CN112280847A - Application of MST1 gene in detecting and/or regulating excessive apoptosis of myocardial cells - Google Patents

Application of MST1 gene in detecting and/or regulating excessive apoptosis of myocardial cells Download PDF

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CN112280847A
CN112280847A CN202011171063.5A CN202011171063A CN112280847A CN 112280847 A CN112280847 A CN 112280847A CN 202011171063 A CN202011171063 A CN 202011171063A CN 112280847 A CN112280847 A CN 112280847A
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mst1
apoptosis
detecting
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gene
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苏冬梅
马旭
关丽娜
石翠格
李乾
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Institute Of Science And Technology National Health Commission
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    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Abstract

The invention belongs to the field of molecular biology, and provides a preparation for detecting excessive apoptosis of myocardial cells and a method for detecting excessive apoptosis of the myocardial cells. Also provides application of MST1 gene expression plasmid in preparing a preparation for improving the myocardial cell apoptosis rate in a high-sugar environment, application of siRNA interference plasmid of MST1 gene in preparing a preparation for reducing the myocardial cell apoptosis rate in the high-sugar environment, and a method for regulating the myocardial cell apoptosis rate. The invention utilizes the relation between MST1 gene and myocardial cell excessive apoptosis, especially myocardial cell excessive apoptosis in high sugar environment, and the provided preparation for detecting myocardial cell excessive apoptosis can efficiently and accurately detect the apoptosis condition of myocardial cell in high sugar environment, thereby making accurate judgment on whether excessive apoptosis occurs in the development of myocardial cell cultured in vitro or cardiac tissue in mother during fetal development.

Description

Application of MST1 gene in detecting and/or regulating excessive apoptosis of myocardial cells
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to application of an MST1 gene in detecting and/or regulating excessive apoptosis of myocardial cells, in particular to application in excessive apoptosis of myocardial cells caused by a high-sugar environment.
Background
CongenitaL Heart Disease (CHD) refers to a group of congenitaL malformations of morphological, structural and functional abnormalities caused by disturbance of the development of the heart and blood vessels during fetal life. Congenital heart disease is one of the leading causes of infant death. In asia, 9.3 out of 1000 live babies had CHD.
The exact cause of CHD remains unclear. For years, research has proved that CHD is a polygenic genetic disease, mainly a typical complex trait disease caused by cardiovascular disease caused by combined action of embryonic stage genetic factors and environmental factors, and the heritability of CHD is 55-65%. Therefore, the study of CHD pathogenesis from the genetic molecular level is one of the most important ways to understand the complex causes of CHD in humans. The discovery of CHD pathogenic/predisposing genes and the deep research of the functions and action mechanisms of CHD become the hot topic of the research on polygenic diseases which are currently and later paid attention for a long time.
The development of the heart is genetically and environmentally influenced by factors including biochemistry, blood sugar, blood pressure, radiation, temperature, changes in oxygen, and teratogens. Studies have found that the higher the blood glucose level of a hyperglycemic patient in the first trimester of pregnancy, the greater the chance of developing a fetal abnormality. The probability of cardiovascular malformation of the fetus of a hyperglycemic pregnant woman is five times that of normal pregnancy.
It is suggested by the scholars that excessive apoptosis of myocardial cells may be one of the causes of structural functional changes of fetal heart caused by hyperglycemia in pregnancy. Although there is no absolute correspondence between the excessive apoptosis of the cardiomyocytes and the congenital heart disease, that is, the excessive apoptosis of the cardiomyocytes may cause the embryonic heart to develop abnormally, the excessive apoptosis of the cardiomyocytes does not necessarily cause the congenital heart disease to occur; however, understanding the excessive apoptosis of embryonic cardiomyocytes can provide a high reference value for the diagnosis of congenital heart diseases. At present, the molecular mechanism by which high sugar causes excessive apoptosis of myocardial cells is not very clear, and no marker or method for indirectly measuring excessive apoptosis of myocardial cells exists in the prior art.
The mammalian sterile line 20-related kinase (MST) is a serine/threonine protein kinase ubiquitinated in vivo in mammalian systems, and mainly comprises four members of MST1, MST2, MST3 and MST4, wherein the MST1 kinase is related to various biological functions such as cell proliferation, growth, apoptosis and organ size, however, little is known about the expression of MST1 and the role of MST1 in the occurrence of fetal heart abnormalities and myocardial cell apoptosis caused by hyperglycemia in the pregnancy of mothers.
Yes-related protein 1(YAP1) is the core protein of the Hippo pathway and plays an important role in cell growth, proliferation and apoptosis. Phosphorylation of Ser127 of YAP1 protein results in retention of YAP1 in the cytoplasm, cytoplasmic YAP1 may undergo further phosphorylation and ubiquitination-dependent degradation, and phosphorylation of Ser397 of YAP1 protein may also mediate ubiquitination of YAP 1. The Ser127 and Ser397 residues of the YAP1 protein were reported to be regulated by LATS1/2 phosphorylation. However, no report is found on the change of the phosphorylation level of YAP1 in the high-sugar environment and the effect of YAP1 in inducing myocardial apoptosis in the high-sugar environment.
Survivin is a representative member of the Inhibitor of Apoptosis Protein (IAP) family, and is highly expressed in many tumor types. It has been reported that abnormal expression levels of Survivin in the blood have been studied as potential biomarkers for several tumorigenesis. Although Survivin has been well studied in tumors, its relationship to cardiomyocyte apoptosis in a high glucose environment has not been reported at all.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides application of the MST1 gene in detecting and/or regulating excessive myocardial cell apoptosis, in particular application in detecting and/or regulating excessive myocardial cell apoptosis caused by a high-sugar environment.
The first aspect of the invention provides a preparation for detecting excessive apoptosis of myocardial cells, wherein the preparation comprises a component for detecting the mRNA expression amount and/or the protein expression amount of the MST1 gene.
In a second aspect of the present invention, there is provided a method for detecting excessive apoptosis in a cardiomyocyte, comprising the step of detecting expression of MST1 gene in the cardiomyocyte.
The third aspect of the invention provides an application of MST1 gene expression plasmid in preparing a preparation for improving the myocardial cell apoptosis rate in a high-sugar environment.
The fourth aspect of the invention provides an application of siRNA interference plasmid of MST1 gene in preparing a preparation for reducing the myocardial cell apoptosis rate under high-sugar environment.
