CN113151177B - Breast or breast cancer tissue acellular matrix and preparation method and application thereof - Google Patents
Breast or breast cancer tissue acellular matrix and preparation method and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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Abstract
The invention discloses a breast or breast cancer tissue acellular matrix, a preparation method and application thereof, and belongs to the field of biomedical engineering. The method comprises the following steps: 1) Freezing breast or breast cancer tissue, and cutting into membrane shape; 2) Repeatedly freezing and thawing for 2-4 times by using liquid nitrogen; 3) Standing or shaking the mixture in the lysate for 12-24 hours, and washing away the lysate; 4) Removing residual cell nucleus components by using nuclease, washing by running water, and rinsing by using buffer solution; 5) Freeze-drying, packaging and sterilizing. The method has good cell removal effect and little damage to the multi-extracellular matrix fiber. The breast or breast cancer tissue acellular matrix can induce the epithelial-mesenchymal transition of cancer cells, and can prepare a cell model of the epithelial-mesenchymal transition of the cancer cells based on the principle, thereby having good application prospect.
Description
Technical Field
The invention belongs to the field of biomedical engineering.
Background
Acellular matrix (ECM), also known as acellular matrix, is a solid form obtained after removal of cells in tissue, and can be used to make surgical patches or carriers for seed cell growth in tissue engineering.
There are a variety of organ sources of decellularized matrix including liver, lung, heart, bladder, dermis, and the like. Due to the structure of the tissue, the cell arrangement, structure, etc., the methods for preparing the decellularized matrix of various organs are different. For example: zhang Peiling et al report a method for preparing a acellular matrix of cartilage comprising enzymatic hydrolysis with 0.5% pancreatin for 24 hours, during which pancreatin is replaced, then enzymatic hydrolysis with 1U/mL nuclease for 4 hours, and further treatment with 1% Triton X-100 for 24 hours (Zhang Peiling et al, preparation of a biomimetic scaffold of cartilage acellular matrix and its effect on chondrogenic differentiation of bone marrow stromal stem cells, tissue engineering and reconstructive surgery, 2021, 2 months, volume 17, stage 1). Patent application CN 111494718A reports a method for preparing a decellularized matrix of the lung, comprising the steps of perfusing a protective agent from the trachea and then perfusing a detergent from the blood vessel, wherein the detergent is selected by perfusing SLES first and perfusing Triton X-100.
Breast and breast cancer tissues have unique structures, individual differences exist in gland compactness and fat content, breast cancer is a highly heterogeneous tumor, and the tissue structures and cell arrangements of breast cancer tissues of different patients and different molecular subtypes also differ. Therefore, the preparation of acellular matrix of breast cancer tissue is difficult. There is no report of using breast cancer tissue for preparing acellular matrix.
Epithelial mesenchymal transition (Epithelial mesenchymal transition, EMT) is a biological process by which epithelial cells change morphology, cell structure, and lose epithelial cell characteristics, resulting in invasive and migratory mesenchymal phenotypes. EMT is involved in the development and progression of tumors and plays an important role in promoting tumor metastasis invasion. Some antitumor drugs act upon EMT intervention. By constructing cancer cell EMT models, it can be used for screening drugs for assisting in the intervention of EMT.
At present, the construction of an EMT model of cancer cells mainly depends on adding specific components into a culture medium. For example: the addition of TGF-1 beta in the culture medium can obviously reduce the expression of the epithelial marker molecule E-cadherein of gastric cancer cells, and the elevated expression of the interstitial marker molecules N-cadherein and Vimentin can establish an EMT model (Zhu Yaodong. Celastrus orbiculatus extract regulates the action and mechanism research of HSP27 for inhibiting the epithelial and mesenchymal transition of gastric cancer, university of Yangzhou doctor academy paper, 2015); after LPS is added into a triple negative breast cancer cell culture environment for culture, the expression of Vimentin and beta-catenin of cells is up-regulated, and the expression of E-cadherein is down-regulated, and an EMT model can be obtained (Chen Zhouhua. The effect of baicalin on inhibiting invasion and epithelial-mesenchymal transition of triple negative breast cancer and related mechanism research. Doctor's academy of Hunan traditional Chinese medicine university, 2018).
There is no report of using acellular matrix for constructing cancer cell EMT model.
Disclosure of Invention
The invention aims to solve the problems that: (1) A method of preparing decellularized matrix of breast or breast cancer tissue is provided; (2) Provides the use of triple negative breast cancer acellular matrix in preparing cancer cell epithelial mesenchymal transition model.
