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A mesenchymal stem cell exosome overexpressing ptgds gene and its preparation method and application

A mesenchymal stem cell and overexpression technology, applied in the field of gastric cancer drug preparation, to achieve the effect of improving gastric cancer progression, high targeting, and inhibiting tumor growth

Active Publication Date: 2019-09-10
九天览月生物科技(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although existing reports have disclosed that overexpression of PTGDS in tumor cells can effectively control the growth of gastric cancer cells in vitro and has the potential to treat gastric cancer (patent application number: 201611203449.3), but the existing technology still lacks effective targeted therapy for gastric cancer. drug

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  • A mesenchymal stem cell exosome overexpressing ptgds gene and its preparation method and application
  • A mesenchymal stem cell exosome overexpressing ptgds gene and its preparation method and application
  • A mesenchymal stem cell exosome overexpressing ptgds gene and its preparation method and application

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preparation example Construction

[0036] The present invention provides a kind of mesenchymal stem cell exosomes overexpressing PTGDS gene, the preparation method of said mesenchymal stem cell exosomes overexpressing PTGDS gene comprises the following steps:

[0037] 1) Transfect PTGDS-overexpressing adenovirus into mesenchymal stem cells, and obtain PTGDS-overexpressing mesenchymal stem cells through screening;

[0038] 2) Inoculate the PTGDS overexpressed mesenchymal stem cells obtained in step 1) into a low-sugar DMEM medium containing fetal bovine serum for the first expansion culture, and when the cells are fused to 70-80%, use a serum-free medium Carry out the second expansion culture, collect the supernatant after 48 hours, centrifuge to remove floating living cells, and obtain the supernatant containing exosomes;

[0039] 3) Separating and purifying the exosome-containing supernatant obtained in step 2) by using a sucrose density gradient centrifugation method to obtain exosomes from mesenchymal stem c...

Embodiment 1

[0066] The main materials and sources used are as follows:

[0067] MSC culture reagents low-sugar DMEM, fetal bovine serum (Gibco company product), trypsin (Sigma company product), carbon dioxide incubator (Forma company), serum-free medium (Shanghai Yikesai company;

[0068] Inverted microscopes, fluorescence microscopes, biological microscopes, electron microscopes, ultra-clean benches, desktop centrifuges;

[0069] PTGDS overexpression adenoviral vector (Suzhou Fubaiao Biotechnology Co., Ltd.), heavy water (D 2 O, Shanghai Chuangsai Company), analytically pure sucrose (Guangzhou Chemical Reagent Factory), rabbit anti-human CD9 antibody (U.S. Bioworld Technology Company), rabbit anti-human CD63 antibody (U.S. Epitomics Company), BCA protein quantification kit, horseradish Oxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (Beijing Kangwei Century Company), HRP chemiluminescent substrate, 100-kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membra...

Embodiment 2

[0081] The main materials and sources are as follows:

[0082] Cell culture reagents: 1640DMEM (product of Gibco Company), fetal bovine serum (product of Gibco Company), trypsin (product of Sigma Company), carbon dioxide incubator (Forma Company), serum-free medium (Shanghai Yikesai Company);

[0083] Inverted microscopes, biological microscopes, ultra-clean benches, desktop centrifuges;

[0084] 24 (6) well culture plate (Corning Company), Transwell migration plate (Corning Company);

[0085] Gastric cancer cell line SGC-7901 was purchased from Shanghai Cell Institute, Chinese Academy of Sciences;

[0086] PTGDS-modified exosomes can inhibit the growth and migration of gastric cancer cells in vitro:

[0087] The specific process is as follows:

[0088] (1) The effect of PTGDS-modified exosomes on the formation of tumor cell clones in vitro, the results are as follows Figure 4 Shown: Gastric cancer cell SGC-7901 cells were planted in a 6-well plate at a cell density of 10...

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Abstract

The invention relates to a mesenchymal stem cell exosome for overexpressing a PTGDS (prostaglandin-H2 D-isomerase) gene as well as a preparation method and application of the mesenchymal stem cell exosome, belonging to the technical field of stomach cancer medicine preparation. The mesenchymal stem cell exosome for overexpressing the PTGDS gene, which is provided by the invention, is obtained by transfecting a mesenchymal stem cell by adopting PTGDS-overexpressed adenovirus and then performing culturing, separating and purifying. The mesenchymal stem cell exosome for overexpressing the PTGDS gene, which is provided by the invention, effectively targets a stomach cancer cell, is capable of inhibiting the growth and migration of the stomach cancer cell and then inhibiting a tumor from growth.

Description

technical field [0001] The invention relates to the technical field of preparation of gastric cancer drugs, in particular to a mesenchymal stem cell exosome overexpressing PTGDS gene and its preparation method and application. Background technique [0002] Gastric cancer is the fifth most common malignant tumor in the world, and its death rate ranks third. In my country, gastric cancer is the third most common cancer, and it is already the second leading cause of cancer death. At present, the clinical drugs for treating gastric cancer mainly include chemotherapy drugs such as 5-fluorouracil. These drugs can significantly inhibit the progress of tumors, but they have strong side effects due to their poor specificity to tumor tissues. Therefore, we urgently need a "biological agent" that has both anti-gastric cancer cell growth and targeted therapeutic effects. [0003] PGD2 is a kind of prostaglandin and is an important cell signaling molecule. Its synthesis is mainly throug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/861A61K35/28A61P35/00
Inventor 张斌尤本帅许文荣钱晖董海新张颢金呈强
Owner 九天览月生物科技(天津)有限公司