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Cell nucleus extraction method

A technology of cell nuclei and cardiomyocytes, applied in the field of biomedicine

Inactive Publication Date: 2019-07-09
FUDAN UNIV
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Problems solved by technology

However, the molecular pharmacological and physiological functions of receptors restricted to the nuclear envelope of the heart remain poorly explored, largely due to challenges associated with in vivo accessibility and survival of said receptors after isolation, Highly pure intact nuclei from cardiac cells for in vitro studies

Method used

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  • Cell nucleus extraction method
  • Cell nucleus extraction method
  • Cell nucleus extraction method

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Embodiment 1

[0047] Extract nuclei as follows:

[0048] Step 1. Obtaining isolated canine cardiomyocytes, including: preparing oxygenated compound buffer solution and related instruments and perfusion devices; routinely processing experimental animals to obtain isolated experimental animal hearts; perfusion and digestion of isolated experimental animal hearts, obtaining cardiomyocytes;

[0049] Step 2, subcellular fractionation, which includes: homogenization of isolated experimental animal myocardial tissue and cardiomyocytes; differential centrifugation of tissue and cell homogenate to obtain separated crude nuclei, membranes and cytoplasm; discontinuous sucrose density gradient centrifugation After centrifugation after obtaining the coarse nuclear precipitation layer, the separated nucleus was obtained and showed an elongated almond shape or a round shape, and was positive for DNA staining; all sub-steps in step 2 were carried out on ice or at 4 degrees.

[0050] More specifically, inc...

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Abstract

The invention belongs to the technical field of biomedicine and relates to a cell nucleus extraction method, in particular to a method for separating cytosol, membranes and nuclei from isolated canineventricular muscle and isolated canine ventricular cardiomyocytes. The cell nucleus extraction method includes the steps of 1, obtaining isolated cardiomyocytes and 2, carrying out subcellular fractionation. The cell nucleus extraction method has the advantages that the method helps obtain a great number of human functional nuclei for fundamental research and clinical application; screening and enrichment of specific types of human cells can be safely and effectively realized, so that the obtained cells have higher biological safety; the method detects biological characteristics and functionsof the isolated nuclei comprehensively, provides reliable data for research and application of nuclear separation, and provides a set of referential method system for extracting and evaluating the nuclei of other novel cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a method for extracting cell nuclei, in particular to a method for separating and extracting cell nuclei from isolated experimental animal heart myocardium and ventricular myocyte samples. Background technique [0002] The prior art discloses the molecular mechanisms by which the nuclear envelope contains the genome and is responsible for the replication and transcription of DNA and the maturation of nascent RNA. Studies have shown that an increasing number of functions are attributed to the nuclear membrane, including the separation of DNA by receptors and their agents, ion channels, and ion pumps and transporters located within the nuclear membrane itself. It is known in the art that despite the structural and functional importance of nuclei, complete isolation of nuclei from cardiac cells and complete purification to allow in vitro studies remains poor due to challenges assoc...

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2509/10
Inventor 张雪梅王文伟祁晓燕张静
Owner FUDAN UNIV
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