In a fifth aspect of the invention, there is provided a method of modulating the rate of cardiomyocyte apoptosis, the method comprising decreasing the rate of cardiomyocyte apoptosis and increasing the rate of cardiomyocyte apoptosis;
when the apoptosis rate of the myocardial cells needs to be improved, the method comprises the operation of transferring the MST1 gene expression plasmid into the myocardial cells;
when the reduction of the apoptosis rate of the myocardial cells is needed, the method comprises the operation of transferring the siRNA interference plasmid of the MST1 gene into the myocardial cells.
The invention provides a preparation for detecting excessive apoptosis of myocardial cells by utilizing the relation between MST1 gene and excessive apoptosis of myocardial cells, particularly excessive apoptosis of myocardial cells under a high-sugar environment, and the preparation can efficiently and accurately detect the apoptosis condition of the myocardial cells under the high-sugar environment, thereby accurately judging whether excessive apoptosis occurs in the myocardial cells cultured in vitro or the development of myocardial tissues during the development of fetus in a mother body. The invention also comprehensively utilizes the relation between the YAP1 gene and the MST1 gene expression, and makes more accurate judgment for the judgment of the excessive apoptosis of the myocardial cells.
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FIG. 1 is a graph comparing the expression of MST1 in fetal mouse myocardial tissue/cardiomyocytes. Wherein the content of the first and second substances,
FIG. 1-A shows immunohistochemical detection of expression of MST1 protein in fetal mouse myocardial tissue. Wherein, the left graph is the result of the embryonic myocardial tissue of a control group fetal mouse, and the right graph is the result of the embryonic myocardial tissue of a high-glucose group fetal mouse; the double arrows in the figure represent the thickness of the ventricular wall, and the black horizontal scale in the lower left corner of the left figure represents 50 μm.
FIG. 1-B shows the extraction of control fetal rat myocardial tissue protein and detection of MST1 protein expression by Western blot. In the figure, the left band is the result of the embryonic myocardial tissue of the control fetal mouse, and the right band is the result of the embryonic myocardial tissue of the hyperglycemic fetal mouse.
FIG. 1-C shows the extraction of H9C2 cell proteins from control and high-sugar groups, and the detection of MST1 protein expression by Western blot. In the figure, the left band is the result of the H9C2 cell of the control group, and the right band is the result of the H9C2 cell of the hyperglycogen group.
FIG. 1-D shows the extraction of fetal rat myocardial tissue RNA and detection of MST1 mRNA expression by qRT-PCR. Wherein, the left histogram in the figure is the result of the MST1 mRNA expression in the control fetal mouse embryonic cardiac muscle tissue, and the right histogram is the result of the MST1 mRNA expression in the high glucose fetal mouse embryonic cardiac muscle tissue.
FIGS. 1-E show the extraction of H9C2 cellular RNA and detection of MST1 mRNA expression by qRT-PCR. Wherein the left histogram in the figure is the result of MST1 mRNA expression in control H9C2 cells, and the right histogram is the result of MST1 mRNA expression in hyperglycemic H9C2 cells.
FIG. 2 is a graph of the results of the correlation between the abnormal expression of MST1 and cardiomyocyte apoptosis. Wherein the content of the first and second substances,
FIG. 2-A shows the transfer of MST1siRNA into control H9C2 cells and detection of the expression of MST1 protein by Western blot. The left band is the result of transfer of control siRNA and the right band is the result of transfer of MST1 siRNA.
FIGS. 2-B and 2-C show the analysis of apoptosis by Hoechst33342 staining after transfer of MST1siRNA into H9C2 cells. Wherein, the left panel/band shows the result of transferring the control siRNA into the control group H9C2 cells, the middle panel/band shows the result of transferring the control siRNA into the high sugar group H9C2 cells, and the right panel/band shows the result of transferring the MST1siRNA into the high sugar group H9C2 cells.
FIG. 2-D shows the expression of MST1 protein detected by Western blot after the MST1 expression plasmid was transferred into H9C2 cells as a control. The left band shows the result of the transfer of the blank plasmid, and the right band shows the result of the transfer of the expression plasmid of MST 1.
FIGS. 2-E and 2-F show the analysis of apoptosis by Hoechst33342 staining after transfer of the MST1 expression plasmid into control H9C2 cells. The left panel/band shows the result of the transfer into the blank plasmid, and the right panel/band shows the result of the transfer into the expression plasmid of MST 1.
FIG. 3 is a graph of the relative results of YAP1 as a downstream effector gene of MST1 involved in regulating MST 1-induced apoptosis in cardiomyocytes. Wherein the content of the first and second substances,
FIG. 3-A shows the detection of YAP1 protein expression by immunohistochemistry. Wherein, the graph on the left side is the myocardial tissue immunohistochemical result of the fetal rat in the control group, and the graph on the right side is the myocardial tissue immunohistochemical result of the fetal rat in the high-sugar group; the black horizontal scale in the lower left corner of the left graph represents 50 μm.
FIG. 3-B shows the expression of YAP1 protein detected by Western blot after extracting fetal rat myocardial tissue protein. The left graph shows the expression of YAP1 protein in control fetal rat myocardial tissue, and the right graph shows the expression of YAP1 protein in high-glucose fetal rat myocardial tissue.
FIG. 3-C shows the expression of YAP1 protein in cultured H9C2 cells detected by Western blot. The left graph shows the expression of YAP1 protein in H9C2 cells of the control group, and the right graph shows the expression of YAP1 protein in H9C2 cells of the high-sugar group.
FIG. 3-D shows RNA extracted from fetal rat myocardial tissue and YAP1 mRNA expression detected by qRT-PCR. Wherein, the left histogram in the figure is the expression result of YAP1 mRNA of fetal rat embryonic cardiac tissue of control group, and the right histogram is the expression result of YAP1 mRNA of fetal rat embryonic cardiac tissue of high glucose group.
FIGS. 3-E show the extraction of H9C2 cellular RNA and the detection of YAP1 mRNA expression by qRT-PCR. The histogram on the left is the expression of YAP1 mRNA in control H9C2 cells, and the histogram on the right is the expression of YAP1 mRNA in high carbohydrate H9C2 cells.
FIG. 3-F shows the expression of YAP1 protein detected by Western blot after control siRNA and MST1siRNA were transferred into cultured H9C2 cells. The left band is the result of transferring control siRNA (control siRNA) into control group H9C2 cells, the middle band is the result of transferring control siRNA into high-sugar group H9C2 cells, and the right band is the result of transferring MST1siRNA into high-sugar group H9C2 cells.
