摘要
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＆#215;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。
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 ＆#215;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＆amp;nbsp;（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 fr
作者
权大君
曹政
杨媚
李莎
QUAN Dajun CAO Zheng YANG Mei LI Sha(Renmin Hospital of Wuhan University, Wuhan 430060, China)
出处
《山东医药》
CAS
北大核心
2016年第47期21-24,共4页
Shandong Medical Journal
基金
国家自然科学基金面上项目(81270249)
国家自然科学基金面上项目(81570306)
湖北省科技支撑计划项目(2013BCB013)
