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Interference sequence-modified TGF-beta1 silent leukemia cell exosome and preparation method and application thereof

A technology of leukemia cells and interfering sequences, applied in the biological field, can solve the problems of weak immunogenicity and poor clinical efficacy.

Inactive Publication Date: 2017-09-15
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most patients do not benefit from treatment
Although unmodified tumor-derived exosomes can induce certain antigen-specific immune responses, their immunogenicity is relatively weak, and the clinical efficacy is still not optimistic.

Method used

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  • Interference sequence-modified TGF-beta1 silent leukemia cell exosome and preparation method and application thereof
  • Interference sequence-modified TGF-beta1 silent leukemia cell exosome and preparation method and application thereof
  • Interference sequence-modified TGF-beta1 silent leukemia cell exosome and preparation method and application thereof

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preparation example Construction

[0055] The present invention also provides the preparation method of the TGF-β1 silenced leukemia cell exosomes modified by the interference sequence comprising the following steps:

[0056] Step 1, culturing leukemia cells;

[0057] Step 2, preparing leukemia cells modified by interfering sequences;

[0058] Step 3, extraction and purification of exosomes.

[0059] Step 4, detecting the anti-leukemia effect of the modified leukemia cell exosomes targeting the sensitized dendritic cell vaccine as a protective tumor vaccine;

[0060]Step five, the effect detection of the dendritic cell vaccine targeted and sensitized by the modified leukemia cell exosomes in tumor-bearing mice.

[0061] Furthermore, Step 1 is specifically:

[0062] a. Configure 1640 complete medium containing 10% fetal bovine serum, which contains 100U / ml penicillin sodium and 100ug / ml streptomycin sulfate, and freeze the cryopreservation tube with L1210 cells from the liquid nitrogen tank After taking it o...

Embodiment 1

[0090] Example 1 Preparation of L1210-derived exosomes with down-regulated TGF-β1 content

[0091] 1. Construction of TGF-β1shRNA lentivirus

[0092] 1. Preparation of TGF-β1shRNA lentiviral vector

[0093] The TGF-β1 interference lentiviral vector was designed and synthesized by Shanghai Hanheng Biotechnology Co., Ltd. According to the TGF-β1 nucleotide sequence and siRNA in Genebank, three optimal target sequences were designed. Named shRNA1, shRNA2, shRNA3 respectively. The sequence is as follows. Simultaneously, an siRNA unrelated to the TGF-β1 gene sequence was synthesized as a negative control (Negative control), named shRNA NC. After sequence homology analysis, Shanghai Sangon Bio-synthesized double-stranded DNA Oligo containing interfering sequences, both ends of which contain enzyme cleavage site cohesive segments, which were ligated into the pHBLV-U6-Scramble-Zsgreen expression vector after enzyme cleavage. Transfer the ligated plasmid into Escherichia coli DH5a...

Embodiment 2

[0177] Example 2 Dendritic cell vaccine (DC LEX-TGF-β1si )

[0178] Induction of dendritic cells (DCs)

[0179] Isolation and culture of mouse bone marrow DC:

[0180] 1) Take three 6-8 week old mice, kill them, soak in 75% alcohol for 1 minute.

[0181] 2) Cut the back skin of the mouse, pull the skin below the knee joint, cut off the knee joint and iliac joint, and put it in PBS.

[0182] 3) Prepare two 10cm petri dishes, place the tissue in the petri dish, and trim the tissue to facilitate the exposure of the bone end.

[0183] 4) Put the treated tissue into another 10cm culture dish and add 1640 medium.

[0184] 5) Prepare a 10ml syringe, fill it with 1640 medium, cut both ends of the femur, and flush the bone marrow cavity with a 1ml syringe, and a long strip of bone marrow or red liquid can be seen flowing out.

[0185] 6) Collect the washing liquid into a 50ml centrifuge tube, rinse the culture dish, centrifuge, 4 degrees, 1200rpm, 5min

[0186] 7) Discard the sup...

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Abstract

The invention relates to an interference sequence-modified TGF-beta1 silent leukemia cell exosome and a preparation method and application thereof. Through expression of TGF-beta1 of a lentivirus vector silent exosome source cell with shRNA, the leukemia cell exogenic exosome, namely LEXTGF-beta1si of which the TGF-beta1 content is regulated down is produced and purified. The transformed leukemia cell exogenic exosome-sensitized dendritic cell is prepared into a DCLEX-TGF-beta1si vaccine. An in vitro and animal model experiment shows that the transformed vaccine is capable of generating more powerful specific anti-leukemia immunoreaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a TGF-β1-silenced leukemia cell exosome modified by an interference sequence and a preparation method and application thereof. Background technique [0002] Relapse after treatment of leukemia patients is the main factor affecting the long-term survival of patients, and the root cause is the residual leukemia cells in the body after treatment. The traditional methods mainly rely on high-dose consolidation chemotherapy and allogeneic hematopoietic stem cell transplantation to eliminate the residual leukemia cells in patients in remission, in which the anti-leukemia effect of allogeneic lymphocytes in the transplantation is very important, but this non-specific immunity Graft-versus-host disease is often accompanied by severe graft-versus-host disease, which is the main cause of death in hematopoietic stem cell transplantation. Immunotherapy can specifically remove residua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61K39/0011C12N5/0694C12N15/86C12N2510/00C12N2740/15043
Inventor 郝思国黄方
Owner XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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