FIG. 3-G shows the expression of YAP1 protein detected by Western blot after MST1 expression plasmid was transferred into cultured H9C2 cells of control group. Wherein, the left band is the result of YAP1 protein expression after transferring into blank plasmid, and the right band is the result of YAP1 protein expression after transferring into MST1 expression plasmid.
FIG. 3-H shows the expression of YAP1 protein detected by Western blot after YAP1 expression plasmid was transferred into cultured H9C2 cells of control group. The left band is the result of the transfer into the blank plasmid, and the right band is the result of the transfer into the YAP1 expression plasmid.
FIG. 3-I shows the effect of apoptosis analysis YAP1 overexpression on myocardial apoptosis. The left band is the result of transferring blank plasmid into control group H9C2 cells, the middle band is the result of transferring MST1 expression plasmid and blank plasmid into control group H9C2 cells together, and the right band is the result of transferring MST1 and YAP1 expression plasmid into control group H9C2 cells together.
FIG. 4 is a graph of the relative results of YAP1 as a downstream effector gene of MST1 involved in regulating MST 1-induced apoptosis in cardiomyocytes. Wherein the content of the first and second substances,
FIG. 4-A shows the expression of YAP1 protein and the change in its phosphorylation level detected by Western blot after the MST1 expression plasmid was transferred into H9C2 cells as a control. The left band is the result of transfer into the blank plasmid, and the right band is the result of transfer into the expression plasmid of MST 1.
FIG. 4-B shows the expression of Last1/2 protein and the change in its phosphorylation level detected by Western blot after the MST1 expression plasmid was transferred into H9C2 cells as a control. The left band is the result of transfer into the blank plasmid, and the right band is the result of transfer into the expression plasmid of MST 1.
FIG. 4-C shows that MST1siRNA interference plasmid is transferred into H9C2 cells, and the expression of Last1/2 protein and the change of phosphorylation level thereof are detected by Western blot. Wherein, the left band is the result of transferring the control siRNA plasmid into the H9C2 cells of the control group, the middle band is the result of transferring the control siRNA plasmid into the H9C2 cells of the high sugar group, and the right band is the result of transferring the MST1siRNA interference plasmid into the H9C2 cells of the high sugar group.
FIG. 4-D shows the expression of Last1/2 protein and its phosphorylation changes in control and high-sugar groups, which are extracted from fetal mouse myocardial tissue protein and detected by Western blot. The left band is the result of the myocardial tissue of the control fetal rat, and the right band is the result of the myocardial tissue of the high-glucose fetal rat.
FIG. 5 is a graph of the results associated with YAP1 regulating Survivin expression to inhibit cardiomyocyte apoptosis. Wherein the content of the first and second substances,
FIG. 5-A shows the total proteins extracted from myocardial tissues of control and high-sugar fetal mice, and the quantitative analysis of Survivin and cyclin D protein expression by Western blot. The left band is the result of the myocardial tissue of the control fetal rat, and the right band is the result of the myocardial tissue of the high-glucose fetal rat.
FIG. 5-B shows the total protein of H9C2 cells extracted from control and high-sugar groups, respectively, and the quantitative analysis of Survivin and cyclin D protein expression by Western blot. The left band is the result of the control H9C2 cells, and the right band is the result of the hyperglycemic H9C2 cells.
FIG. 5-C shows the expression of Survivin protein detected by Western blot after YAP1 expression plasmid was transferred into H9C2 cells as a control. The band on the left side shows the result of the transfer into YAP1 expression plasmid, and the band on the right side shows the result of the transfer into YAP1 expression plasmid.
FIGS. 5-D show the total protein of H9C2 cells extracted from control and high-sugar groups, respectively, and the expression of Survivin protein was detected by Western blot. The left band is the result of transferring the control group H9C2 cells into the blank plasmid, the middle band is the result of transferring the high saccharide group H9C2 cells into the blank plasmid, and the right band is the result of transferring the high saccharide group H9C2 cells into the YAP1 expression plasmid.
FIG. 5-E shows the statistics of the rate of myocardial apoptosis following the addition of Survivin inhibitor YM-155 to the culture broth of hyperglycemic H9C2 cells. In the order from left to right, the first bar-shaped band is the result of transferring control group H9C2 cells into blank plasmid (not adding YM-155), the second bar-shaped band is the result of transferring high saccharide group H9C2 cells into blank plasmid (not adding YM-155), the third bar-shaped band is the result of transferring high saccharide group H9C2 cells into YAP1 expression plasmid (not adding YM-155), and the fourth bar-shaped band is the result of transferring high saccharide group H9C2 cells into YAP1 expression plasmid and adding YM-155.
Detailed Description
In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific embodiments. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
In the present invention, unless otherwise specified, the term "high sugar environment" means: in the myocardial cells cultured in vitro, the glucose concentration in the culture solution is 33mM (mmol/L); or a glycemic environment with a maternal blood glucose concentration of at least 16.7mM, while in the embryo. The term "myocardial cell apoptosis" refers to: in the myocardial cells cultured in vitro, compared with the normal culture solution environment with the glucose concentration of 5.3-5.5mM in the culture solution, the proportion of the apoptotic myocardial cells is between 15% and 30%; or in the embryo, compared with the blood sugar environment with the maternal blood sugar concentration of normal value (3.1-5.6mM), the proportion of the apoptotic myocardial cells is between 15% and 35%.
The first aspect of the invention provides a preparation for detecting excessive apoptosis of myocardial cells, wherein the preparation comprises a component for detecting the mRNA expression amount and/or the protein expression amount of the MST1 gene.
According to a first aspect of the invention, the formulation is particularly suitable for detecting excessive apoptosis of cardiomyocytes caused by a high sugar environment.
According to the first aspect of the present invention, the component for detecting the mRNA expression level of MST1 gene may include a component for extracting total RNA from cardiomyocytes; a component for reverse transcription of RNA into cDNA; and a component for quantitatively determining the mRNA expression level of the MST1 gene by qRT-PCR using cDNA as a template and the following MST1 gene amplification primers:
forward primer (sequence 1 in sequence listing):
5′-CATGGCTCAGGTGAACAGTAT-3′；
reverse primer (sequence 2 in sequence table):
5′-GGTCTCTGGGTCCAAAGTATAAC-3′。
wherein, the component for extracting total RNA from the myocardial cells; a component for reverse transcription of RNA into cDNA; and the components for quantitative determination of mRNA expression of the MST1 gene by using the MST1 gene amplification primer and qRT-PCR by using cDNA as a template are all reagent components commonly used in the field, can be obtained from various commercial channels, and are not described in detail in the invention.