The technical scheme of the invention is as follows:
a preparation method of a breast or breast cancer tissue acellular matrix comprises the following steps:
1) Freezing breast or breast cancer tissue, and cutting into membrane shape;
2) Repeatedly freezing and thawing for 2-4 times by using liquid nitrogen;
3) After cracking for 12-24 hours in the cracking liquid, washing away the cracking liquid;
4) Removing residual cell nucleus components by nuclease digestion, washing with running water and rinsing with buffer solution;
5) Freeze-drying, packaging and sterilizing;
the lysate is Triton X-100 and 10-30 mM NH with the volume ratio of 0.25-1% 4 Mixed solution of OH solution.
Further, the lysate is Triton X-100 and 20mM NH with a volume ratio of 0.5% 4 Mixed solution of OH solution.
Further, the nuclease of step 4) is DNase I.
Further, the concentration of DNase I in step 4) is 100mg/ml;
and/or, the nuclease digestion time in step 4) is 3 hours;
and/or, the temperature of nuclease digestion in step 4) is 37 ℃.
The acellular matrix prepared by the method.
A method of preparing a cancer cell epithelial-mesenchymal transition model, the method comprising the steps of:
1) Inoculating cancer cells onto the surface of wet triple negative breast cancer acellular matrix;
2) Incubating to allow the cells to adhere sufficiently to the decellularized matrix;
3) Adding a culture medium for culture.
Further, the DNA residue of the acellular matrix of the triple negative breast cancer is lower than 50ng/mg.
Furthermore, the triple negative breast cancer acellular matrix is prepared by taking triple negative breast cancer as a raw material and utilizing the preparation method of the breast or breast cancer tissue acellular matrix.
Further, the cancer cell is a breast cancer cell.
Further, the cancer cells are MCF-7 cells.
The beneficial effects are that:
1) The preparation method of the acellular matrix has high efficiency of removing the mammary gland cell components. No cell residues were observed by Masson staining, and the DNA content after decellularization was below 50ng/mg.
2) The acellular matrix preparation method disclosed by the invention has small influence on the microstructure of the extracellular matrix.
3) The acellular matrix of the triple negative breast cancer tissue can promote the cancer cells to generate epithelial mesenchymal transition, and the expression level of E-cadherein is reduced, and the expression level of Vimentin is increased; can be used for constructing cancer cell epithelial mesenchymal transition model.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: HE staining before and after decellularization and DAPI staining.
Fig. 2: quantification of DNA before and after decellularization of normal breast tissue (B), luminela (LA) breast cancer tissue, and Triple Negative (TN) breast cancer tissue.
Fig. 3: appearance of normal breast tissue after decellularization and Masson staining results before and after decellularization. (a) general appearance after decellularization of normal breast tissue (b) Masson staining before decellularization of normal breast tissue (c) Masson staining after decellularization of normal breast tissue (bar=100 μm).
Fig. 4: the expression of E-cadherein and Vimentin of tumor cells cultured with B-ECM, LA-ECM and TN-ECM was measured (P < 0.05, P <0.001, & lt, & gt is statistically different from 2D, and # is statistically different from B-ECM, and delta is statistically different from LA-ECM, and O is statistically different from TN-ECM).
Detailed Description
EXAMPLE 1 preparation of mammary and Breast cancer acellular matrix
1. Tissue origin
The normal breast tissue and breast cancer tissue used in this experiment were all obtained from breast surgery at Huaxi hospital at university of Sichuan. The breast tissue is derived from an early breast cancer patient undergoing improved radical treatment of breast cancer, and normal breast tissue far from the tumor tissue is obtained during the treatment. Breast cancer tissue was obtained from primary breast cancer surgery patients who did not receive radiotherapy and chemotherapy, and endocrine therapy. All the specimens were informed consent from the patient prior to acquisition. Transferring the materials to a laboratory by a liquid nitrogen tank, and storing in a refrigerator at-80 ℃ for later use.
