大鼠骨骼肌挫傷后ASCT2 mRNA時序性表達研究
發(fā)布時間：2018-08-12 10:23
【摘要】：目的： 應(yīng)用實時定量PCR技術(shù)檢測大鼠骨骼肌挫傷后細胞內(nèi)ASCT2mRNA表達,分析其與挫傷的時間關(guān)系,旨為法醫(yī)學早期損傷時間的推斷探尋新的思路。 方法： (1)選取成年雄性Sprague-Dawley大鼠96只,并且將其隨機分成3組,包括：生前損傷組(72只)、正常對照組(6只)、死后損傷組(18只)。生前損傷組動物經(jīng)麻醉、固定于鼠板和褪毛后,將重力錘自由落下打擊右后肢大腿內(nèi)側(cè)肌群致骨骼肌挫傷,分別于傷后4、8、12、16、20、24、28、32、36、40、44、48小時迅速頸椎脫臼處死,并取材。在生前損傷12、24、36小時組動物左后肢大腿內(nèi)側(cè)骨骼肌取材,以比較正常肌肉和損傷肌肉之間ASCT2mRNA表達水平。死后損傷組動物按照處死并打擊致挫傷之后的時間間隔不同,分別6、12、18小時三個時間點取材。正常對照組動物未經(jīng)打擊,直接頸椎脫臼處死；(2)使用Invitrogen TRIzol Reagent試劑盒提取肌肉組織中總RNA,并且使用紫外分光光度計對總RNA純度進行檢驗,并根據(jù)紫外分光光度計所測得OD260/OD280的比值來評價核酸純度；(3)將RNA逆轉(zhuǎn)錄合成cDNA,再以cDNA為模板、RPL13為內(nèi)參基因進行Real.time PCR,采用2＾-△△CT法比較其與正常肌肉組織中ASCT2mRNA的相對表達量。 結(jié)果： (1)根據(jù)紫外分光光度計所測得結(jié)果提示：總RNA完整性較理想,符合后續(xù)實驗的要求；(2)目的基因與內(nèi)參基因的擴增效率分別為89.5%和90.1%,相關(guān)系數(shù)均大于理想值0.98,表明直線性較好,定量準確,擴增效率較高且較為一致,表明RPL13作為ASCT2mRNA的內(nèi)參基因較為合適；(3)損傷組肌肉組織中ASCT2mRNA在挫傷后12、16、36、40小時表達量分別為正常組的196.40%、189.15%、182.54%和136.65%,損傷后4、8、20、24、28、32、44、48小時ASCT2mRNA表達量與正常組相比無統(tǒng)計學差異(P0.05),說明ASCT2mRNA在損傷12至16小時其表達量迎來第一個高峰,隨后又降至正常水平,而到了損傷后36至40小時表達量又迎來第二個高峰,之后回落并趨于穩(wěn)定；(4)生前損傷組正常肌肉ASCT2mRNA的表達相對于正常對照組,二者并無差異(P0.05)；(5)正常對照組與死后損傷組的各個時間點之間的ASCT2mRNA表達量并不存在差異(P0.05)。 結(jié)論： (1)ASCT2mRNA表達水平在生前損傷組正常肌肉和正常對照組肌肉之間不存在差異,說明同一個體未受損傷的肌肉組織可以作為評價挫傷肌肉組織的參照檢材；(2)與正常組相比,死后損傷組的RNA盡管出現(xiàn)了降解,但是在死后的6-18小時內(nèi)ASCT2mRNA的表達水平趨于穩(wěn)定,說明不影響實驗結(jié)果。(3)大鼠骨骼肌挫傷后48小時內(nèi)ASCT2mRNA呈時序性表達,有希望成為推斷骨骼肌損傷時間的新指標。
[Abstract]:Objective: to detect the expression of ASCT2mRNA in rat skeletal muscle contusion cells by real-time quantitative PCR, and to analyze the relationship between the expression of ASCT2mRNA and the time of contusion, so as to find a new way to infer the time of early injury in forensic medicine. Methods: (1) 96 adult male Sprague-Dawley rats were randomly divided into 3 groups: antemortem injury group (72 rats), normal control group (6 rats) and postmortem injury group (18 rats). After anaesthesia, fixation on the rat plate and hair loss, the injured animals in the antemortem group dropped the gravity hammer freely and hit the medial thigh muscle group of the right hind limb to cause contusion of skeletal muscle. The contusion of skeletal muscle in the right hind limb was carried out in 48 hours after the injury, and the cervical vertebrae was dislocated and executed for 48 hours. The medial thigh skeletal muscle of the left hind limb was collected from the animals in the group of 12 ~ 24 ~ 36 hours of antemortem injury to compare the expression level of ASCT2mRNA between the normal muscle and the injured muscle. In postmortem injury group, the time interval after death was different, and the time interval was 612 hours and 18 hours, respectively. The normal control group was killed without attack. (2) Invitrogen TRIzol Reagent kit was used to extract total RNAs from muscle tissue, and ultraviolet spectrophotometer was used to test the purity of total RNA. The purity of nucleic acid was evaluated according to the ratio of OD260/OD280 measured by UV spectrophotometer. (3) reverse transcription of RNA was used to synthesize cDNA. then cDNA was used as internal reference gene for Real.time PCR. The relative expression of ASCT2mRNA was compared with that in normal muscle tissue by 2 ^ -CT. Results: (1) according to the results of ultraviolet spectrophotometer, the integrity of total RNA is ideal, which meets the requirements of subsequent experiments; (2) the amplification efficiency of target gene and internal reference gene were 89.5% and 90.1%, respectively, and the correlation coefficient was higher than that of ideal value 0.98, which indicated that the linearity was better, the quantity was accurate, the amplification efficiency was higher and the amplification efficiency was consistent, which indicated that RPL13 was more suitable as the internal reference gene of ASCT2mRNA. (3) the expression of ASCT2mRNA in muscle tissue of the injured group was 182.54% and 136.65% of the normal group at 12 ~ 1616 ~ 36 ~ 40 hours after contusion, respectively. The expression of ASCT2mRNA in the muscle tissue of the injury group was not significantly different from that of the normal group at 48 hours after injury (P0.05), which indicated that the expression of ASCT2mRNA reached the first peak at 12 to 16 hours after injury. Then it decreased to normal level, and then reached the second peak 36 to 40 hours after injury, then decreased and tended to stabilize; (4) the expression of ASCT2mRNA in normal muscle in antemortem injury group was not different from that in normal control group (P0.05). (5) there was no difference in ASCT2mRNA expression between normal control group and postmortem injury group (P0.05). Conclusion: (1) there is no difference in the expression of ASCT2mRNA between normal muscle and normal muscle in antemortem injury group, indicating that the muscle tissue without injury in the same body can be used as a reference material for evaluating contusion muscle tissue. (2) compared with the normal group, the expression of RNA in postmortem injury group tended to be stable within 6-18 hours of postmortem injury, indicating that it did not affect the experimental results. (3) the expression of ASCT2mRNA in rat skeletal muscle was time-sequentially expressed within 48 hours after contusion of skeletal muscle. Hopefully, it will be a new indicator for estimating the time of skeletal muscle injury.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：D919.4