According to the first aspect of the present invention, the component for detecting the protein expression amount of MST1 gene may include a component for extracting protein from cardiomyocytes, and a component for subjecting the extracted protein to Western blot operation to determine MST1 protein.
The components for extracting proteins from cardiomyocytes and the components for performing Western blot on the extracted proteins to determine the MST1 proteins are all reagent components commonly used in the field, and can be obtained from various commercial sources, and are not described in detail in the invention.
According to the first aspect of the present invention, the formulation further comprises a component for detecting the protein expression level of YAP1 gene.
Wherein, the component for detecting the protein expression amount of the YAP1 gene can comprise a component for extracting protein from myocardial cells and a component for performing Western blot operation on the extracted protein to identify YAP1 protein. The components for extracting proteins from cardiac muscle cells and the components for identifying YAP1 proteins by Western blot operation of the extracted proteins are all common reagent components in the field, and can be obtained from various commercial sources, and are not described in detail in the invention.
According to a first aspect of the invention, the formulation further comprises a component for detecting the phosphorylation level of Last 1/2.
The component for detecting the phosphorylation level of Last1/2 can be any reagent component for detecting the phosphorylation level of Last1/2 in the field, and can be obtained from various commercial sources, and the details are not repeated in the invention.
According to the first aspect of the invention, the preparation further comprises a component for detecting the content of the anti-apoptotic protein Survivin.
The component for detecting the content of the anti-apoptotic protein Survivin can comprise a component for extracting protein from myocardial cells and a component for performing Western blot operation on the extracted protein to identify the content of the anti-apoptotic protein Survivin. The components for extracting proteins from cardiac muscle cells and the components for identifying YAP1 proteins by Western blot operation of the extracted proteins are all common reagent components in the field, and can be obtained from various commercial sources, and are not described in detail in the invention.
In a second aspect of the present invention, there is provided a method for detecting excessive apoptosis in a cardiomyocyte, comprising the step of detecting expression of MST1 gene in the cardiomyocyte.
According to a second aspect of the invention, the method is particularly suitable for detecting excessive apoptosis of cardiomyocytes caused by a high sugar environment.
According to the second aspect of the present invention, the operation of detecting the expression of MST1 gene in the cardiomyocyte includes an operation of detecting the mRNA expression level of MST1 gene in the cardiomyocyte; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in the cardiomyocytes cultured in vitro, when the expression level of mRNA of MST1 gene is increased by at least 10-fold, e.g., 10-15-fold, preferably 12.5-fold, as compared to the cardiomyocytes cultured in a normal culture medium environment with a glucose concentration of 5.3-5.5mM in the culture medium, the cardiomyocytes undergo apoptosis; alternatively, the first and second electrodes may be,
when the expression level of mRNA of MST1 gene is increased at least 10-fold, for example, 10-15-fold, preferably 12.5-fold, in the embryo as compared with the blood glucose environment where the maternal blood glucose concentration is normal (3.1-5.6mM), cardiomyocytes are excessively apoptotic.
According to the second aspect of the present invention, the operation of detecting the mRNA expression level of MST1 gene in the cardiomyocytes may be a conventional operation for detecting the mRNA expression level in the art.
In a preferred embodiment, the operation of detecting the mRNA expression level of MST1 gene in the cardiomyocytes may comprise: firstly, extracting total RNA in myocardial cells, then reversely transcribing the total RNA into cDNA, and carrying out quantitative determination by using the obtained cDNA as a template and the following MST1 gene amplification primers by adopting qRT-PCR:
forward primer (sequence 1 in sequence listing):
5′-CATGGCTCAGGTGAACAGTAT-3′；
reverse primer (sequence 2 in sequence table):
5′-GGTCTCTGGGTCCAAAGTATAAC-3′。
according to the second aspect of the present invention, the operation of detecting the expression of MST1 gene in the cardiomyocyte includes an operation of detecting the protein expression level of MST1 gene in the cardiomyocyte; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in the cardiomyocytes cultured in vitro, when the protein expression level of MST1 gene is increased by at least 6.5-fold, e.g., 6.5-10.5-fold, preferably 9-fold, as compared to the cardiomyocytes cultured in a normal culture medium environment with a glucose concentration of 5.3-5.5mM in the culture medium, the cardiomyocytes undergo apoptosis; alternatively, the first and second electrodes may be,
when the expression level of the protein of MST1 gene is increased at least 6-fold, for example, 6-10-fold, preferably 9-fold, in the embryo as compared with the blood glucose environment where the maternal blood glucose concentration is normal (3.1-5.6mM), cardiomyocytes are excessively apoptotic.
According to the second aspect of the present invention, the operation of detecting the protein expression level of MST1 gene in cardiomyocytes may be a conventional operation for detecting the protein expression level in the art, for example, a Western blot operation.
According to a second aspect of the invention, the method further comprises the operation of detecting the phosphorylation level of Last 1/2.
Wherein, the operation of detecting the phosphorylation level of Last1/2 can be any operation mode used for detecting the phosphorylation level of Last1/2 in the field, including but not limited to kinase activity analysis method, Western blot method, enzyme-linked immunosorbent assay (ELISA) method, intracellular flow cytometry and immunocytochemistry/immunohistochemistry (ICC/IHC) method, mass spectrometry and the like; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in the cardiomyocytes cultured in vitro, the cardiomyocytes were excessively apoptotic when the Last1/2 phosphorylation level was increased at least 7-fold, e.g., 7-10-fold, preferably 9-fold, compared to the cardiomyocytes cultured in a normal culture medium environment with a glucose concentration in the culture medium of 5.3-5.5 mM.
In the embryo, myocardial cells are over-apoptotic when the phosphorylation level of Last1/2 is increased at least 7-fold, e.g., 7-10-fold, preferably 9-fold, compared to the glycemic environment with maternal blood glucose concentration of 3.1-5.6 mM.
According to a second aspect of the invention, the method further comprises the operation of detecting the level of the anti-apoptotic protein Survivin.
The operation for detecting the content of the anti-apoptotic protein Survivin can be an operation mode which is conventional in the art and is used for detecting the protein expression amount, for example, an operation mode which adopts Western blot; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in cardiomyocytes cultured in vitro, the cardiomyocytes undergo excessive apoptosis when the level of the anti-apoptotic protein Survivin is reduced by at least 50%, e.g. 50% to 70%, preferably 64%, compared to cardiomyocytes cultured in a normal culture environment with a glucose concentration in the culture medium of 5.3 to 5.5 mM.
In the embryo, the cardiomyocytes are over-apoptotic when the level of the anti-apoptotic protein Survivin is reduced by at least 50%, e.g. 50-70%, preferably 64% compared to the glycemic environment with maternal blood glucose concentration of 3.1-5.6 mM.
