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Application of Rspo1 to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells

A technology of bone marrow mesenchymal and cardiomyocyte-like cells, applied in the field of regenerative medicine, can solve problems such as no reports on the effect, and achieve a significant effect of promoting differentiation

Inactive Publication Date: 2019-04-02
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but Rspo1 The role in the differentiation of BMSCs has not been reported so far

Method used

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  • Application of Rspo1 to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells
  • Application of Rspo1 to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells
  • Application of Rspo1 to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Isolation and culture of rat BMSCs.

[0050] Under sterile conditions, expose the bone marrow cavity of the femur and tibia of the hindlimb of Sprague-Dawley rats, wash the bone marrow cavity with medium (L-DMEM + 10% fetal bovine serum + 1% double antibody, the same below) until the bone marrow cavity changes from red to White.

[0051] Pipette the bone marrow suspension into the 25cm 2 Culture bottle, placed in humidity ≥ 95%, 37°C, 5% CO 2 cultured in an incubator.

[0052] Three days later, when the confluence of BMSCs reached about 90%, the first passage was carried out. Discard the culture medium, wash it twice with PBS, add 1.5ml of 0.25% trypsin (containing EDTA) solution, digest at 37°C for 3 minutes, the cells become round and float under the microscope, add the culture medium to stop the digestion.

[0053] Transfer the cells into a centrifuge tube with a pipette and centrifuge at 1000rpm for 5min. Discard the supernatant, add the culture medi...

Embodiment 2

[0057] Example 2: Identification of rat BMSCs.

[0058] Take the P3 cells cultured in Example 1, add 0.25% trypsin without EDTA, digest at 37°C, and wash with PBS. The obtained cells were used to detect the specific surface molecules and cell cycle of BMSCs by flow cytometry.

[0059] Prepare the cell suspension with 1ml PBS and count the cells so that the cell density is 1×10 5 cells / ml, centrifuge at 1000rpm for 5min, discard the supernatant. Cell suspension was prepared in 200 μl PBS, CD29-APC, CD90-FITC, CD45-PE antibodies (Abcam Company) were added, and incubated at room temperature for 30 min in the dark. Wash with PBS, resuspend the cells in 200 μl buffer, and detect the specific surface molecules CD29, CD90 of BMSCs and CD45 of hematopoietic cells by flow cytometry, and the results are as follows figure 2 shown.

[0060] figure 2 It shows that the expression rates of BMSCs-specific surface molecules CD29 and CD90 are (99.54±0.13)% and (95.74±1.39)% respectively,...

Embodiment 3

[0069] Example 3: Using adenovirus as a carrier Rspo1 Rat BMSCs were transfected.

[0070] The P3 cells of Example 1 were taken, washed with PBS, added with 0.25% trypsin containing EDTA, and digested at 37°C for 3 minutes. Cells in 8×10 4 The density of each well is planted in a 6-well plate, the humidity is ≥95%, 37 ° C, 5% CO 2 Cultivate in the incubator for 24h.

[0071] Divide BMSCs into 3 groups: control group, only add 2ml of medium without double antibody; negative control group, add 1ml of negative control virus solution + 1ml of medium without double antibody; Rspo1 overexpression group, add concentration of 2×10 8 PFU / ml with gene Rspo1 1ml of virus solution + 1ml of double antibody-free medium.

[0072] The three groups of BMSCs were incubated for 24 hours, then the medium was changed, and the incubation was continued for 2 days, observed and photographed with a fluorescence microscope.

[0073] according to Image 6 Fluorescence microscope observation resu...

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Abstract

The present invention provides an application of Rspo1 to induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells. Adenoviruses are used as a vector, Rspo1 is transfected into the rat bone marrow mesenchymal stem cells, after induced differentiation by 5-azacytidine, expression of cardiomyocyte-specific proteins in the cardiomyocyte-like cells is detected, and besides, the expression is significantly increased, indicating that the Rspo1 promotes the differentiation of the bone marrow mesenchymal stem cells into the cardiomyocyte-like cells and a conversion rate of the cardiomyocyte-like cells is as high as 53%.

Description

technical field [0001] The invention belongs to the technical field of regenerative medicine, and relates to a method for inducing differentiation of cardiomyocytes, in particular to a method for using Rspo1 A method for directing bone marrow mesenchymal stem cells to differentiate into cardiomyocytes. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells that exist in the bone marrow and have strong self-renewal ability and multi-directional differentiation potential. Under specific physiological environment or in vitro induction conditions, BMSCs can differentiate into cells of multiple germ layers, such as cardiomyocytes, bone and chondrocytes, adipocytes, nerve cells, and liver cells. [0003] BMSCs are rich in sources, relatively harmless to donors, low in immunogenicity, easy to introduce exogenous genes, there is no ethical issue of embryonic stem cells, and there is no controversy over the gene stability of induced...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/861
CPCC12N5/0657C12N15/86C12N2500/40C12N2501/415C12N2510/00C12N2710/10343C12N2800/107
Inventor 杨丽红解军杨巧艳程肖霞宋慧芳何生张娟于保锋刘丹
Owner SHANXI MEDICAL UNIV
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