A method for screening probiotics with the function of enhancing intestinal cell tight junction at the cellular level

An intestinal cell and cell-level technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems that cannot accurately reflect the function of probiotics to regulate the intestinal barrier

Active Publication Date: 2020-01-21
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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  • A method for screening probiotics with the function of enhancing intestinal cell tight junction at the cellular level
  • A method for screening probiotics with the function of enhancing intestinal cell tight junction at the cellular level
  • A method for screening probiotics with the function of enhancing intestinal cell tight junction at the cellular level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Comparison of different cell processing methods

[0076] First compare the segmented incubation method provided by the present invention with the traditional co-incubation method, such as figure 1 As shown in the figure, A indicates that RPMI 1640 cell culture medium without serum and antibiotics is added separately and incubated with HT-29 cells for 3 hours and then washed away; B indicates that PT is added to RPMI 1640 cell culture medium without serum and antibiotics. -Gliadin and HT-29 cells are incubated for 3 hours and then washed away; C means PT-BSA is added to RPMI 1640 cell culture medium without serum and antibiotics and washed away after 3 hours of incubation with HT-29 cells; D means in Add PT-gliadin to the RPMI 1640 cell culture medium without serum and antibiotics and add different kinds of probiotics at the same time. Incubate with HT-29 cells for 3 hours and wash it off; E means first use serum-free and antibiotic-free but with PT-gliadin RPMI...

Embodiment 2

[0084] Example 2: Comparison of tight junction protein expression levels obtained by different cell processing methods and animal experiments

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

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

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

[0088] ③Mix the samples with the fluorescent dye SYBR Green super mix (Qiagen, Germany), and use the real-time fluorescent quantitative gene amplification instrument CFX96 TM The detection was performed on the Real-Time System (Bio-Rad, Hercules, CA). Each sample was set up with 3 parallel holes, and the housekeeping gene β-Actin was used as the reference. The results were analyzed by the method of 2-ΔΔCq.

[0089] (2) Chang...

Embodiment 3

[0097] Example 3: Effects of different treatments on the transmembrane resistance of different intestinal epithelial cells

[0098] Spread HT-29 cells flat in a permeable nested chamber (3640-Clear, Corining Corporate), add 400μL of RPMI 1640 cell culture medium without serum and antibiotics to the inner chamber, and add 1mL without serum and antibiotics to the outer chamber Culture the RPMI 1640 cell culture medium until the monolayer of cells covers the entire membrane surface. After washing away the culture medium, add 4mg / mL PT-gliadin and 1×10 to the chamber of the co-incubation group. 8 400μL of RPMI 1640 cell culture medium without serum and antibiotics in CFU / mL probiotics, add 1 mL of RPMI 1640 cell culture medium without serum and antibiotics to the outer chamber and continue culturing for 3 hours; and add it to the chamber of the segmented incubation group first 400μL of RPMI 1640 cell culture medium containing 4mg / mL PT-gliadin without serum and antibiotics was incuba...

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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 probiotics 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 acceleration of modern life, more and more Chinese people are also suffering from inflammatory bowel disease (IBD), which was popular in Western countries in the past. This disease includes 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. The destruction of intestinal cell tight junctions (TJ) is an important factor in inducing inflammatory bowel disease. Therefore, enhancing the intestinal cell tight junctions has also become a protection against inflammation. An effective way of enteropathy. [0003] The exact pathogenesis of inflammatory bowel disease is still unclear, and traditional antibiotics, immunomodulators, etc. hav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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