The third aspect of the invention provides an application of MST1 gene expression plasmid in preparing a preparation for improving the myocardial cell apoptosis rate in a high-sugar environment.
According to the third aspect of the present invention, the vector of the MST1 gene expression plasmid may be any plasmid vector commonly used in the art, as long as it can be used to successfully transfer the MST1 gene into cardiomyocytes and exert its biological activity, and the present invention is not limited thereto.
The fourth aspect of the invention provides an application of siRNA interference plasmid of MST1 gene in preparing a preparation for reducing the myocardial cell apoptosis rate under high-sugar environment.
According to the fourth aspect of the present invention, the interference sequence of the siRNA interference plasmid of MST1 gene is:
siRNA (sequence 7 in sequence listing): 5'-GCCGAGCCTTCCACTACAATA-3' are provided.
In a fifth aspect of the invention, there is provided a method of modulating the rate of cardiomyocyte apoptosis, the method comprising decreasing the rate of cardiomyocyte apoptosis and increasing the rate of cardiomyocyte apoptosis; when the apoptosis rate of the myocardial cells needs to be improved, the method comprises the operation of transferring the MST1 gene expression plasmid into the myocardial cells; when the reduction of the apoptosis rate of the myocardial cells is needed, the method comprises the operation of transferring the siRNA interference plasmid of the MST1 gene into the myocardial cells.
Wherein, the siRNA interference plasmid interference sequence of the MST1 gene is as described above.
According to a fifth aspect of the invention, the method is particularly useful for modulating excessive apoptosis of cardiomyocytes caused by a high sugar environment.
According to a fifth aspect of the invention, the method further comprises: when the apoptosis rate of the myocardial cells needs to be improved, the method comprises the operation of transferring siRNA interference plasmids of YAP1 genes into the myocardial cells; when it is desired to reduce the rate of myocardial cell apoptosis, the method includes the operation of transferring the YAP1 expression plasmid into myocardial cells.
The present invention will be described in detail below with reference to specific examples.
Various reagents, materials and the like used in the following examples are commercially available products unless otherwise specified; unless otherwise specified, all the tests and detection methods used in the following examples are conventional in the art and can be obtained from textbooks, tool books or academic journals.
In the following examples, all concentrations are mass percent concentrations unless otherwise specified.
Example 1
A hyperglycemic pregnant rat model (or hyperglycemic group) is established according to the following operation mode:
female and male SD (Sprague-Dawley) rats (10-11 weeks old) were housed in metabolic cages with free access to food and water at 22 + -2 deg.C, 55 + -5% humidity, and 12 hours light/12 hours dark cycle.
Diabetes was induced in rats using Streptozotocin (STZ) to obtain high carbohydrate group test rats. The specific operation is as follows:
female SD rats and male SD rats were housed in cages. If plugs were observed the following morning, the day that was considered to be day 0.5 of pregnancy. After the mating plug is seen, the pregnant mice are fasted for 12h and injected with STZ intraperitoneally according to the standard of 50 mg/kg. After 72 hours, blood is taken from the tail tip, the blood sugar level of the mother mouse is measured by a glucometer, and the pregnant mouse with the blood sugar level reaching or being more than 16.7mM is taken as a hyperglycemic pregnancy model group (called hyperglycemic group for short). On the 15.5 th day of pregnancy, euthanizing the pregnant rat, cesarean section, and freezing and storing fetal rat embryo heart as the high carbohydrate material.
The control group was as follows:
the procedure was the same as that described above except that the STZ of the high saccharide group was changed to an equivalent amount of citric acid buffer. Also, after 72 hours, the blood glucose levels monitored were between 4-6 mM. When pregnant mice are pregnant for 15.5 days, the pregnant mice are euthanized, cesarean section is carried out, fetal mouse embryo hearts are taken for cryopreservation, and the fetal mouse embryo hearts are used as control group materials.
H9C2 cell culture was performed as follows:
the control cell culture was as follows:
H9C2 cells were incubated at 37 ℃ with 5% CO2In an incubator, the cells were cultured in a cardiomyocyte medium (purchased from Gibco, USA) containing 10% fetal bovine serum, 100mg/ml penicillin and 100mg/ml streptomycin.
The culture method of the high carbohydrate group cells comprises the following steps:
the cells were cultured in the same manner as the control group except that glucose was added to H9C2 cell culture medium (here, H9C2 cell culture medium includes the aforementioned cardiomyocyte culture medium and supplemented with 10% fetal bovine serum, 100mg/ml penicillin and 100mg/ml streptomycin) to a final concentration of 33 mM.
Immunoblotting (Western blot) was performed as follows:
the cryopreserved fetal mouse embryonic hearts or cultured H9C2 cells were removed, lysed, and the proteins were collected and electrophoresed using 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were incubated with the following antibodies, respectively: MST1 antibody (Abcam, USA), YAP1 antibody (Abcam), YAP1(Ser397) antibody (cell signaling), YAP1(Ser127) antibody, (cell signaling), LATS1/2 antibody (MyBioSource), LATS1/2(Thr1079/1041) antibody (Biorbyt), Survivin antibody (Abcam) and beta-actin (Sigma-Aldrich) antibody were incubated overnight. After 1 hour incubation with the corresponding secondary antibody, exposure was performed using SuperSignal West Pico chemiluminescent substrate kit (Pierce Biotechnology, usa).
Immunohistochemistry was performed as follows:
after the heart of the fetal rat is embedded by paraffin, paraffin embedded sections are obtained, the sections are taken out, the xylene is used for dewaxing, and gradient alcohol solution is used for dehydration. After washing with water, the sections were boiled in 0.1M citric acid buffer (pH 6.1) for 10 minutes, and thenThen cooling to room temperature. The sections were washed with phosphate buffer and placed in 0.3% H2O2To inhibit endogenous peroxidase activity. After washing again, the sections were incubated with normal serum for 1 hour, then incubated overnight with anti-YAP 1 and MST1 antibodies, respectively (purchased from Abcam, UK). After washing, incubation with biotinylated secondary antibody for 1 hour followed by incubation with avidin and biotinylated peroxidase complex. Finally, the sections were incubated in a peroxidase substrate (diaminobenzidine tetrachlorochloride) solution until the desired staining intensity was reached. Sections were washed with water, dehydrated, and mounted for viewing.
The gene expression plasmid construction and transfection procedures were as follows:
the MST1 expression plasmid and the YAP1 expression plasmid were constructed from Polepolar.