2. Preparation method
1. Taking out normal breast and breast cancer specimens from a refrigerator at-80 ℃ and rapidly transferring the specimens to a frozen microtome for embedding OTC (optimum cutting temperature compound, the optimal slicing temperature mixture is a commercial product);
2. tumor tissue pieces were trimmed to about 1cm x 1cm pieces, and serial sections were prepared to give a patch thickness of 150 μm; directly trimming normal mammary tissue blocks, slicing into film and sheet;
washing with PBS for three times, each for 20min, and removing superfluous oil, blood stain, impurities and embedding agent components around the membrane;
4. repeatedly freezing and thawing the membrane in liquid nitrogen for 3 times, wherein each time lasts about 30 minutes;
5. the tissue after freeze thawing was transferred to a lysate (Triton X-100 at 0.5% by volume and 20mM NH) 4 Mixed solution of OH solution) for 18h, at 37℃with a constant temperature shaker at 125rpm;
6. rinsing a large amount of PBS solution for three times, each time for 30min, and centrifuging to change the solution;
7.100mg/ml DNase I treatment for 3h to remove residual nuclear components, shaking table at 37deg.C, 125rpm;
8. washing with flowing water for 2h, rinsing with a large amount of PBS solution for three times, each time for 30min, and centrifuging to change the liquid;
9. the material is freeze-dried after washing is satisfied, and the ethylene oxide is sterilized after packaging.
3. Evaluation of Decellularization
1. HE staining before and after decellularization and Masson staining
HE Staining and Masson Staining were performed using a Hematoxylin-Eosin/HE Staining Kit (Solarbio, beijing) and a Masson Stain Kit (Solarbio, beijing), respectively.
2. DNA quantification
Using Quant-iT TM dsDNA Reagent and Kits (Invitrogen, UK) to detect the residual DNA content in tissue,the measurements were repeated 3 times. The detection method refers to the product instruction book.
4. Results
1. Histological characterization showed complete tissue decellularization
Prior to decellularization, both HE staining and DAPI staining of normal breast tissue revealed a large number of cells and cell debris distributed in the breast parenchyma and interstitium. HE staining of ECM after decellularization showed a large amount of eosinophilic interstitial component, and no blue stained nuclei. DAPI staining results suggested a nuclear structure in ECM with no apparent blue-violet fluorescence (fig. 1).
2. Histological quantification indicated complete tissue decellularization
The residual DNA content after decellularization was detected by PicoGreen kit. The results of repeated detection are shown in FIG. 2, wherein the average DNA content of normal breast tissue, luminal A-type breast cancer tissue and triple-negative breast cancer tissue is (411.13 +/-35.17) ng/mg, (504.96 +/-42.52) ng/mg, (504.13 +/-38.63) ng/mg respectively, and the average DNA content after decellularization is (17.46+/-11.23) ng/mg, (46.91 +/-18.14) ng/mg, (44.79+/-16.29) ng/mg respectively. The residual DNA content in all three ECMs met the criterion of decellularization (< 50 ng/mg).
3. The decellularization treatment process has little influence on the extracellular matrix fiber structure
The ECM membrane obtained after physical + chemical + enzymatic treatment was white non-uniform translucent with tacky surface and soft texture (fig. 3 a). Fig. 3b shows Masson staining of normal breast tissue prior to decellularization, which shows that the breast tissue is composed of acini and stroma. FIG. 3c is a Masson stain after decellularization treatment, with no cell residue in the field of view, consisting of blue-stained collagen fibers. The contrast shows that the ECM fiber conformation is substantially consistent before and after decellularization treatment, and the fiber is preserved intact without obvious breakage after decellularization.
Experimental results show that the method combines physical-chemical and biological enzyme methods to perform decellularization treatment on normal breast tissue and breast cancer tissue membranes, successfully removes cell components in the tissues, and has little influence on extracellular matrix structures.
Example 2 Effect of decellularized matrix on epithelial and mesenchymal phenotypes of tumor cells
Acellular matrix was prepared from normal breast tissue (B), luminea (LA) breast cancer tissue and Triple Negative (TN) breast cancer tissue using the method of example 1, respectively, and named in turn: B-ECM, LA-ECM and TN-ECM.
1. Experimental procedure
1. Preparation of acellular matrix before inoculation: the ethylene oxide sterilized B-ECM, LA-ECM, TN-ECM were taken and soaked in serum free MEM basal medium for at least 3h in 24 well plates. Cells were again washed 2 times with MEM complete medium before seeding and transferred to 48-well plates. The ECM was left to dry slightly in a sterile operating table, preferably with no medium in the well plate, and the ECM was kept wet.
The preparation method of the MEM complete culture medium comprises the following steps: each 100ml of complete medium contains 90ml of MEM basal medium, 10% fetal bovine serum, 0.11g/L of sterile sodium pyruvate, 0.01mg/ml bovine insulin, 1% penicillin and streptomycin, and is stored at 4 ℃ after being prepared.
2. Preparation of cells: breast cancer cells MCF-7 (purchased from ATCC biological cell bank) were cultured in MEM complete medium. MCF-7 cells grown in log phase were prepared into 5X 10 cells using MEM complete medium 6 Cell suspension per ml.
