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Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis

A bone marrow mesenchymal and stem cell technology, applied in the field of biomedicine, can solve the problems of low survival rate and poor treatment effect, and achieve the effect of improving survival rate, good effect and wide application range

Inactive Publication Date: 2009-02-11
FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the disadvantages of low survival rate and poor therapeutic effect of bone marrow mesenchymal stem cells, the seed cells for the treatment of ischemic cardiomyopathy in the current clinical cell transplantation, and provides a method that can improve the survival of bone marrow mesenchymal stem cells and further enhance its curative effect. medicine

Method used

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  • Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis
  • Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis
  • Use of SIP in preparing medicine for preventing mesenchymal stem cells apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. In vitro test of S1P inhibiting bone marrow mesenchymal stem cell apoptosis

[0038] Proceed as follows

[0039] (1), MSCs isolation and culture

[0040] Sprague-Dawley rats, male or female, were taken, anesthetized with ketamine hydrochloride (60-80mg / kg) intraperitoneally, then soaked in 75% alcohol for 3-5 minutes for disinfection; Culture medium (IMDM+15% FBS) (both IMDM and FBS were purchased from Gibcol Biological Company) washed the bone marrow cavity 10 times, sucked and blown the bone marrow repeatedly with a micropipette to form a dispersed single-cell suspension, inoculated it in a culture bottle, and placed it at 37 °C, 5% CO 2 The cells were cultured in an incubator; after 5 days, the cells could grow to more than 80% confluence, and passaged at a ratio of 1:3 (0.25% trypsin) (trypsin was purchased from Sigma-Aldrich); the first-generation cells that grew well were used for experiments.

[0041] (2), experimental grouping and processing

[00...

Embodiment 2

[0048] Example 2, Bone Marrow Mesenchymal Stem Cell Apoptosis Detection Test

[0049] The test method is as follows:

[0050] (1) Experimental grouping is the same as in Example 1.

[0051] (2) According to the instructions of the Annexin V / PI Apoptosis Detection Kit (Beijing Baosai Biotechnology Co., Ltd.), the cells of each group obtained in Example 1 were digested with trypsin, washed twice with cold PBS, and then washed with Annexin V / PI. V solution was incubated at room temperature for 30 minutes, then PI solution was added to incubate for 5 minutes, and then the apoptosis rate was detected by flow cytometry (Annexin-V / PI flow cytometry can distinguish early apoptosis from mid-late apoptosis of cells. Early apoptosis The PS on the cell membrane is everted to the outer layer of the cell membrane, showing Annexin-V positive, but the cell membrane is intact, and PI is negative, where Annexin V+ / PI- represents early cell apoptosis).

[0052] The results of the normal contro...

Embodiment 3

[0053] Embodiment 3, the test of the influence of S1P on caspase-3 activity

[0054] (1) Test treatment:

[0055] (a) Normal control group: normal culture with complete medium (IMDM+15% FBS); (b) apoptosis model group: treated with serum-free (IMDM) and hypoxia (placed in a closed hypoxia tank, built-in consumption Oxygen), 37°C, 5% CO 2 Incubate for 6 hours; (c) S1P (purchased from Sigma, USA) treatment group: MSCs were pretreated with 10uM S1P for 1 hour, and then incubated for 6 hours under hypoxic and serum-free conditions; (d) S1P (10μM) + apoptosis Model + LY294002 (25uM) group (LY294002 was purchased from cellsignal); (e) S1P (10 μM) + apoptosis model group + U0126 (20uM) group (U0126 was purchased from cellsignal).

[0056] (2) Test method: After 6 hours of hypoxia without serum, the cells of each group were digested with trypsin (0.25%), the cells were collected, and the cell protein was extracted, and the protein concentration was measured by the Coomassie brillian...

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Abstract

The invention discloses the application of S1P in preparing a medicament for inhibiting mesenchymal stem cells from apoptosis, in preparing a medicament for curing ischemic heart disease and a medicament for curing limb ischemia. The invention can significantly improve the survival rate of mesenchymal stem cells in clinical transplantation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a new application of S1P. Background technique [0002] Sphingosine 1-phosphate (English name: sphingosine 1-phosphate, referred to as S1P) is a biologically active sphingomyelin metabolite derived from serum and has important physiological functions. It can function as a second messenger in cells, It can also bind to specific receptors on the cell surface to play important biological functions. The chemical structural formula of sphingosine-1-phosphate is: [0003] [0004] Its molecular formula is: C 18 h 38 NO 5 P, its molecular weight is: 379.47. At present, patent applications "Application of 1-phosphate-sphingosine (S1P) receptor agonists for the treatment of brain degenerative diseases" (application number: 200480037861.1) and "combination of S1P receptor agonists / modulators and immunosuppressive drugs Anti-lymphocyte Antibody Induction" (Application No.: 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/661A61P9/10
Inventor 陈曦刘学彬
Owner FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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