Rat MST1 and/or YAP1 expression plasmids were transferred to H9C2 cells using the transfection reagent Lipofectamine 3000, according to the instruction manual (Thermo Fisher Scientific, USA), and the transfected H9C2 cells were used for apoptosis analysis and Western blot for the following procedures 48 hours after transfection.
The RNA extraction and qRT-PCR were performed as follows:
total RNA was extracted from H9C2 cells using TRIzol reagent (purchased from Invitrogen, USA).
RNA was reverse transcribed into cDNA using the Access-RT-PCR system, and then quantitative analysis was performed using fluorescent real-time quantitative qRT-PCR.
The PCR amplification system is as follows:
Figure BDA0002747320820000151
Figure BDA0002747320820000161
the procedure for PCR amplification was:
after mixing the PCR tubes with the added samples, Real-time PCR was performed using a Step One Real-time fluorescent quantitative PCR instrument system (ABI, USA). Two-step method for PCR reactionThe reaction is carried out under the reaction condition of 95 ℃ for 60 seconds; then 95 ℃ for 15 seconds followed by 60 ℃ for 60 seconds, repeating 40-50 cycles. The data obtained were processed with the software supplied by ABI (applied biosystems) at 2–ΔΔCtThe Ct value in the formula represents the number of PCR cycles required to reach a detection threshold beyond the fluorescence signal.
The primers for PCR amplification were as follows:
MST1 gene:
forward primer (sequence 1 in sequence listing):
5′-CATGGCTCAGGTGAACAGTAT-3′；
reverse primer (sequence 2 in sequence table):
5′-GGTCTCTGGGTCCAAAGTATAAC-3′。
YAP1 gene:
forward primer (sequence 3 in sequence listing):
5′-TCGGCAGGCAATACGGAATA-3′；
antisense primer (sequence 4 in sequence table):
5′-CATGCTGAGGCCACTGTCTGT-3′。
beta-actin gene:
forward primer (sequence 5 in sequence listing): 5'-TCGTGCGTGACATTAAGGAG-3', respectively;
reverse primer (sequence 6 in sequence table): 5'-ATGCCAGGGTACATGGTGGT-3' are provided.
The RNA interference (RNAi) operates as follows:
the sequence composition of small interfering RNA (siRNA) of MST1 gene is 5'-GCCGAGCCTTCCACTACAATA-3' (sequence 7 in the sequence table). The negative control sequence was: 5'-CGTCACATGGGCTTTCACC-3' are provided. Recombinant plasmids containing the small interfering RNA sequence of the MST1 gene and a negative control sequence were purchased from Youbao Bio, wherein the plasmid vector was pSilencer 4.1-CMV Neo (Ambion, USA). The MST1 interference plasmid was transferred into H9C2 cells using Lipofectamine 3000(Thermo Fisher Scientific, USA) according to the manufacturer's recommendations. 48H after transfection, the transfected H9C2 cells were used for subsequent manipulations.
The mode of operation of the apoptosis detection of Hoechst33342 staining was as follows:
H9C2 cell nuclear morphology change was observed by staining with Hoechst33342 (purchased from Sigma) and apoptosis was observed by fluorescence microscopy. The experiment was repeated 5 times. Randomly selecting 5 visual fields, counting the number of apoptotic cell nuclei and calculating the apoptosis ratio.
The data obtained were statistically analyzed as follows:
data are expressed as mean ± standard deviation (mean ± s.d.) and represent results from three independent parallel experiments. Statistical significance of differences between means was tested by ANOVA (variance). Mean values were significantly different and were tested by multiple comparisons using T-test. Probability value P <0.05 represents significant difference, denoted by "+"; p <0.01 indicates that the difference was very significant, indicated by "×".
The results are as follows:
correlation of MST1 gene expression with the occurrence of high sugar-induced cardiac dysplasia:
after HE staining of the fetal rat embryonic cardiac muscle tissue obtained as described above, it was found that the cardiac dysplasia of the fetal rat of the hyperglycemic pregnant rat was more thinly characterized by a thinner ventricular wall than the control group (not shown).
As a result of immunohistochemical examination shown in FIG. 1-A, the expression of MST1 protein was increased in the fetal rat myocardial tissue (myocardial tissue in the thinner part of the ventricular wall) of the pregnant hyperglycemic rat as compared with the fetal rat myocardial tissue (left side) of the control group (according to FIG. 1-A, the expression of MST1 protein was increased by about 8.5 times). As a result of Western blot analysis shown in FIG. 1-B, it was also confirmed that the expression level of MST1 protein was increased in fetal rat myocardial tissue of the pregnant hyperglycemic rat (according to FIG. 1-B, the expression level of MST1 protein was increased by about 9-fold). Meanwhile, as shown in FIG. 1-C, the Western blot detection results of the in vitro cell assay also confirmed that the expression of MST1 protein was increased in cardiomyocytes cultured in vitro under high glucose environment (according to FIG. 1-C, the expression level of MST1 protein was increased by about 9 times in H9C2 cells). As shown in FIG. 1-D, at the transcription level, mRNA expression of MST1 was also significantly increased in fetal rat myocardial tissue of pregnant hyperglycemic rats as compared with that of control fetal rat fetal myocardial tissue by qRT-PCR detection (according to FIG. 1-D, mRNA expression of MST1 was increased by about 12 times). As shown in FIGS. 1-E, the mRNA expression of MST1 gene in cardiomyocytes was observed to change under high sugar conditions by in vitro cell assay, and at the transcription level, the mRNA expression of MST1 in cardiomyocytes was also significantly increased by qRT-PCR assay compared to the control group (according to FIGS. 1-E, the mRNA expression level of MST1 was increased by about 12.5 times).
Effect of aberrant expression of MST1 on cardiomyocyte apoptosis:
in order to deeply research the regulation effect of MST1 on myocardial cell apoptosis, RNAi interference plasmids are constructed, and the expression change of MST1 protein in myocardial cells is detected through in vitro cell experiments. As shown in FIG. 2-A, after the MST1siRNA interference plasmid was transferred into the control group H9C2 cells cultured in the aforementioned manner, a significant decrease in the expression level of MST1 protein was found (according to FIG. 2-A, the expression of MST1 protein was reduced by 65%). As a result of apoptosis staining analysis shown in FIGS. 2-B and 2-C, it was found that apoptotic cells were decreased after transfer of MST1siRNA into H9C2 cells (about 15% of apoptotic cells were decreased after transfer of MST1siRNA into H9C2 cells according to FIGS. 2-B and 2-C), indicating that interference with expression of MST1 in H9C2 cells partially suppressed the promotion of cardiomyocyte apoptosis by high sugars. In addition, as shown in FIG. 2-D, Western bot results showed a significant increase in MST1 protein expression (about 9-fold increase in MST1 protein expression according to FIG. 2-D) after transfer of the MST1 expression plasmid into control H9C2 cells cultured in the manner described above. As can be seen from the results of apoptosis staining shown in FIGS. 2-E and 2-F, the transfer of the MST1 expression plasmid into the control group H9C2 cells cultured in the aforementioned manner induced apoptosis of cardiac muscle cells (the apoptotic cell rate was increased by about 15% according to FIGS. 2-E and 2-F).
