The method of sodium-calcium exchanger 1 promoter combined with salvianolic acid b to induce directional myocardial differentiation of iPSCs

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, the failure of clinical application to be widely carried out, ischemia and hypoxia in the local myocardial microenvironment, and achieve the effect of broad application prospects

Active Publication Date: 2019-10-18
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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  • The method of sodium-calcium exchanger 1 promoter combined with salvianolic acid b to induce directional myocardial differentiation of iPSCs
  • The method of sodium-calcium exchanger 1 promoter combined with salvianolic acid b to induce directional myocardial differentiation of iPSCs
  • The method of sodium-calcium exchanger 1 promoter combined with salvianolic acid b to induce directional myocardial differentiation of iPSCs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of iPSCs

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

[0023] 2) Cultivation and passage of iPSCs: The trophoblast obtai...

Embodiment 2

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

[0025] 1) Construction and identification of lentivirus Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2:

[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 was designed as follows:

[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 sodium-calcium exchanger 1 promoter was used as a template for amplification, the PCR product was detected by electrophoresis, and the target band was recovered and purified by gel cutting. Recombine the vector Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2 with the amplified sodium-calcium exchanger 1 promoter under the action of endonuclease, take 2 μL of the recombination reaction solution to trans...

Embodiment 3

[0035] Example 3 Cell immunofluorescence and morphological identification

[0036] After induction culture for 20 days, wash with PBS 3 times, fix with 4% paraformaldehyde, wash with PBS, counterstain cell nuclei with DAPI for 10 min, and fix with resin glue. Observe under a fluorescence microscope and an inverted microscope, and observe 10 fields of view in each well, a total of 30 fields of view.

[0037] NCX1 expression after NCX1 lentivirus transfection induced pluripotent stem cells, such as figure 1 shown. The results showed that NCX1 was effectively expressed in iPSCs through qualitative analysis by fluorescence microscopy.

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

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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 in particular relates to a method for inducing iPSCs directional 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, wide range of myocardial necrosis, low homing efficiency of cell transplantation, and ischemia and hypoxia in the local myocardial microenvironment. be widely developed. Induced pluripotent stem cells (iPSCs) are a kind of pluripotent stem cells obtained by reprogramming somatic cells with high proliferation and multipotent differentiation potential similar to embryonic stem cells (ECSs). Lacking the immunogenicity and moral and ethical restrictions of embryonic stem cells, induced pluripotent stem cells are expected to become seed cells with great clinical application pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
Inventor 郭军江灿
Owner JINAN UNIVERSITY
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