3. Inoculating: MCF-7 cell suspensions with a total volume of 10. Mu.l were micro-uniformly inoculated onto the prepared surfaces of B-ECM, LA-ECM, TN-ECM with a micro gun in 2-3 times, and 3 replicates were set per group using a normal plate as a 2D control group. Inoculating cells, and placing at 37deg.C and 5% CO 2 The incubator was incubated for 2 hours to allow the cells to adhere well to the ECM scaffold. After 2 hours, MEM complete medium was added, after which the medium was changed daily.
4. Extraction of total RNA of cells: after 5 days of incubation, total RNA of MCF-7 cells cultured with different ECMs was extracted using Eastep Total RNA Purification Kit (Promega, US) for subsequent PCR quantitative detection experiments.
5. Extraction of total cell proteins: after 5 days of incubation, MCF-7 protein was extracted using RIPA lysate (Yase, china).
6. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of mRNA expression level of EMT marker molecules of MCF-7 cells: usingThe mRNA expression levels of the epithelial marker E-cadherein and the interstitial marker Vimentin under each ECM culture are quantitatively detected by PCR (polymerase chain reaction) in a 1-Step RT-qPCR System kit row. Experimental procedures reference is made to the specification. The sequences of the primers are as follows:
7. western blot detection of EMT marker protein expression level of MCF-7 cells: the assays were performed using the Yase company-related kit, the experimental procedure being referred to in the specification, wherein the primary antibodies were purchased from abcam (US) company and the secondary antibodies were purchased from Solarbio (Beijing) company.
2. Experimental results
1. Expression of breast cancer MCF-7 cell EMT marker
Tumor infiltration, recurrence or metastasis often are closely related to EMT of tumor cells, and in order to further explore the influence of ECM on EMT of MCF-7 cells, experiments respectively detect mRNA and protein expression changes of MCF-7 cell epithelial marker molecule E-cadherein and interstitial marker molecule Vimentin after 5 days of culture under three ECM environments by RT-qPCR and Western blot.
The PCR results of FIG. 4A show that the mRNA levels of E-cadherein were significantly lower in the TN-ECM group than in the B-ECM and LA-ECM, whereas the mRNA levels of Vimentin were significantly higher, the differences were all statistically significant (P < 0.001). Western blot (FIG. 4B) results also showed that E-cadherein was lowest in the TN-ECM group, while Vimentin was highly expressed in the TN-ECM group.
The above results indicate that TN-ECM induces EMT in MCF-7 cells, while LA-ECM has the effect of maintaining the epithelial phenotype of MCF-7 cells. Therefore, ECM of triple negative breast cancer can be used as a scaffold for cell culture, and cancer cells can be inoculated to prepare an EMT model of cancer cells.
In conclusion, the method can effectively remove the cell components in the breast and breast cancer tissues, and has little influence on the extracellular matrix structure. And the prepared triple negative breast cancer ECM has the effect of promoting the generation of EMT by tumor cells, and can be used for constructing an EMT model of cancer cells.
SEQUENCE LISTING
<110> Huaxi Hospital at university of Sichuan
<120> acellular matrix of breast or breast cancer tissue, and preparation method and application thereof
<130> GYKH1124-2021P0112887CC
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Claims (2)
1. A method for preparing a cancer cell epithelial-mesenchymal transition model, comprising: the method comprises the following steps:
1) Inoculating cancer cells to the decellularized stroma surface of wet triple negative breast cancer tissue; the cancer cells are MCF-7 cells; the preparation method of the triple negative breast cancer tissue acellular matrix comprises the following steps:
(1) Freezing triple negative breast cancer tissue, and cutting into film slices;
(2) Repeatedly freezing and thawing for 2-4 times by using liquid nitrogen;
(3) After cracking for 12-24 hours in the cracking liquid, washing away the cracking liquid;
(4) Removing residual cell nucleus components by nuclease digestion, washing with running water and rinsing with buffer solution;
(5) Freeze-drying, packaging and sterilizing;
the lysate is Triton X-100 and 20mM NH with a volume ratio of 0.5% 4 A mixed solution of OH solutions; the nuclease is DNase I, the concentration of the DNase I is 100mg/ml, the nuclease digestion time is 3h, and the nuclease digestion temperature is 37 ℃;
2) Incubating to allow the cells to adhere sufficiently to the decellularized matrix;
3) Adding a culture medium for culture.
2. The method of claim 1, wherein: the DNA residue of the acellular matrix of the triple negative breast cancer is lower than 50ng/mg.
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