Construction and application of cardiac muscle cell protein difference expression atlas

A cardiomyocyte differential expression technology, applied in the field of constructing the protein differential expression map of icariin-induced stem cell-directed differentiation cardiomyocytes, can solve the problems that have not yet been studied

Inactive Publication Date: 2008-12-24
ZHEJIANG UNIV
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Problems solved by technology

However, it is not enough to study only from the perspective of genes. Only by studying the process of protein transcription and translation from genes can we truly reveal the nature and laws o

Method used

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  • Construction and application of cardiac muscle cell protein difference expression atlas
  • Construction and application of cardiac muscle cell protein difference expression atlas
  • Construction and application of cardiac muscle cell protein difference expression atlas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of differential protein expression map of ICA-induced directed differentiation of stem cells into cardiomyocytes

[0023] (1) To establish a model of drug-induced stem cell directed differentiation into cardiomyocytes: ES cells were cultured in hanging drops to form embryoid bodies, and drug icariin was added to induce their directed differentiation into cardiomyocytes.

[0024] (2) Preparation of two-dimensional electrophoresis protein samples: collect the above-mentioned differentiated cells in different differentiation phases, rinse with phosphate buffer, blot the residual liquid, add lysis buffer and mix well, freeze and thaw repeatedly in liquid nitrogen for 3 times, add 50 μg / ml RNase and 200 μg / ml DNase, place at 4°C for 15 minutes, centrifuge at 16100 rpm, discard the supernatant, wash twice with 1% dithiothreitol (DTT) pre-cooled acetone, dry at room temperature for 1 hour, and dissolve the hydration solution The protein concentration wa...

Embodiment 2

[0029] Example 2 ICA Induced Directed Differentiation of Mouse ES Cells into Cardiomyocytes in Vitro

[0030] 1. Differentiation culture of ES cells induced by ICA

[0031] (1) Hanging drop for three days

[0032] Undifferentiated ES cells were made into single-cell suspension with ES cell differentiation medium, counted, and diluted to 2.0×10 with high-glucose DMEM medium containing 20% ​​fetal bovine serum. 4 cells / ml, 30 μl / drop of cell solution (each hanging drop contains 600 ES cells) was added dropwise on the inner surface of the culture dish cover for hanging drop differentiation and cultured for 3 days until embryoid bodies (embryonic bodies, EBs) were formed.

[0033] (2) Suspension for two days

[0034]The bottom of the culture dish was paved with 1% agar solution in advance, and the formed EBs were transferred to a culture dish containing high-sugar DMEM and 20% fetal bovine serum medium for suspension for 2 days.

[0035] (3) adherent culture

[0036] Transfer ...

Embodiment 3

[0040] Example 3 Two-dimensional gel electrophoresis separation of protein expression points of ICA-induced cardiomyocyte differentiation of ES cells

[0041] Isoelectric focusing electrophoresis is performed first to separate the total protein according to the isoelectric point, and then SDS-PAGE is further separated according to the molecular size. The electrophoretic map obtained by staining is a two-dimensional distribution of protein maps. The target protein was discovered and determined by analyzing the protein expression changes of ES cells differentiated into cardiomyocytes at different differentiation stages and after ICA induction by comparing protein maps.

[0042] (1) Sample preparation

[0043] A. Cell samples

[0044] Aspirate the culture solution, and collect the cells (spontaneously differentiated and differentiated cardiomyocytes induced by ICA for 3 days and 7 days in adherent culture) with a cell scraper. Add D-Hanks solution, centrifuge at 1500 rpm for 10...

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Abstract

The invention provides a buildup method of a protein difference expression atlas when the committed differentiation of stem cells is induced by drugs, which detailedly establishes the protein difference expression atlas formed by that the stem cell is induced by the ICA and directionally differentiated to be a cardiac muscle cell; a model that the drug induces the stem cell to be directionally differentiated to be the cardiac muscle cell is established so as to prepare a two-dimensional electrophoresis protein sample; electrophoresis is gelled bidirectionally, images are collected and analyzed so as to confirm the difference protein. A comparison protein atlas is used for screening and identifying the difference expression protein when the ES cell is directionally differentiated to be the cardiac muscle cell by the inducing of the ICA; the difference expression protein is used as a drug effect target and applied to the preparation of a novel inducer which has high-efficiency and low-toxicity and is used for prompting the stem cell to be differentiated.

Description

technical field [0001] The invention belongs to the field of pharmacology and medical technology, and relates to the construction and application of a protein differential expression map of drug-induced stem cell directional differentiation, in particular to the construction of a protein differential expression map of icariin-induced stem cell directional differentiation cardiomyocytes, and the identification of icariin Characteristic changes in the expression proteome of the induced and spontaneously differentiated groups. Background technique [0002] Embryonic stem cells (ES cells) can be directedly differentiated into cardiomyocytes with typical structure and function in vitro, and this differentiation system has become an important tool for studying cardiac gene expression and function. At present, the functions of many genes and the regulation mechanism of signal transduction network in the process of directed differentiation of ES cells into cardiomyocytes are still n...

Claims

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Application Information

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IPC IPC(8): G01N27/64G01N27/447
Inventor 朱丹雁楼宜嘉李欢张翔南周立民梁星光
Owner ZHEJIANG UNIV
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