Application of Lactobacillus paracasei to CAR (chimeric antigen receptor)-T cell therapy

A Lactobacillus and paracheese technology, applied in the field of microorganisms, can solve the problems of less discussion on the immune regulation of probiotic strains, and no regulation mechanism of probiotic strains is proposed, so as to achieve the effects of regulating inflammatory response, enhancing immune function, and overcoming adverse reactions

Active Publication Date: 2019-10-08
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the above studies seldom explored whether probiotic strains can be applied to immune regulation after tumor therapy, especially CR

Method used

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  • Application of Lactobacillus paracasei to CAR (chimeric antigen receptor)-T cell therapy
  • Application of Lactobacillus paracasei to CAR (chimeric antigen receptor)-T cell therapy
  • Application of Lactobacillus paracasei to CAR (chimeric antigen receptor)-T cell therapy

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Embodiment 1

[0041] Lactobacillus paracasei ( Lactobacillus paracasei ) Isolation, purification and identification of MIT-16

[0042] 1. Isolation and purification of Lactobacillus paracasei strain

[0043] Take the intestinal content of the mouse, and serially dilute it 10 times to 10 -6 , take 200 μL of each dilution concentration and spread it on the solution containing 1% CaCO 3 On the MRS lactic acid bacteria screening medium, cultured in an anaerobic tank at 37°C for 72 hours, picked a single colony with a calcium-dissolving circle on the plate, continued to streak and purify it on the MRS solid medium, and cultured in an anaerobic tank at 37°C for 48 hours to obtain purer colonies. MRS medium for screening colonies see figure 1 . The strain of the present invention contains CaCO 3 The diameter of the colony on the MRS medium is 1-2mm, white, raised, and the transparent circle around the colony is a calcium-dissolving circle. The purified colonies were identified by Gram stai...

Embodiment 2

[0049] Lactobacillus paracasei ( Lactobacillus paracasei ) Determination of Adhesion Properties of MIT-16

[0050] The human colon cancer cell line HT-29 was cultured overnight in DMEM medium containing 10% serum, 5% CO2, and 37°C. Cells were collected and seeded in 6-well plates, 1×10 per well 5 cells, grown overnight to a monolayer. Wash with PBS three times, take the resuspension of Lactobacillus paracasei, and adjust the concentration of DMEM medium to 1×10 8 CFU / mL, add 200 μL bacterial solution and 1800 μL DMEM culture solution to a 6-well plate, place at 37°C, 5% CO 2 Incubate for 2 hours in the incubator. After washing with sterile PBS three times, 500 μL of 0.25% trypsin was added to each well to digest for 2 min, and then DMEM solution containing 10% fetal bovine serum was added to terminate the digestion. Take out the cell slides in the 6-well plate, carry out Gram staining, observe under the microscope, randomly select 50 fields of view, count the total number...

Embodiment 3

[0052] Lactobacillus paracasei ( Lactobacillus paracasei ) Bacteriostatic effect of MIT-16 on opportunistic pathogenic bacteria

[0053] The activated Lactobacillus paracasei MIT-16 strain was cultured anaerobically at 37° C. for 48 hours in MRS solid medium. Pick a single colony in MRS liquid medium, culture it anaerobically at 37°C overnight, measure OD 600 value, dilute the bacterial solution to 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL. While culturing Lactobacillus paracasei MIT-16, activate Salmonella in LB liquid medium, culture at 37°C for 12h, measure OD 600 value, dilute the bacterial solution to 10 5 CFU / mL. Spread the Salmonella bacteria liquid on LB solid medium and culture overnight at 37°C. The bacteriostasis experiment of Lactobacillus paracasei was carried out by Oxford cup method. Take 200 μL of Lactobacillus paracasei MIT-16 bacteria liquid in an Oxford cup, culture overnight at 37°C, and measure the size of the inhibition zone.

[0054] ...

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Abstract

The invention relates to an application of Lactobacillus paracasei to CAR (chimeric antigen receptor)-T cell therapy, belongs to the technical field of microorganisms and particularly discloses Lactobacillus paracasei MIT-16 and an application of the Lactobacillus paracasei to the immunological therapy process of canine tumors. The Lactobacillus paracasei MIT-16 with higher acid-producing abilityis separated and screened from an intestinal tract of a mouse, has good acid resistance, stronger bactericidal capacity and remarkable immune regulation functions, particularly plays an important rolein immune response regulation in tumor therapy and can be used as an immune regulation biotherapy agent.

Description

technical field [0001] The present invention relates to the therapeutic application of a strain of Lactobacillus paracasei combined with CAR-T cells, in particular to a strain of Lactobacillus paracasei with excellent performance ( Lactobacillus paracasei , MIT-16) and its application with CAR-T cell technology, which belongs to the field of microbial technology. Background technique [0002] In recent years, canine neoplastic diseases have a tendency to increase clinically, becoming one of the most important diseases that seriously endanger pet dogs and working dogs. Currently, there is no effective specific immunotherapy for the treatment of canine tumors. Antibody and adoptive cell therapy (ACT) are the two directions of tumor immunotherapy that are currently attracting the most attention. CAR-T, which expresses chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cells, is a type of adoptive cell therapy. Compared with traditional treatment methods, C...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K35/747A61P29/00A61P31/04A61P37/04C12R1/225
CPCA61K35/747A61P29/00A61P31/04A61P37/04C12N1/205C12R2001/225
Inventor 张兴晓姜琳琳张建龙陈国忠肖童齐越赵尘培于馨朱洪伟黄清荣
Owner LUDONG UNIVERSITY
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