| Objective: Experiments were performed at the biological cell and molecular level to verify the correlation between ROCK2 and Sudden coronary death,and to provide a new reference for predicting Sudden coronary death.Methods: By liposome transfection,mouse coronary artery smooth muscle cells(CVSMc)were expressed to enhance green fluorescent protein-labeled ROCK2protein(EGFP-m ROCK),and the expression of ROCK2 was indicated by the expression of EGFP.After that,a model of Sudden coronary death was constructed,and the expression of ROCK2 and cell contraction in the control group,transfection group,coronary heart disease group,Sudden coronary death,and Slx-2119 inhibitor group were monitored in real time,qualitatively and in situ by super-resolution fluorescence microscope.And the effect of ROCK2 on cell apoptosis in a Sudden coronary death was detected with a totipotent cytometer.In addition,at the molecular level of the cell model of Sudden coronary death,real-time fluorescence quantitative PCR(RT-q PCR)was used to detect ROCK2 m RNA expression and Western Blot was used to detect ROCK2 protein expression.Results:(1)After transfection,mouse CVSMc could normally express green fluorescent protein-labeled ROCK2(EGFP-ROCK2);and the amount of EGFP-ROCK2 expressed by mouse CVSMc within 12 h of transfection under a fluorescence microscope increased slowly with time.(2)Intervention of MCP-1(Monocyte chemoattractant protein-1)(10 ng / m L)within 24 hours of CVSMc,the relative expression of TF m RNA at 2 hours was higher than that of other groups(P<0.05).After MCP-1(10 ng / m L)was used to intervene in CVSMc for 2 h,different concentrations of AT-Ⅱ(Angiotensin II)were used for 1 h.When AT-Ⅱ was 0.1 μM,the relative expression of ROCK2 m RNA was higher than that of other groups(P<0.05).Then,after 2 hours of intervention with MCP-1(10 ng / m L),AT-II 0.1 μM interfered with CVSMc.Within 6 hours,the relative expression of ROCK2 m RNA was higher in the 1 and 3 hours than in the control group(P <0.05).There was no significant difference in expression between the two(P> 0.05).After that,within 12 hours of transfection,the relative expression of ROCK2 m RNA was gradually increased in different time groups(P <0.05).However,when CVSMc was transfected for 8 hours,MCP-1(10 ng / m L)used for 2 hours and AT-II 0.1 μM used for 1 hour,the relative expression of ROCK2 m RNA was significantly increased(P <0.05).(3)Under super-resolution fluorescence microscope,in situ,real-time and qualitative: the expression of ROCK2 in the model of Sudden coronary death was higher than that of the blank control group and coronary heart disease group;at the same time,the cells shrank,the volume became smaller,spindle and triangular cells increased.(4)Compared with the control group,apoptosis in the coronary heart disease group and the SCD group increased,and the Pearson chi-square values were 12.144 and 93.377,respectively,P <0.05.Compared with the coronary heart disease group,the apoptosis in the SCD cell model increased,and the Pearson chi-square value was 40.659,P<0.05.There was no statistically significant difference in apoptosis between the control group and SLX-2119(ROCK2-specific inhibitor group),P>0.05.(5)Quantitative detection at the molecular level: Compared with the control group,the relative expression of ROCK2 m RNA in coronary heart disease group and SCD group increased,P <0.05;the gray value of ROCK2/GAPDH increased,P <0.05.Compared with the coronary heart disease and SLx-2119 inhibitor groups,the relative expression of ROCK2 m RNA in the SCD group increased,P <0.05;the gray value of ROCK2/GAPDH increased,P <0.05.Compared with the control group,the relative expression of ROCK2 m RNA in SLx-2119 inhibitor group had no significant difference,P> 0.05;there was no significant statistical difference in ROCK2/GAPDH gray value,P> 0.05.Conclusions: ROCK2’s expression increases at the cells and molecules level of Sudden coronary death;ROCK2 as a core factor of coronary spasm is an important cause of cells and molecules. |
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