Antibacterial and antitumor application of NK cell exosome and related miRNA

A technology of NK cells and exosomes, applied in DNA/RNA fragments, antitumor drugs, antibacterial drugs, etc., can solve the problem of inability to obtain exosomes, and achieve low cost, enhanced antibacterial effect, and enhanced therapeutic effect. Effect

Active Publication Date: 2018-05-08
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The biggest bottleneck in the application of NK cell exosomes in clinical treatment is that NK cells cannot be expanded well in vitro, so that a sufficient number of exosomes cannot be obtained

Method used

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  • Antibacterial and antitumor application of NK cell exosome and related miRNA
  • Antibacterial and antitumor application of NK cell exosome and related miRNA
  • Antibacterial and antitumor application of NK cell exosome and related miRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Acquisition of aNK exosomes and their structural characteristics

[0031] Using the existing method, the NK system was expanded using transmembrane IL-21 trophoblasts: the peripheral venous blood from volunteers was isolated to obtain PBMCs, and trophoblasts expressing transmembrane IL-21 after irradiation and IL-2 were added. , during which complete culture solution was added and cultivated for 18 days.

[0032] The aNK cell culture fluid cultured for 18 days was collected, and the purity of the aNK cells detected by flow cytometry reached more than 95%, and the culture fluid was free of bacteria and mycoplasma contamination.

[0033] 1.1a The NK cell culture solution was centrifuged at 400g for 5 minutes in a bench-top low-speed centrifuge to remove the cell pellet, and the supernatant was collected and stored at 4°C for later use.

[0034] 1.2 The exosomes in the culture medium were purified using a hollow fiber tangential filtration system (Spectrum Labo...

Embodiment 2

[0038] Example 2: aNK exosome miRNA sequencing

[0039] According to the aNK exosomes obtained in Example 1, Nanjing Shihe Gene Biotechnology Co., Ltd. was commissioned to sequence the miRNA in aNK exosomes. NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) was used for library construction, and sequencing adapters were added to the samples to be sequenced. The samples were sequenced using the Illumina X-tenPE150 platform, with at least 300M reads for each sample. In order to reduce false positives, independent sequences with a signal-to-noise ratio greater than 100 were included in the calculation of miRDeep log-odds score, and the range of miRDeep log-odds score was set to -10 to 10. In order to discover new miRNAs in aNK exosomes, miRDeep score=0 is used as the threshold, and all miRNAs exceeding the threshold are regarded as brand new miRNAs, such as Figure 4 As shown, 732 known miRNAs (SEQ ID NO.1412-SEQ ID NO.2144) were found in the sequencing. In ...

Embodiment 3

[0040] Example 3: the antibacterial properties of aNK exosomes and the antibacterial properties of aNK exosomes combined with NK cells

[0041] In order to determine whether exosomes can inhibit bacterial growth, Citrobacter (C.R.), Escherichia coli, Staphylococcus aureus, Salmonella typhi (CT18), Staphylococcus aureus (LAC), and Acinetobacter baumannii (XH386) were selected as Detection object. The aNK exosomes obtained in Example 1 were mixed with the above-mentioned 6 bacteria in different proportions, and the number of bacteria was quantified by measuring the optical density (OD). The result is as Figure 5 As shown, the growth of Escherichia coli, S. aureus, S. typhi (CT18) and S. aureus (LAC) showed concentration-dependent growth with increasing exosome concentration compared with the negative control without exosomes. significant inhibition. At the same time, the number of bacteria was quantified by detecting the luciferase activity in Citrobacter. As the concentrati...

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Abstract

The invention discloses antibacterial and antitumor application of exosomes secreted by NK cells (aNK) activated by transmembrane IL-21 and multiple miRNAs contained in the exosomes, and provides a new method for bacterial infection and antitumor treatment. The method has the advantages that 1, the method is different from antibiotic treatment, and exosome antibiosis has lower side effects; 2, effects of antibacterial treatment can be enhanced through joint use; 3, the exosomes can be stored at low temperature, the miRNAs can be artificially synthesized, costs are low and large-scale production can be achieved; 4, the exosomes and the multiple miRNAs have inhibitory effects on a variety of bacteria, and have wider antibacterial spectra; 5, the miRNAs act on a variety of shared signal pathways of tumor cells, can inhibit a variety of tumors, and have a wide range of antitumor application.

Description

technical field [0001] The present invention relates to the exosome (exosome) secreted by NK cells (aNK) activated by transmembrane IL-21, and the application of related miRNA in antibacterial and antitumor. Background technique [0002] Exosomes are a type of nanoscale membranous vesicles secreted by cells that carry cytoplasmic components. They are secreted by various cells in the body and widely distributed in body fluids such as saliva, plasma, and milk. Exosomes contain a variety of biologically active substances such as proteins, mRNAs, and miRNAs. Exosomes deliver miRNAs and proteins to other cells in the form of membrane fusion, serving as a bridge for intercommunication between cells. [0003] NK cells are the main effector cells in the innate immune system and have been used in the treatment of tumors. They kill infected or cancerous cells by releasing perforin and granzymes. The exosomes secreted by NK cells contain some active substances of NK cells and also ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P31/04A61P31/10A61P35/00A61P35/02A61P35/04A61K31/7105
CPCA61K31/7105A61P35/00C12N15/113C12N2310/141Y02A50/30
Inventor 徐以兵胡薇蕾寿鑫王国胜陈海琼
Owner ZHEJIANG UNIV
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