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Application of Y-27632 inhibitor to CD44 positive intestinal stem cell sorting

A technology of Y-27632 and MY-27632, which is applied in the application field of Y-27632 inhibitor in the sorting of CD44-positive intestinal stem cells, can solve the problems of expensive transgenic mice, reduced number of sorted stem cells, and affecting cell viability, etc. Achieve the effects of simple operation, high product purity and improved survival rate

Inactive Publication Date: 2015-08-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, during the sorting process, it is impossible to add sufficient amount of Y-27632 inhibitor in the sheath fluid to inhibit the empty nest apoptosis of intestinal stem cells, which is the main technical limitation of sorting Lgr5 positive intestinal stem cells
Also, since C57 / B6J Lgr5-IRES-CRE-eGEFP The expression of mouse CRE gene needs to be induced by tamoxifen, and 10% of Lgr5 intestinal stem cells are immediately apoptotic after being induced by tamoxifen, which will reduce the number of sorted stem cells; secondly, compared with inbred For wild-type mice, C57 / B6J Lgr5-IRES-CRE-eGEFP Transgenic mice are expensive and the breeding conditions are harsh; third, the binding site of the Lgr5 monoclonal antibody currently on the market is only in the cell, and it is necessary to rupture the cell membrane through methods such as antibody labeling, which will inevitably affect cell activity
Therefore, the use of Lgr5 as a target protein to sort intestinal stem cells is limited by the above technical conditions

Method used

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  • Application of Y-27632 inhibitor to CD44 positive intestinal stem cell sorting
  • Application of Y-27632 inhibitor to CD44 positive intestinal stem cell sorting
  • Application of Y-27632 inhibitor to CD44 positive intestinal stem cell sorting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1C57

[0088] Example 1 Isolation and phenotype identification of CD44 positive cells in C57BL / 6 mice

[0089] 1. Under sterile conditions, obtain the whole small intestine of C57BL / 6 mice (Experimental Animal Center, Academy of Military Medical Sciences of the Chinese People's Liberation Army) that was used in other people's experiments. -hanks fluid to wash away intestinal contents.

[0090] 2. Gently scrape the surface of the intestinal lumen with the sterilized coverslip to remove the villi in the epithelium.

[0091] 3. Cut the intestinal tissue into 3mm pieces with sterile scissors, place them in a 50mL centrifuge tube, and rinse repeatedly with pre-cooled D-hanks until the supernatant is clear.

[0092] 4. Add separation solution and incubate for 30 minutes with shaking at 4°C.

[0093] 5. After the incubation, wait for the tissue to settle to the bottom of the centrifuge tube, carefully aspirate the separation solution; centrifuge at 1000 rpm for 5 minutes, and discard the ...

Embodiment 2

[0103] Induction culture of embodiment 2CD44 positive cells

[0104] 1. Same as steps 1-13 in Example 1, magnetic bead sorting of CD44-positive cells in C57BL / 6 mice (positive selection), with CD44-negative cells (negative selection) as a control.

[0105] 2. Resuspend the Matrigel that has been melted on ice in the ratio of 10 μL Matrigel per 100 sorted cells. This step should be performed on ice to prevent the Matrigel from solidifying due to temperature rise.

[0106] 3. After the cells are premixed, add the Matrigel-cell suspension at a ratio of 10 μL Matrigel / well to a 96-well culture plate preheated at 37°C (100 sorted cells / 10 μl Matrigel / well); when inoculating, The Matrigel should be centered on the bottom of the plate, avoiding the glue touching the walls.

[0107] 4. Incubate the inoculated cells in a 37°C incubator for 5 minutes to finalize the Matrigel; prepare a complete medium.

[0108] 5. After the incubation, add 100 μL of complete medium to each well and st...

Embodiment 3

[0110] Example 3 Differentiation of CD44 positive cells into 4 types of intestinal epithelial cells

[0111] 1. After 2 weeks of in vitro culture, discard the complete medium; add pre-cooled neutral formaldehyde solution (200 μL / well) to the 96-well plate to fix the “mini-intestine” differentiated from CD44-positive cells, 4°C, 30min .

[0112] 2. Wash off the neutral formaldehyde fixative with pre-cooled DPBS (2-3 times); add pre-configured 0.025% Triton X-100 solution (200 μL / well) to the 96-well plate, and incubate at 4°C for 10 minutes .

[0113] 3. Blow Matrigel with pre-cooled DPBS solution to dissolve and release the inner "mini intestine"; centrifuge at 1000rpm for 2min.

[0114] 4. After washing the "mini intestine" pellet 2-3 times with DPBS, transfer it to a new EP tube.

[0115] 5. Incubate the "mini intestine" pellet with 1 mL of PBS solution containing 5% BSA at 4°C for 30 min.

[0116] 6. Add the pre-configured primary antibody working solution, including: a...

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Abstract

The invention relates to an application of a Y-27632 inhibitor to CD44 positive intestinal stem cell sorting. CD44 is taken as a mark for sorting intestinal stem cells. By using the Y-27632 inhibitor and magnetic beads to sort the CD44 intestinal stem cells, a method for separating the intestinal stem cells is more convenient and simpler to operate. A membrane antigen is applied to the sorting of the intestinal stem cells in a recess without damaging cell structures; moreover, sufficient intestinal stem cells can be obtained once with the separation method, so that a material basis is laid for carrying out subsequent applications.

Description

technical field [0001] The invention relates to the technical field of stem cell isolation and culture, in particular to the application of a Y-27632 inhibitor in the sorting of CD44-positive intestinal stem cells. Background technique [0002] In recent years, high expression of Lgr5(GPCR49) protein in intestinal crypts has been widely accepted as a marker for identifying intestinal stem cells. In 2009, Sato and other scholars in Japan learned from the parent C57 / B6J Lgr5-IRES-CRE-eGEFP Cells highly expressing Lgr5 protein were isolated from the intestinal tract of mice, and a complete in vitro induction and differentiation system was established. They confirmed that cells with high expression of Lgr5 protein at the base of mouse intestinal crypts can independently proliferate and differentiate into a complete small intestinal epithelial structure in vitro. [0003] At present, the main technology used in the world to isolate intestinal stem cells is flow cytometry. Howe...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/071
Inventor 常鹏宇
Owner JILIN UNIV
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