本文選題：印記基因　切入點(diǎn)：骨骼肌損傷　出處：《武漢體育學(xué)院》2017年碩士論文

【摘要】：目的:本研究的目的是通過(guò)測(cè)定印記基因Grb10和H19在骨骼肌急性損傷后的表達(dá)情況,來(lái)探究在骨骼肌損傷后的修復(fù)過(guò)程中,印記基因Grb10和H19與骨骼肌修復(fù)之間的關(guān)系。對(duì)象和方法:30只SD成年雄性大鼠,6周齡,戊巴比妥鈉麻醉,50mg/kg體重標(biāo)準(zhǔn)進(jìn)行腹腔注射。大鼠麻醉后,參照Kami、陳世益等大鼠骨骼肌鈍傷的實(shí)驗(yàn)?zāi)Ｐ椭谱鞣椒ā⒃炷３晒Φ拇笫筮M(jìn)行編號(hào)后,使用Excel隨機(jī)數(shù)產(chǎn)生法,將大鼠隨機(jī)分為五組,每組6只。取材時(shí)間分別在損傷后第2天、第4天、第6天、第8天和第10天。其中,實(shí)驗(yàn)組為鈍傷造模成功的大鼠左側(cè)腓腸肌中段組織,對(duì)照組為大鼠右側(cè)未損傷腓腸肌中段組織。取材后進(jìn)行常規(guī)包埋,固定,切片。通過(guò)HE染色觀察骨骼肌急性鈍傷后修復(fù)過(guò)程中的形態(tài)學(xué)變化;采取實(shí)時(shí)熒光定量PCR測(cè)定H19、Grb10 mRNA的表達(dá)轉(zhuǎn)錄情況;采用免疫印跡測(cè)定Grb10蛋白的表達(dá)情況。結(jié)果:通過(guò)HE染色切片的結(jié)果可以觀察到:骨骼肌損傷造模成功后取材發(fā)現(xiàn),肌纖維排列紊亂,第2天細(xì)胞核數(shù)量開(kāi)始增多,第4天則明顯多于對(duì)照組,第6天、第8天和第10天細(xì)胞核數(shù)量明顯比第四天減少;從實(shí)時(shí)熒光定量PCR測(cè)定結(jié)果來(lái)看:Grb10和H19 mRNA在損傷后的第2到第10天,對(duì)照組和實(shí)驗(yàn)組中均有表達(dá)。損傷后第2天,Grb10 mRNA的表達(dá)量有所升高(P0.05),到第4天其表達(dá)量持續(xù)升高并達(dá)到峰值(P0.01),第6天開(kāi)始其表達(dá)量明顯下降,第8天和第10天表達(dá)量趨于穩(wěn)定,與對(duì)照組基本持平(P0.05);骨骼肌損傷后第2天,H19 mRNA的相對(duì)表達(dá)量與對(duì)照組相比明顯下降,第4天,H19 mRNA相對(duì)表達(dá)量比對(duì)照組又有明顯升高(P0.01)。第6天,H19 mRNA相對(duì)表達(dá)量比第4天有明顯下降,第6天、第8天和第10天的H19 mRNA相對(duì)表達(dá)量基本趨于穩(wěn)定。Grb10的免疫印跡結(jié)果顯示:Grb10蛋白在骨骼肌損傷后的第2天到第4天呈上升趨勢(shì),第4天達(dá)到峰值(P0.01),之第6至10天,Grb10蛋白表達(dá)量比第4天明顯降低,并逐漸趨于穩(wěn)定。結(jié)論:1)印記基因Grb10、H19在骨骼肌急性損傷后的修復(fù)過(guò)程中,二者的表達(dá)量發(fā)生顯著變化,因此推斷印記基因Grb10、H19對(duì)骨骼肌急性損傷后的修復(fù)產(chǎn)生了一定的影響。2)印記基因Grb10、H19在骨骼肌急性損傷后的修復(fù)過(guò)程中,二者表達(dá)量的趨勢(shì)相似,因此推斷印記基因Grb10與H19對(duì)骨骼肌急性損傷后的修復(fù)可能存在一定的相關(guān)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.
【學(xué)位授予單位】：武漢體育學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R873

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