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Exosome containing ECRG4 mRNA and preparation method and application of exosome

An ECRG4-HEK293, exosome technology, applied in the field of cell biology, can solve the problems of changing the morphological structure of exosomes, limited packaging efficiency, etc., and achieve the effects of inhibiting growth and proliferation, increasing expression, promoting and guiding effects

Active Publication Date: 2018-11-06
SOUTHWEST MEDICAL UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first three methods mainly change the physical structure of exosomes through certain stimulating factors, so that exosomes are passively loaded with exogenous genes. This method has a huge risk of changing the shape and structure of exosomes, and the packaging efficiency is limited.

Method used

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  • Exosome containing ECRG4 mRNA and preparation method and application of exosome
  • Exosome containing ECRG4 mRNA and preparation method and application of exosome
  • Exosome containing ECRG4 mRNA and preparation method and application of exosome

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Embodiment 1

[0070] 1. Construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: Extract total RNA from human umbilical vein endothelial cells by Trizol method, and amplify ECRG4 gene by reverse transcription PCR. After cutting and ligation with T4 DNA ligase, it was transformed into Escherichia coli, positive clones were screened, plasmids were extracted, and identified by double enzyme digestion and DNA sequencing. Among them, the ECRG4 primer was designed using the human ECRG4 gene sequence (GenBank: AF325503.1) as a template, the upstream primer contained an XhoI restriction site, and the downstream primer contained a BamHI restriction site. The base sequence of the upstream primer is: 5'-ATGACTCGAGTGCTCGCGCCCCGCCGCCATG-3'; the base sequence of the downstream primer is: 5'-TCATGGATTCTGTGGCAAGTCATGGTTAGT-3'.

[0071] 2. Cell preparation: resuscitate HEK293T cells, use DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (referred to as DMEM medium) at 37°C, 5% CO 2 Cult...

Embodiment 2

[0078] 1. Construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: the specific operation is the same as in Example 1.

[0079] 2. Cell preparation: the specific operation is the same as in Example 1.

[0080] 3. Preparation of lentivirus: the specific operation is the same as in Example 1.

[0081] 4. Establishment of ECRG4-HEK293 cell line: resuscitate HEK293 cells, use DMEM medium (referred to as DMEM medium) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C, 5% CO2 Cultured in a constant temperature incubator, after 1-2 passages, inoculate 0.8×10 6 Put the cells into a 6cm culture dish; the next day, mix the virus solution obtained in step 2 with the DMEM medium at a volume ratio of 3:1, and then add polybrene so that the concentration of polybrene is 8 μg / ml to obtain The second mixed solution: add the second mixed solution to HEK293 cells, centrifuge at 1800rpm and 37°C for 45 minutes, replace with DMEM medium after 12 hours of cultivation, and perform f...

Embodiment 3

[0087] 1. Construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: the specific operation is the same as in Example 1.

[0088] 2. Cell preparation: the specific operation is the same as in Example 1.

[0089] 3. Lentivirus preparation: prepare solution a containing 450 μl of Opti-MEM and 18 μl of lipofectamine2000; prepare 450 μl of Opti-MEM, 2 μg of plasmid pMD2.G, 2 μg of plasmid psPAX2 and 2 μg of plasmid pLVX-IRES-ZsGreen1- Solution b of ECRG4; add solution b to solution a, and mix gently to obtain the first mixed solution. After standing at room temperature for 5 minutes, add the first mixed solution to the HEK293T cells obtained in step 1, and place at 37°C, 5% CO 2 In a constant temperature incubator, rinse twice with PBS after 6 hours, replace with DMEM medium, collect the culture night after 36 hours, centrifuge at a temperature of 4°C and a speed of 2000g for 7 minutes, and take the supernatant as the virus solution;

[0090] 4. Establishment of ECRG4-HEK293 cell line: th...

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Abstract

The invention discloses an exosome containing ECRG4 mRNA and a preparation method and application of the exosome. The exosome is loaded with ECRG4 mPNA generated by a cell for overexpression of an ECRG4 gene, and can convey the ECRG4 mPNA in a recipient cell, so that the expression level of the recipient cell ECRG mRNA is remarkably improved, and the proliferation / growth of a tumor cell, the angiogenesis and the expression of inflammatory response related genes are inhibited; meanwhile, the compatibility of the exosome and the recipient cell is high, the immunoreaction and the inflammatory response of a recipient are not caused, and toxic and side effects of gene treatment can be remarkably reduced; the exosome can remarkably inhibit the proliferation / growth of the tumor cell; and the exosome has a positive effect of treating tumor diseases.

Description

technical field [0001] The present invention relates to the field of cell biology, in particular to an exosome containing ECRG4 mRNA and its preparation method and application. Background technique [0002] Oral squamous cell carcinoma (OSCC) is a malignant tumor that seriously threatens human health. Exploring safe and efficient treatment options is a hot spot in medical research. Human esophageal cancer related gene 4 (Esophageal Cancer Related Gene 4, ECRG4) is a tumor suppressor gene with extensive tumor suppressor functions cloned from normal human esophageal epithelium using mRNA differential display technology. ECRG4 is widely distributed in various tissues and organs of the human body, including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. In the occurrence of many tumors such as esophageal cancer, colon cancer, breast cancer, lung cancer, and kidney cancer, the expression of ECRG4 is significantly down-regulated, and its expression lev...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K48/00A61P35/00
CPCA61K48/005A61P35/00C12N5/0686C12N15/86C12N2510/02C12N2740/15043
Inventor 党喜同毛亮周锐
Owner SOUTHWEST MEDICAL UNIVERISTY
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