YAP1 was involved as an effector gene downstream of MST1 in regulating MST 1-induced apoptosis of cardiomyocytes:
as shown in fig. 3-a, when YAP1 was detected by immunohistochemistry in high-sugar fetal mouse myocardial tissue and normal control fetal mouse myocardial tissue, it was found that YAP1 protein expression was reduced in the high-sugar fetal mouse myocardial tissue compared to the normal control fetal mouse myocardial tissue (the YAP1 protein expression was reduced by 60% according to fig. 3-a). Western blot assay results as shown in FIG. 3-B also confirmed the reduced expression of YAP1 protein in myocardial tissue of high-glucose fetal mice (62% reduction of YAP1 protein expression according to FIG. 3-B). As shown in FIG. 3-C, YAP1 protein expression was significantly reduced in the H9C2 cells of the high carbohydrate group (YAP1 protein expression was reduced by 64% according to FIG. 3-C) as compared to the H9C2 cells cultured in the culture manner of the aforementioned control group. The mRNA expression of the YAP1 gene in the myocardial tissue of the high-sugar fetal rat and the myocardial tissue of the normal control fetal rat through qRT-PCR detection as shown in FIG. 3-D can be found to be insignificant compared with the mRNA expression of the YAP1 gene in the myocardial tissue of the high-sugar fetal rat in the myocardial tissue of the normal control fetal rat. As shown in fig. 3-E, the mRNA reduction of YAP1 gene was also not significant in H9C2 cells of the high carbohydrate group compared to H9C2 cells of the control group as detected by qRT-PCR. As shown in FIG. 3-F, it was found by Western blot assay that YAP1 protein expression was elevated in the high saccharide group H9C2 cells into which MST1siRNA was transferred (YAP1 protein expression was elevated by 40% according to FIG. 3-E) compared with the high saccharide group H9C2 cells into which control siRNA was transferred, indicating that the transfer of MST1siRNA could partially counteract the down-regulation of YAP1 protein expression by high saccharide. As can be seen from the results shown in FIGS. 3-G and 3-H, YAP1 protein expression was significantly reduced (by about 40%) after overexpression of MST1 in H9C2 cells of the control group, indicating that MST1 is involved in regulating YAP1 protein expression. As shown in fig. 3-I, when YAP1 expression plasmid was transferred into H9C2 cells of the control group, the apoptosis analysis results showed that overexpression of YAP1 partially resisted the induction of apoptosis of cardiac muscle cells by MST1 (the apoptotic cell rate decreased by about 11% as shown in fig. 3-I); thus, YAP1 is a downstream effector gene of MST1 and regulates MST 1-induced myocardial apoptosis.
MST1 regulated YAP1 expression required the involvement of Last 1/2:
as shown in FIG. 4-A, the Western blot assay result shows that the phosphorylation levels of serine at 127 th site and 397 th site of YAP1 protein are increased after MST1 is over-expressed in the H9C2 cells of the control group; wherein the phosphorylation of serine at position 127 is increased by about 31% and the phosphorylation of serine at position 397 is increased by about 33%. As shown in FIG. 4-B, it was found from the results of Western blot detection of the change in phosphorylation levels of Last1/2 that the phosphorylation levels of Last1/2 were increased in the H9C2 cells (MST1 overexpressed) of the control group into which the MST1 expression plasmid was transferred, as compared with the H9C2 cells of the control group into which the blank plasmid was transferred (about 9-fold increase in phosphorylation levels of Last1/2 according to FIG. 4-B. from the results shown in FIG. 4-C, it was found that the upregulation of phosphorylation levels of Last1/2 by high sugars was partially inhibited after MST1siRNA was transferred into the H9C2 cells of the high sugar group, as shown in FIG. 4-D, it was found that phosphorylation levels of Last1/2 were significantly increased in the myocardial tissue of fetal mice of the high sugar group, as compared with the myocardial tissue of the control group, as shown in FIG. 4-D.
YAP1 modulation of Survivin expression inhibited cardiomyocyte apoptosis:
as shown in FIG. 5-A, the Western blot assay showed that the expression of the anti-apoptotic protein Survivin was reduced by about 64% in the myocardial tissue of the high-sugar fetal mouse obtained in the manner described above (compared to the myocardial tissue of the control fetal mouse obtained in the manner described above). As shown in FIG. 5-B, Western blot analysis revealed that the expression of Survivin, an anti-apoptotic protein, was reduced by about 64% in the high carbohydrate group H9C2 cells cultured in the manner described above (compared to the control group H9C2 cells cultured in the manner described above). As shown in FIG. 5-C, the expression of YAP1 protein was increased after transferring YAP1 expression plasmid into the control group H9C2 cells cultured in the aforementioned manner. From the results shown in FIGS. 5-D, it can be seen that, after transferring YAP1 expression plasmid into the hyperglycome H9C2 cells cultured in the aforementioned manner, the expression of the anti-apoptotic protein Survivin was increased compared to the hyperglycome H9C2 cells transferred with the blank plasmid, indicating that YAP1 overexpression could partially counteract the downregulation of Survivin by hyperglycemia. As can be seen from the results shown in FIGS. 5-E, when Survivin inhibitor YM-155 was added to the culture medium of high carbohydrate group H9C2 cells, the inhibitory effect of YAP1 on cardiomyocyte apoptosis was inhibited. Therefore, Survivin is a downstream effector gene of YAP1 and regulates the apoptosis of myocardial cells induced by YAP 1.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> institute of science and technology of the national institute of health and wellness
Application of <120> MST1 gene in detecting and/or regulating excessive apoptosis of myocardial cells
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catggctcag gtgaacagta t 21
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtctctggg tccaaagtat aac 23
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcggcaggca atacggaata 20
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
catgctgagg ccactgtctg t 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcgtgcgtga cattaaggag 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgccagggt acatggtggt 20
<210> 7
<211> 21
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<213> Artificial Sequence (Artificial Sequence)
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gccgagcctt ccactacaat a 21

Claims (10)

1. A formulation for detecting excessive apoptosis in cardiac myocytes, comprising:
the preparation comprises a component for detecting the mRNA expression quantity and/or the protein expression quantity of the MST1 gene.