本文編號：2178761
[Abstract]:Objective: to detect the expression of ASCT2mRNA in rat skeletal muscle contusion cells by real-time quantitative PCR, and to analyze the relationship between the expression of ASCT2mRNA and the time of contusion, so as to find a new way to infer the time of early injury in forensic medicine. Methods: (1) 96 adult male Sprague-Dawley rats were randomly divided into 3 groups: antemortem injury group (72 rats), normal control group (6 rats) and postmortem injury group (18 rats). After anaesthesia, fixation on the rat plate and hair loss, the injured animals in the antemortem group dropped the gravity hammer freely and hit the medial thigh muscle group of the right hind limb to cause contusion of skeletal muscle. The contusion of skeletal muscle in the right hind limb was carried out in 48 hours after the injury, and the cervical vertebrae was dislocated and executed for 48 hours. The medial thigh skeletal muscle of the left hind limb was collected from the animals in the group of 12 ~ 24 ~ 36 hours of antemortem injury to compare the expression level of ASCT2mRNA between the normal muscle and the injured muscle. In postmortem injury group, the time interval after death was different, and the time interval was 612 hours and 18 hours, respectively. The normal control group was killed without attack. (2) Invitrogen TRIzol Reagent kit was used to extract total RNAs from muscle tissue, and ultraviolet spectrophotometer was used to test the purity of total RNA. The purity of nucleic acid was evaluated according to the ratio of OD260/OD280 measured by UV spectrophotometer. (3) reverse transcription of RNA was used to synthesize cDNA. then cDNA was used as internal reference gene for Real.time PCR. The relative expression of ASCT2mRNA was compared with that in normal muscle tissue by 2 ^ -CT. Results: (1) according to the results of ultraviolet spectrophotometer, the integrity of total RNA is ideal, which meets the requirements of subsequent experiments; (2) the amplification efficiency of target gene and internal reference gene were 89.5% and 90.1%, respectively, and the correlation coefficient was higher than that of ideal value 0.98, which indicated that the linearity was better, the quantity was accurate, the amplification efficiency was higher and the amplification efficiency was consistent, which indicated that RPL13 was more suitable as the internal reference gene of ASCT2mRNA. (3) the expression of ASCT2mRNA in muscle tissue of the injured group was 182.54% and 136.65% of the normal group at 12 ~ 1616 ~ 36 ~ 40 hours after contusion, respectively. The expression of ASCT2mRNA in the muscle tissue of the injury group was not significantly different from that of the normal group at 48 hours after injury (P0.05), which indicated that the expression of ASCT2mRNA reached the first peak at 12 to 16 hours after injury. Then it decreased to normal level, and then reached the second peak 36 to 40 hours after injury, then decreased and tended to stabilize; (4) the expression of ASCT2mRNA in normal muscle in antemortem injury group was not different from that in normal control group (P0.05). (5) there was no difference in ASCT2mRNA expression between normal control group and postmortem injury group (P0.05). Conclusion: (1) there is no difference in the expression of ASCT2mRNA between normal muscle and normal muscle in antemortem injury group, indicating that the muscle tissue without injury in the same body can be used as a reference material for evaluating contusion muscle tissue. (2) compared with the normal group, the expression of RNA in postmortem injury group tended to be stable within 6-18 hours of postmortem injury, indicating that it did not affect the experimental results. (3) the expression of ASCT2mRNA in rat skeletal muscle was time-sequentially expressed within 48 hours after contusion of skeletal muscle. Hopefully, it will be a new indicator for estimating the time of skeletal muscle injury.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：D919.4
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