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Application of isl1 gene and its expression product in promoting expression of hcn4

A gene and protein expression technology, applied in the application of protein in the diagnosis of diseases related to abnormal sinoatrial node, in the field of Isl1 gene, it can solve the problems of difficulty in distinguishing the structure of pacemaker cells, difficult separation and purification, and limited understanding.

Active Publication Date: 2019-09-10
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
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Problems solved by technology

[0006] But relatively speaking, because the deletion of known pacing-related genes will cause a large number of early death of embryos, the pacemaker cells are also difficult to distinguish in terms of tissue structure, and the number of pacing cells is limited, so it is difficult to separate and purify. The understanding of the cardiac pacing conduction system, especially the mechanisms regulating the formation and function of cardiac pacemaker cells is very limited

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  • Application of isl1 gene and its expression product in promoting expression of hcn4
  • Application of isl1 gene and its expression product in promoting expression of hcn4
  • Application of isl1 gene and its expression product in promoting expression of hcn4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0124] Example 1Isl1 and HCN4 are expressed in cardiac SAN cells

[0125] In order to study the role of Isl1 in the formation and function of SAN, the expression of Isl1 in the process of SAN development was first analyzed, and several SAN cells and atrial cardiomyocyte antibodies (HCN4 Tbx3 Cx40 purchased from abcam company) were used as markers.

[0126] HCN4 is a pacemaker cell channel protein, which begins to be expressed in the early heart development and plays a key role in the differentiation and development of SAN. At E9.5, Isl1 is expressed in the sinus venosus (SV) of the myocardium, and HCN4 is highly expressed in the germinal region of pacemaker cells in early embryos ( figure 1 A).

[0127] At E10.5, Isl1 and HCN4 were co-expressed in the embryonic SAN head ( figure 1 B) and tail ( figure 1 C), the venous valve (vv), and the atrial myocardium (DM) surrounding the vestibular mesentery ( figure 1 A, B, arrows).

[0128] During late embryonic development and ear...

Embodiment 2

[0132] Example 2 Reduction of Isl1 expression in Isl1 compound mutant mouse embryos leads to reduction of sinus arrhythmia and SAN cells

[0133] 2.1 Construction of an Isl1 compound mutant mouse model with incomplete expression of Isl1 (Isl1nLacZ / f Neo+ ), its phenotype is milder than that of whole-body knockout mice, and can survive for a little longer (death at E12.5 days), but its phenotype is heavier than that of Isl1 incompletely expressed mice.

[0134] The Isl1 compound mutant mouse model uses the Isl1 nuclear LacZ knock-in mouse line (Isl-nLacZ), and the Isl1 low expression mouse line (Isl1f neo / +, the mouse Isl1 intron contains a neomycin site, which can interfere with the normal expression of Isl1).

[0135] Results: Isl1 complex mutant mice (Isl1 nLacZ / f neo ) expresses lower amounts of Isl1 than Isl1-incompletely expressing mouse lines and causes embryonic death at E11.5 (54). X-gal staining of tissue sections showed that the color development of Isl1-nLaz in ...

Embodiment 4I

[0148] Example 4Isl1 is still essential to differentiated cardiomyocytes

[0149] In Isl1 compound mutant embryos, the expression of Isl1 was significantly reduced in differentiated SAN pacemaker cells (E9.5-E11.5), cardiac progenitor cells in the second heart region, and SV, SAN myocardium. Therefore, Troponin-Cre (cTnT-Cre) mice were used to specifically knock out Isl1 in differentiated cardiomyocytes to observe the role of Isl1 in differentiated sinus node cells.

[0150] The phenotype of cTnT-Cre knockout Isl1 mice (cTnT-Cre; Isl1 mouse line) is similar to that of Isl1 compound mutant mice. The mutant mice die at the early embryonic stage at E9.5-E11.5. Marked slow heart rate and premature beats, see Figure 7 .

[0151] Conclusion: The experiments showed that the expression of Isl1 is still required for the pacing function of cardiomyocytes even after the cardiomyocytes have matured.

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Abstract

The invention discloses an application of Isl1 gene and its expression product in HCN4 expression promotion, concretely provides a use of the Isl1 gene, protein or its promoter in the preparation of medicinal compositions for promoting the HCN4 gene or protein expression, or in the preparation of HCN4 transcription activators, and also provides recombinant animal cells foe inducible expression of Isl1, and a method for constructing heart disease model animals. The sl1 gene and its expression product can be used to research congenital heart disease animal models and can also be used for early intervention of the congenital heart disease.

Description

technical field [0001] The present invention relates to the field of diagnosis and treatment of congenital heart disease, in particular, the present invention relates to the application of Isl1 gene and protein in the diagnosis of abnormal sinus node related diseases. Background technique [0002] The cardiac pacing conduction system is a special myocardial tissue that generates and transmits electrical impulses and plays an important role in regulating the rhythmic contraction of the heart. Abnormalities in the development and function of the pacing conduction system can lead to arrhythmias and sudden cardiac death. [0003] In China, more than 500,000 cases of sudden cardiac death occur every year. Arrhythmias are the leading cause of sudden cardiac death. [0004] The cardiac pacing conduction system consists of the pacemaker (sinus node / SAN), atrioventricular node (AVN) and Purkinje fibers. In mice, cardiac inflow tracts begin to register pacing signals at embryonic d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K38/17G01N33/68C12N5/10C07K14/47A01K67/02A61P9/06
Inventor 孙云甫梁兴群
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE