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Exosome as well as preparation method and application thereof in preparing medicine for treating lung cancer

A technology of exosomes and additives, applied in the field of biomedicine, can solve the problems of unseen exosomes and insufficient anti-tumor activity, and achieve enhanced anti-lung tumor activity, enhanced cytotoxicity, and enhanced effect of effect

Active Publication Date: 2017-09-01
贺飞 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of ATP on exosomes has not yet been studied.
[0008] In summary, the antitumor activity of CIK cell-derived exosomes has not yet been fully confirmed
In addition, the synergistic effect of KRN7000 and ATP on the culture of CIK cells and its activity on CIK cell-derived exosomes has not been reported

Method used

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  • Exosome as well as preparation method and application thereof in preparing medicine for treating lung cancer
  • Exosome as well as preparation method and application thereof in preparing medicine for treating lung cancer
  • Exosome as well as preparation method and application thereof in preparing medicine for treating lung cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The cultivation of embodiment 1CIK cell

[0061] 1. Cord blood mononuclear cell extraction:

[0062] Fresh umbilical cord blood was anticoagulated with heparin, and umbilical cord blood mononuclear cells (CBMNC) were separated by Ficoll-Hypaque method. Mix cord blood and phosphate buffer saline (PBS solution) in equal volumes, and slowly add it to Ficoll-Hypaque (purchased from GE) at a volume ratio of 1:1, centrifuge at 20°C, 400×g for 25 minutes; carefully draw The CBMNC band layer in the centrifuge tube was resuspended with PBS solution and centrifuged at 200×g for 15 minutes; the supernatant was removed, and the pellet was resuspended with PBS solution and centrifuged at 200×g for 15 minutes to obtain cord blood single nuclei cell.

[0063] 2. Cell culture:

[0064] According to different groups, the above-mentioned CBMNCs were resuspended and cultured. See Table 1 for the grouping situation, the addition amount of various components (ATP corresponds to the final...

Embodiment 2CI

[0080] Example 2Extraction and identification of CIK cell-derived exosomes

[0081] 1. Extraction of exosomes

[0082] Exosomes were extracted from the cell culture fluid obtained according to the grouping in Table 1, specifically:

[0083] Collect 20mL of the above cell culture supernatant, centrifuge at 2000×g for 30min, take the supernatant, discard cell debris and other precipitates; centrifuge at 4°C and 10000×g for 60min to obtain a concentrated solution containing exosomes; Centrifuge the concentrated solution at 4°C and 100,000×g for 60 minutes to collect the bottom precipitate; resuspend the obtained precipitate in PBS buffer, and centrifuge again at 100,000×g for 60 minutes to collect the bottom precipitate, which is CIK cell exosomes; The body was resuspended with 2ml of PBS buffer, sterilized by filtration with a 0.22μm filter membrane, and stored at -80°C.

[0084] 2. Identification of Exosomes

[0085] ① Morphological observation of exosomes:

[0086] The exo...

Embodiment 3

[0095] Example 3 Effect of KRN7000 and ATP on the expression of cytotoxic protein in exosomes of CIK cells

[0096] The levels of FasL and perforin in exosomes were detected by protein immunoassay:

[0097] Use RIPA lysate to lyse the exosomes in each group for 30 minutes, and use a homogenizer to homogenize the protein (15s×3 times); after standing for 30 minutes, centrifuge the above homogenate at 4°C and 12,000g for 15 minutes; For protein quantification with the BCA kit, adjust the total protein concentration to the same level; add 5× loading buffer and boil for 5 minutes; load the above solution, conduct shrink gel electrophoresis at 80V for about 30 minutes, and conduct separation gel electrophoresis at 120V About 60min; the protein was transferred to PVDF membrane by electrotransfer (250mA, 2h), and blocked with blocking solution for 1h at room temperature; After incubation overnight, the membrane was washed with TBST (10min×3 times); after that, it was incubated with ...

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Abstract

The invention relates to exosome as well as a preparation method and application thereof in preparing medicines for treating lung cancer. The preparation method comprises a step of stimulating cells to secrete the exosome by using KRN7000 and ATP (Adenosine Triphosphate). By adopting the preparation method provided by the invention, efficient in-vitro exosome preparation can be achieved. The invention further provides the exosome prepared by using the method. The exosome has relatively good cytotoxic effects and lung tumor resistance activity. The invention further provides application of the exosome in preparing medicines for treating lung cancer.

Description

technical field [0001] The invention relates to an exosome, its preparation method and its application in the preparation of a drug for treating lung cancer, belonging to the technical field of biomedicine. Background technique [0002] As malignant tumors pose an increasingly serious threat to human survival and health, new models and methods of tumor diagnosis and treatment are also emerging. Exosomes play an important role in the mechanism of tumor occurrence and development. It is an emerging research field in oncology. Many breakthroughs have been made in recent years, and it has a good prospect in tumor treatment. [0003] Exosomes are vesicle bodies secreted by a variety of living cells, which contain various key components such as proteins and RNAs. Exosomes play an important role in various physiological and pathological processes, including malignant tumors. . Exosomes can be used for immunotherapy of tumors. Studies by Rao et al. have shown (Rao et al, Hepatolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786A61K35/15A61P35/00
CPCA61K35/15C12N5/0634C12N2501/2302C12N2501/24C12N2501/515
Inventor 贺飞李涛张凡李振华杨乐吴东栋滕铁山姬新颖
Owner 贺飞
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