2. The formulation of claim 1, wherein:
the preparation is suitable for detecting excessive apoptosis of myocardial cells caused by high-sugar environment.
3. The formulation according to claim 1 or 2, characterized in that:
the component for detecting the mRNA expression quantity of the MST1 gene comprises:
a component for extracting total RNA from cardiomyocytes;
a component for reverse transcription of RNA into cDNA;
the components for quantitative determination of mRNA expression level of MST1 gene by qRT-PCR using cDNA as template and the following MST1 gene amplification primers:
a forward primer: 5'-CATGGCTCAGGTGAACAGTAT-3', respectively;
reverse primer: 5'-GGTCTCTGGGTCCAAAGTATAAC-3', respectively;
preferably, the component for detecting the protein expression amount of the MST1 gene includes a component for extracting a protein from a cardiomyocyte, and a component for subjecting the extracted protein to a Western blot procedure to determine the MST1 protein.
4. The formulation of claim 3, wherein:
the preparation also comprises a component for detecting the protein expression quantity of the YAP1 gene;
preferably, the preparation also comprises a component for detecting the phosphorylation level of Last 1/2;
further preferably, the preparation also comprises a component for detecting the content of the anti-apoptotic protein Survivin.
5. A method for detecting excessive apoptosis in cardiomyocytes, comprising:
the method comprises the operation of detecting the expression of MST1 gene in the myocardial cells.
6. The method of claim 5, wherein:
the method is suitable for detecting excessive apoptosis of the myocardial cells caused by a high-sugar environment;
preferably, the operation of detecting the expression of the MST1 gene in the myocardial cell comprises an operation of detecting the mRNA expression level of the MST1 gene in the myocardial cell; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
when the expression level of mRNA of MST1 gene is increased by at least 10 times, preferably 10 to 15 times, and more preferably 12.5 times in the cardiomyocytes cultured in vitro as compared with the cardiomyocytes cultured in the normal culture medium environment with a glucose concentration of 5.3 to 5.5mM in the culture medium, the cardiomyocytes are excessively apoptotic; alternatively, the first and second electrodes may be,
when the expression level of mRNA of MST1 gene is increased by at least 10 times, preferably 10-15 times, more preferably 12.5 times, as compared with the blood sugar environment of maternal blood sugar concentration of 3.1-5.6mM, the myocardial cells are over-apoptotic in the embryo;
preferably, the operation of detecting the expression of the MST1 gene in the myocardial cell comprises an operation of detecting the protein expression level of the MST1 gene in the myocardial cell; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
when the protein expression level of MST1 gene is increased by at least 6.5 times, preferably 6.5 to 10.5 times, and more preferably 9 times in the cardiomyocytes cultured in vitro as compared with the cardiomyocytes cultured in the normal culture medium environment with a glucose concentration of 5.3 to 5.5mM in the culture medium, the cardiomyocytes are excessively apoptotic; alternatively, the first and second electrodes may be,
when the expression level of the protein of MST1 gene is increased at least 6-fold, preferably 6-10-fold, more preferably 9-fold in embryo compared with the blood sugar environment of maternal blood sugar concentration of 3.1-5.6mM, the cardiomyocytes are over-apoptotic;
preferably, the method further comprises the operation of detecting the phosphorylation level of Last 1/2; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in the cardiomyocytes cultured in vitro, when the phosphorylation level of Last1/2 is increased by at least 7-fold, preferably 7-10-fold, and more preferably 9-fold, as compared to the cardiomyocytes cultured in a normal culture medium environment in which the glucose concentration in the culture medium is 5.3 to 5.5 mM;
in the embryo, when the phosphorylation level of Last1/2 is increased at least 7-fold, preferably 7-10-fold, and more preferably 9-fold, compared with the blood sugar environment of maternal blood sugar concentration of 3.1-5.6mM, the cardiomyocytes are over-apoptotic;
preferably, the method further comprises the operation of detecting the content of the anti-apoptotic protein Survivin; and judging whether the myocardial cells are excessively apoptotic according to the following criteria:
in the cardiomyocytes cultured in vitro, the cardiomyocytes are excessively apoptotic when the content of the anti-apoptotic protein Survivin is reduced by at least 50%, preferably 50% to 70%, more preferably 64%, compared to the cardiomyocytes cultured in a normal culture medium environment with a glucose concentration in the culture medium of 5.3 to 5.5 mM;
in the embryo, when the content of the anti-apoptotic protein Survivin is reduced by at least 50%, preferably 50% to 70%, and more preferably 64%, as compared with the blood glucose environment in which the maternal blood glucose concentration is 3.1 to 5.6mM, the cardiomyocytes are excessively apoptotic.
Application of MST1 gene expression plasmid in preparation of preparation for improving myocardial cell apoptosis rate in high-sugar environment.
The application of siRNA interference plasmid of MST1 gene in preparing preparation for reducing myocardial cell apoptosis rate in high sugar environment;
preferably, the interference sequence of the siRNA interference plasmid of the MST1 gene is as follows:
5′-GCCGAGCCTTCCACTACAATA-3′。
9. a method of modulating the rate of apoptosis in a cardiomyocyte, comprising:
the method comprises reducing the rate of cardiomyocyte apoptosis and increasing the rate of cardiomyocyte apoptosis;
when the apoptosis rate of the myocardial cells needs to be improved, the method comprises the operation of transferring the MST1 gene expression plasmid into the myocardial cells;
when the reduction of the apoptosis rate of the myocardial cells is needed, the method comprises the operation of transferring the siRNA interference plasmid of the MST1 gene into the myocardial cells;
preferably, the interference sequence of the siRNA interference plasmid of the MST1 gene is as follows:
5′-GCCGAGCCTTCCACTACAATA-3′。
10. the method of claim 9, wherein:
the method is suitable for regulating and controlling excessive apoptosis of the myocardial cells caused by a high-sugar environment;
preferably, when the increase of the apoptosis rate of the myocardial cells is needed, the method further comprises the operation of transferring the siRNA interference plasmid of the YAP1 gene into the myocardial cells;
preferably, when it is desired to reduce the rate of cardiomyocyte apoptosis, the method further comprises the operation of transferring the YAP1 expression plasmid into cardiomyocytes.
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