Method for screening probiotics with function of enhancing intestinal cell tight junction at cell level

An intestinal cell, cell-level technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as the inability to accurately reflect the function of probiotics in regulating the intestinal barrier

Active Publication Date: 2017-03-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, using the method of co-incubation of digested gliadin, probiotics and animal cells to simulate the relieving effect of probiotics on inflammatory bowel disease in vitro can no longer accurately reflect the function of probiotics in regulating the intestinal barrier in vivo, and it is necessary to establish a compensation method. A new method with defects in traditional methods to meet the needs of rapid and accurate screening of probiotics with enhanced intestinal cell tight junction function in vitro

Method used

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  • Method for screening probiotics with function of enhancing intestinal cell tight junction at cell level
  • Method for screening probiotics with function of enhancing intestinal cell tight junction at cell level
  • Method for screening probiotics with function of enhancing intestinal cell tight junction at cell level

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Example 1: Comparison of Different Cell Treatment Methods

[0076] At first the subsection incubation method provided by the invention is compared with the traditional co-incubation method, as figure 1 As shown, A in the figure means adding RPMI 1640 cell culture medium without serum and antibiotics and incubating with HT-29 cells for 3 hours before washing off; B means adding PT to RPMI 1640 cell culture medium without serum and antibiotics -gliadin was washed away after co-incubating with HT-29 cells for 3 hours; C indicated that PT-BSA was added to RPMI 1640 cell culture medium without serum and antibiotics and washed away after co-incubating with HT-29 cells for 3 hours; D indicated that the PT-gliadin was added to the RPMI 1640 cell culture medium without serum and antibiotics, and at the same time, different kinds of probiotics were added to incubate with HT-29 cells for 3 hours, and then washed away; E indicated that PT-gliadin was used without serum and antibiot...

Embodiment 2

[0084] Example 2: Comparison of tight junction protein expression levels obtained in different cell treatments with tight junction protein levels in animal experiments

[0085] (1) Changes in the relative expression of tight junction-related protein genes in cells:

[0086] ①Use the total RNA extraction reagent TRIzol (Invitrogen, Carlsbad, CA) to extract total RNA from HT-29 cells according to the manual;

[0087] ②Use Prime Script, a reverse transcription kit containing gDNA clearance TM RT reagent Kit (Takara, Tokyo, Japan), reverse transcribed RNA into cDNA according to the manual;

[0088] ③The samples were mixed with fluorescent dye SYBR Green super mix (Qiagen, Germany), and the real-time fluorescent quantitative gene amplification instrument CFX96 TM Real-Time System (Bio-Rad, Hercules, CA) was used for detection. Three parallel wells were set up for each sample, and the housekeeping gene β-Actin was used as a reference. The results were analyzed by the 2-ΔΔCq meth...

Embodiment 3

[0097] Example 3: Effects of different treatment methods on the transmembrane resistance values ​​of different intestinal epithelial cells

[0098] Spread HT-29 cells in a permeable nested chamber (3640-Clear, Corining Corporate), add 400 μL of RPMI 1640 cell culture medium without serum and antibiotics to the inner chamber and add 1 mL of serum and antibiotics to the outer chamber The RPMI 1640 cell culture medium was cultured until the monolayer of cells covered the entire membrane surface. After washing away the culture solution, add 4mg / mL PT-gliadin and 1×10 8 CFU / mL of RPMI 1640 cell culture solution without serum and antibiotics 400 μL of probiotics, 1 mL of RPMI 1640 cell culture solution without serum and antibiotics was added to the outer chamber to continue culturing for 3 hours; Incubate the cells with 400 μL of RPMI 1640 cell culture medium without serum and antibiotics containing 4 mg / mL PT-gliadin for 3 hours, then wash the cells in the chamber with phosphate b...

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Abstract

The invention discloses a method for screening probiotics with the function of enhancing intestinal cell tight junction at the cell level. The method includes: firstly utilizing digested protein to treat cells, washing off residual digested gliadin, then adding a certain amount of probiotics to conduct co-incubation for a certain period of time, and then washing off the probiotics so as to determine the probiotics' ability of improving cell tight junction destroyed by digested gliadin. An in vitro experimental model involved in the invention can reflect the actual repair effect of probiotics on gliadin caused intestinal epithelial injury, and the obtained result is in line with probiotics' ability of repairing intestinal barrier damage already formed in the intestinal tract, thereby providing a more convenient and accurate detection method for determining whether probiotics and other microorganisms can enhance intestinal cell tight junction at the cell level.

Description

technical field [0001] The invention relates to the technical field of probiotic screening, in particular to a method for quickly judging whether probiotics can enhance the tight junction of intestinal cells in vitro. Background technique [0002] With the accelerated pace of modern life, more and more Chinese people are also suffering from inflammatory bowel disease (IBD), which was more prevalent in Western countries in the past, including Crohn's disease (CD) and ulcerative colitis (UC). It has been reported that the integrity of the intestinal barrier is the guarantee of human health, and the destruction of intestinal cell tight junctions (TJ) is an important factor in triggering inflammatory bowel disease. Effective approach to venereal enteropathy. [0003] The exact pathogenesis of inflammatory bowel disease is still unclear, and traditional antibiotics and immunomodulatory agents have strong side effects, so probiotics have been proposed by more researchers as a lo...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12R1/01C12R1/225
CPCC12N1/02C12N1/20
Inventor 陈卫王刚许奇田丰伟张秋香刘小鸣范大明赵国忠张白曦翟齐啸毛丙永郭敏杨波陆文伟赵建新张灏
Owner JIANGNAN UNIV
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