Application of miR-150 and exosome loaded with miR-150 simulant to preparation of medicine for treating colorectal cancer
The technology of mir-150mimic and 1.mir-150 is applied in the application field of preparing drugs for the treatment of colorectal cancer, which can solve the problems of drug resistance clinical treatment, disturbance of the body's microenvironment, poor prognosis of colorectal cancer patients, etc. Adverse factors, prolonged patient survival, efficacy with safety
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[0051] In one embodiment of the present invention, the preparation method of exosomes loaded with miR-150mimic comprises the following steps:
[0052] (1) Extraction of carrier exosomes: Exosomes from the cell supernatant were extracted using the ExoQuickTG Exosome Extraction Kit, and resuspended in PBS to obtain an exosome suspension;
[0053] (2) Acquisition of miRNA: artificially synthesize miR-150 mimics;
[0054] (3) Transfer of miR-150mimic into carrier exosomes: transfer the artificially synthesized miR-150 mimics in step (2) into the exosome suspension in step (1) by chemical methods to obtain miR-150mimic-loaded of exosomes.
[0055] In one embodiment of the present invention, in step (3), the chemical method comprises the following steps:
[0056] ①Preparation of siRNA-ExoFectin mixture: prepare Exofectin suspension exosome transduction reagent and miR-150mimic suspension; mix the two and incubate to obtain siRNA-ExoFectin mixture;
[0057] ② Transduction of siRNA...
Embodiment 1
[0065] This embodiment provides a method for preparing therapeutic exosomes for intestinal cancer metastasis, comprising the following steps:
[0066] (1) Screening of exosomal miRNAs related to colon cancer metastasis:
[0067] According to the analysis of peripheral blood exosomal miRNA chip results of 30 clinical samples (10 normal subjects, 10 cases of colon cancer without metastasis and 10 cases of colorectal cancer liver metastases), it was found that patients with colon cancer liver metastases were similar to those without colon cancer metastases. In contrast, the expression of miR-150 in peripheral blood exosomes decreased, while the expression of miR-548ai and miR-3190-5p increased (see figure 1 ). figure 1 A indicates that the expressions of miR-548ai and miR-3190 are gradually increased in patients with colorectal cancer and liver metastasis of colorectal cancer; figure 1 B indicates that compared with normal people, the expression of miR-150 in peripheral blood e...
Embodiment 2
[0114] This embodiment provides a method for preparing therapeutic exosomes for intestinal cancer metastasis, comprising the following steps:
[0115] (1) Cell culture
[0116] 1) Recovery of cells:
[0117] a) Take out the cryopreservation tube of the desired cell line from the liquid nitrogen, quickly transfer it to a constant temperature water bath at 37°C, shake it to melt it, and sterilize the cryopreservation tube with 70% alcohol, draw it into a 15ml centrifuge tube in an ultra-clean bench, and add 2ml Medium blowing evenly;
[0118] b) centrifugal 1000r / min, 5min;
[0119] c) After centrifugation, aspirate the supernatant, add 1ml of cell culture medium, and carefully pipette evenly;
[0120] d) Transfer the cell suspension to a cell culture flask, add an appropriate volume of cell culture medium, and place it at 37°C with 5% CO 2 Cultivated in a constant temperature incubator;
[0121] e) Observe under the microscope the next day, if the cells adhere to the wall ...
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