Objective To observe the expression of HCN4 protein,If and its effect on the pulsation frequency of car-diomyocytes in cardiac fibroblasts transfected with human TBX18 gene.Methods The hearts were extracted after decapi-tation of the newborn SD rats,and the fibroblasts and cardiomyocytes were isolated and cultured.Adenovirus vector Ad-GFP-TBX18 was constructed and the virus titer was purified to 1 ×1010 TU/mL.The fibroblasts were divided into groups A,B and C at the time of passage 2 to 5.After 24 hours of culture,groups A and B were transfected with Ad-GFP-TBX18 and pHBAd-MCMV- GFP,respectively,and group C without any treatment.The mRNA expression of TBX18 mRNA was detected by RT-PCR.The hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4)protein was detec-ted by Western blotting.The hyperpolarization activated inward current (If)was recorded by voltage clamp.The fibroblasts of each group were co-cultured with neonatal rat cardiomyocytes to observe the frequency of cardiomyocytes in neonatal rats. Results The relative expression levels of TBX18 mRNA in the groups A,B and C were 98.32 ±6.21,1.03 ±0.05 and 1.02 ±0.02,respectively.The relative expression of TBX18 mRNA in the group A was higher than that in the groups B and C (all P<0.05).There was no significant difference in the relative expression of TBX18 mRNA between group B and group C.The relative expression of HCN4 protein in the groups A,B and C were 0.49 ±1.21,0.12 ±0.02,and 0.09 ±0.01,respectively.The relative expression of HCN4 protein in the group A was higher than that in the groups B and C (P<0.05).There was no significant difference in the relative expression of HCN4 protein between group B and group C. About 30% of the cells in group A were able to detect If,whereas in the groups B and C we did not detect If.At the same time,the frequency of cardiomyocyte pulsation in group A was higher than that in the groups B and C (all P<0.05 ). There was no significant difference in the frequency of cardiomyocytes between groups B and C.Conclusion HCN4 protein is highly expressed in rat cardiac fibroblasts transfected with human TBX18 gene and the If currents are present,which may promote the pulsation of neonatal rat cardiomyocytes.%目的：观察转染人TBX18基因的大鼠心脏成纤维细胞HCN4蛋白表达、超极化激活内向离子电流（If）及对心肌细胞搏动频率的影响。方法取新生SD大鼠，断头处死后取心脏，分离培养成纤维细胞及心肌细胞。构建人TBX18基因的腺病毒载体Ad-GFP-TBX18，包装并纯化病毒滴度至1×1010 TU／mL。取传代2～5代时对数生长期新生大鼠成纤维细胞分为A、B、C组，培养24 h后A、B组分别转染Ad-GFP-TBX18、腺病毒载体pHBAd-MCMV-GFP（阴性对照），C组不做任何处理。采用RT-PCR法检测各组细胞TBX18 mRNA。采用Western blotting法检测各组超极化激活环核苷酸门控阳离子通道4（HCN4）蛋白。采用电压钳记录各组细胞超极化激活内向离子电流（If）。将各组细胞与新生大鼠心肌细胞共培养，观察新生大鼠心肌细胞搏动频率。结果 A、B、C组TBX18 mRNA相对表达量分别为98．32±6．21、1．03±0．05、1．02±0．02。A组TBX18 mRNA相对表达量高于B、C组（P均＜0．05）；B、C 组TBX18 mRNA相对表达量差异无统计学意义。A、B、C组HCN4蛋白相对表达量分别为0．49±1．21、0．12±0．02、0．09±0．01。A组HCN4蛋白相对表达量高于B、C组（P均＜0．05）；B、C组HCN4蛋白相对表达量差异无统计学意义。A组约有30％的细胞可以检测到If，而B、C组中没有细胞检测到If。同一时点A组新生大鼠心肌细胞搏动频率高于B、C组（P均＜0．05）；B、C组新生大鼠心肌细胞搏动频率差异无统计学意义。结论转染人TBX18基因的大鼠心脏成纤维细胞高表达HCN4蛋白，存在If电流，并能促进共培养新生大鼠心肌细胞的搏动。
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