印记基因（Grb10、H19）在骨骼肌急性损伤后修复过程中表达的研究

发布时间：2018-04-01 06:52

本文选题：印记基因　切入点：骨骼肌损伤　出处：《武汉体育学院》2017年硕士论文

【摘要】：目的:本研究的目的是通过测定印记基因Grb10和H19在骨骼肌急性损伤后的表达情况,来探究在骨骼肌损伤后的修复过程中,印记基因Grb10和H19与骨骼肌修复之间的关系。对象和方法:30只SD成年雄性大鼠,6周龄,戊巴比妥钠麻醉,50mg/kg体重标准进行腹腔注射。大鼠麻醉后,参照Kami、陈世益等大鼠骨骼肌钝伤的实验模型制作方法。将造模成功的大鼠进行编号后,使用Excel随机数产生法,将大鼠随机分为五组,每组6只。取材时间分别在损伤后第2天、第4天、第6天、第8天和第10天。其中,实验组为钝伤造模成功的大鼠左侧腓肠肌中段组织,对照组为大鼠右侧未损伤腓肠肌中段组织。取材后进行常规包埋,固定,切片。通过HE染色观察骨骼肌急性钝伤后修复过程中的形态学变化;采取实时荧光定量PCR测定H19、Grb10 mRNA的表达转录情况;采用免疫印迹测定Grb10蛋白的表达情况。结果:通过HE染色切片的结果可以观察到:骨骼肌损伤造模成功后取材发现,肌纤维排列紊乱,第2天细胞核数量开始增多,第4天则明显多于对照组,第6天、第8天和第10天细胞核数量明显比第四天减少;从实时荧光定量PCR测定结果来看:Grb10和H19 mRNA在损伤后的第2到第10天,对照组和实验组中均有表达。损伤后第2天,Grb10 mRNA的表达量有所升高(P0.05),到第4天其表达量持续升高并达到峰值(P0.01),第6天开始其表达量明显下降,第8天和第10天表达量趋于稳定,与对照组基本持平(P0.05);骨骼肌损伤后第2天,H19 mRNA的相对表达量与对照组相比明显下降,第4天,H19 mRNA相对表达量比对照组又有明显升高(P0.01)。第6天,H19 mRNA相对表达量比第4天有明显下降,第6天、第8天和第10天的H19 mRNA相对表达量基本趋于稳定。Grb10的免疫印迹结果显示:Grb10蛋白在骨骼肌损伤后的第2天到第4天呈上升趋势,第4天达到峰值(P0.01),之第6至10天,Grb10蛋白表达量比第4天明显降低,并逐渐趋于稳定。结论:1)印记基因Grb10、H19在骨骼肌急性损伤后的修复过程中,二者的表达量发生显著变化,因此推断印记基因Grb10、H19对骨骼肌急性损伤后的修复产生了一定的影响。2)印记基因Grb10、H19在骨骼肌急性损伤后的修复过程中,二者表达量的趋势相似,因此推断印记基因Grb10与H19对骨骼肌急性损伤后的修复可能存在一定的相关性。
[Abstract]:Aim: to investigate the relationship between imprinted genes Grb10 and H19 and skeletal muscle repair in the course of skeletal muscle injury by measuring the expression of imprinted genes Grb10 and H19 after acute skeletal muscle injury.Participants and methods 30 adult Sprague-Dawley male rats were injected intraperitoneally with pentobarbital sodium anaesthesia 50 mg / kg body weight standard.After anaesthesia, the experimental model of blunt skeletal muscle injury in rats was made with reference to Kamiand Chen Shiyi.After numbering the successful rats, the rats were randomly divided into five groups with 6 rats in each group by Excel random number generation method.The sampling time was 2 days, 4 days, 6 days, 8 days and 10 days after injury.The experimental group was the middle segment of the left gastrocnemius muscle and the control group was the middle segment of the right side of the gastrocnemius muscle.After the material is taken, the routine embedding, fixing and slicing are carried out.The morphological changes of skeletal muscle after acute blunt injury were observed by HE staining, the expression of H19Grb10 mRNA was measured by real-time fluorescence quantitative PCR, and the expression of Grb10 protein was detected by Western blot.Results: the results of HE staining showed that the muscle fibers were disordered, the number of nuclei began to increase on the second day, the number of nuclei on the 4th day was significantly higher than that of the control group, and on the 6th day, the number of the muscle fibers was disordered, and on the 4th day it was obviously more than that of the control group.The number of nuclear cells on the 8th and 10th day was significantly lower than that on the fourth day, and the results of real-time fluorescence quantitative PCR showed that the expression of mRNA in the control group and the experimental group was observed from the 2nd to the 10th day after injury.On the second day after injury, the expression of Grb10 mRNA increased a little, but on the 4th day, the expression level continued to increase and reached the peak value (P0.01A). At the 6th day, the expression level of GRB 10 mRNA decreased obviously, and the expression level on the 8th day and 10th day tended to be stable.On the second day after skeletal muscle injury, the relative expression of H19 mRNA was significantly lower than that of the control group, and the relative expression of H19 mRNA on the 4th day was significantly higher than that of the control group.On the 6th day, the relative expression of H19 mRNA was significantly lower than that on the 4th day, and on the 6th day,On the 8th and 10th day, the relative expression of H19 mRNA tended to be stable. The results of Western blot showed that the expression of the protein showed an upward trend from the 2nd to the 4th day after skeletal muscle injury.On the 4th day, the peak value of P0.01 was reached, and the expression of Grb10 protein decreased significantly from the 6th to the 10th day compared with that on the 4th day, and gradually became stable.Conclusion during the repair of skeletal muscle after acute injury, the expression of Grb10H19, a molecular marker gene, changed significantly.It is inferred that the imprinting gene Grb10H19 has a certain effect on the repair of skeletal muscle after acute injury. 2) the trend of the expression of Grb10H19 is similar to that of Grb10H19 during the repair of skeletal muscle after acute injury.It is concluded that the imprinting gene Grb10 may be related to the repair of skeletal muscle after acute injury.
【学位授予单位】：武汉体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R873

【参考文献】