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Sca-1+/CD34- uterine stem cells and separation method thereof

A CD34-, CD34 technology, applied in the field of stem cells, can solve problems such as the treatment mechanism and effect of uterine stem cell ischemic diseases that have not been reported.

Inactive Publication Date: 2013-09-18
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there is no report on the mechanism and effect of this kind of uterine stem cells in angiogenesis and the treatment of ischemic diseases.

Method used

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  • Sca-1+/CD34- uterine stem cells and separation method thereof
  • Sca-1+/CD34- uterine stem cells and separation method thereof
  • Sca-1+/CD34- uterine stem cells and separation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Sca-1 + / CD34 － Isolation of Uterine Stem Cells 。

[0027] 1) Mice aged 8-10 weeks were anesthetized with isoflurane, intubated, ventilated to maintain breathing, and anesthetized with 2-3% isoflurane.

[0028] 2) The anesthetized mouse was placed in a supine position, the thoracotomy was performed in the middle, the pericardium was torn apart, and the right atrium was incised, and the aorta was continuously perfused with 0.9% normal saline at physiological pressure, and the tissue was washed until it flowed out from the right atrium (no blood contamination) ) of saline.

[0029] 3) Open the abdomen, take out the uterus, and mince it well.

[0030] 4) Digest with 0.25% trypsin, 2mg / ml collagenase, 0.01% DNase at 37°C for 1 hour to minimize the destruction of cell surface markers.

[0031] 5) From the above-mentioned digested tissue, use magnetic bead-labeled anti-CD34 antibody (stemcell, Canada) to screen out CD34 by magnetic bead screening method － cel...

Embodiment 2

[0035] Example 2: Sca-1 + / CD34 － Cultivation of uterine stem cells.

[0036] The cells screened in Example 1 were inoculated in a 35mm culture dish, and 1% methylcellulose basal medium, 10% fetal bovine serum, 4.5×10 -4 M thioglycerol, 25 μg / mL ascorbic acid, 2 mM glutamine, 200 μg / mL saturated transferrin, the culture environment was kept at a humidity ≥ 95%, temperature 37 ° C, CO 2 Concentration 5%.

Embodiment 3

[0037] Example 3: Sca-1 + / CD34 － Differentiation of Uterine Stem Cells.

[0038] Differentiation medium composition: IMDM medium, 10% fetal bovine serum and 25% endothelial cell primary medium.

[0039] Sca-1 + / CD34 － Uterine stem cells, Sca-1 － / CD34 － Cells and cell extracts of bone marrow mesenchymal stem cells (MSCs) were added to Matrigel, and human umbilical cord blood endothelial cells (HUVECs) and smooth muscle cells (SMCs) were cultured separately, and cultured in ordinary medium as a control group, and The length of neovascularization was used to evaluate the ability of HUVECs to form vascular structures.

[0040] The results showed that Sca-1 + / CD34 － The ability of uterine stem cells to induce endothelial cells to form vascular structures is better than that of Sca-1 － / CD34 － and bone marrow MSC are stronger, see figure 2 , image 3 .

[0041] figure 2 A-B shows Sca-1 + / CD34 － Migration results of human umbilical vascular endothelial cells (H...

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Abstract

Sca-1+ / CD34- uterine stem cells, isolated from the uterus of mammals, express CD34 and Sca-1 in the form of CD34-, Sca-1+, and the isolation method is to use magnetic beads to label anti-CD34 antibody from digested CD34-cells were screened out from uterine tissue; Sca-1+ cells were separated from CD34-cells by magnetic bead-labeled anti-SCa-1 antibody to obtain Sca-1+ / CD34-uterine stem cells. Experiments have confirmed that Sca-1+ / CD34- uterine stem cells have the ability to induce endothelial cells to form vascular structures, can form a large number of functional blood vessels, and can restore tissue perfusion to a certain extent and reduce tissue damage; in vivo levels have confirmed that Sca-1 The + / CD34-uterine stem cells have a therapeutic effect on ischemic diseases and can be used to prepare medicines for preventing or treating ischemic diseases.

Description

technical field [0001] The present invention relates to a stem cell, in particular to a stem cell extracted from the uterus. The present invention also relates to the separation method of the uterine stem cells and the application of the uterine stem cells. Background technique [0002] With the change of people's lifestyle, ischemic diseases have become one of the diseases with the highest incidence in the world in recent years, and the incidence has been rising. Ischemic disease is a disease that causes poor tissue perfusion due to vascular stenosis or occlusion, and eventually causes organ dysfunction or even failure. disease, cerebral infarction, etc. Under normal conditions, angiogenesis is the body's own compensatory mechanism for ischemia and hypoxic injury. In the early stage of myocardial ischemia, capillary density increases compensatoryly, and new blood vessels are formed around blocked or narrowed coronary arteries to establish collateral circulation, thereby ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074A61K35/48A61P9/10
Inventor 李仁科解军刘志贞孙卓
Owner SHANXI MEDICAL UNIV
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