Method for inducing self-renewal of mammary stem cells

A technology of mammary gland stem cells and mammary glands, which is applied in the field of small molecule RNA, can solve problems such as ignorance of the role, and achieve the effect of enhancing the ability of cloning formation

Inactive Publication Date: 2015-03-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Scott Valastyan et al. found that miR-31 inhibits the migration of breast cancer cells by targeting the expression of RDX and RhoA. However, the role of miR-31 in normal breast development is still unknown

Method used

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  • Method for inducing self-renewal of mammary stem cells
  • Method for inducing self-renewal of mammary stem cells
  • Method for inducing self-renewal of mammary stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation of mammary gland stem cells

[0026] 1. Take the fresh mammary gland tissue of the mouse, remove the lymph nodes, put it into a 10cm cell culture dish, put it on ice, and cut the mammary gland tissue as much as possible with a scalpel and tweezers.

[0027] 2. Put the breast tissue above into a 15mL centrifuge tube, add an appropriate amount of collagenase / hyaluronidase digestion solution to it, place it in a hybridization oven at 37°C and rotate it, and digest for 6-8 hours.

[0028] 3. After the mammary gland tissue is completely digested, centrifuge at 350xg for 5-10 minutes, and carefully discard the supernatant.

[0029] 4. Add 2-3mL ammonium chloride solution to the above breast tissue fragments to destroy red blood cells in the breast tissue. Centrifuge at 350xg for 5 minutes and discard the supernatant.

[0030] 5. Add 1-2mL trypsin and mix up and down with a pipette for 1-3min. Immediately add 10 mL of pre-cooled HBSS buffer solution to s...

Embodiment 2

[0035] Example 2Dox induces miR-31 expression and detection of miR-31 in K5rt-TA / TRE-miR-31 transgenic mouse mammary gland tissue

[0036] 1. Use water containing 2 g / L Dox to feed 3-week-old K5rt-TA / TRE-miR-31 double positive mice, while feeding K5rt-TA or TRE-miR-31 single positive mice as a control. Mice were sacrificed at 12 weeks for sampling.

[0037] 2. After the mice were killed by dislocation, the lymph nodes were removed with RNase-removing surgical scissors and surgical forceps, and the mammary gland tissue was placed in a DEPC-treated 1.5mL centrifuge tube, and 1mL Lysis Binding Buffer was added to it. Homogenize with a homogenizer for 10-15 minutes until the tissue is completely lysed, then add 100 μL HomegenateAdditive to the above homogenate, shake gently several times, and place on ice for 10 minutes.

[0038] 3. Add 1mL of phenolic extract to the above mixture, shake gently for 1min, and centrifuge at 10,000g for 5min until the layers are separated.

[0039] 4...

Embodiment 3

[0047] Example 3 Effect of miR-31 on Mammary Stem Cells In Vitro Clonogenic Ability

[0048] 1. Lin obtained by sorting the method in Example 1 - CD24 + CD29 high The cell population was equally divided into 3 parts, each containing 10 5 cell.

[0049] 2. Using the method of electroporation, 2 cells were electrotransfected with 4 μg of Scramble RNA, and the other cell was electroporated with 4 μg of miR-31 inhibitor (5’-CAGCUAUGCCAGCAUCUUGCCU-3’; Shanghai GenePharma Co., Ltd).

[0050] 3. These cells were resuspended in EpiCult-B culture medium, and cultured on cell culture scaffolds (Millipore Cor., Billerica, USA). The culture medium is EpiCult-B culture medium adding growth-promoting factors, including 10ng / mL recombinant human Epidermal Growth Factor (rhEGF; Catalog #02633, Stem Cell Technologies), 10 ng / mL recombinant human Basic Fibroblast Growth Factor (rh-bFGF; Catalog # 02634, Stem Cell Technologies), 4 μg / mL (0.0004%) Heparin (Catalog #07980, Stem Cell Technolog...

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Abstract

The invention provides a method for inducing self-renewal of mammary stem cells. The invention discloses a new application of microRNA31. Experiments show that through over-expression of microRNA31, the proportion of mammary stem cells is obviously regulated up, clone formation ability is enhanced and therefore the self-renewal of the mammary stem cells is promoted.

Description

technical field [0001] The present invention relates to a small molecular RNA, in particular to the application of a small molecular RNA—miR-31 in promoting the self-renewal of mammary gland stem cells. Background technique [0002] Transgenic technology is to introduce foreign genes into prokaryotic or eukaryotic cells to study gene expression regulation and gene function. But the function of a gene in a single cell in vitro does not necessarily represent the function of the gene in the whole. Since the 1960s, life science researchers have tried to find an appropriate method to introduce specific genes into developing mammals in order to study the function and regulation of genes. Transgenic animals refer to animal strains established by introducing exogenous genes into early embryonic cells by experimental methods to integrate them into the cell genome. The first successful establishment of transgenic animals is transgenic mice (transgenic mice). Since the establishment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/68A01K67/027G01N15/14
Inventor 于政权李丰银李想王珊孟庆勇
Owner CHINA AGRI UNIV
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