at high temperature and high pressure after being placed in 75% medical alcohol overnight, and can be used for cell and animal experiments.
To ensure the decellularization result, this example performed a series of quality controls on the decellularized matrix.
1. Gross observation of the decellularized tissue specimen: the specimen became transparent white (fig. 1);
and 2, DNA content detection: 1mg of ECM contained DNA at a level not exceeding 50ng (FIG. 2);
3. histological staining: h & E and Masson staining (fig. 3);
4. scanning Electron Microscope (SEM): comparing the microstructure of the tissue before and after decellularization, further verifying that the tissue after decellularization has no cells (figure 4);
5. immunohistochemistry (IHC-P): the major ECM structures such as Collagen type I, Collagen type IV (Collagen1,4), fibronectin (fibronectin), laminin (laminin) and the like are mainly observed (FIG. 5).
The oral squamous carcinoma tissue acellular matrix can be used as a culture scaffold, for example: cutting into small pieces, sterilizing, inoculating cells, and performing three-dimensional cell co-culture; digesting the acellular material by pepsin to obtain a polypeptide mixture, and preparing a hydrogel solution for three-dimensional culture; digesting with enzyme such as pepsin to obtain polypeptide mixture or soaking to obtain leachate, and adding into culture medium for two-dimensional culture.
The oral squamous carcinoma tissue acellular matrix can also be used for constructing oral squamous carcinoma organoids.
The oral squamous carcinoma tissue acellular matrix can also be used for related experiments such as tumor drug screening and the like.
Example one
3D co-culture: after the acellular matrix is disinfected, oral squamous carcinoma cells cal-27 are inoculated on the scaffold, and the samples are collected after 5 days, and the distribution state of cal-27 in dECM is observed by SEM.
The experimental results are as follows: the cal-27 cells are distributed in dECM, and form a three-dimensional reticular structure with dECM through structures such as cellosilk, and the like, so that the acellular matrix is proved to have good biocompatibility and can be used as a three-dimensional platform for cell culture. (FIG. 6)
Example two
Drug resistance experiments: after digestion of normal oral tissue, early dECM (E-dECM) and late dECM (L-dECM) into polypeptide mixtures by pepsin, the 3 matrix solutions were diluted to concentrations of: 0.5mg/ml, and cisplatin drug was diluted with the above solution with concentration gradients set at 0, 4, 8, 12 μ M. Cal-27 is expressed at 5X 103The concentration of each well is put in a 96-well plate, after the culture is carried out for 24h by using a DMEM high-sugar culture medium, the original culture medium is removed, the matrix solution and the solution containing 0, 4, 8 and 12 mu M are respectively added, the ordinary serum-free DMEM high-sugar culture medium is used as a control group, after the culture is carried out for 24h, the culture medium is removed, the culture medium containing CCK-8 reagent is added after the PBS is washed, after the incubation is carried out for 4h, the absorbance value OD of each well is detected, and the absorbance value OD is converted into the survival percentage. Each set was set with 3 parallel wells and the experiment was repeated 3 times.
The experimental results are as follows: late dECM is most tolerant to late dECM, in turn early dECM, normal group, 2D group, and the worst cis-platinum tolerance. The strongest ECM resistance of the advanced tumor was demonstrated, which explains to some extent the increased difficulty of treatment of advanced tumors (P <0.05) (fig. 7).
The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.
Claims (10)
1. A preparation method of an oral squamous carcinoma tissue acellular matrix is characterized by comprising the following steps:
(1) cleaning oral squamous carcinoma tissue and cutting into blocks;
(2) treating with 1% Triton-X100 on a shaking table for 12h to obtain a primary acellular tissue;
(3) treating with 1% SDS for 12h on a shaker;
(4) shaking the mixture on a shaking table for 24 hours by using DNAse I with the concentration of 100U/ml;
(5) washing with PBS for 12h on a shaking table to obtain a acellular matrix;
(6) the acellular matrix is placed in a 0.1U/L streptomycin solution for closed preservation.
2. The method for producing an oral squamous carcinoma tissue acellular matrix according to claim 1, characterized in that: in the step (1), the oral squamous carcinoma tissue is washed 3 times by PBS, and the oral squamous carcinoma tissue is cut into blocks at 4 ℃.
3. The method for producing an oral squamous carcinoma tissue acellular matrix according to claim 1, characterized in that: and (3) performing the steps (2) to (5) at room temperature.
4. The method for producing an oral squamous carcinoma tissue acellular matrix according to claim 1, characterized in that: and (3) after the step (2), the step (3) and the step (4) are finished, washing the acellular tissue by using PBS.
5. The method for producing an oral squamous carcinoma tissue acellular matrix according to claim 1, characterized in that: the preservation temperature in the step (6) is 3-5 ℃ or-90 ℃ to-70 ℃.
6. The method for producing an oral squamous carcinoma tissue acellular matrix according to claim 1, characterized in that: the rotating speed of the shaking table in the steps (2) to (5) is 225 rpm.
7. An oral squamous carcinoma tissue acellular matrix prepared by the method of any of claims 1-6.
8. Use of the oral squamous carcinoma tissue acellular matrix of claim 7 as a culture scaffold.
9. Use of the oral squamous carcinoma tissue acellular matrix of claim 7 for oral squamous carcinoma organoid construction.
10. Use of the oral squamous carcinoma tissue acellular matrix of claim 7 for screening of tumor drugs.
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| US20140228246A1 (en) * | 2011-10-04 | 2014-08-14 | Mitra Biotech Private Limited | Ecm composition, tumor microenvironment platform and methods thereof |
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