Research approach for element for forcing knubble microenvironment and application thereof
A technology of tumor microenvironment and factors, which is applied in the research of contributing factors of tumor microenvironment and its application field, and can solve problems such as inability to achieve good results
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Embodiment 1
[0030] Example 1: Isolation of Esophageal Cancer Fibroblasts
[0031]Six pairs of primary esophageal cancer and adjacent non-tumor tissues were obtained from Sun Yat-sen University Cancer Center (Guangzhou, China) during tumor resection of patients with esophageal cancer. None of the patients had received any adjuvant therapy before surgery. Cut the esophageal cancer tissue and its corresponding adjacent normal esophageal tissue into about 1mm with a scalpel 3 Small pieces were digested with 0.1% collagenase type IV for 30 minutes at 37°C. The digested cells were filtered through a 20 μm sterile sieve to collect a single cell suspension. The cells were washed twice with DMEM medium and centrifuged at 1,500 rpm for 5 minutes each time. Then resuspend the cells with 5ml DMEM medium containing 20% FBS, and inoculate them in a 6cm cell culture dish. After incubating in a 37°C incubator for 30 minutes, according to the difference in cell adhesion, that is, the attachment time ...
Embodiment 2
[0033] Example 2: Immunofluorescence staining
[0034] Cells were seeded on 24mm×24mm coverslips and cultured for 24 hours, then fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were washed with PBS, a PBS solution containing 5% bovine serum albumin (PBS-B) was added, and the cells were blocked at 37° C. for 30 minutes to remove non-specific staining. Add primary antibodies (fibronectin, E-cadherin, CD68, cytokeratin, etc.) and incubate overnight at 4°C. Wash the cells with PBS, add FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine isothiocyanate) labeled fluorescent secondary antibody, and incubate at room temperature for 1 hour in the dark. DAPI (4,6-diamino-2-phenylindole) was added for counterstaining, and the results were observed and photographed under a fluorescent microscope.
[0035] In order to confirm that the TF derived from esophageal cancer tissue is a single fibroblast without the contamination of macrophages and esop...
Embodiment 3
[0037] Embodiment 3: Flow cytometry detects DNA content
[0038] Collect about 1-2 x 10 6 Cells were fixed with 70% ethanol and stained with propidium iodide (PI) for flow cytometric analysis. Modfit LT 2.0 analyzes the changes of the cell cycle; calculates the DNA index (DI) for NF, TF and tumor cell ploidy analysis, and the DI value is the ratio of the average DNA content in the G1 phase of the tested cells to the DNA content in the G1 phase of normal peripheral blood lymphocytes. Three experiments were carried out to verify the results. figure 2 B, C, and D are related experimental results. figure 2 B is a histogram of flow cytometry, showing that the ratio of cells in S and G2-M phases of the TF cell cycle is significantly higher than that of NF. figure 2 C. figure 2 D is the experimental result diagram of the DNA content of two samples of NF, TF, and tumor cells detected by flow cytometry. The results show that the ploidy of TF and NF cells are all diploid cells, ...
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