Time-dependent expression of SNAT2 mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation
Time-dependent expression of SNAT2 mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation
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DOI：
10.1007/s12024-013-9482-y
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发表时间：
2013-12-01
影响因子：
1.8
通讯作者：
Wang, Ying-yuan
中科院分区：
文献类型：
--
作者：
Du, Qiu-xiang;Sun, Jun-hong;Wang, Ying-yuan
采用实时荧光定量聚合酶链反应（real-time polymerase chain reaction，PCR）检测骨骼肌挫伤后SNAT 2 mRNA的表达，以判断骨骼肌挫伤的年龄。将78只雄性SD大鼠分为对照组和挫伤组。挫伤后4、8、12、16、20、24、28、32、36、40、44和48 h（n = 6），用致死剂量的戊巴比妥处死大鼠。另24只大鼠分别于死后6、12、18和24 h行脑挫伤（n = 6）。使用TRIzol试剂从肌肉标本中分离总RNA并逆转录成第一链cDNA。利用AlleleID 6软件设计SNAT 2和RPL 13 mRNA的序列特异性引物和TaqMan荧光探针，采用实时荧光定量PCR检测SNAT 2 mRNA的表达水平。伤后4、16、20和24 h，与对照组相比，RPL 13 mRNA标准化的SNAT 2 mRNA表达水平分别增加了2.07（P <0.05）、2.53（P <0.05）、2.68（P < 0.05）和2.06倍（P < 0.05）。而在挫伤后24 ~ 48 h，SNAT 2 mRNA的表达水平与RPL 13 mRNA相比无显著性差异（P > 0.05）。同一损伤大鼠左侧肢体正常骨骼肌SNAT 2 mRNA表达水平与对照组无明显差异。死后各时间点SNAT 2 mRNA均未发生降解（P > 0.05）。这一结果表明，通过实时PCR测定SNAT 2 mRNA水平可能有助于估计伤口年龄。
To estimate the age of skeletal muscle contusion, the expression of SNAT2 mRNA in contused skeletal muscle of rats was detected by real-time polymerase chain reaction (PCR). In total, 78 Sprague-Dawley male rats were divided into control and contusion groups. At 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h (n = 6) after contusion, the rats were sacrificed with a lethal dose of pentobarbital. Another 24 rats received contusion injuries at 6, 12, 18, and 24 h (n = 6) after death. Total RNA was isolated from muscle specimens using the TRIzol reagent and reverse-transcribed into first-strand cDNA. Sequence-specific primers and TaqMan fluorogenic probes for SNAT2 mRNA and RPL13 mRNA were designed using the AlleleID 6 software, and the expression levels of SNAT2 mRNA were determined by real-time PCR. At 4, 16, 20, and 24 h after contusion, expression levels of SNAT2 mRNA normalized to RPL13 mRNA increased by 2.07 (P < 0.05), 2.53 (P < 0.05), 2.68 (P < 0.05), and 2.06 fold (P < 0.05) respectively, versus that in the control group. However, there was no significant change in the expression level of SNAT2 mRNA from 24 to 48 h (P > 0.05) after contusion, when normalized to RPL13 mRNA. There was no change in the expression level of SNAT2 mRNA between the normal skeletal muscle from the left limb of the same injured rat and the control. Also, no degradation of SNAT2 mRNA was detected in the postmortem samples (P > 0.05). This result suggests that the determination of SNAT2 mRNA levels by real-time PCR may be useful for estimating wound age.