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Method of combining salvianolic acid B to induce directed myocardiac differentiation of iPSCs by sodium-calcium exchanger 1 promoter

A technology of salvianolic acid and sodium-calcium exchange, applied in the field of medicine, can solve the problems of a wide range of myocardial necrosis, low efficiency of cell transplantation and homing, and the clinical application cannot be widely carried out, and achieves the effect of broad application prospects.

Active Publication Date: 2017-08-04
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] There are still many difficulties in stem cell therapy for acute myocardial infarction, such as the selection of seed cells, wide range of myocardial necrosis, low homing efficiency of cell transplantation, and ischemia and hypoxia in the local myocardial microenvironment. be widely developed

Method used

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  • Method of combining salvianolic acid B to induce directed myocardiac differentiation of iPSCs by sodium-calcium exchanger 1 promoter
  • Method of combining salvianolic acid B to induce directed myocardiac differentiation of iPSCs by sodium-calcium exchanger 1 promoter
  • Method of combining salvianolic acid B to induce directed myocardiac differentiation of iPSCs by sodium-calcium exchanger 1 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of iPSCs

[0022] 1) Preparation of the trophoblast layer (MEF): 0.1% (volume fraction) of gelatin is added to the T25 culture flask, placed in a 37°C cell culture incubator for 20 minutes, then sucked out, and 5-6mL preheated 37°C MEF culture medium is added At the same time, the mouse embryonic fibroblasts (MEF) (purchased from the cell bank of the Shanghai Chinese Academy of Sciences) were quickly taken out of liquid nitrogen, placed in a 37°C water bath and quickly thawed and immediately wiped and stored with 75% alcohol with a volume fraction of alcohol. After the tube is taken to the ultra-clean table, transfer the cell suspension in the cryopreservation tube to a 15 mL centrifuge tube containing MEF medium, centrifuge at 1000 rpm for 5 minutes, discard the supernatant and resuspend it and add it to a T25 culture flask, and place it in CO 2 In a constant temperature incubator, iPSCs can be added to the feeder layer after 24 hours of cultivation; ...

Embodiment 2

[0024] Example 2 Preparation of Sodium-Calcium Exchanger 1 Promoter

[0025] 1) Lentivirus Neo-Promoter_419bp> Luciferase:IRES:DsRed_Express2 construction and identification:

[0026] Primer design: According to the cDNA sequence of the sodium-calcium exchanger 1 promoter gene in Genebank, the primer sequence of the sodium-calcium exchanger 1 promoter is designed as:

[0027] Upstream primer: 5'-GCAACAGACATACAAACTAAAGAAT-3';

[0028] Downstream primer: 5'-GGAGCAACATAGTTAAGAATACC-3'.

[0029] PCR amplification: under the action of the above primers, the cDNA of the promoter of sodium-calcium exchanger 1 is used as a template for amplification, the PCR product is detected by electrophoresis, and the gel is cut to recover and purify the target band. Add the vector Neo-Promoter_419bp> Luciferase:IRES:DsRed_Express2 was recombined with the amplified sodium-calcium exchanger 1 promoter under the action of endonuclease, 2μL of the recombination reaction solution was used to transform 50μL of ...

Embodiment 3

[0035] Example 3 Cellular immunofluorescence and morphological identification

[0036] After 20 days of induction culture, washed with PBS for 3 times, fixed with 4% paraformaldehyde, washed with PBS, counterstained with DAPI for 10 minutes, and fixed with resin glue. Observe under a fluorescence microscope and an inverted microscope, with 10 fields of view in each hole, a total of 30 fields of view.

[0037] The expression of NCX1 after NCX1 lentivirus transfected induced pluripotent stem cells, such as figure 1 Shown. The results showed that the qualitative analysis of fluorescence microscope showed that NCX1 was effectively expressed in iPSCs.

[0038] Sodium-calcium exchanger 1 promoter combined with salvianolic acid B induces morphological changes after iPSCs differentiation, such as figure 2 Shown; where, A: control group; B: salvianolic acid B (1μM); C: salvianolic acid B (10μM); D: salvianolic acid B (100μM); E: NCX1 group; F: NCX1 + salvianol Acid B (1 μM); G: NCX1 + Salv...

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Abstract

The invention discloses a method of combining salvianolic acid B to induce directed myocardiac differentiation of iPSCs by a sodium-calcium exchanger 1 promoter and belongs to the technical field of medicines. The method comprises the following steps: constructing NCX1 lentivirus particles and transfecting the NCX1 lentivirus particles to iPSCs; and performing assistance with combined induction of salvianolic acid B. The concentration of the salvianolic acid B is 1-100[mu]M. The method disclosed by the invention verifies that the sodium-calcium exchanger 1 promoter combined with the salvianolic acid B can effectively induce directed myocardiac differentiation of iPSCs. The salvianolic acid B can effectively promote NCX1 expresion and promote remarkable improvement of expressions of cardiac markers alpha-actin and cardiac troponin (cTnT). According to the shape of the iPSCs, fusiform change of a myocardial cell sample can be seen. IPSCs as a seed cell for repairing acute myocardial infarction have a wide application prospect.

Description

Technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a method for inducing iPSCs directed myocardial differentiation with a sodium-calcium exchanger 1 promoter combined with salvianolic acid B. Background technique [0002] There are still many difficulties in stem cell therapy for acute myocardial infarction, such as the selection of seed cells, a wide range of myocardial necrosis, low cell transplantation homing efficiency, and local myocardial microenvironment ischemia and hypoxia. Therefore, its clinical application is still not Can be widely carried out. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells obtained by reprogramming somatic cells with high proliferation and multidirectional differentiation potential similar to embryonic stem cells (ECSs). It lacks the immunogenicity and moral and ethical restrictions of embryonic stem cells, induced pluripotent stem cells are expected to become seed cells w...

Claims

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Application Information

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IPC IPC(8): C12N5/10
Inventor 郭军江灿
Owner JINAN UNIVERSITY
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