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Objective@#To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes.@*Methods@#The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups: control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry.@*Results@#At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs. (0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h: 0.32±0.06 vs. 0.46±0.03; 72 h: 0.31±0.01 vs. 0.63±0.04; 96 h: 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h post-transfection with Tnni3-siRNA, the percentage of G1 phase, S phase and G2 phase cells was (71.25±3.82)%, (18.28±2.78)% and (9.94±1.09)%, respectively.There was a significant increase in the proportion of G2 phase cells [(9.94±1.09)% vs. (4.54±0.99)%] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA, an increased expression of Cyclin A1 protein (1.89±0.09 vs.1.00±0.00) and a decreased expression of Cyclin B1 protein (0.47±0.06 vs.1.00±0.00) were observed in Tnni3-siRNA-transfected H9C2 cells, respectively, and the differences were statistically significant (t=6.35, 17.12, 15.32, all P<0.01).@*Conclusions@#Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis, suppress cell proliferation, and led to G2 cell cycle arrest.
ABSTRACT
Objective To investigate the effect of silencing troponin Ⅰ3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes.Methods The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups:control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group).At 48 h,72 h after transfection,the cells were collected,and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3,and Western blot was used to detect the protein expressions of Tnni3,Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry.Results At 48 h post-transfection with Tnni3-siRNA,H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27 ± 0.05 vs.1.00 ± 0.00) and protein (0.18 ± 0.03 vs.1.00 ± 0.00) compared with those transfected with NC-siRNA,and the differences were statistically significant (t =25.26,47.40,all P ＜ 0.01).Apoptotic cells were observed in the NC-siRNA group and the Tnai3-siRNA group.At 72 h post-transfection,the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30 ± 1.85) ％ vs.(0.33 ± 0.15) ％] compared with those transfected with NC-siRNA,an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39 ±0.13 vs.1.00 ±0.00),and the differences were statistically significant (t =10.24,5.19,all P ＜ 0.01).Compared with NC-siRNA-transfected H9C2 cells,a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h:0.32 ± 0.06 vs.0.46 ± 0.03;72 h:0.31 ± 0.01 vs.0.63 ±0.04;96 h:0.36 ± 0.01 vs 0.75 ± 0.04),and the differences were statistically significant (t =3.62,13.45,16.39,all P ＜ 0.01).At 72 h post-transfection with Tnni3-siRNA,the percentage of G1 phase,S phase and G2 phase cells was (71.25 ± 3.82) ％,(18.28 ± 2.78) ％ and (9.94 ± 1.09) ％,respectively.There was a significant increase in the proportion of G2 phase cells [(9.94 ± 1.09) ％ vs.(4.54 ±0.99) ％] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA,an increased expression of Cyclin A1 protein (1.89 ±0.09 vs.1.00 ±0.00) and a decreased expression of Cyclin B1 protein (0.47 ± 0.06 vs.1.00 ± 0.00) were observed in Tnni3-siRNA-transfected H9C2 cells,respectively,and the differences were statistically significant (t =6.35,17.12,15.32,all P＜0.01).Conclusions Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis,suppress cell proliferation,and led to G2 cell cycle arrest.
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OBJECTIVE:To explore the effectiveness and safety of bicyclol combined with ganciclovir in the treatment of infan-tile cytomegaloirus hepatitis. METHODS:One hundred and twenty children with cytomegaloirus hepatitis in department of pediatrics of our hospital during May 2012-Aug. 2015 were selected and divided into observation group and control group according to random number table,with 60 cases in each group. Both groups received conventional treatment such as protecting liver,vitamin C,vitamin K and Compound glycyrrhizin injection 20 mL,ivgtt,qd. Control group additionally received Ganciclovir injection(induction period:5 mg/kg,q12 h,dripping time >1 h,for 7 d;maintenance period:5 mg/kg,q24 h,for 7 d);observation group was additionally giv-en Bicyclol tablet 0.5 mg/kg,bid,on the basis of control group. Clinical efficacies of 2 groups were observed as well as liver enzyme level,jaundice level before and after treatment. The rate of negative CMV and the occurrence of ADR were compared. RESULTS:Clinical total response rate of observation group was 93.3%,which was significantly higher than 80.0% of control group,with statisti-cal significance(P0.05). After treatment,liver enzyme level and jaundice level of 2 groups were decreased significantly,and observation group was significantly lower than control group,with statistical significance(P0.05). CONCLUSIONS:Bicyclol combined with ganci-clovir shows significant therapeutic efficacy for infantile cytomegaloirus hepatitis,and can effectively reduce the levels of liver en-zymes,eliminate jaundice,protect liver function,promote virus clearance with good safety.